The binding properties of rabbit antibodies against various penicillins as studied by immunoprecipitation, indirect haemagglutination and radiometric immunoassay.

Rabbit antibodies raised against protein conjugates of azidocillin (AzO), ampicillin (AmpO), benzylpenicillin (BPO) and crude 6-aminopenicillanic acid (6-APA) gave rise to two precipitation lines in immunoprecipitation tests against penicillin protein conjugates where the carrier was unrelated to the immunogens. This pattern verifies extensive cross-reactions between penicillins, which have previously been reported. The use of AzO-coated rabbit red cells in indirect haemagglutination (IHA) inhibition tests showed good reactivity (a) between the inhibitor AzO and antibodies raised against AzO, AmpO and BPO, (b) between the inhibitor AmpO and antibodies raised against AmpO and BPO, (c) between the inhibitor BPO and antibodies raised against BPO. When BPO-coated red cells were used, AzO, AmpO and BPO inhibited the IHA activity of antibodies against the BPO. Varying IHA results were obtained using differently conjugated rabbit red cells. Thus antibodies against AmpO gave higher titres when using AzO- than BPO-coated red cells while for antibodies against BPO the opposite was noted. The radiometric assay utilizing AzO-serum albumin as tracer as well as reference substance resulted in significantly higher values for antisera against AzO and AmpO compared with those for antisera against crude 6-APA. Antisera against BPO were not analysed in this respect. Furthermore, there was poor correlation between the results obtained with the indirect IHA technique and the radiometric immunoassay.


Differential induction of Epstein-Barr virus-related antigens in human lymphoblastoid cells by virus-mediated cell-to-cell contact.

Differential induction of Epstein-Barr virus (EBV)-related antigens was observed after Sendai virus-medicated fusion of producer and non-producer cells with various human and mouse cells. The EBV-determined early nuclear antigen (ENA), early antigen (EA) and viral capsid antigen (VCA) could be induced at a high rate in producer cells (P3HR-1 and B95-8), normally expressing these antigens at very low frequency, as early as 12-24 h after fusion with each other or with human amnion FL cells. In contrast, only one antigen, ENA, was induced in producer cells following fusion with non-producer cells. This limited induction occurred also in non-producer cells fused with FL cells, but not after fusion with each other. EBV induction did not result from fusion of either producer or non-producer cells with mouse cells (L-M (TK-) Cl1D and MCB-L). The differential induction was not the result of heterokaryon formation as determined by autoradiography. The implications of these findings are discussed.


RNA associated with non-histone chromosomal proteins in rat liver and in ascites hepatoma.

The purpose of the experiments was to determine if changes in the post-transcriptional processing of RNA in the hepatoma also affect the low molecular weight nuclear RNA which is transcribed from families of related genes and associated with non-histone chromosomal proteins. Separation of non-histone chromosomal proteins on Sephadex G-200 into three fractions separated the low molecular weight RNA associated with these proteins into metabolically stable RNA firmly bound to the first eluted fractions of high molecular weight non-histone chromosomal proteins, and into a metabolically active RNA which is eluted with the third protein fraction and can be separated from the low molecular weight non-histone chromosomal proteins by chromatography on DEAE-Sephadex A 25 (fraction III RNA). In liver as well as in hepatoma, this fraction III RNA represents about 50% of the RNA associated with the non-histone chromosomal proteins. Fraction III RNA from both tissues has an approximate molecular weight of 13,000, is rich in guanylic acid, is lacking dihydropyrimidines and is copied from the repetitive sequences of DNA. The content of uridylic acid is much higher in fraction III RNA isolated from hepatoma than in the same RNA isolated from liver, and competitive hybridization has shown that hepatoma fraction III RNA contains not only new base sequences which are not present in liver fraction III RNA, but also lacks some sequences which are present in the liver RNA. The technique of RNA/DNA hybridization in the presence of competing RNA has shown that, in hepatoma, the cytoplasmic RNA competes with more than 60% of the fraction III RNA for the hybridization sites on repetitive DNA. No competition was found when liver cytoplasmic RNA was used. The low ratio of competing hepatoma cytoplasmic RNA or of liver or hepatoma nuclear RNA which is required to displace fraction III RNA from its hybridization with DNA indicates that this RNA is synthesized and in hepatoma is also released into the cytoplasm as a part of larger RNA molecules. The detection of the nucleotide sequences found in liver associated with non-histone chromosomal proteins in the cytoplasm of hepatoma cells is evidence for extensive disruption of the post-transcriptional control in hepatoma.


The detection of virus DNA sequences in a herpes type 2 transformed hamster cell line (333-8-9).

Herpes simplex virus type 2 DNA labelled in vitro with 125I has been used as a probe to search for virus DNA sequences in the 333-8-9 line of transformed primary hamster cells and in clines derived from the line. Virus DNA sequences were present when the cells were examined initially but these sequences were lost on subsequent handling. No virus sequences were detected in clones derived from the line. It is suggested that the presence of virus DNA is an unstable characteristic of the cell line and that these sequences may be lost at cell division. The possibility that a small fragment of the genome remains cannot be excluded.


Clonal derivatives of a herpes type 2 transformed hamster cell line (333-8-9): cytogenetic analysis, tumorigenicity and virus sequence detection.

The characteristics of the Syrian hamster cell line (333-8-9), putatively transformed by herpes virus type 2 (HSV-2), strain 333, have been examined. The line has been cloned and the clones characterized cytogenetically and morphologically. The original line and the clones have been assayed for tumorigenicity and for the presence of herpes-virus-specific nucleic acid sequences using in situ hybridization with 125I HSV-2 DNA. The line 333-8-9 is hyperdiploid with a mode of 49 chromosomes and contains a spectrum of cytogenetic marker chromosomes present in varying frequencies. The clones demonstrate a segregation of these marker chromosomes and other abnormal chromosomes. Cloned lines have distinct stem-cell types with the characteristic abnormal chromosomes present in a high percentage of cells. Some of the marker chromosomes have been identified by Giemsa  banding techniques. All the clones have distinctive, persistent in vitro morphologies, ranging from purely epithelial to purely fibroblastic. They differ from the line 333-8-9 and between themselves in tumorigenicity, varying from highly tumorigenic to entirely non-tumorigenic. Herpes virus 2 specific sequences have been detected in the original cell line and in all but two of the clones. There does not appear to be any obvious correlation between the three parameters of: (1) detection of HSV-2 information; (2) marker chromosome occurrence; and (3) tumorigenicity.


Characterization of an early cytotoxicity-inducing factor in sera of Japanese quails after inoculation with Rous sarcoma virus.

By pre-treatment with serum of normal spleen cells used in the microcytotoxicity assay, a humoral factor which induces cytotoxic activity in normal spleen cells was demonstrated in about 40% of sera of Japanese quails as early as 3 days after inoculation with Rous sarcoma virus (RSV). This cytotoxicity-inducing activity was not present either in the IgM or the IgG fraction obtained by Sephadex gel filtration. In sera of quails with experimentally induced agammaglobulinemia, the cytotoxicity-inducing activity was demonstrated at the same frequency as in normal animals. Thus, it seems unlikely that the early cytotoxicity-inducing factor is an immunoglobulin. On the other hand, membrane fractions extracted with 3 M potassium chloride from the RSV-induced quail tumor (QT) cells used as target cells in the microcytotoxicity assay exhibited cytotoxicity-inducing activity. After spontaneous regression of an RSV-induced tumor the serum of the regressor quail contained antibodies specific to the QT cell extract. This serum removed the cytotoxicity-inducing activity of both QT cell extract and serum sampled 3 days after RSV inoculation. In contrast, serum without antibody to the QT cell extract failed to absorb the cytotoxicity-inducing activity of the extract. It is therefore suggested that soluble tumor antigens can act as an early cytotoxicity-inducing factor.


Tumorigenicity of Epstein-Barr virus (EBV)-transformed lymphoid line cells in autologous squirrel monkeys.

Eight squirrel monkeys (Saimiri sciureus) challenged with EBV or EBV-transformed SLCL were naturally or experimentally infected with Plasmodium knowlesi or Pl. brasilianum. Most of the animals had been splenectomized and unilaterally nephrectomized. Three of these monkeys received one dose of 6 to 12 X 10(8) autologous SLCL. These lines were derived from saimiri lymphoid cells permanently transformed by B-EBV in vitro. All three animals developed multiple undifferentiated malignant lymphomas and died 8 to 10 days post inoculation. Necropsy tumor specimens were EBNA-positive and contained 7 to 21 EBV genome equivalents per cell. EBNA- and EA-positive SLCL were established in vitro from four tumor explants of two monkeys. These results demonstrate that in vitro EBV-transformed SLCL are able to cause tumor formation in autologous squirrel monkeys. Five monkeys received high transforming doses of B-EBV or S-EBV, derived from one of the tumorigenic SLCL. None of the animals developed any sign of tumor formation during an observation period of up to 130 days. Three of these monkeys showed no detectable EBV-related seroconversion while the sera of two monkeys had become anti-EBNA-positive when tested at days 28 and 130 respectively post inoculation. Two additional monkeys received neither EBV nor SLCL. They showed no clinical evidence of tumor development or spontaneous seroconversion over a period of more than 1 year.


N-acetylserine in horse muscle  acylphosphatase.

A ninhydrin-negative peptide fraction obtained from tryptic digest of carboxymethyl acylphosphatase was isolated by chromatography on a column of PA 28 Beckman resin and analysed for the amino acid composition. Degradation with carboxypeptidase B and A indicated that the sequence of this peptide was: X-Thr-Ala-Arg. The amino-terminal residue was identified as N-acetylserine by high voltage electrophoresis. It is therefore suggested that the sequence of the NH2-terminal portion of CM-acylphosphatase is N-acetyl-Ser-Thr-Ala-Arg. Digestion with carboxypeptidase A and B indicated also that the COOH-terminal portion of CM-acylphosphatase is-Arg-Tyr-OH.


Appearance of Epstein-Barr virus-determined nuclear antigen in human epithelial cells following fusion with lymphoid cells.

When human epithelial FL cells were fused with lymphoid cells harboring Epstein-Barr virus (EBV) genome, EBV-determined nuclear antigen (EBNA) immunofluorescence became evident in high frequency in FL nuclei of heterokaryons several days after fusion. The implications of the findings are discussed in relation to the presence of EBV genome in nasopharyngeal carcinoma cells.


The effect of culture on ultrastructure of dissociated rabbit adenohypophysial cells.

Cells from the rabbit pars distalis and pars intermedia have been cultured in several ways. In clump cultures the glandular adenohypophysial cells of the primary clump remain identifiable for long periods. Both acidophil and mucoid cells can be maintained in this way for over 50 days. During this time two different modes of granule extrusion are observed, one being found only in acidophil cells and the other being exclusive to mucoid cells. From this it is concluded that specific release of secretory products from these cells can occur in the absence of hypothalmic control. A special form of collagenous fibre is produced in clump cultures. Monolayer cell cultures are derived either from isolated adenohypophysial cells or from secondarily aggregated clumps, but in either case the predominant cells are fibroblasts, which persist for over 50 days (as isolated cultures) and for over 90 days (as spreads from clumps). Rounded cells can be recognized as a separate type in isolated cell culture.


Ultrastructural features of human cerebral cortex.

Neuronal and glial elements have been examined in the temporal or frontal cortex of four brains with normal parenchyma. The identifying features recognized in animal brains have been found valid in human cortex for stellate and pyramidal neurons and their dendrites, spines, synapses, initial segments of axons, nodes of Ranvier and axon terminals; also for astrocytes, oligodendrocytes of various electron densities, pericytes, blood vessels and basement lamina, and for the relation of subarachnoid space to arterioles. However, most microglial cells have unexpectedly sparse and pale cytoplasm with few organelles, and the distribution of glial nuclear chromatin is not entirely in accordance with that described in animal brains. Fusiform neurons have been identified, and their cytoplasm resembles that of stellate cells. Peculiarities of human cortex include a nuclear inclusion that is present in stellate neurons only. Two neuronal cell-body profiles were myelinated, and a blood capillary was seen to penetrate a neuron. Somatic spines were found occasionally with or without synapses. Ribosomes can be present in axons, espicially at nodes of Ranvier, and a small portion of axon terminals contain a variety of dense bodies of which some are derived from mitochondria. Endoplasmic reticulum can occur in a compressed stack in neurons, and an unusual vesicular organelle has been seen in dendrites, axons and somata. Astrocytes as well as oligodendrocytes and occasional microglial cells act as satellites to neurons, and the exchange of a membrane-bound body between a neuron and asatellite glial cell is illustrated. Some dark pycnotic neurons were present, and it was remarkable that a closely apposed neuron could be entirely normal. These findings are compared with published descriptions of cortical ultrastructure in the brains of laboratory animals.


Synthesis of (beta-methyl-3H-)-benzylpenicillin and (beta-methyl-3H)-6-aminopenicillanic acid.

The synthesis of benzylpenicillin and 6-aminopenicillanic acid, labeled with tritium in the beta-methyl group, is described. Benzylpenicillin S-sulfoxide benzyl ester is refluxed in benzene and tritiated water and is successively debenzylated and deoxygenated to (beta-methyl-3H)-benzylpenicillin. Removal of the side chain with a bacterial acylase gives (beta-methyl-3H)-6-aminopenicillanic acid.


Comparison of herpes simplex virus isolates using a quantitative selection assay for transformation.

Transformation potential of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) was quantitatively compared in a system selecting for biochemical transformants. The highest frequency of transformation was obtained with HSV-2 strain 333. The transformation rates for two strains of HSV-1 were lower and distinctly different from HSV-2 and from each other. The dose of inactivating ultraviolet light received by the virus and the multiplicity of infection proved to be critical components of the procedure. Clones of these biochemically transformed cells expressed HSV-specific antigens, altered cell morphology, and increased levels of thymidine kinase activity. Isolates of HSV-1 recovered from the same site 11 years apart yielded similar transforming frequencies. These results suggest that this assay should prove useful in further characterizing the biological properties of HSV.


Particles morphologically resembling mouse hepatitis virus in nude mouse uterus.

Particles morphologically resembling mouse hepatitis virus were observed in uterine gland lumens and endometrial cells of 8 nude mice. Although 3 mice had been experimentally inoculated with baboon type C viral-infected materials, no ultrastructural evidence of oncornavirus-associated structures was observed.


Respiratory load compensation in infants.

We have studied the respiratory compensation for elastic loads in 15 term and preterm infants. Elastic loads, approximately equal to the infant's effective elastance, were applied to the airway for five breaths while tidal volume and mask pressure were monitored. Motion of the rib cage and abdomen were monitored simultaneously with magnetometers. The studies were done both in active or REM sleep and in quiet or non-REM sleep. During quiet sleep the load immediately reduced the tidal volume by about 50% but a progressive increase in tidal volume occurred over the next four loaded breaths. During active sleep load compensation was disorganized with respect to both tidal volume and frequency, and compensation was significantly less. Active sleep was also characterized by marked rib cage distortion. We suggest that during active sleep there is tonic inhibition of the intercostal muscles, allowing the diaphragm to distort the rib cage. This distortion impairs load compensation by a direct mechanical effect and indirectly by initiating an intercostal-phrenic reflex.


Presence of herpesvirus-associated antigens on the surfaces of transformed, tumor, and metastatic cells and enhanced antigenicity of transformed cells using 5-iodo-2'-deoxyuridine.

Using a direct immunofluorescence technique, herpes simplex virus type 1 (HSV-1)-transformed cells and cell lines subsequently derived from tumors and metastases were examined for the presence of HSV-1-associated surface antigens. Fluorescein-labeled immunoglobulins derived from antisera were used and included anti-HSV-1 (from hamsters), sera from tumor-bearing hamsters, and antibodies prepared in rabbits against cell material shed early into the medium after infection of rabbit kidney cells with HSV-1. All immunoglobulins showed the greatest reactivity with cell lines derived from tumors and metastatic lesions. There appeared to be little or no reactivity on the surfaces of the transformed cells. Transformed cells treated with 5-iodo-2'-deoxyuridine for 48 h, however, showed an enhanced reactivity which approached that seen with the tumor-derived cell lines.


Effects of crp mutations on adenosine 3',5'-monophosphate metabolism in Salmonella typhimurium.

Wild-type Salmonella typhimurium could not grow with exogenous cyclic adenosine 3',5'-monophosphate (AMP) as the sole source of phosphate, but mutants capable of cyclic AMP utilization could be isolated provided the parental strain contained a functional cyclic AMP phosphodiesterase.All cyclic AMP-utilizing mutants had the growth and fermentation properties of cyclic AMP receptor protein (crp) mutants, and some lacked cyclic AMP binding activity in vitro. The genetic defect in each such mutant was due to a single point mutation, which was co-transducible with cysG. crp mutants isolated by alternative procedures also exhibited the capacity to utilize cyclic AMP. crp mutants synthesized cyclic AMP at increased rates and contained enhanced cellular cyclic AMP levels relative to the parental strains, regardless of whether or not cyclic AMP phosphodiesterase was active. Moreover, adenylate cyclase activity in vivo was less sensitive to regulation by glucose, possibly because the enzyme II complexes of the phosphotransferase system, responsible for glucose transport and phosphorylation, could not be induced to maximal levels. This possibility was strengthened by the observation that enzyme II activity (measured both in vitro by sugar phosphorylation and in vivo by sugar transport and chemotaxis) was inducible in the parental strain but not in crp mutants. The results suggest that the cyclic AMP receptor protein regulates cyclic AMP metabolism as well as catabolic enzyme synthesis.


Regulation of tyramine oxidase synthesis in Klebsiella aerogenes.

Tyramine oxidase in Klebsiella aerogenes is highly specific for tyramine, dopamine, octopamine, and norepinephrine, and its synthesis is induced specifically by these compounds. The enzyme is present in a membrane-bound form. The Km value for tyramine is 9 X 10(-4) M. Tyramine oxidase synthesis was subjected to catabolite repression by glucose in the presence of ammonium salts. Addition of cyclic adenosine 3',5'-monophosphate (cAMP) overcame the catabolite repression. A mutant strain, K711, which can produce a high level of beta-galactosidase in the presence of glucose and ammonium chloride, can also synthesize tyramine oxidase and histidase in the presence of inducer in glucose ammonium medium. Catabolite repression of tyramine oxidase synthesis was relieved when the cells were grown under conditions of nitrogen limitation, whereas beta-galactosidase was strongly repressed under these conditions. A cAMP-requiring mutant, MK54, synthesized tyramine oxidase rapidly when tyramine was used as the sole source of nitrogen in the absence of cAMP. However, a glutamine synthetase-constitutive mutant, MK94, failed to synthesize tyramine oxidase in the presence of glucose and ammonium chloride, although it synthesized histidase rapidly under these conditions. These results suggest that catabolite repression of tyramine oxidase synthesis in K. aerogenes is regulated by the intracellular level of cAMP and an unknown cytoplasmic factor that acts independently of cAMP and is formed under conditions of nitrogen limitation.


Repression of Escherichia coli carbamoylphosphate synthase: relationships with enzyme synthesis in the arginine and pyrimidine pathways.

Cumulative repression of Escherichia coli carbamoylphosphate synthase (CPSase; EC 2.7.2.9) by arginine and pyrimidine was analyzed in relation to control enzyme synthesis in the arginine and pyrimidine pathways. The expression of carA and carB, the adjacent genes that specify the two subunits of the enzyme, was estimated by means of an in vitro complementation assay. The synthesis of each gene product was found to be under repression control. Coordinate expression of the two genes was observed under most conditions investigated. They might thus form an operon. The preparation of strains blocked in the degradation of cytidine and harboring leaky mutations affecting several steps of pyrimidine nucleotide synthesis made it possible to distinguish between the effects of cytidine and uridine compounds in the repression of the pyrimidine pathway enzymes. The data obtained suggest that derivatives of both cytidine and uridine participate in the repression of CPSase. In addition, repression of CPSase by arginine did not appear to occur unless pyrimidines were present at a significant intracellular concentration. This observation, together with our previous report that argR mutations impair the cumulative repression of CPSase, suggests that this control is mediated through the concerted effects of regulatory elements specific for the arginine and pyrimidine pathways.


Isolation and heteroduplex mapping of a lambda transducing bacteriophage carrying the structural genes for carbamoylphosphate synthase: regulation of enzyme synthesis in Escherichia coli K-12 lysogens.

A N-lambda bacteriophage transducing the structural genes for Escherichia coli K-12 carbamoylphosphate synthase (glutamine) (CPSase; EC 2.7.2.9) has been isolated and analyzed both genetically and physically. The whole int-N region is substituted for a short chromosomal segment corresponding almost exactly to the car locus. The study of CPSase, ornithine carbamoyltransferase, and aspartate carbamoyltransferase regulation in carriers of lambdadcar confirms the previously reported participation of the argR gene product in the control of CPSase synthesis and points to the existence of a regulatory molecule involved in the control of both CPSase and aspartate carbamoyltransferase synthesis. The general usefulness of using N- lambda transducing bacteriophages for the recovery of large amounts of gene products is discussed.


Peptidoglycan biosynthesis in Micrococcus luteus (sodonensis): transglycosidase and phosphodiesterase activities in membrane preparations.

Two enzyme activities involved in the biosynthesis of peptidoglycan in Micrococcus luteus (sodonensis), a transglycosidase and a phosphodiesterase, have been demonstrated in isolated membrane preparations. The transglycosidase activity promotes the in vitro synthesis of an uncross-bridged peptidoglycan that is completely susceptible to lysozyme. This in vitro-synthesized peptidoglycan consists of 76% "soluble" and 24% "insoluble" material. The soluble peptidoglycan is primarily a single low-molecular-weight species of approximately 20 disaccharide peptide units. "Insoluble" peptidoglycan, which likely represents newly synthesized material incorporated into an existing cell wall, was solubilized by butanol extraction, and the two were compared. The phosphodiesterase activity demonstrated in this system cleaves uridine diphosphate-N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-lysyl-D-alanyl-D-alanine to yield N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-lysyl-D-alanyl-D-alanine plus uridine 5'-monophosphate plus inorganic phosphate. This phosphodiesterase activity, not detected under normal transglycosidase assay conditions, is a recycling mechanism and acts indirectly through formation and subsequent cleavage of a lipid-linked intermediate.


Characterization of intracytoplasmic hydrocarbon inclusions from the hydrocarbon-oxidizing Acinetobacter species HO1-N.

The ultrastructure of Acinetobacter sp. strain HO1-N grown on hydrocarbon and nonhydrocarbon substrates was compared using thin sections and freeze-etching. Hydrocarbon-grown cells were characterized by the presence of intracytoplasmic membrane-bound hexadecane inclusions. This membrane did not exhibit a typical unit membrane structure but appeared as a monolayer. The freeze-etch technique revealed the internal structure of the hexadecane inclusions and provided evidence for the presence of a smooth-surfaced limiting membrane. Freeze-etching also revealed intracytoplasmic membranes in the hexadecane-grown cells. These ultrastructural modifications were not present in nonhydrocarbon-grown cells. The hexadecane inclusions were isolated from Acinetobacter. Negative-staining of the inclusions revealed electron-transparent vesicles approximating the size of the inclusions seen in whole cells. Freeze-etching of the purified inclusions revealed membrane-bound vesicles. The purified inclusions exhibited a relatively high value of lipid phosphorus to protein. The lipid composition and the electrophoretic banding pattern of the inclusions on sodium dodecyl sulfate-polyacrylamide gels were determined and compared with other membrane fractions (outer membrane and cytoplasmic membrane) previously isolated from this organism.


D-Ribulose-1,5-bisphosphate carboxylase and polyhedral inclusion bodies in Thiobacillus intermedius.

The growth-related parameters of Thiobacillus intermedius, cultured in glutamate-CO2-S2O32- medium, have been determined. After centrifugation at 48,000 X g for 1 h, 24% of the D-ribulose-1,5-bisphosphate carboxylase (RuBPCase) activity of the disrupted-cell suspensions obtained from CO2-S2O32--and glutamate-CO2-S2O3(3)- grown cells could be sedimented, and the specific activities of this enzyme in the supernatant fractions were almost equivalent. The enzyme was stable in T. intermedius starved of thiosulfate in the presence and absence of glutamate, but a progressive decrease was evident in several growth cycles, each cycle supported by resupplementation of cells with thiosulfate. Polyhedral inclusion bodies were present in CO2-S2O3(2)- and glutamate-CO2S2O3(2)- grown cells. The number of polyhedral bodies per cell increased during mixotrophic growth approximately in proportion to the observed increase in the specific activity of RuBPCase. RuBPCase could not be detected in T. intermedius grown heterotrophically on yeast extract, nor could polyhedral bodies be found.


Control of the synthesis of alkaline phosphatase and the phosphate-binding protein in Escherichia coli.

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunological techniques, we have compared the synthesis of the phoA protein (alkaline phosphatase) and the phoS protein (phosphate-binding protein) in response to the level of phosphate in the medium in different genetic backgrounds containing the known alkaline phosphatase control mutations. Both proteins are produced in excess phosphate media in a phoR1a- strain, whereas neither protein is produced in a phoB- strain even under derepression conditions. In four different phoR1c- strains, however, the phoA product cannot be detected in extracts of cells obtained from any growth condition, whereas the phoS product is produced in both excess and limiting phosphate media. It is not yet known if phoR1c- mutants are a special class of mutations within the phoB gene or whether they occur in a separate cistron involved in alkaline phosphatase regulation. From these results we conclude that the expression of the phoA gene is not always co-regulated with expression of the phoS gene product. We have determined that the phoS protein is a component of periplasmic protein band P4 described by Morris et al. (1974). The phoS product lacks sulfur-containing amino acids and is extractable by treatment with polymyxin sulfate. The other component of band P4 contains methionine and/or cysteine and is not extracted by polymyxin sulfate treatment. Like the phoS and phoA proteins, its synthesis is sensitive to the concentration of phosphate in the growth medium. In addition, the existence of a new class of periplasmic proteins synthesized at maximum rate in high phosphate media is demonstrated.


Association of messenger ribonucleic acid with 70S monosomes from down-shifted Escherichia coli.

The complexed 70S ribosomes (monosomes) that accumulate in Escherichia coli after an energy source shift-down were examined in an electron microscope. In all cases, the ribosomes lie at or near one end of a ribonucleic acid (RNA) strand. This messenger RNA (mRNA) has a mean length of 168 nm and a length-average length of 200 nm, sufficient to code for polypeptides of a weight-average molecular weight of 20,000. The length distribution indicates that these strands are a reasonable representation of the population of monocistronic mRNA's of E. coli. The mRNA strands disappear entirely upon digestion with pancreatic ribonuclease, phosphodiesterase I, or polynucleotide phosphorylase. The susceptibility to digestion by 3'-exonucleases indicate that the ribosomes lie at the 5' end of the mRNA strands. These results are consistent with the hypothesis that down-shifted cells have a translational defect at a point subsequent to the binding of ribosomes to mRNA but prior to the formation of the first peptide bond, such that ribosomes remain bound at or near their points of initial attachment to mRNA.


Natural and altered induction of the L-fucose catabolic enzymes in Klebsiella aerogenes.

Mutants of Klebsiella aerogenes W70 were isolated that had gained the ability to utilize the uncommon pentose D-arabinose as their sole source of carbon and energy. In contrast to the D-arabinose-negative, parent strain, these mutants were found to be either constitutive for certain enzymes of the L-fucose catabolic pathway or inducible for such enzymes when incubated in the presence of D-arabinose. The mutants used L-fucose isomerase to convert D-arabinose to D-ribulose, which is an intermediate and inducer of the ribitol catabolic pathway. The D-ribulokinase of the ribitol pathway was then induced. This enzyme catalyzed the phosphorylation of D-ribulose at the 5-carbon position. Mutants that were negative for D-ribulokinase could still dissimilate D-arabinose slowly by using all three enzymes, the isomerase, kinase, and aldolase, of the L-fucose pathway. Using condition negative mutants, we were able to demonstrate that the natural induction of the L-fucose pathway enzymes by L-fucose required the activity of a functional L-fucose isomerase and a functional L-fuculokinase but not an L-fuculose-1-phosphate aldolase. A metabolic intermediate, L-fuculose-1-phosphate, was thereby shown to be a probable inducer of at least the isomerase and kinase of the L-fucose catabolic pathway. Similar experiments, with D-arabinose-positive mutants, which were induced for the L-fucose pathway enzymes upon incubation with D-arabinose, revealed that the activities of the L-fucose isomerase and the L-fuculokinase were also required for the induction of the L-fucose enzymes. These D-arabinose-positive mutants apparently produced an altered regulatory protein that accepted both L-fuculose-1-phosphate and D-ribulose-1-phosphate as inducers. Examination of constitutive mutants revealed that L-fucose isomerase and L-fuculokinase were both synthesized constitutively, with the aldolase apparently under separate control.


Achromobacter cycloclastes nitrite reductase. The function of copper, amino acid composition, and ESR spectra.

1. Dialysis against cyanide at pH 7 of Achromobacter cycloclastes nitrite reductase [EC 1.7.99.3] of a dissimilatory type led to the removal of about 50% of the copper from the enzyme molecule, with a concomitant decrease of the enzymatic activities. It was inferred that enzyme-bound copper atoms play an essential role in the catalytic activities of the enzyme. 2. The amino acid composition of the enzyme was determined after acid hydrolysis. 3. ESR spectra of the frozen solution and lyophilized powder of the nitrite reductase predominantly showed the presence of two kinds of copper: Type 1 Cu2+, which had narrow and sharp hyperfine splitting, and Type 2 Cu2+, which had broader hyperfine splitting. The bond between the oxidized enzyme and nitrite seems to be ionic.


A new form of high molecular weight DNA polymerase in the nuclei of rat ascites hepatoma cells.

A new form of high molecular weight DNA polymerase [EC 2.7.7.7] (polymerase N) was isolated from the nuclei of rat ascites hepatoma cells. Polymerase C, which was isolated previously from whole cell extract, was also isolated from the nuclei (Tsuruo, T. and Ukita, T. (1974) Biochim. Biophys. Acta 353, 146-159). Polymerase N was not found in the cytoplasmic fraction of the cell, while polymerase C existed in both the nucleus and cytoplasm. The molecular weight of polymerase N (8.7 S) was larger than that of polymerase C (7.4 S). On freezing and thawing, polymerase N was converted to polymerase C. In the nucleus the amount of polymerase N was larger than that of polymerase C. These data suggest that polymerase N, which was specifically present in the nucleus, was a complex form of polymerase C. In in vitro assay, polymerase N showed properties similar to those of polymerase C. Oligoribonucleotide was an effective initiator for the polymerization reaction by polymerase N. The DNA synthesis on single stranded fd phage DNA was greatly stimulated by the concomitant synthesis of RNA.


A new kinetic property characteristic of the actomyosin-nucleoside-triphosphatase.

The nucleoside triphosphatase [EC 3.6.1.15] activity of actomysin and that of myosin are measured by varying the concentration of nucleoside triphosphate and that of CaCl2 or MgGl2. The results thus obtained are examined by asking a question of which is responsbile for the activity, the true substrate and the active enzyme in terms of the reaction scheme shown in p. 719. The answers found for the above question are summarized in Table I (see p. 720). It is emphasized that the summmary (Table I) corresponds very well to the fact that myosin alone does not superprecipitate in the presence of either calcium or magnesium ions, whereas actomyosin does superprecipitate in the presence of magnesium ions and not in the presence of calcium ions. Obviously, the true substrate type of reaction scheme represents a kinetic property characteristic of the superprecipitation-coupled nucleoside-triphosphatase. It is also noted of the summary (Table I) that actin is capable of not only activating Mg-nucleoside-triphosphatase but also switiching the reaction scheme from the active enzyme type to the true substrate type. It is known that trinitrophenylation of myosin results in activation of the Mg-ATPase activity of myosin. However, it is now found that trinitrophenylation is not capable of switiching the reaction scheme, that is to say that the Mg-ATPase reaction of trinitrophenyl-myosin stays with the active enzyme type of reaction scheme and that of acto-trinitrophenyl-myosin with the true substrate type of reaction scheme. Effect of actin on the function of myosin seems, therefore, very unique.


Isolation and characterization of the plasma membranes from rat ascites hepatomas and from normal rat livers, including newborn, regenerating, and adult livers.

Plasma membranes (PM) were isolated from island-forming types of rat ascites hepatoma (AH 130, AH 602, and AH 7974) and from their free-cell sublines (AH 130FN and AH 7974F), and were characterized in terms of electron-microscopic morphology, marker enzyme activities, and lipid contents. The results were compared with those of the PM isolated in a similar way from newborn, regenerating, and adult livers. The marker enzyme activities, such as Na+, K+-insensitive Mg2+-ATPase [EC 3.6.1.3] (Mg2+-ATPase) and 5'-nucleotidase [EC 3.1.3.5], as well as the phospholipid composition of the PM isolated from hepatomas by Wallach's nitrogen gas cavitation method were similar to those obtained with the PM isolated by a modification of Emmelot's method, although the former method gave a much lower yield in terms of protein than the latter. Based on the modified Emmelot method, sufficiently pure PM preparations could be obtained from the hepatomas in the form of large membrane sheets without any contamination by other identifiable components, as determined with an electron microscope, and with high specific activities of the marker enzymes, such as Na+, K+-sensitive ATPase [EC 3.6.1.3] (Na+, K+ -ATPase), Mg2+ -ATPase, and 5'-nucleotidase. As for the characteristics of the hepatoma PM, lower specific activity of 5'-nucleotidase and higher fatty aldehyde molar percentages in total phospholipids were noted in all the PM from the hepatomas in comparison with normal liver PM of various origins. The PM from the hepatomas showed an increased amount of cholesterol (mumole per mg protein), whereas actively growing newborn and regenerating livers gave rather lower amounts in comparison with that of normal adult liver.


Urinary excretion of retinol-binding protein (RBP) in rabbit chronically poisoned with cadmium. Isolation and partial characterization of rabbit RBP.

Studies were conducted to induce urinary excretion of retinol-binding protein (RBP) in the rabbit by long-term administration of cadmium, and to isolate and characterize rabbit RBP. Two rabbits were exposed to Cd at a dose of 0.8 and/or 1.5mg/kg of body weight by means of subcutaneous injections five times per week. One rabbit excreted large amounts of protein (0.8 to 1.6 g/day) from days 80 to 118. The electrophoretic patterns of the urinary protein showed two fluorescent bands specific for protein-bound retinol in the alpha-region; these were presumed to be RBP. Rabbit RBP was isolated from the pooled urine (10.3 liters) by a sequence of procedures which included gel filtration on Sephadex G-100 and chromatography on DEAE- and SP-Sephadex. Purified RBP (20 mg) was found to be homogeneous by physical and immunological criteria. The RBP had alpha-mobility, with a molecular weight of approximately 20,000. The properties of rabbit RBP resembled those of human RBP simultaneously isolated from the urine of patients with "Itai-Itai" disease in many ways: ultraviolet and fluorescence spectra, and amino acid compositions. A monospecific anti-rabbit RBP antiserum was raised in a goat. There was no immunological cross-reactivity between rabbit and human RBP. The molecular size of the retinol-containing protein in fresh rabbit serum was estimated to be about 60,000 to 70,000 by gel filtration on Sephadex G-200. Rabbit RBP in the serum was also shown to be immunologically identical with purified RBP from the urine.


Phosphatidylserine synthetase mutants of Escherichia coli. Genetic mapping and membrane phospholipid composition.

Mutants of Escherichia coli K-12 defective in CDP-diglyceride:L-serine phosphatidyltransferase (phosphatidylserine synthetase) can be isolated by a rapid autoradiographic screening assay described previously (Raetz, C. R. H. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 2274-2278). Four organisms of this kind have now been characterized. The gene (designated pss) which is altered in these mutants is closely linked to the nadB locus near minute 49 on the E. coli chromosome. Strains carrying the pss-8 mutation do not grow at elevated temperatures and have low levels of an altered synthetase in cell extracts. An analysis of several hundred transductants and temperature-resistant revertants reveals that the pss-8 mutation is responsible both for the enzyme defect and for the phenotype. When a pss-8 mutant is shifted to the nonpermissive temperature, the cells stop dividing and form long filaments. After 3 hours at 44 degrees the level of phosphatidylethanolamine drops from 66 to 32% (percentage of the total lipid phosphorus), while the combined levels of phosphatidylglycerol and cardiolipin rise from 34 to 68%.


Restoration of a regulatory response to low density lipoprotein in acid lipase-deficient human fibroblasts.

Previous studies have shown that cultured fibroblasts derived from patients with genetic defects in lysosomal acid lipase (i. e. the Wolman Syndrome and Cholesteryl Ester Storage Disease) are defective in their ability to hydrolyze the cholesteryl esters contained in plasma low density lipoprotein (LDL). As a result, these mutant cells show a reduced responsiveness to the regulatory actions of LDL, as evidenced by a decreased LDL-mediated suppression of the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase and by a decreased LDL-mediated activation of cellular cholesteryl ester formation. In the current studies, the Wolman Syndrome and Cholesteryl Ester Storage Disease cells were grown in the same Petri dish with mutant fibroblasts derived from a patient with the homozygous form of Familial Hypercholesterolemia. Whereas pure monolayers of either the Familial Hypercholesterolemia cells (lacking cell surface LDL receptors) or the acid lipase-deficient cells (lacking cholesteryl ester hydrolase activity) responded poorly to LDL, the mixed monolayers developed lipoprotein responsiveness as measured by an enhancement of both LDL-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and LDL-mediated stimulation of cholesteryl ester formation. This effect was shown to result from the release of the lysosomal acid lipase from the Familial Hypercholesterolemia homozygote cells into the culture medium and its subsequent uptake by the acid lipase-deficient cells. The acquisition of this acid lipase activity enhanced the ability of the Wolman Syndrome and Cholesteryl Ester Storage Disease cells to respond to the lipoprotein by suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase and activation of cellular cholesteryl ester formation. These data emphasize the importance of the lysosomal acid lipase in the cellular metabolism of LDL cholesteryl esters and, in addition, demonstrate that delivery of this enzyme to genetically deficient cells can enhance the regulatory response to the lipoprotein.


Cell cycle-specific activity of type I and type II cyclic adenosine 3':5'-monophosphate-dependent protein kinases in Chinese hamster ovary cells.

Types I and II cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinases have been studied during the cell cycle of Chinese hamster ovary cells. Chinese hamster ovary cells were synchronized by selective detachment of mitotic cells from monolayer cultures. Protein kinases were separated by DEAE-cellulose chromatography and were similar to the types of cAMP-dependent protein kinases studied in skeletal muscle and in heart extracts. The total amount of protein kinases activity per cell was substantial, both in mitosis and at the G1/S boundary. During mitosis, the relatively high activity of protein kinase was due to a predominance of type I protein kinase. During early G1, the activity of type I protein kinase decreased and there was little detectable type II activity. A rapid increase in the activity of type II was evident at the G1/S boundary. The administration of puromycin (50 mug/ml) from 1 to 5 hours after selective detachment of mitotic cells abolished the activity of type II cAMP-dependent protein kinase seen at the G1/S border, but had no observable effect on the activity of type I protein kinase. The data presented demonstrate cell cycle-specific activity patterns of type I and type II protein kinase Type I protein kinase activity is high in mitosis and is constant throughout the cell cycle. Increased type II protein kinase activity seems to be related to the initiation of DNA synthesis in S phase. The data suggest a translational control of type II cAMP-dependent protein kinase activity.


Dissociation and reassociation of the phosphorylated and nonphosphorylated forms of adenosine 3':5' -monophosphate-dependent protein kinase from bovine cardiac muscle.

Adenosine 3':5' -monophosphate (cyclic AMP) -dependent protein kinase from bovine heart muscle catalyzes the phosphorylation of its regulatory, cyclic AMP-binding subunit. Phosphorylation enhances net dissociation of the enzyme by cyclic AMP. Chromatography on omega-aminohexyl-agarose was used to study the effects of phosphorylation on cyclic AMP binding and subunit dissociation and reassociation. This method permitted rapid separation of the catalytic subunit from the cyclic AMP -binding protein and holoenzyme. Phospho- and dephosphoprotein kinases were found to dissociate to the same extent at any given concentration of cyclic AMP and completely at saturation. At equilibrium, the amount of cyclic AMP bound was the same for both forms of enzyme and was directly proportional to the degree of dissociation of the holoenzyme. In the absence of cyclic AMP, phospho- and dephospho-cyclic AMP-binding proteins reassociated completely with the catalytic subunit. However, the rate of reassociation of the dephospho-cyclic AMP-binding protein was at least 5 times greater than the phospho-cyclic AMP-binding protein. Retardation of reassociation was directly proportional to the extent of phosphorylation. We conclude that the degree to which the cyclic AMP-binding protein is phosphorylated markedly affects its intrinsic ability to combine with the catalytic subunit to regenerate the inactive cyclic nucleotide-dependent kinase and that the state of phosphorylation of this subunit may be important in detemining the proportion of dissociated (active) and reassociated (inactive) protein kinase at any given time.


Modification of Escherichia coli DNA ligase by cleavage with trypsin.

Limited treatment of Escherichia coli DNA ligase with trypsin results in rapid loss of DNA joining activity. However, the ability to react with DPN to form the covalent enzyme-AMP intermediate is unaffected. The cleaved enzyme is also unable to catalyze the formation of DNA-adenylate, the second covalent intermediate in the ligase-catalyzed reaction. These findings demonstrate that portions of the DNA ligase molecule that are required for phosphodiester bond formation are not required for at least one of the partial reactions catalyzed by this enzyme.


Nucleotide sequence of 5 S ribosomal RNA precursor from Bacillus subtilis.

The complete nucleotide sequence of a 179-nucleotide precursor (p5A) of 5 S ribosomal RNA from Bacillus subtilis is presented. In addition to the 116-nucleotide mature segment, the p5A molecule contains 21 additional nucleotides at its 5' end and 42 at its 3' terminus. Structural features within the p5A molecule which possibly interact with a specific maturation endonuclease (RNase M5) are  identifiable. These include 2-fold rotational symmetry about the cleavage sites, which may approximately place the p5A molecule on the RNase M5 surface, and translational (positional) symmetry, which may precisely orient substrate phosphodiester bonds with respect to catalytic amino acids. Certain features of the 3' -terminal, precursor-specific portion of p5A possibly are involved in the termination of DNA transcription.


The kinetic mechanism of rat kidney gamma-glutamylcysteine synthetase.

A detailed kinetic investigation was made of the binding mechanism of gamma-glutamylcysteine synthetase purified from rat kidney. The results of initial rate and inhibition studies are consistent with a partially random mechanism in which ATP is the obligatory first substrate and both amino acids bind in a random order to the enzyme-ATP complex. Formation of the enzyme-substrate quaternary complex is necessary prior to release of products. This mechanism is consistent with previous binding studies with the enzyme and while it does not rule out participation of enzyme-bound gamma-glutamyl phosphate as an intermediate in catalysis, such an intermediate cannot be a discrete covalent complex.


Differential response to adrenocorticotropic hormone analogs of bovine adrenal plasma membranes and cells.

The ability of three analogs of ACTH1-24 ([Gln5, Phe9] ACTH1-24, [Gln5, Ala9[Acth1-24, and [Gln5, Lys8, Phe9[ ACTH1-24) embodying tryptophan substitutions to activate the adenylate cyclase system of a bovine adrenal plasma membrane preparation was compared to the effect of the analogs on adenosine 3':5'-monophosphate (cyclic AMP) accumulation and steroidogenesis in viable bovine adrenocortical cells. The results were not comparable. Whereas the analogs antagonized the ACTH1-24-activated membrane cyclase they stimulated cyclic AMP accumulation as well as steroid production of the cells. None of the analogs inhibited steroidogenesis of ACTH1-24-stimulated cells, but two of them, at very high dose levels, inhibited cyclic AMP production. The ability of the analogs to stimulate steroidogenesis of the adrenal cells half-maximally decreased in the order tryptophan greater than phenylalanine greater than alanine, indicating that the aromaticity of the indole ring of tryptophan is necessary for maximal interaction between hormone and receptor. Both the absolute and relative steroidogenic potencies were the same for several analogs when assayed with rat adrenal cells. Although only a small fraction of the cell's potential to produce cyclic AMP was necessary to induce maximum steroid production, the relative activities of a series of analogs were the same for steroidogenesis as for cyclic AMP accumulation. Furthermore, the concentration of cyclic AMP necessary for full steroidogenesis was practically identical for a series of peptides that differed widely in potency. These findings support the postulate that cyclic AMP accumulation and steroidogenesis in adrenocortical cells are coupled processes. The differential behavior of bovine adrenal plasma membranes and bovine adrenocortical cells toward ACTH analogs indicates that structure-function studies using cyclase assays may not reflect events that take place in the intact adrenal or in cell preparations derived therefrom.


Infulence of hormones and medium composition on the degradation of phosphoenolpyruvate carboxykinase (GTP) and total protein in Reuber H35 cells.

Reuber H35 cells were pulse-labeled with radioactive leucine and the influence of hormones, serum, and amino acids on protein degradation was investigated during a subsequent chase period. Radioactive, immunoprecipitable phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) had a half-life of 5 to 6 hours which was not influenced by either N6, O2-dibutyryl adenosine 3':5'-monophosphate, dexamethasone, or insulin. The rate of phosphoenolpyruvate carboxykinase degradation was the same under steady state conditions as during the approach to a new steady state following hormonal induction or deinduction of the enzyme. Therefore, hormonal regulation of enzyme activity in vivo is the result of changes in the rate of enzyme synthesis. The rate of proteolysis for total cell proteins was increased under nutritional step-down conditions produced by the removal of serum or amino acids, or both, from the medium. This effect was completely prevented by insulin. Cycloheximide and puromycin, but not actinomycin D or cordycepin, inhibited protein degradation under step-down conditions but did not further decrease the basal rate of proteolysis measured in the presence of either insulin or serum plus amino acids. There was a good correlation between changes in proteolysis produced by serum and amino acids and changes in the degradation rate of phosphoenolpyruvate carboxykinase. Also, inhibition of proteolysis with cycloheximide and puromycin was accompanied by a decrease in the degradation rate for enzyme antigen. It is suggested that nutritional step-down leads either to the synthesis or activation of a proteolytic system.


NAD+-malic enzyme. Regulatory properties of the enzyme from Ascaris suum.

The regulatory properties of the NAD-dependent malic enzyme from the mitochondria of Ascaris suum have been studied. The malate saturation curve exhibits sigmoidicity and the degree of this sigmoidicity increases as the pH is increased. Fumarate was the only compound tested that stimulated the enzyme activity, whereas oxalacetate was the most powerful inhibitor. Activation by low levels of fumarate was found to be competitive with malate. It is proposed that this stimulation has physiological significance in controlling the dismutation reaction in the parasite. The branched-chain volatile fatty acid excretion products, tiglate, 2-methylbutanoate, and 2-methylpentanoate, inhibited the enzyme activity and this inhibition was competitive with malate. The Ki values for these compounds are in the physiological range of their concentrations; therefore, it is suggested that they may aid in controlling the malic enzyme activity in vivo. Oxalacetate inhibition of malic enzyme activity was competitive with malate, and the Ki values decreased with an increase in pH. Two alternatives are proposed which could account for the lack of oxalacetate decarboxylation by the ascarid malic enzyme.


Asparagusate dehydrogenases and lipoyl dehydrogenase from asparagus mitochondria. Physical, chemical, and enzymatic properties.

Asparagusate dehydrogenases I and II and lipoyl dehydrogenase have been obtained in homogeneous state from asparagus mitochondria. They are flavin enzymes with 1 mol of FAD/mol of protein. Asparagusate dehydrogenases I and II and lipoyl dehydrogenase have s20,w of 6.22 S, 6.39 S, and 5.91 S, respectively, and molecular weights of 111,000, 110,000, and 95,000 (sedimentation equilibrium) or 112,000, 112,000, and 92,000 (gel filtration). They are slightly acidic proteins with isoelectric points of 6.75, 5.75, and 6.80. Both asparagusate dehydrogenases catalyzed the reaction Asg(SH)2 + NAD+ equilibrium AsgS2 + NADH + H+ and exhibit lipoyl dehydrogenase and diaphorase activities. Lipoyl dehydrogenase is specific for lipoate and has no asparagusate dehydrogenase activity. NADP cannot replace NAD in any case. Optimum pH for substrate reduction of the three enzymes are near 5.9. Asparagusate dehydrogenases I and II have Km values of 21.5 mM and 20.0 mM for asparagusate and 3.0 mM and 3.3 mM for lipoate, respectively. Lipoyl dehydrogenase activity of asparagusate dehydrogenases is enhanced by NAD and surfactants such as lecithin and Tween 80, but asparagusate dehydrogenase activity is not enhanced. Asparagusate dehydrogenases are strongly inhibited by mercuric ion, p-chloromercuribenzoic acid, and N-ethylmaleimide. Amino acid composition of the three enzymes is presented and discussed.


Reversible inactivation of phenylalanine hydroxylase by catecholamines in cultured hepatoma cells.

Phenylalanine hydroxylase in Reuber H4 hepatoma cell cultures can be rapidly inactivated by the addition of epinephrine, norepinephrine, dopamine, or 3,4-dihydroxyphenylalanine, in order of decreasing effectiveness, to the culture medium. The enzyme was 50% inactivated in 1 hour by 25 muM (R)-epinephrine or 45 muM (R)-norepinephrine in the medium. High concentrations of epinephrine caused a 70% inactivation in 15 min. Phenylalanine hydroxylase appears to be reversibly inactivated by epinephrine within the cells; since washing the compound off the cell cultures resulted in a rapid restoration of enzyme activity (40% in 1 hour), cycloheximide had little effect on the initial rate of recovery of enzyme activity and the same amount of phenylalanine hydroxylase antigen per cell was isolated from treated and normal cultures. Both (S)- and (R)-epinephrine inactivated the enzyme, and 0.1 mM desmethylimipramine, an inhibitor of amine transport, significantly decreased the effect of epinephrine on the hydroxylase activity. The possibility, suggested by the above results, that epinephrine might be directly inactivating phenylalanine hydroxylase within the cells was supported by the finding that purified rat liver phenylalanine hydroxylase would be 50% inactivated by 1.5 muM epinephrine in 10 min.


Mechanism of inactivation of phenylalanine hydroxylase by p-chlorophenylalanine in hepatome cells in culture. Two possible models.

The mechanism by which p-chlorophenylalanine specifically reduces phenylalanine hydroxylase activity in rat liver in vivo and in Reuber H4 hepatoma cells in culture has been investigated. Chromatography on hydroxylapatite of liver extract from rats injected with p-chlorophenylalanine showed that the compound differentially affected the three normal phenylalanine hydroxylase isoenzymes (I, II, and III); isoenzymes II and III were completely absent after the treatment, but isoenzyme I was only reduced in quantity compared with normal adult rats. Normal Reuber H4 cells only possess isoenzyme I; treatment with p-chlorophenylalanine yielded a reduced level of enzyme activity which appeared to be noraml isoenzyme I by both chromatographic and kinetic criteria. There is evidence, based on immunochemical techniques, that cultures grown in the presence of p-chlorophenylalanine have significantly reduced levels of phenylalanine hydroxylase antigen, and that p-chlorophenylalanine inactivates phenylalanine hydroxylase at or near the time of enzyme synthesis. The bulk of enzyme synthesized prior to the addition of the compound appears unaffected by it. There is no indication that protein synthesis itself is affected by p-chlorophenylalanine. In addition, p-chlorophenylacetate was found to inactivate phenylalanine hydroxylase in an apparently identical manner with p-chlorophenylalanine, which almost certainly eliminates from consideration any mechanism of inactivation specifically requiring an amino acid. Finally, effects of cycloheximide and chlorophenylalanine were compared. Taken together, the data lead to two possible models for the inactivation of the enzyme. The model most consistent with all data requires (predicts) the existence of a proenzyme form of phenylalanine hydroxylase which can be specifically inactivated by p-chlorophenylalanine.


Phosphorylation of distinct regions of f1 histone. Relationship to the cell cycle.

The phosphorylation of different amino acids in distinct regions of f1 histone was studied in highly synchronized Chinese hamster cell populations (line CHO). The purified, 32P-labeled f1 histone was bisected into NH2-terminal and COOH-terminal fragments with N-bromosuccinimide. Tryptic phosphopeptides from these fragments were resolved using sequential high voltage electrophoretic steps on paper. No phosphorylation was observed in early G1-arrested cells. Interphase phosphorylation began in late G1 in the COOH-terminal portion of the molecule on serine. This event continued throughout S phase and persisted into mitosis. However, in mitosis additional phosphorylation was observed in the COOH-terminal portion of the molecule on threonine, and for the only time in the CHO cell cycle the NH2-terminal portion of the molecule was also phosphorylated on both serine and threonine. The peptide studies thus predicted that a minimum of four sites (two serine and two threonine) were phosphorylated in the f1 histone of mitotic CHO cells. This was confirmed using electrophoresis in long polyacrylamide gels.


Regulation of receptor concentration by homologous hormone. Effect of human growth hormone on its receptor in IM-9 lymphocytes.

When cultured human lymphocytes of the IM-9 line were exposed to human growth hormone (hGH) at 37 degrees, washed for 2 hours, and incubated with 125I-hGH, the binding of 125I-hGH was reduced. The magnitude of the reduction in binding was dependent on the concentration of growth hormone present as well as the duration of the exposure. As little as 2 X 10(-11) M (0.5 ng/ml) growth hormone had a discernible effect. Growth hormone at 2 X 10(-10) M (5.0 ng/ml), which is a low resting concentration of hormone in vivo and occupies about 20% of the receptors at steady state at 30 degrees, produced a 50% reduction in binding while 20 mg/ml, which occupies about 50% of the receptors under steady state conditions, produced an 80% loss of receptors. Further increases in growth hormone concentration produced little further effect on receptor loss. Thus, the loss of receptors at a given concentration of growth hormone (up to 20 ng/ml) in the preincubation at 37 degrees was greater than the occupancy produced by that concentration of growth hormone receptors under steady state conditions at 30 degrees. Analysis of the data indicated that the decrease in binding of 125I-hGH was due to a loss of receptors per cell without any change in affinity of receptor for hormone or in cell number. The concentration of insulin receptors on these cells was affected by the insulin concentration in the medium, and the concentration of growth hormone receptors was affected by growth hormone, but neither hormone had any effect on the heterologous receptors. Exposure of the cells to cycloheximide (0.1 mM) produced a progressive but smaller loss of growth hormone receptors, and the effect of cycloheximide was additive to the receptor loss induced by growth hormone, suggesting that cycloheximide inhibited synthesis of receptors while growth hormone accelerated loss of receptors. When growth hormone was removed from the medium, receptor concentrations were restored rapidly; half of the loss was restored by 6 to 8 hours and the full complement of receptors was restored by 24 hours following removal of the hormone. If the growth hormone was removed and replaced with cycloheximide, the return of the receptors was delayed until the cycloheximide was removed. Thus restoration of the receptors appeared to require the synthesis of new proteins. These data indicate that in the IM-9 lymphocytes the concentration of growth hormone receptors is very sensitive to regulation by growth hormone and also add further support to the suggestion that hormones in general actively regulate the concentration of their own receptors.


Hepatic pyruvate kinase. Regulation by glucagon, cyclic adenosine 3'-5'-monophosphate, and insulin in the perfused rat liver.

A reversible interconversion of two kinetically distinct forms of hepatic pyruvate kinase regulated by glucagon and insulin is demonstrated in the perfused rat liver. The regulation does not involve the total enzyme content of the liver, but rather results in a modulation of the substrate dependence. The forms of pyruvate kinase in liver homogenates are distinguished by measurements of the ratio of the enzyme activity at a subsaturating concentration of P-enolpyruvate (1.3 mM) to the activity at a saturating concentration of this substrate (6.6 mM). A low ratio form of pyruvate kinase (ratio between 0.1 and 0.2) is obtained from livers perfused with 10(-7) M glucagon or 0.1 mM adenosine 3':5'-monophosphate (cyclic AMP). A high ratio form of the enzyme is obtained from livers perfused with no hormone (ratio = 0.35 to 0.45). The regulation of pyruvate kinase by glucagon and cyclic AMP occurs within 2 min following the hormone addition to the liver. Insulin (22 milliunits/ml) counteracts the inhibition of pyruvate kinase caused by 5 X 10(-11) M glucagon, but has only a slight influence on the enzyme properties in the absence of the hyperglycemic hormone. The low ratio form of pyruvate kinase obtained from livers perfused with glucagon or cyclic AMP is unstable in liver extracts and will revert to a high ratio form within 10 min at 37 degrees or within a few hours at 0 degrees. Pyruvate kinase is quantitatively precipitated from liver supernatants with 2.5 M ammonium sulfate. This precipitation stabilizes the enzyme and preserves the kinetically distinguishable forms. The kinetic properties of the two forms of rat hepatic pyruvate kinase are examined using ammonium sulfate precipitates from the perfused rat liver. At pH 7.5 the high ratio form of the enzyme has [S]0.5 = 1.6 +/- 0.2 mM P-enolpyruvate (n = 8). The low ratio form of enzyme from livers perfused with glucagon or cyclic AMP has [S]0.5 = 2.5 +/- 0.4 mM P-enolpyruvate (n = 8). The modification of pyruvate kinase induced by glucagon does not alter the dependence of the enzyme activity on ADP (Km is approximately 0.5 mM ADP for both forms of the enzyme). Both forms are allosterically modulated by fructose 1,6-bisphosphate, L-alanine, and ATP. The changes in the kinetic properties of hepatic pyruvate kinase which follow treating the perfused rat liver with glucagon or cyclic AMP are consistent with the changes observed in the enzyme properties upon phosphorylation in vitro by a clyclic AMP-stimulated protein kinase (Ljungström, O., Hjelmquist, G. and Engström, L. (1974) Biochim. Biophys. Acta 358, 289--298). However, other factors also influence the enzyme activity in a similar manner and it remains to be demonstrated that the regulation of hepatic pyruvate kinase by glucagon and cyclic AMP in vivo involes a phosphorylation.


Structure of high density lipoprotein. The immunologic reactivities of the COOH- and NH2-terminal regions of apolipoprotein A-I.

Only 5 to 10% of the apolipoprotein A-I (ApoA-I) of intact high density lipoprotein (HDL) is detectable by radioimmunoassay. In addition, when isolated ApoA-I is recombined with lipids in vitro, its immunologic reactivity is decreased by 30 to 95%. Thus, ApoA-I is less reactive immunologically in the presence of lipids. Our aim was to ascertain whether the COOH- or NH2-terminal regions of ApoA-I were equally reactive in intact HDL2. CNBr fragments of ApoA-I were produced by the method of Baker et al. (Baker, H.N., Jackson, R.L., and Gotto, A.M. (1973) Biochemistry 12, 3866-3871) and iodinated with lactoperoxidase. Double-antibody radioimmunoassays were set up using anti ApoA-I antisera and 125I-CNBr I (COOH-terminal region) or 125I-CNBr II (NH2-terminal). Both labels were bound by the antisera. Affinity columns were prepared by binding CNBr I or CNBr II to Sepharose 4B. Antibodies specific against CNBr I or CNBr II were isolated by means of these columns, suggesting that ApoA-I had at least two antigenic sites. In other assays using labeled fragments and anti ApoA-I antisera, 125I-CNBr I was displaced by CNBr I, ApoA-I , and HDL2 but not CNBr II. Conversely, 125I-CNBr II was displaced by CNBr II, ApoA-I, and HDL2 but not by CNBr I. Thus the assays were region-specific. The reactivities of isolated ApoA-I and the ApoA-I in intact HDL2-ApoA-I) were compared in these assays. On a molar basis, HDL2-ApoA-I was consistently more reactive (2- to 5-fold) in the 125I-CNBr I than in the 125I-CNBr II assays. The findings suggest (a) that the two terminal regions of ApoA-I are immunologically distinct, (b) that the two regions can be assayed independently of each other in intact HDL2, and (c) that the COOH-terminal region is more reactive immunologically than is the NH2-terminal. The results are compatible with a more "exposed" position for the COOH-terminal region on the surface of HDL2.


Phosphorylation of histone catalyzed by a bovine brain protein kinase.

The use of polyethyleneimine-cellulose thin layer sheets to follow the phosphorylation of histone and decomposition of ATP catalyzed by an adenosine 3':5'-monophosphate (cyclic AMP)-stimulated protein kinase, protein kinase I, has made possible a more detailed analysis of the time course of these reactions than has been achieved previously be observing only recovered phosphorylated protein. When [gamma-32P] ATP was employed, significant error was introduced by the presence of 32Pi at the solvent front on these sheets, and this limited the accuracy of the available information. However, the analysis of assays performed with [U-14C] ATP was straightforward and appeared to have an accuracy comparable to that of the present standard assay. This appears to be the first use of [U-14C] ATP to assay protein kinases. Our physical characterization of protein kinase I showed it to be a homogeneous protein species by polyacrylamide gel electrophoresis, sodium dodecyl sulfate gel electrophoresis and analytical ultracentrifugation. Kinetic studies with protein kinase I indicated the absence of histone phosphatase and cyclic AMP phosphodiesterase activity. Furthermore, the ATPase activity seen is believed to be intimately associated with the protein kinase action, particularly in view of the observed dependence of the rate of Pi production on the presence of cyclic AMP. The kinetic data for the phosphorylation of histone catalyzed by protein kinase I under full stimulation by cyclic AMP are consistent with a double displacement mechanism.


The molar ratio of the two major polypeptide components of human high density lipoprotein.

Human high density lipoprotein (HDL) and its subfractions (HDL2 and HDL3) were separated by ultracentrifugation and the molar ratio of the two major polypeptide chains apo-Gln-I and apo-Gln-II was determined by fluorescence tagging of sodium dodecyl sulfate-denatured proteins combined with polyacrylamide disc gel electrophoresis. Using purified apo-Gln-I and apo-Gln-II standards, it was found that holo HDL, holo HDL2, and holo HDL3 from all plasma samples contained a molar ratio of apo-Gln-I to the disulfide-bound dimer of apo-Gln-II of 2:1, that is a 1:1 ratio in terms of each species of polypeptide chain. The method described is useful for making repeated and rapid measurements on microgram quantities of intact lipoproteins.


The structure of Paracoccus denitrificans cytochrome c550.

The crystal structure of Paracoccus (formerly Micrococcus) denitrificans cytochrome c550 has been solved by x-ray diffraction to a resolution of 2.45 A. In both amino acid sequence and molecular structure it is evolutionarily homologous with mitochondrial cytochrome c from eukaryotes and photosynthetic cytochrome c2 from purple non-sulfur bacteria. All of these cytochromes c have the same basic folding pattern, with surface insertions of extra amino acids in c550. Various strains of c2 have all, some, or none of the extra insertions observed in c550. The hydrophobic heme environment, position of aromatic rings, and structure and environment of the heme crevice, are virtually identical in cytochromes c55o, c, and c2. Radical changes observed at all regions on the molecular surface except the heme crevice argue for the importance of the crevice and the exposed edge of the heme in the transfer of electrons to and from the cytochrome molecule.


Sex-specific occurrence of androgen receptors in rat brain.

The metabolism and binding of [1, 2, 6, 7-3H] testosterone in male and female rat brain has been studied in an attempt to find an explanation for the relative androgen unresponsiveness characterizing the female hypothalamo-pituitary axis involved in regulation of hepatic steroid metabolism. The most significant sex differences in the pattern of [3H] testosterone metabolites recovered from several brain regions (including pituitary, pineal gland, and hypothalamus) after intraperitoneal administration of [3H] testosterone were the predominance of testosterone and androstenedione in male brain compared to the quantitative importance of 5alpha-androstane-3alpha, 17beta-diol, 5alpha-androstane-3beta, 17beta-diol, epitestosterone, and dihydroepitestosterone in female brain. One possible explanation for the androgen unresponsiveness of female rats is, therefore, the faster metabolism of testosterone to inactive compounds in female brain. Experiments both in vivo and in vitro showed the presence of high affinity, low capacity binding sites for [3H] testosterone in male pituitary, pineal gland, and hypothalamus (Kd values in the region of 1 X 10(-10) to 1 X 10(-9) M and number of binding sites 1.0 to 1.4 X 10(-14) mol per mg of protein). The steroid - macromolecular complexes generally had a pI of 5.1, were excluded from Sephadex G-200, were heat-labile, and were sensitive to protease. Competition experiments indicated the following order of ligand affinities: testosterone is greater than 5alpha-dihydrotestosterone and estradiol is greater than androstenedione is greater than corticosterone. No steroid-binding proteins of similar nature were found in pituitary, pineal gland, or hypothalamus from female rats. On the basis of these results it is suggested that the androgen unresponsiveness of female rats referred to above relates to the absence of receptor protein for androgens in female rat brain. In support of this hypothesis, 28-day-old female rats, which are known to be affected by androgens with regard to liver enzyme activities, were shown to contain receptor proteins for androgen in the brain. In conclusion, the relative androgen unresponsiveness of the female hypothalamo-pituitary axis is probably explained by the absence of receptor proteins for androgen in female hypothalamus and pituitary. The fast metabolism of testosterone in female rat brain also serves to decrease the availability of active androgen to potential receptor sites. It may be speculated that the presence of androgen receptors in male brain is the result of neonatal programming ("imprinting") by testicular androgen.


Biosynthesis of adrenocorticotropic hormone in mouse pituitary tumor cells.

A double antibody immunoprecipitation technique using affinity-purified adrenocorticotropic hormone (ACTH) antiserum was employed to investigate the biosynthesis of ACTH in a mouse pituitary tumor cell line. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of cell extracts resolved four forms of ACTH with apparent molecular weights of 4,500, 13,000, 23,000, and 31,000. These four forms of ACTH can be detected by radioimmunoassay of cell extracts or by immunoprecipitation of cell extracts following incubation of cultures in [3H] tryptophan, [3H] lysine, or [3H] tyrosine. The double antibody immunoprecipitation scheme developed is specific, quantitative, and reproducible. ACTH biosynthesis was examined in both steady and pulse-labeling experiments using [8H] tyrosine or [3H] lysine. The results of these experiments are consistent with the proposal that Mr=31,000 ACTH is the biosynthetic precursor for all three smaller forms of ACTH and that Mr=23,000 ACTH is a biosynthetic intermediate. Both Mr=13,000 ACTH and Mr=4,500 ACTH are derived from the intracellular processing of Mr=31,000 ACTH.


High molecular weight forms of adrenocorticotropic hormone are glycoproteins.

Mouse pituitary tumor cells (AtT-20/D-16v) were incubated in medium containing [3H] glucosamine or [3H] mannose. By analyzing immunoprecipitates of cell extracts and culture medium it was shown that [3H] glucosamine and [3H] mannose were incorporated into all three high molecular weight forms of ACTH; label was not incorporated into Mr=4,500 ACTH (which is thought to be similar to the 39 amino acid polypeptide form of ACTH, alpha(1-39)). Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis the apparent molecular weights of these glycoprotein forms of ACTH were 31,000, 23,000, and 13,000. Gel filtration in 6 M guanidine HCl indicated that the molecular weights of these forms of ACTH were substantially lower; sodium dodecyl sulfate-polyacrylamide gel electrophoresis has often been found to overestimate the molecular weight of glycoproteins. A significant fraction of the high molecular weight ACTH in tumor cell extracts binds to columns of concanavalin A-agarose and can be eluted with 0.2 M alpha-methyl-D-mannopyranoside; porcine alpha(1-39) does not bind to concanavalin A-agarose. High molecular weight glycoprotein ACTH can be detected in extracts of mouse and bovine pituitary by using concavalin A affinity chromatography.


A relation between (NAD+)/(NADH) potentials and glucose utilization in rat brain slices.

Changes in several parameters involved in the control of metabolism were correlated with changes in glucose utilization in rat brain slices incubated under conditions which reduced glucose oxidation by 40 to 70%. The parameters included: the concentrations of ATP, ADP, AMP, and the adenylate energy charge; the cytoplasmic oxidation-reduction state ([NAD+]/[NADH]), determined from the [pyruvate]/[lactate] equilibrium; the mitochondrial oxidation-reduction state, determined from the [NH4+] ]2-oxoglutarate]/[glutamate] Equilibrium; the cytoplasmic and mitochondrial oxidation-reduction potentials (in volts), calculated from the respective [NAD+]/ [NADH] ratios using the Nernst equation; and the difference between the cytoplasmic and mitochondrial [NAD+]/[NADH] potentials. The conversion of [3, 4-14C] glucose to 14CO2 and of [U-14C] glucose to acetylcholine and to lipids, proteins, and nucleic acids by the brain slices were also determined. The values obtained by subtracting the mitochondrial from the cytoplasmic [NAD+1/[NADH] potentials correlated more closely with glucose utilization than did other parameters, under the conditions studied. For the synthesis of acetylcholine, the correlation coefficient was 0.96, and for the production of 14CO2 from [3, 4-14C] glucose it was 0.82.


Human erythrocyte pyrimidine nucleoside monophosphate kinase. Partial purification and properties of two allelic gene products.

Human pyrimidine nucleoside monophosphate kinase is a polymorphic enzyme having two allelic gene products, UMPK 1 and UMPK 2, in several populations. A procedure is described for the partial purification of this enzyme from human red blood cells resulting in a 1500-fold purification of the enzyme for UMPK 1 and 583-fold for UMPK 2. The purified enzyme preparation catalyzed the phosphorylation of UMP, CMP, and dCMP, and used ATP as the preferred phosphate donor. The heavy metals, mercury, and copper, were found to be strong inhibitors of pyrimidine nucleoside monophosphate kinase activity. EDTA was found to protect the enzyme from inactivation by the heavy metals, and 2-mercaptoethanol stabilized the enzyme during purification. UMPK 1 and UMPK 2 were found to have similar kinetic properties; however, UMPK 2 had a slower electrophoretic mobility and greater thermolability than UMPK 1.


Cyclic 3':5'-nucleotide phosphodiesterase. Ca2+ confers more helical conformation to the protein activator.

The ultraviolet spectrum of a protein activator of cyclic nucleotide phosphodiesterase and adenylate cyclase purified to homogeneity from bovine brain displayed absorption peaks at 252, 259, 265, 269, and 277 nm. The activator contained no phosphate and did not serve as a substrate for cyclic adenosine 3':5'-monophosphate- or cyclic guanosine 3':5'-monophosphate-dependent protein kinases. The activator binds Ca2+, and the active form appears to be a Ca2+ activator complex (Lin, Y.M., Liu, Y.P., and Cheung, W.Y. (1974) J. Biol. Chem. 249, 4943-4954). Optical rotatory dispersion measurement showed that the Ca2+-free activator exhibited a reduced mean residue rotation ([m']231) of -5700, corresponding to 39% of helical content. In the presence of Ca2+, the [m']231 was increased to -7500, corresponding to 57% of helical content. The Ca2+ -induced conformational change was corroborated by a chemical method. In the presence of Ca2+, the activator was more resistant to trypsin inactivation, presumably because proteins with more helical structures are more resistant to tryptic attack. The activator is rich in aspartate and glutamate. Chemical block of some of the carboxyl groups with glycine ethyl ester or methoxyamine diminished the [m']231 of the activator and its activity, suggesting that blockade of some of the carboxyl groups in the activator unfolded the molecule, leading to a loss of activity. We conclude that Ca2+, which confers more helical structure to the activator, converts the inactive, less helical structure to the active, more helical structure, and that chemical modification of the activator leading to unfolding of the molecule abolishes its biological activity.


Role of phospholipids in the calcium-dependent ATPase of the sarcoplasmic reticulum. Enzymatic and ESR studies with phospholipid-replaced membranes.

Three types of partially purified ATPase enzymes having different phospholipid contents and compositions have been prepared: (a) an enzyme whose phospholipid moiety has been replaced predominantly by dioleoyl lecithin (DOL-enzyme), with about the same phospholipid content as the original sarcoplasmic reticulum, (b) dipalmitoyl lecithin-replaced enzyme whose phospholipid content is 30% of that of DOL-enzyme (DPL-enzyme), and (c) a partially delipidated enzyme with about the same phospholipid content as DPL-enzyme but with the original sarcoplasmic reticulum phospholipid composition (del-enzyme). The temperature dependence of Ca2+-activated ATPase activity of these preparations showed clearcut differences; with DOL-enzyme there was no appreciable break in the Arrhenius plot in the 3-40 degrees range; DPL-enzyme showed a break at 29 degrees, and del-enzyme and sarcoplasmic reticulum one at 18 degrees. Transition temperatures obtained from ESR studies with the use of spin-labeled stearic acid incorporated into the membranes agreed with those derived from ATPase assays. Thermo-dynamic analysis of the ATP hydrolysis rates shows that DPL-enzyme has considerably larger values of activation enthalpy and activation entropy below the transition temperature (29 degrees) than those of the other preparations, while all enzyme preparations show similar free energies of activation. The ESR data show that below their transition temperatures DPL-enzyme, and to a lesser degree del-enzyme, have a strongly restricted motion of their phospholipid molecules as compared with either DOL-enzyme or sarcoplasmic reticulum. Studies on the formation and decomposition of phosphoenzyme have been carried out with the three types of ATPase preparations. At 0 degrees, the rate of inorganic phosphate liberation is 8 times lower in DPL-enzyme than in del-enzyme with little difference in the steady state level of phosphoenzyme. In DOL-enzyme, the level of phosphoenzyme and the rate of inorganic phosphate liberation are 1.8 and 3.5 times higher than the corresponding values obtained with del-enzyme. Addition of ADP to the phosphorylated intermediate of DPL-enzyme induces a fast reversal of the phosphorylation reaction. These results indicate that the physical state of the phospholipid molecules associated with the enzyme affects the decomposition of phosphoenzyme, with little effect on the phosphorylation reaction and its reversal.


Thyroid-stimulating hormone binding to beef thyroid membranes. Role of N-acetylneuraminic acid.

The effect of sugars on 125I-thyroid-stimulating hormone binding to beef thyroid membranes was studied to determine their role in thyroid-stimulating hormone (TSH) binding. At 0.1 M concentration, N-acetylneuraminic acid produced a 3- to 7-fold increase in TSH binding, was the only sugar to enhance TSH binding, and did so whether binding was determined in the cyclase medium or under conditions of optimum binding. The enhanced TSH binding remained after the membranes were removed from the high NeuAc concentration and an effect was observed at concentrations of 10 mM NeuAc. NeuAc did not alter the kinetics of TSH binding but the pH optimum for TSH binding shifted from pH 5.5 to 7.5 in the presence of NeuAc. Incubation of the membranes with increasing concentrations of NeuAc resulted in increased sialic acid content of the membranes. The NeuAc concentration curve of membrane sialic acid and TSH binding were roughly parallel. The capacity of the low affinity site increased from 0.74 to 2.5 nmol/mg of protein in the presence of NeuAc. The apparent affinity (0.88 X 10(6) M-1) of this site was unaffected by NeuAc. With the high affinity site, NeuAc increased both the apparent affinity and capacity from 2.2 X 10(8)M-1 to 5.5 X 10(8) M-1 and 1.6 to 3.1 pmol/mg of protein, respectively. Neuraminidase or neuraminidase plus beta-galactosidase incubation of the membranes removed approximately 60% of the sialic acid from the membranes within 15 to 30 min but did not affect TSH binding. Large quantities of sialic acid were detected in the soluble fractions during isolation of the membranes, 4 to 5% of which was ultrafilterable and not associated with high molecular weight proteins. It is concluded that among the sugars tested, NeuAc exhibits an unique effect on TSH binding that may have physiological significance. The inability to alter TSH binding by enzymatic removal of endogenous sialic acid suggests that either NeuAc resistant to hydrolysis is sufficient to maintain TSH binding or that NeuAc important in TSH binding is removed during membrane preparation but is replaced by incubation with exogenous NeuAc.


Isolation of stimulatory modulator of guanosine 3':5'-monophosphate-dependent protein kinase from mammalian heart devoid of inhibitory modulator of adenosine 3':5'-monophosphate-dependent protein kinase.

The stimulatory and inhibitory activities in the crude preparation of protein kinase modulator from dog heart were separated by Sephadex G-100 gel filtration, and the stimulatory modulator was further purified by DEAE-cellulose chromatography. The isolated stimulatory modulator, as the crude modulator preparation, stimulated the activity of the purified guanosine 3':5'-monophosphate (cGMP)-dependent protein kinases of both mammalian and arthropod origins in the presence of cGMP. The cGMP-dependent protein kinases were not activated by cGMP in the absence of either the isolated stimulatory modulator or the crude modulator. The stimulatory modulator, unlike the crude modulator had no effect on the activity of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase. The stimulatory modulator was a protein since its activity was destroyed by trypsin but was resistant to hydrolysis by DNase, RNase, phospholipase C, and lysozyme. The isolated inhibitory modulator, presumably the same as the protein inhibitor of cAMP-dependent protein kinase reported by Walsh et al. (Wash. D.A., Ashby, C.D., Gonzalez, C., Calkins, D., Fischer. E.H., and Krebs, E.G. (1971) J. Biol. Chem. 246, 1977-1985), depressed the cAMP-stimulated activity of cAMP-dependent protein kinase as did the crude preparation of protein kinase modulator. The isolated inhibitory modulator, unlike the crude preparation, was without effect on cGMP-dependent protein kinase. The present findings provide evidence to support that in mammals there are separate proteins for the stimulatory and the inhibitory activities of protein kinase modulator, in contrast to the modulator from an arthropod tissue (lobster tail muscle, Donnelly et al. (Donnelly, T.E., Jr., Kuo, J.F., Reyes, P.L., Liu, Y.P., and Greengard, P. (1973) J. Biol. Chem. 248, 190-198) which has been shown to possess both activities.


Function and structure of RNA polymerase from vesicular stomatitis virus.

The RNA-dependent RNA polymerase associated with vesicular stomatitis virus was isolated to apparent homogeneity by a newly developed procedure, which includes stepwise removal of proteins from virions by successive treatment with high concentrations of cesium sulfate and cesium chloride, followed by glycerol gradient centrifugation or chromatography on phosphocellulose or DEAE-Sephadex column. The polymerase thus purified contained L (large protein) and NS proteins as the intrinsic subunits and multiple species of enzyme were found which differ in the molar ratio of L to NS. Since the enzyme with the highest activity was composed of equimolar amounts of the two subunits and exhibited the sedimentation coefficient of approximately 11 S in a buffer containing 0.2 M NaCl, the structure of active protomer was suggested to be (L)1(NS)1. In accordance with this conclusion, enzyme preparations deficient in the content of NS protein, were activated by the addition of preparations deficient in the content of NS protein. The purified RNA polymerase catalyzed the synthesis of poly(A), which was covalently attached to the 3' termini of RNA products, and RNA, only in the presence of all 4 substrates. The present finding might be the first which indicates that the transcriptase itself catalyzes post-transcriptional modification of mRNA by adding poly(A) sequences to the 3'-OH termini. The molecular mechanism of the switch from transcription to poly(A) synthesis, however, remains to be investigated.


Size-dependent allosteric effects of monovalent cations on rabbit liver fructose-1,6-bisphosphatase.

Effects of monovalent cations on the neutral rabbit liver fructose-1,6-bisphosphatase are multifunctional and dependent on their nonhydrated ionic size. (a) The maximal velocity is increased by addition of monovalent cations with the optimum stimulation occurring with a nonhydrated ionic radius of 1.2 A in the presence of a chelating agent such as EDTA. (B) Activation curves are sigmoidal with n values varying from 1.5 to 2.3 as ionic radius of monovalent cation increases. The apparent Ka values from 16.0 to 180 mM, obtained for various monovalent cations, have a linear relationship to ionic radii of cations. (c) At lower concentrations of fructose 1,6-bisphosphate monovalent cations show the inhibitory effect and the apparent Km for fructose 1,6-bisphosphate is increased as the concentration of monovalent cation is increased. A linear relationship is obtained between the slopes of increase in the Km and the reciprocals of ionic volume of monovalent cations. (d) The apparent Ka for Mg2+ is also increased as the concentration of monovalent cation is increased, and a linear relationship is obtained again between the increases in Ka and the reciprocals of ionic volume of monovalent cations. The cooperative nature for Mg2+ saturation is decreased as the Ka increases. (e) The apparent Ki for AMP is also linearly altered as the concentration of monovalent cation is varied. However, the alteration of the Ki is unusual, that is, the smaller cations than K+ increase the Ki (Li+ greater than Na+ greater than NH4+), whereas the larger cations decrease the value ((CH2CH2OH)3N+ greater than Cs+ greater than Rb+). The effect of K+ is insignificant. Alterations in the Ki are also linearly related to the reciprocals of ionic volume of monovalent cations. The cooperative nature for AMP inhibition is decreased or increased as the Ki increased or decreased. (f) In the absence of the chelating agent, the curves for Mg2+ saturation and AMP inhibition were hyperbolic without monovalent cations. By addition of monovalent cation the Ka for Mg+2+ or Ki for AMP is increased and cooperative natures for binding of both ligands are induced. For nonspherical monovalent cations, the application of "functional ionic radius" is proposed. Functional ionic radii of NH4+, (CH2OH)3CNH3+, and (CH2CH2OH)3N+ are estimated to be 1.17, 2.55, and 2.87 A, respectively. The presence of two distinct sites for the actions of monovalent cations is suggested.


Kinetic resolution of ligand binding to the alpha and beta chains within human hemoglobin.

Nitric oxide has been used as a chain-specific, spin label of unliganded heme groups present in kinetic mixtures of human hemoglobin and n-butyl isocyanide. In these experiments, deoxyhemoglobin was reacted with n-butyl isocyanide for a controlled time and then mixed rapidly with a high concentration of nitric oxide to fill residual, unoccupied heme sites. The final mixture was frozen immediately after formation to prevent any displacement of bound isonitrile. The EPR spectrum of the frozen sample was resolved into alpha and beta nitric oxide components; these reflect the relative proportions of alpha- and beta-heme sites which were unoccupied by n-butyl isocyanide. Individual time courses for the alpha and beta subunits were obtained by varying the time between the formation of the isonitrile/hemoglobin mixture and its reaction with nitric oxide. At pH 7.0 only the beta chain time course exhibits an initial rapid phase; the alpha chain time course is monophasic, exhibiting almost, exponential behavior. This result shows unequivocally that the beta-hemes within deoxyhemoglobin react much more rapidly with n-butyl isocyanide than the alpha hemes.


Stimulation of ornithine decarboxylase synthesis and its control by polyamines in regenerating rat liver and cultured rat hepatoma cells.

Ornithine decarboxylase has been induced in log phase hepatoma cells grown in suspension culture. Induction with N6, O2'-dibutyryl cyclic adenosine 3':5'-monophosphate produced a 4-fold increase in enzyme activity by 3 hours which was followed by a return to base levels by 6 hours. Induction  with dexamethasone, a potent synthetic glucocorticoid, exhibited a slow steady rate of increase in enzyme activity, reaching a plateau level of approximately 5- to 6-fold stimulation by about 12 hours. Induced cell and regenerating rat liver ornithine decarboxylase were shown to be indistinguishable by titration with antibody monospecific to the latter and by heat stability. L-[14C]Leucine incorporation into immunoprecipitable enzyme protein after induction in vitro or partial hepatectomy showed an increase which, when coupled with the increase in enzymatic activity, indicated de novo synthesis of enzyme protein. Physiological concentrations of the naturally occurring polyamines, spermidine and spermine, abolish cyclic AMP induction whereas they have no effect on dexamethasone induction. Both inductions were abolished by cycloheximide; in contrast, inhibition by actinomycin D was complete for dexamethasone induction and only partial with respect to cyclic AMP induction. The different time pattern of induction seen with cyclic AMP and dexamethasone, the partial inhibition of the cyclic AMP induction seen with actinomycin D, as well as the absence of inhibition of the dexamethasone induction by polyamines, indicate that these inducers might affect different aspects of the control of the same enzyme.


Enzymological studies on hereditary avian muscular dystrophy.

White and red muscles of normal and genetically dystrophic chickens were compared with regards to activity levels of three soluble enzymes, glyceraldehyde-3-phosphate dehydrogenase, creatine phosphokinase, and acetyl phosphatase. In dystrophic white muscle (pectoral), activity of the two sulfhydryl enzymes, glyceraldehyde-3-phosphate dehydrogenase and creatine phosphokinase, was preferentially lost from the sarcoplasm resulting in decreased specific activities. By contrast, acetyl phosphatase was preferentially retained and showed increased specific activity. Dystrophic white muscle had decreased sulfhydryl content in the soluble proteins, severe reduction in muscle mass, fatty infiltration, and fragmentation of fibers. Red dystrophic muscles (thigh) were minimally involved in accordance with the known sparing of red fibers. Enzyme activities were correlated with histological observations. The results suggested that the disease process in dystrophic white muscle may be related to alterations in the sulfhydryl groups of proteins. The data are correlated with the beneficial effects of our treatment of hereditary avian dystrophy with the sulfhydryl compound, penicillamine (Chou, T.H., Hill, E.J., Bartle, E., Woolley, K., LeQuire, V., Olson, W., Roelofs, R., and Park, J.H. (1975) J. Clin. Invest. 56, 842-849).


Guanosine 3':5'-monophosphate-dependent protein kinase from silkworm, properties of a catalytic fragment obtained by limited proteolysis.

Although guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase (protein kinase G) which was partially purified from silkworm pupae was not dissociated by cyclic GMP into catalytic and regulatory subunits as described for adenosine 3':5'-monophosphate-dependent protein kinase (protein kinase A) (Takai, Y., Nakaya, S., Inoue, M., Kishimoto, A., Nishiyama, K., Yamamura, H., and Nishizuka, Y. (1976) J. Biol. Chem. 251, 1481-1487), limited proteolysis with trypsin resulted in the formation of catalytic and cyclic GMP-binding fragments which showed molecular weights of approximately 3.4 X 10(4) and 3.6 X 10(4), respectively (the molecular weight of native protein kinase G was 1.4 X 10(5)). The catalytic fragment did not bind cyclic GMP and was fully active in the absence of the cyclic nucleotide. The fragment did not show an absolute requirement for a sulfhydryl compound and high concentrations of Mg2+ (50 to 100 mM), both of which were necessary for the maximal activation of native protein kinase G. The catalytic fragment was not inhibited by the cyclic GMP-binding fragment nor by the regulatory subunit of protein kinase A. Inversely, the cyclic GMP-binding fragment was unable to inhibit the catalytic subunit of protein kinase A. Protein inhibitor, which was described for protein kinase A, was inert for the catalytic fragment.


Intra-osseous glomus tumour: a case report.

A rare case of intra-osseous glomus tumour is described, together with a review of eight other reported cases. Electron microscopic studies suggest that glomus cells, the histogenesis of which has not been completely resolved, originate from smooth muscle. Studies in this case support that hypothesis: they showed the important role of contraction of the glomus cells in eliciting the peculiar type of pain.


Laryngeal granular cell myoblastoma two case reports with radiographic demonstration.

Laryngeal granular cell myoblastoma is a rare, benign lesion. Two cases were studied by contrast laryngography and demonstrated similar, non-specific findings of a smooth, sessile mass.