Mortality patterns among workers exposed to chloromethyl ethers--a preliminary report.

Chloromethyl methyl ether (CMME) has been used extensively as a crosslinking agent for ion-exchange resins. Commercial grades of CMME are contaminated to the extent of 2-8% with bischloromethyl ether, an alkylating agent which has been shown to be a very potent lung carcinogen in animals. Reports by other investigators in this and other countries have implicated CMME as a lung carcinogen in chemical workers. The purpose of the study reported here was to examine the lung cancer mortality experience with respect to intensity and duration of exposure in six of the seven chemical companies that account for virtually all of the CMME use in the United States. The study included about 1800 workers who were exposed in the period 1948 to 1972 and about 8000 workers not exposed to CMME from the same plants who served as controls. Exposed workers were characterized according to job description and duration of exposure. In several plants the intensity of exposure was numerically graded for each job category with adjustment for temporal changes in the plant processes. Social Security records were used to identify deaths among workers who had left the companies and death certificates have been obtained for virtually all known deaths. The age-adjusted death rate for respiratory cancer in the CMME exposed group as a whole was 2.5 times that in the control group, whereas death rates due to other causes were comparable. There was also a gradation of lung cancer risk according to intensity and duration of exposure and the time elapsed since the onset of exposure.


Exposure to halogenated hydrocarbons in the indoor environment.

The indoor environment has frequently been ignored as a significant source of exposure to air pollutants. To date there are a number of documented examples of levels of indoor air pollutants greatly exceeding those levels which commonly occur in the outdoor environment. Among these instances are airborne buildup of polynuclear aromatics and cadmium from cigarette smoke, lead from burning candles, and vinyl chloride from use of aerosols containing this substance as a propellant. These examples suggest that there may be additional sources of indoor air pollutants, particularly halogenated hydrocarbons from aerosol products, which have heretofore not been generally recognized as important. The present paper endeavors to review those instances where halogenated hydrocarbons in the indoor air environment may build up to concentrations of potential public health concern. These considerations may be especially relevant in future years as increasing efforts are being made to insulate buildings more efficiently as a means to conserve energy. The available data strongly suggest that halogenated hydrocarbons are an important class of air pollutants in the indoor environment and that their presence in the outdoor environment should also be carefully examined. In this regard, halogenated hydrocarbons in the outdoor environment may also contaminate indoor air spaces.


Acute liver injury by vinyl chloride: involvement of endoplasmic reticulum in phenobarbital-pretreated rats.

A single 6-hr exposure to vinyl chloride monomer (5%) produces extensive vacuolization of centrolobular liver parenchyma and focal midzonal necrosis in the hepatic lobuole in phenobarbital-pretreated rats. Ultrastructurally, vacuolization consists of dilation of cysternae of rough endoplasmic reticulum and in the same cells smooth endoplasmic reticulum coalesces into discreet aggregates resembling denatured membranes. The findings support the hypothesis that vinyl chloride is hepatotoxic because it is converted into a toxic metabolite by components of the mixed function oxidase system of liver endoplasmic reticulum.


Screening tests for pulmonary function abnormality.

Simple tests based on a voluntary forced expiration can detect pulmonary impairment of occupational or environmental origin earlier than can be done using clinical or radiographic examination. These tests are easily performed and repeated, require little time and can be readily set up in the field. Flow impairment ("obstructive") has conventionally been measured by the forced expiratory volume--1 sec (FEV1); earlier changes, in the small airways are more likely to be detected by the maximal mid-expiratory flow (MMF) and the maximum expiratory flow-volume (MEFV) curve at low lung volumes. Volume impairment ("restrictive") is detected by the forced vital capacity (FVC) from which the preceding measurements are made.


Effects of vinyl chloride exposures to rats pretreated with phenobarbital.

Male rats were exposed to 10 consecutive days, 6 hr/day, to vinyl chloride vapors at an average concentration of 13,500 ppm. The exposed rats were divided into three groups of eight rats each: one group was pretreated with 3-methylcholanthrene, one group was pretreated with phenobarbital, and the third group received no treatment. Half the animals in each group were sacrificed 18 hr after the last exposure and half were sacrificed 4 days later. In a second experiment, four rats pretreated with phenobarbital were exposed to vinyl chloride vapors at a concentration of 17,300 ppm for 2 days and sacrificed about 9 A.M. on the third day. In both experiments control animals, also treated with phenobarbital or 3-methylcholanthrene, were exposed to air only. At the time of sacrifice, lungs, kidneys, spleen, heart, and a small piece of liver from each animal were preserved for histological examination. The remainder of the liver was processed for assay of microsomal enzyme activity. The following parameters were investigated: growth rate, organ weights, morphological changes, and both benzphetamine-N-demethylase activity and cytochrome P-450 content of microsomes prepared from the livers. In both experiments the only marked difference noted in any group was a decrease in the growth rate of the animals exposed to vinyl chloride and treated with phenobarbital. This decreased growth rate was particularly apparent on the third day of the vinyl chloride exposures. Occasional morphological changes were also seen in the livers of the animals treated with phenobarbital and exposed to vinyl chloride.


Pulmonary function defects in nonsmoking vinyl chloride workers.

Pulmonary function was assessed by spirometry and maximum expiratory flow-volume curves to determine whether exposure to an occupational environment contaminated with vinyl chloride (VC) fumes and poly(vinyl chloride) (PVC) dust is associated with an increased risk of respiratory impairment. Data were analyzed for smoking, duration of exposure, and age. The high prevalence of impaired flow (57.5%) could not be attributed to smoking. Prevalence in nonsmokers was 36.4% when exposure was less than 10 yr, 42% when exposure was between 10 and 20 yr, and 80% when exposure exceeded 20 yr. The last is virtually the same rate as for smokers exposed more than 20 years. The same trend is shown with increasing age. Unlike younger workers, when smokers and nonsmokers more than or equal to 40 years of age are compared, prevalence rates of air flow impairment are not statistically different. The present investigation is one of the few in which the effects of occupational exposure could be separated from and were found to predominate over the effects of smoking.


Aging of polymers.

This paper provides background on the basic chemical processes which can occur during the exposure of polymers to typical end-use environments such as air, sunlight, water vapor, and various atmospheric pollutant gases.


Additives in plastics.

The polymers used in plastics are generally harmless. However, they are rarely used in pure form. In almost all commercial plastics, they are "compounded" with monomeric ingredients to improve their processing and end-use performance. In order of total volume used, these monomeric additives may be classified as follows: reinforcing fibers, fillers, and coupling agents; plasticizers; colorants; stabilizers (halogen stabilizers, antioxidants, ultraviolet absorbers, and biological preservatives); processing aids (lubricants, others, and flow controls); flame retardants, peroxides; and antistats. Some information is already available, and much more is needed, on potential toxicity and safe handling of these additives during processing and manufacture of plastics products.


Industrial preparation of poly(vinyl chloride).

Vinyl chloride (VCM) is unloaded from railroad tank cars or tank trucks into pressurized storage spheres. VCM, emulsifiers, and catalysts are metered into polymerization vessels wherein PVC is produced through a chemical reaction in an aqueous medium under controlled conditions of temperature and pressure. After the reaction reaches a predetermined completion, the contents are transferred to a secondary vessel wherein steam is injected and the VCM containing vapors are pumped to a recovery system. The VCM-containing vapors are compressed, cooled, condensed, decanted, and recycled to the process for reuse. The stripped PVC resin water slurry is then pumped to blending tanks where the batches from multiple reaction vessels are blended for product uniformity. From the plant tanks the PVC resin water slurry is pumped to a dewatering centrifuge, where approximately 90% of the water is removed and subsequently discharged to the industrial sewer system. The PVC resin wet cake is conveyed from the centrifuge to a flash dryer where essentially all the remaining water is removed. At this point, the dry resin is buoyant in an air stream and enters a two-stage collection system for separation of conveying air. The PVC resin is then screened and air-conveyed to storage for bulk shipment, compounding, or bagging.


Public health implications of components of plastics manufacture. Flame retardants.

The four processes involved in the flammability of materials are described and related to the various flame retardance mechanisms that may operate. Following this the four practical approaches used in improving flame retardance of materials are described. Each approach is illustrated with a number of typical examples of flame retardants or synthetic procedures used. This overview of flammability, flame retardance, and flame retardants used is followed by a more detailed examination of most of the plastics manufactured in the United States during 1973, their consumption patterns, and the primary types of flame retardants used in the flame retardance of the most used plastics. The main types of flame retardants are illustrated with a number of typical commercial examples. Statistical data on flame retardant market size, flame retardant growth in plastics, and price ranges of common flame retardants are presented.


Cancer mortality in U.S. counties with plastics and related industries.

Counties in the United States have been identified with chemical establishments whose primary manufacturing processes use vinyl chloride. Site-specific cancer mortality comparisons have revealed an excess of multiple myeloma in males associated with two of the manufacturing categories, synthetic rubber and synthetic fibers. A causal relationship between these manufacturing categories and multiple myeloma could not be established. An industry-based assessment of the occupational contribution to this excess is needed to evaluate the etiologic importance of this relationship.


Preliminary studies on the fate of inhaled vinyl chloride monomer (VCM) in rats.

Rats were exposed to vinyl chloride monomer gas (VCM) in a closed recirculating system. The rate at which VCM was removed from the system via metabolism was determined for rats exposed to initial concentrations of VCM ranging from 50 to 1167 ppm. Upon exposure to initial concentrations of 50 to 105 ppm, the rate of metabolism was 8.04 plus or minus 3.40 x 10(-3) min-1. Upon exposure to initial concentrations ranging from 202 to 1167 ppm, the rate constants were less; the mean value being 2.65 plus or minus 1.35 x 10(-3) min-1. Regardless of concentration, the disappearance followed apparent first order kinetics. Pretreatment of rats with pyrazole prior to exposure to initial concentrations of 65 and 1234 ppm VCM caused 71 and 87% reductions in the rate of metabolism. Ethanol caused 96% and 83% reductions in the rate of VCM metabolism by rats exposed to 56 and 97 ppm VCM, respectively. Ethanol was less effective in blocking the rate of metabolism by rats exposed to high concentrations of VCM; 46 and 36% in rats exposed to 1025 and 1034 ppm VCM. In rats exposed to an initial concentration of 65 ppm VCM, SKF-525-A administration caused no inhibition of the rate of VCM metabolism; however, a 19% inhibition was seen in rats exposed to 1038 ppm. The nonprotein sulfhydryl content of the liver (glutathione and cysteine) of rats exposed to VCM concentrations ranging from 50 to 15,000 ppm VCM is reduced without a relationship to dose. With repeated daily exposure the degree of reduction is reduced. Preliminary results indicate that the primary metabolites of VCM react with the nonprotein sulfhydryl. Final metabolic products excreted in the urine appear to be S-(2-hydroxyethyl) cysteine and S-(2-carboxymethyl)cysteine and the respective N-acetyl derivatives. Monochloroacetic acid was identified as another potential metabolite. Considering the results in toto, it is hypothesized that VCM is readily and extensively metabolized. Metabolism via the primary pathway, postulated to involve alcohol dehydrogenase, is swamped by exposures to concentrations exceeding 220 ppm. In rats exposed to concentrations at and exceeding this level, metabolism occurs via a secondary pathway(s), postulated to be epoxidation and/or peroxidation. These results are considered pertinent is assessing the potential hazard at low level exposures to VCM.


General aspects in polymer synthesis.

The term, synthetic polymer, denotes a macromolecule composed of simple structural units and usually derived from monomers by a process of polymerization. This process is possible only if certain chemical, thermodynamical, and mechanistic conditions are satisfied. The monomer must have a functionality of two or higher. The polymerization process must be characterized by a negative free energy change. The monomer must have activable or reactive functions. Activable structures are polarizable multiple bonds, and bonds entailing heteroatoms in ring compounds. Suitable reactive functions are those that participate in: carbonyl addition, substitution, multiple bond addition, and free-radical coupling. According to their growth mechanisms, polymerization reactions are divided into two major groups: chain-growth polymerization and step-growth polymerization. Polyaddition and polycondensation occur in either group, but polyinsertion is a chain-growth process exclusively. Radical, ionic, and heterogeneous initiations are entailed in chain growth, whereas both nucleophilic and electrophilic initiation prevails in step-growth. The chemical structure of polymers is determined mainly by constitutional and configuration parameters. Principal constitutional ones are related to interlinking of chains and structural units, composition, substituents, endgroups, and the molecular weight. Configurational ones refer particularly to the position of substituents on a central chain atom relative to the neighboring structural unit and thus to the tacticity of the polymer.


Effect of X-irradiation on vitamin B12 binding capacity to intrinsic factor.

The effects of whole-body X-irradiation on vitamin B12-protein complex formation in gastric juice after oral administration of [57Co]-B12 have been studied. Two proteins with B12-binding activity have been isolated by gel filtration from gastric juice. 57Co-activity, recovered from B12-protein complex in gastric juice, is found to be about 30% less in the X-irradiated rat. In serum, vitamin B12 is mainly associated with alpha1-globulin. Radioactivity distribution in serum globulins after intraperitoneal injection of [57Co]-B12 was similar in control and X-irradiated rats.


Dietary vitamin E and the effects of inhaled nitrogen dioxide on rat lungs.

Groups of rats were fed diets containing supplemented adequate, or deficient amounts of alpha-tocopherol. Some rats from each group were exposed for 2 h to high-level concentrations of nitrogen dioxide in their breathing air. A comparison of wet and dry lung weights did not indicate that exposure of the gas was a cause of edema. Proportion of lung weight to total body weight was increased in all animals with the lipid content diminishing according to the amount of dietary apha-tocopherol available. Dietary intake of the vitamin did not seem to protect the lipid content of lungs of the supplemented dietary group from oxidation. No difference in total peroxides was noted between any of the groups. Inflation and deflation compliance measurements were greater for both exposed and non-exposed supplemented animals when compared to the adequate and deficient groups.


Some properties of a unique cadmium-binding moiety in the soluble fraction of rat testes.

A 30,000 molecular weight testicular Cd-binding peak (30,000 MW Cd-BP) previously implicated in Cd-induced testicular injury was unstable during storage with respect to apparent molecular weight determined by Sephadex G-75 chromatography. Storage of testicular cytosol labeled with 109Cd in vivo or in vitro for several days at 4 degrees C under nitrogen resulted in disappearance of the 30,000 MW Cd-BP and increased 109Cd uptake in other protein fractions. Rechromatography of the previously isolated 30,000 MW Cd-BP after storage gave rise to a 109Cd peak eluting in the higher molecular weight region. The latter effect was prevented by 1 mM dithiothreitol, suggesting that sulfhydryl groups were involved in the apparent aggregation. The 30,000 MW Cd-BP found in testes of rats was not present in testes of roosters, nor in liver and kidney of either species, providing further evidence of a correlation between the occurrence of 30,000 MW Cd-BP protein in the tissue and susceptibility to Cd-injury. The inability of parenterally administered HgCl2 to induce testicular injury compared to the same dose of CdCl2(0.011 mmol/kg) is apparently related to the poor uptake of Hg in the testes (one-eighteenth that of Cd) rather than to an inability of Hg to bind to the 30,000 MW Cd-BP. Our studies indicate that binding of Cd to this unique 30,000 MW testicular component, as yet unidentified, is a possible basis for the unique sensitivity of the testis to Cd injury.


Effect of ethanol on the metabolism of the white blood cells.

Ethanol at a concentration of 40 mg% in the medium was found to inhibit the respiratory uptake of oxygen by polymorphonuclear cells obtained from rat peritoneal fluid. The stimulated oxygen uptake by these cells under conditions of phagocytosis was also inhibited with this concentration of ethanol. The quantities of hydrogen peroxide produced by these cells were also found to have been diminished. The possible significance of these findings is discussed.


Polysome translational state during the cell cycle.

HeLa cells were synchronized with a double thymidine block. Ribosomal subunits, monomers and polyribosomes have been quantitatively analysed at hourly intervals, during interphase, and every 15 min, during mitosis. This analysis was performed on linear 7-47% sucrose gradients. From the beginning of G1 up to the end of S phase, a certain equilibrium among ribosomal subunits, monomers and polyribosomes is maintained, while from the time of entering G2 to M the translation machinery appears to be mobilized in the sense of polysome formation. Under these conditions, the amount of polysomes per cell during the mitotic cycle is expressed by a bi-phasic pattern showing pre- and post-mitotic peaks with a falling-off during S. The G1 peak, meanwhile, is much lower than the G2 peak. The incorporation of [3H]leucine into nascent polypeptide chains on polysomes, as well as into bulk cell proteins and into nuclear and cytoplasmic proteins considered separately, is also represented by a bi-phasic curve which shows, however, a higher peak in G1 and a lower peak in G2, with two fallings-off during S and M, respectively. Since between the G1 and the G2 amino acid pools there are not strong differences of leucine concentration, the discrepancy between the amount of polysomes and the rate of labelling is discussed on the basis of the differences of polysome shape found at the different stages of the cycle. In young cells, in fact, there is an abundance of small polysomes, while in the old cell large polysomes predominate. It is suggested that, in the old cell, the rate of translation on large polysomes could be relatively lower or that among these heavy aggregates a given number of "frozen" polysomes could be present. The ribosome state is considered as a probable limiting-factor of translation, particularly in mitosis.


Structural studies on the coat protein of alfalfa mosaic virus. Isolation and characterization of the tryptic peptides and the alignment of the cyanogen-bromide fragments.

The reduced and carboxymethylated coat protein of alfalfa mosaic virus (AMV 425) was fragmented by means of cyanogen-bromide cleavage. The tryptic peptides from the protein and its four cyanogen-bromide fragments were isolated on a preparative scale by combinations of column and paper separation techniques. The tryptic digest of the carboxymethylated protein contained 24 peptides and two free amino acids. All peptides have been characterized by amino acid analyses and end-group determinations. Together the tryptic peptides account for a total chain length of 228 amino acids. The data are in good agreement with previous reports from this laboratory.


Liver 3-phosphoglycerate kinase. Physico-chemical characterization of the bovine-liver enzyme.

Homogeneous phosphoglycerate kinase from bovine liver possesses a maximum ultraviolet absorption at 278 nm (A 1%,1Cm 280 equals 6.7; Amax/Amin equals 2.26; e280 equals 31.5 mM(-1) X cm(-1). The enzyme consists of about 420 amino-acid residues and is a slightly acidic protein with an isoelectric point of 6.5 as expected from amino-acid analysis. The most notable features of the chemical composition are two tryptophan, 12 methionine and four half-cystine residues per enzyme molecule. Although phosphoglycerate kinases from mammalian tissues are partially similar to each other, clear differences in serine, glutamic acid, glycine, cysteine, valine, leucine, tyrosine, tryptophan and arginine contents were found. Fingerprinting and column chromatography of tryptic digests of the S-carboxymethylated protein confirm the data of amino-acid analysis. Liver phosphoglycerate kinase is inactivated when modified with either p-chloromercuribenzoate or 5,5'dithio-bis(2-nitrobenzoic acid) (Nbs2). The enzyme has two thiol groups available for reaction with Nbs2 under denaturing conditions, one of which is essential for catalysis. After reduction by NaBH4 four cysteine residues per molecule were determined with Nbs2, sugessting the presence of a disulfide bridge. Using sedimentation equilibrium studies, the molecular weight was found to be 49600. Gel filtration yielded values of 43000-50000. By analytical dodecylsulfate-polyacrylamide gel electrophoresis a molecular weight of 45600 was estimated. Inconsistent with these results in the value 37500 obtained by thin-layer gel chromatography in 6 M guanidine-HCl. Sedimentation velocity experiments revealed a sedimentation coefficient s20,w equals 3.4 S. The Stokes radius was 2.77 nm, the partial specific volume v 0.747 ml x g(-1). The diffusion coefficient was found to be 76.9 mum2 x s(-1) by analytical gel filtration. From these data a molecular weight of 44000 was calculated. Other physical constants of bovine-liver phosphoglycerate kinase are: frictional ratio f/f0 equals 1.18, axial ratio equals 3.3, maximal degree of hydration equals 0.1 g per g of protein. Bovine-layer phosphoglycerate kinase could not be dissociated into smaller subunits by treatments which have caused dissociation of various other proteins (8 M urea, 6 M guanidine-HCl, dodecyl sulfate, carboxymethylation, maleylation). All experiments strongly support the lack of subunit structure of the enzyme. Some characteristics of bovine-liver phosphoglycerate kinase are compared with the corresponding proteins from rabbit muscle, yeast and human erythrocytes.


The binding of atractylate and carboxy-atractylate to mitochondria.

35S-labelled atractylate and carboxy-atractylate are produced biosynthetically and used for studying the binding of these specific ligands to the ADP, ATP carrier in beef heart mitochondria. The following results are obtained. 1. Inhibition of translocation activity goes parallel to the increase of binding by [35S]atractylate. No additional binding is observed after full inhibition of translocation is reached giving evidence that atractylate binds exclusively to the carrier. 2. The maximum number of binding sites of both atractylates is about 1.6 mumol/g protein in beef heart mitochondria and decreases on treatment of the membrane by Pi, freezing, ageing, etc. The dissociation constants of the binding are approximately for atractylate Kd = 5-10(-8) M and for carboxy-atractylate Kd = 10(-8) M. The mass action plots of the concentration dependence for the binding are nonlinear-convex in particular with carboxy-atractylate and more linear with atractylate. Nonlinearity appears to be caused by some retardation of equilibration in the case of very high affinity binding. 3. The binding of atractylate and carboxy-atractylate is relatively fast in intact mitochondria and slower in aged membranes. There is a slower and a faster binding portion. 4. The atractylates remove ADP in a nearly 1:1 stoichiometry from untreated mitochondria. In aged and Pi-treated membranes the ratio deltaADP/deltaatractylate approaches 0. Obviously binding of carrier sites to ADP is more sensitive to alterations than that of the atractylates. The assumption is maintained that the binding site for atractylate is identical with that for ADP and ATP. 5. Bongkrekate prevents binding of both atractylates. However, when added after, it only removes atractylate but not the carboxy compound because of its different tight binding. The removal of atractylate depends on the synergistic effect of bongkrekate with ADP. 6. The binding studies with [35S]atractylate and in particular the interaction with bongkrekate support the reorienting carrier model in which atractylate as an impermeable ligand fixes the binding site of the carrier outside while with bongkrekate the carrier site is turned to the inside.


Translational step inhibited in vivo by aflatoxin B1 in rat-liver polysomes.

Aflatoxin B1 strongly inhibits protein synthesis in rat liver cells. We previously demonstrated that this inhibition could be divided into two steps: up to 5 h aflatoxin blocks protein synthesis directly and specifically at the polysome level; beyond 7 h protein synthesis inhibition appears chiefly as a consequence of transcription impairment due to drug action. This paper confirms the foregoing results and represents an attempt to localize the translational step inhibited in vivo by aflatoxin B1. We used the simulation study developed by Li, Kisilevsky, Wasan and Hammond, 1972 (Biochim. Biophys. Acta, 272, 451-462) to determine precisely the site inhibited in vivo after drug intoxication. This analysis is based on two parameters: the kinetics of polysome labeling to follow the nascent peptide synthesis, and the kinetics of supernatant labeling to follow the completed protein synthesis. Up to 5 h after dosing, aflatoxin specifically inhibits the elongation and/or termination steps during protein synthesis; after longer periods of time inhibition occurs essentially at the initiation step. When the intracellular concentration of aflatoxin is too high, particularly 2 h after dosing, each step of protein synthesis is blocked. Polypeptide synthesis by the postmitochondrial supernatants isolated from aflatoxin-treated animals is impaired in the same proportion as protein synthesis in vivo. The damage caused by aflatoxin is mostly observed on microsomes. However, purified polysomes isolated from aflatoxin-treated rats synthesize proteins in vitro to the same extent as those from controls. These results suggest that aflatoxin metabolite(s) are bound to polysomes with noncovalent bonds. These active metabolites are probably lost during polysome isolation procedures. Finally, relationships between protein metabolism and aflatoxin carcinogenesis are discussed.


Deficiency of joining of Okazaki-type fragments in absence of cellular protein synthesis.

The dependence of integration of newly formed DNA chain ( less than 10 S) into larger DNA on concomitant protein synthesis was studied in a special cellular system. Exponentially growing Ehrlich ascites tumor cells in vivo show decreasing rated and finally complete cessation of protein and DNA synthesis upon transfer into an isotonic but non-nutritive environment (Hanks' balanced salt solution). Both protein and DNA synthesis is stimulated in these cells for a period of 30 min when they are placed into fresh Hanks' balanced salt solution; however, stimulation of protein synthesis is completely prevented in Hanks' balanced salt solution containing cycloheximide. This system allowed us to investigate the formation and fate of newly formed DNA chains ( less than 10 S) in dependence of protein synthesis. Analysis of DNA produced in [3H]thymidine pulses showed that DNA chains smaller than 18 S were still formed during the phase of totally delayed protein synthesis and in the presence of cycloheximide, but they were not converted into DNA molecules sedimenting faster than 18 S under these conditions. Stimulation of protein synthesis for a period of 30 min allowed the short DNA pieces to be chased into larger DNA 30 min post stimulation of protein synthesis. The results clearly indicate that DNA chain growth, by sealing of DNA chains smaller than 18 S, is strongly dependent of concomitant protein synthesis. Direct chain elongation by addition of new deoxyribonucleotides is less dependent on concomitant cellular protein synthesis.


Interactions of heteroaromatic compounds with nucleic acids. A - T-specific non-intercalating DNA ligands.

In the present paper we report the results of a study on the base specificity and affinity of eight dyes potentially able to interact with DNA. These compounds include four triphenylmethane dyes used in histochemistry, auramine, "Hoechst 33258" and two acridines substituted with t-butyl groups. They were selected with regard to their inability to intercalate between the base pairs of helical polynucleotides due to structural limitations. Hydrodynamic studies performed with the DNA complexes of crystal violet and Hoechst 33258 confirmed our assumptions that compounds of this type bind to the outside of DNA. The main results from DNA binding studies indicate that the triphenylmethane dyes except p-fuchsin are bound with high preference to two adjacent A - T pairs while Hoechst 33258 seems to need three A - T pairs as the binding site. Model studies with synthetic polynucleotides revealed that not only a sequence of A - T pairs, but also their structural arrangement in a helix, is crucial for the high affinities observed for most of the ligands when interacting with natural DNA. Methyl green and Hoechst 33258 can be used for increasing the resolution power of cesium chloride density gradients for DNAs with different (A + T) content.


Conformational changes of alamethicin induced by solvent and temperature. A 13C-NMR and circular-dichroism study.

13C nuclear magnetic resonance (NMR) and circular dichroism (CD) have been used for studies on the conformation of alamethicin. The 13C NMR spectrum is assigned with the aid of signals of synthetic partial sequences and selective proton decoupling. The solvent and temperature-dependence of the 13C NMR spectra, T1 measurements and the use of lanthanide-shift reagents allow the differentiation between the amino acids belonging to a rigid alpha-helical portion of the alamethicin sequence and those belonging to a more flexible part. The 13C NMR results are in agreement with results obtained from extended solvent and temperature-dependent CD studies which indicate a highly stabilized nonpolar and intrachenar alpha-helical part. The concentration-dependence of the CD spectrum of alamethicin in a nematic phase revealed aggregation phenomena which might simulate those observed in natural and synthetic membranes. After dissolving alamethicin in aqueous alcohol there is a time-dependence of the ellipticity of the Cotton effects showing a sort of memory effect on the mode of dissolution. Four different conformations can be characterized by CD spectra depending on the solvent and concentration. A model illustrating the dynamic conformations and aggregation phenomena within a membrane is proposed.


Reassociation kinetics of deoxyribonucleic acid in antigen-stimulated mouse-spleen cells.

In order to directly compare the complexity of the genome of lymphoid cells which have been antigenically stimulated, with that of non-immunized and non-lymphoid cells, DNA was pulse labeled and extracted from BALB/c mouse spleen cells at various time intervals after antigenic stimulation in vivo; the reassociation rates of these newly synthesized DNA preparations were compared with those of the total mouse spleen DNA, obtained from same sources and at the same times. DNA labeled for 60 min at 43, 53, or 72 h after antigenic restimulation, reassociated faster than the corresponding total DNA. On the other hand, the ressociation profile of DNA, labeled for 60 min during the first 24 after restimulation did not differ from that of the total DNA extracted at the same time. When labeled thymidine was available for incorporation at a constant concentration over a period of 24 h, reassociation patterns of labeled DNA were identical to those of the corresponding total DNA at all times after restimulation. Newly synthesized nuclear DNA exhibited reassociation profiles identical to those of the corresponding total nuclear DNA at all times tested. Also, no differences between the reassociation rates of nuclear and total cellular DNA were observed. It was concluded that antigenic stimualtion does not induce a major amplification of genes in the stimulated cells, and that the rapidly reassociating DNA species described represent extranuclear (cytoplasmic) DNA.


Interaction of a non-histone chromatin protein (high-mobility group protein 2) with DNA.

1. The interaction with DNA of the calf thymus chromatin non-histone protein termed the high-mobility group protein 2 has been studied by sedimentation analysis in the ultracentrifuge and by measuring the binding of the 125I-labelled protein to DNA. The results have been compared with those obtained previously by us [Eur. J. Biochem. (1974) 47, 263-270] for the interaction of high-mobility group protein 1 with DNA. Although the binding parameters are similar for these two proteins, high-mobility group protein 2 differs from high-mobility group protein 1 in that the former appears to change the shape of the DNA to a more compact form. 2. The molecular weight of high-mobility group protein 2 has been determined by equilibrium sedimentation and a mean value of 26 000 was obtained. 3. A low level of nuclease activity detected in one preparation of high-mobility group protein 2 has been investigated.


Effects of ricin on the ribosomal sites involved in the interaction of the elongation factors.

The effects of ricin on the different steps of the elongation cycle of protein synthesis in a rabbit reticulocyte cell-free system are studied in this paper. The toxin most probably acts by catalytically inactivating the ribosomes, since a single molecule of the toxin can inactivate 300 ribosomes for poly(U)-directed phenylalanine incorporation. The effect of the toxin on the ribosome is irreversible. Ricin specifically inhibits elongation-factor-1-dependent aminoacyl-tRNA binding to ribosomes but has no effect on the non-enzymic binding of aminoacyl-tRNA. Ricin also inhibits formation of the complex elongation-factor-2 - ribosome - nucleotide with GTP, GDP or GMP-P(CH2)P. However, the toxin has no effect on translocation. These apparently conflicting results are discussed in this study.


Trans-N-deoxyribosylase: purification by affinity chromatography and characterization.

trans-N-Deoxyribosylase (EC 2.4.2.6) is usually considered as a single protein catalyzing indifferently the transfer of the deoxyribosyl moiety to and from a purine or a pyrimidine base. Affinity chromatography of an extract from Lactobacillus helveticus with two types of ligands allowed the separation and purification of two distinct trans-N-deoxyribosylases. One catalyzes specifically the deoxyribosyl transfer to and from purine bases exclusively: trans-N-deoxyribosylase-I, the other catalyzes the transfer to and from pyrimidine and purine bases: trans-N-deoxyribosylase-II. A Tris inhibition study showed a markedly different susceptibility of the two enzymes. Preliminary results indicate that the purine-specific enzyme is a polymeric enzyme of molecular weight 86 000 (+/- 4000).


Trans-N-deoxyribosylase: substrate specificity studies. Purine bases as acceptors.

A series of purine bases and analogues were tested as substrates for trans-N-deoxyribosylase (EC 2.4.2.6). It was observed that the pyrimidine ring and its substituents on positions 1, 2 and 6, are of minor importance. On the other hand only a few modifications are tolerated on the imidazole moiety, as follows. 1. A tautomeric proton must be present on the imidazole ring. The "usual" shift is between position 9 and 7. 2. The position of the tautomeric proton governs the site of substitution. 3. For steric reasons no substituent is allowed on position 8.


The biosynthesis of basement-membrane collagen by isolated rat glomeruli.

A technique is described for the rapid isolation of highly purified preparations of viable glomeruli from rat kidney cortex. The synthesis of protein as judged by the incorporation of [14C]proline into non-diffusible material was shown to be linear for up to 6 h. The synthesis of collagen, measured as non-diffusible 4-hydroxy[14C]proline, was also linear over this period but represented only a small proportion of total protein synthesis. Similar studies conducted in vivo confirmed that collagen synthesis accounted for less than 5% of total protein synthesis in glomeruli. When isolated glomeruli were incubated with [14C]proline, it was found that approximately 16% of the hydroxyproline present in the collagenous component occurred as the 3-isomer. When glomeruli were incubated with [14C]lysine over 90% of the hydroxy[14C]lysine synthesised was glycosylated and most of the glycosylated hydroxy[14C]lysine was present as glucosyl-galactosyl-hydroxy[14C]lysine. The size of the basement membrane collagen synthesised by the isolated glomeruli was estimated by treating the 14C-labelled protein with mercaptoethanol and sodium dodecyl sulphate and then chromatographing the 14C-labelled protein on an agarose column equilibrated and eluted with buffer containing 0.1% (w/v) sodium dodecyl sulphate. The initial form of [14C]collagen synthesised was found to consist of polypeptide chains which had molecular weights of approximately 140 000 and which were shown to be distinctly larger than the polypeptide chains from embryonic chick tendon procollagen. Also when glomeruli were labelled with [14C]proline for 2 h and chased with unlabelled proline for 4 h there was a time-dependent conversion of the initially synthesised collagen moiety to collagen polypeptide chains which co-chromatograph with tendon pro-alpha chains (molecular weight approx. 120 000).


On the mechanism of action of alkylguanidines on oxidative phosphorylation in mitochondria.

The effect of octylguanidine and oligomycin on the oxygen uptake of rat liver mitochondria and on the ATPase activity of "sonic" submitochondrial particles has been studied. 1. Octylguanidine inhibits state 3 respiration with glutamate-malate and succinate as substrates, but much lower concentrations are required to inhibit oxygen uptake with the former substrates. State 4 respiration is unaffected by octylguanidine. 2. The titration-curve for the octylguanidine inhibition of glutamate-malate oxidation is hyperbolic and apparently biphasic, half-maximal inhibition is obtained at 30 muM octylguanidine. The octylguanidine-curve for inhibition of succinate oxidation is sigmoid with half-maximal inhibition at about 250 muM. 3. Octylguanidine and oligomycin show additive inhibitory action on state 3 respiration with both glutamate plus malage and succinate as respiratory substrates. 4. Concentrations of oligomycin or octylguanidine, which added separately are ineffective on state 3 respiration, become inhibitory when the two inhibitors are added together. 5. Octylguanidine inhibits the ATPase activity of sonic submitochondrial particles with a hyperbolic titration-curve analogous to that obtained for oligomycin inhibition. The inhibitory actions of octylguanidine and oligomycin on the ATPase activity are additive. 6. It is concluded that octylguanidine acts directly on the ATPase complex and that its binding at the action site is mutually exclusive with the binding of oligomycin. A kinetic explanation is given for the reported higher sensitivity of site I phosphorylation to octylguanidine.


Initiation of transcription within an RNA-polymerase binding site.

1. The 5'-terminal sequence of the RNA transcribed from bacteriophage fd replicative form DNA under the control of promotor region I has been determined to be ppp(Gp)nUpApApApGpApCpCpUpGpApUpUp. . . 2. This sequence is complementary to the 5'-terminal sequence of the minus strand of the corresponding RNA polymerase binding site I, the starting point for RNA synthesis lying approximately in the middle of the binding site. 3. This initial sequence is also transcribed faithfully from isolated complexes of RNA polymerase and binding site I, obtained by DNase digestion of complexes between RNA polymerase and fd replicative form DNA. These highly stable complexes can not be reconstituted from binding site and enzyme. 4. It is concluded that RNA polymerase binding site and initiation site are identical parts of a promoter region, and that no "drift" between these sites is required as a step in RNA chain initiation. An additional non-transcribed outside region is implicated as essential for full promoter function.


Specific deuterium labelling and computerized gas chromatography -- mass spectrometry in studies on the metabolism in vivo of a steroid sulphate in the rat.

The metabolism of 3beta-hydroxy-5alpha-pregnan-20-one sulphate was studied in bile fistula rats and in isolated perfused livers. Computerized gas chromatography--mass spectrometry, in combination with specific deuterium-labelling, was employed to follow the metabolic transformations. Male animals excreted metabolites into bile more rapidly than females, a finding which could be correlated with the preferential formation of glucuronide conjugates in the male liver. The major metabolic pathway in male rats involved the steps: hydrolysis, 2alpha-hydroxylation, oxidoreduction at C-3 and glucuronide conjugation, yielding 2alpha, 3alpha-dihydroxy-5alpha-pregnan-20-one glucuronide as the major metabolite. Only traces of the injected steroid sulphate were detected in bile from male animals. In contrast, the administered compound was the major steroid excreted in bile of female rats, where the main metabolite was identified as 3beta,15beta-dihydroxy-5alpha-pregnan-20-one sulphate. A minor metabolite, 3beta,16alpha-dihydroxy-5alpha-pregnan-20-one, was found as a monosulphate in female rats and as both a disulphate and a glucuronide conjugate in male rats. The deuterium content of the sulphated 15beta-and 16alpha-hydroxylated metabolites was consistent with metabolic pathways involving direct hydroxylation of the injected steroid sulphate. The results obtained from the liver perfusions were essentially the same as those from the experiments with bile fistula animals. This indicates that all the observed metabolic reactions took place in the liver.


Broad-specificity proteinase inhibitors in Scopolia japonica (Solanaceae) cultured cells. Isolation, physicochemical properties, and inhibition kinetics.

Proteinase inhibitors were isolated from Scopolia japonica cultured cells. Isolation procedures involve concentration by a hydrophobic resin of Diaion HP-20, decolorization by Duolite A-7, affinity chromatography on trypsin-Sepharose, and Bio-Gel P-4 chromatography. It was found that the proteinase inhibitors from S. japonica cells are a mixture of at least five components. For the inhibitory components except one, amino acid analyses, measurements of sedimentation equilibrium and optical rotatory dispersion (ORD) were carried out. The inhibitors were shown to be the polypeptides with molecular weights in the range of approximately 4000 to 6000. In addition, one of them was found to have approximately 15% alpha-helical conformation by the Moffitt-Yang analysis of ORD data. The inhibitors were found to have potent inhibitory activity for trypsin, chymotrypsin, plasmin, kallikrein and pepsin but not for papain with synthetic and natural substrates. These inhibitors formed stable complexes with trypsin and chymotrypsin in an equimolar ratio, and their inhibitory mechanisms for both enzymes were of non-competitive type.


Rat-liver cholesterol 7 alpha-hydroxylase. 1. Development of a new assay based on the enzymic exchange of the tritium located on the 7 alpha position of the substrate.

A new assay is described to measure the activity of cholesterol 7alpha-hydroxylase and compared to the conventional 14C method used by other investigators. This method is based on the mechanism of the enzymic hydroxylation, i.e. a direct and stereospecific substitution of the 7alpha-hydrogen by a hydroxyl group. [7alpha-3H]Cholesterol is incubated at 37 degrees C and in the presence of molecular O2, in a medium buffered by postassium phosphate at pH 7.4 and containing liver microsomes (or 9000 X g supernatant), NADPH, MgCl2 and cysteamine. Tween-80 (1.5 mg/ml) is used to introduce enough substrate (300 muM) in the incubation mixture to saturate the enzyme (Km = 100 muM). Under these conditions the tritiated water released into the incubation medium reflects accurately the enzymic activity. The results obtained with this method are similar to the one obtained with a [4-14C]cholesterol technique (r = 0.96; P less than 0.001). The main advantage of the [7alpha-3H]cholesterol method is a complete independence from further metabolism of the first enzymic product, the 7alpha-hydroxycholesterol, the tritiated water representing the entire cholesterol 7alpha-hydroxylase activity.


Fingerprinting studies of the maturation of ribosomal RNA in mammalian cells.

32P-labelled ribosomal RNA of L 5178 Y cells (a mouse cell line) was digested with T1 ribonuclease and fingerprinted by electrophoresis at pH 3.5 on cellulose acetate and homochromatography on DEAE-cellulose thin-layer plate. From this, it can be concluded that 18-S and 28-S RNA have different and characteristic fingerprints and that the number, the size and the frequency of the large T1 oligonucleotides demonstrate that the guanylic residues are randomly interspaced along the molecule. Using a double-labelling technique with 32P-labelled 45-S RNA and 14C-labelled 18-S RNA or 28-S RNA, long T1 oligonucleotides of the 45-S RNA can be divided into three classes: (a) those which are lost during the transition, (b) those which are present in the 18-S RNA and (c) those which are present in the 28-S RNA. These results provide direct evidence for the existence of one common precursor for the two mature ribosomal RNAs. The comparison of the fingerprints of T1-ribonuclease-digested 47-S, 45-S and 41-S RNA shows that the 47-S and 41-S RNA have a characteristic ribosomal pattern. Finally the size, the number and the mobility of the oligonucleotides present in the different RNA precursors but absent from the mature RNA demonstrate that the non-conversed RNA pieces do not have a monotonous and repetitive sequence.


Stimulation of tumor-cell respiration by inhibitors of pyruvate kinase.

In a model system consisting of highly coupled rat liver mitochondria respiring in the presence of substrate, pyruvate kinase, phosphoenolpyruvate, ATP, hexokinase and glucose, the increase in the mitochondrial concentration results in a progressive decrease in the activity of pyruvate kinase. These results are in accord with a role of pyruvate kinase as a determinant of glycolytic activity by competing with mitochondrial oxidative phosphorylation for the available ADP. The addition of adequate amounts of the amino acids, cysteine, alanine and phenylalanine, known as inhibitors of pyruvate kinase, to living Ehrlich ascites tumor cell suspensions results in a stimulation of the respiratory rate and in a decrease of the glycolytic rate of the cells. Concomitant with these changes, there is an accumulation of intracellular phosphoenolpyruvate and ADP, and a decrease in pyruvate and ATP. These results provide additional evidence for paying attention to pyruvate kinase as another key enzyme whose properties and activities may be major determinants for the control of glycolysis and the Crabtree and Pasteur effects of tumor cells.


Cholesterol 7alpha-hydroxylase. 2. Biochemical properties and participation of endogenous cholesterol in the assay in vitro.

70% of the microsome-bound cholesterol is directly accessible to cholesterol 7alpha-hydroxylase in an assay in vitro. After 5 min of incubation this endogenous cholesterol makes a single pool with the exogenously added substrate and modifies its specific radioactivity. Thus, an accurate estimation of the enzymic activity should take the participation of endogenous cholesterol into account. Cholesterol 7alpha-hydroxylase activity is enhanced in vitro by thiol-containing substances like mercaptoethanol, dithiothreitol, or cysteamine. On the contrary, the enzymic activity is inhibited by heavy cations (Hg2+, Pb2+, Cu2+, Zn2+), or --SH-blocking agents (mersalic acid p-chloro-mercuribenzoic acid). Several steroids are potent inhibitors (Ki less than Km) of the enzyme, among them pregnenolone and its acetate derivative and the cholesterol closely related 7-oxocholesterol and 7-dehydrocholesterol. The cholesterol esters are neither substrates nor inhibitors of cholesterol 7alpha-hydroxylase. Only a high concentration (1 mM) of biliary acids or of their glyco or tauro derivatives inhibits cholesterol 7alpha-hydroxylase. The quantitatively less important lithocholic acid and deoxycholic acid are the strongest inhibitors; the most common cholic acid does not affect the enzymic activity even at 1 mM.


Rat-liver cholesterol 7alpha-hydroxylase. 3. New results about its circadian rhythm.

The establishement of the circadian rhythm of cholesterol 7alpha-hydroxylase activity requires protein and RNA synthesis. The spontaneous decrease of the enzymic activity, at the end of the night, allows us to evaluate a half-life time of about two hours. The half-life time goes up to about four hours when the enzymatic activity decay is measured following cycloheximide administration. This difference suggests that an active mechanism is involved in the control of the enzyme degradation. The daily variation of the enzyme activity is regulated via the hypothalamo-hypophysis-adrenal axis. At the cellular level glucocorticoids are the most likely responsible agent. The hepatic cholesterol 7alpha-hydroxylase variations always parallel the plasmatic corticosterone concentration fluctuations, the latter being by far the most important adrenocortical excretion product. These two rhythms are modified in a similar manner under different physio-pathological conditions, such as the inversion of lighting in the animal room or the inversion of feeding time. Of these two parameters, the moment of food intake is the most important and accounts for the synchronisation of the rhythm in the animals. The rhythm is retained after several days of starvation but its amplitude decreases and the individual variations among the animals increase significantly at each time point.


RNA metabolism in nuclei isolated from HeLa cells.

Nuclei with low cytoplasmic contamination, capable of synthesizing RNA for an extended period of time, were prepared from HeLa cells. Besides elongating RNA chains already initiated in vivo, the nuclear preparation initiates the synthesis of new RNA chains. This was shown by labelling the newly synthesized RNA with [gamma-32P]GTP and by detecting the presence of labelled guanosine tetraphosphate among the alkaline hydrolysis products of synthesized RNA. By synthesizing RNA in the presence of each of the four gamma-32P-labelled nucleoside triphosphates, it was possible to conclude that RNA chain synthesis starts predominantly with a purine base. Both nucleolar and nucleoplasmic RNAs are made. The nuclear preparation methylates the nucleolar RNA by utilizing S-adenosyl-L-methionine as a methyl-group donor.


Interconversion between different states of affinity for acetylcholine of the cholinergic receptor protein from Torpedo marmorata.

In receptor-rich membrane fragments from Torpedo, acetylcholine binds, in the presence of 70 muM Tetram, to a homogeneous population of high-affinity sites with Kd = (3.4 +/- 0.8) x 10(08) M. Dissolution of these membrane fragments by sodium cholate causes a decrease of affinity associated with the appearance of medium-affinity (Kd approximately 10(-7) M) and low-affinity (Kd greater than or equal to 10(-6) M) sites. Dissolution by neutral detergents Triton X-100 or Emulphogene preserves the high affinity of the acetylcholine binding sites. In all the soluble states of the receptor protein, Ca2+ ions and local anaesthetics no longer enhance the affinity for acetylcholine. Elimination of sodium cholate by dilution leads to the reassociation of the receptor protein, the recovery of high-affinity sites and the control by Ca2+ ions and local anaesthetics. Purification by affinity chromatography of the receptor protein in Triton X-100 is accompanied by a conversion of a majority of the acetylcholine sites into their state of low affinity. High-affinity sites can no longer be recovered by detergent dilution from these low-affinity ones.


The relation of conformation and association of insulin to receptor binding; x-ray and circular-dichroism studies on bovine and hystricomorph insulins.

Crystal and solution structure studies on insulins of different sequences and of widely different receptor binding affinities are reported. Bovine insulin, studied as a control, has a circular dichroism spectrum which is dependent both on protein concentration and zinc concentration. The spectrum appears to be related to the level of association of the insulin molecules. This implies that when using circular dichroism to compare solution structures of insulin derivatives or species variants one must make the comparison at equivalent levels of association and not merely at the same concentration. Changes in circular dichroism are related to the known crystal structure of zinc insulin hexamers. The chinchilla insulin spectrum shows a reduced zinc dependence in low-salt conditions which correlates with the inability to form crystals in similar conditions. This is attributed to an amino acid substitution at position B4. Crystals are obtained in high-salt conditions and X-ray diffraction patterns show these to be isomorphous with bovine 4Zn insulin crystals. Guinea pig insulin failed to crystallise under conditions which are normally conducive to the formation of crystals of zinc insulin hexamers and the circular dichroism showed no zinc dependence. This is consistent with a monomeric structure. The significance of the association behaviour of chinchilla and guinea pig insulins may be in the storage of the hormone in vivo. Whereas the monomeric form of chinchilla insulin has a structure closely related to bovine insulin, the circular dichroism indicates a gross structural difference for guinea pig insulin. This may be similar to that in des-A21, des-B30-insulin, as both lack the Arg-B22--Asn-A21 carboxylate ion pair. The similarity of structure of chinchilla and bovine insulins is reflected in their receptor binding whereas the low receptor binding of guinea pig insulin probably results from the changes in its conformation rather than an alteration in residues of a receptor binding region.


Mode of action of the macrolide-type antibiotic, chlorothricin. Effect of the antibiotic on the catalytic activity and some structural parameters of pyruvate carboxylases purified from rat and chicken liver.

The macrolide-type antibiotic chlorothricin inhibits pyruvate carboxylases purified from rat liver, chicken liver and Azotobacter vinelandii. Under standard assay conditions the concentration of chlorothricin required for half-maximal inhibition of oxalacetate synthesis is 0.26 mM (rat liver), 0.12 mM (chicken liver), and 0.5 mM (Azobacter vinelandii). Inhibition by chlorothricin appears non-competitive in character when measured as a function of the concentration of the substrates of the pyruvate carboxylase reaction as well as of CoASAc and Mg2+. This pattern of inhibition suggests that this antibiotic interacts at unique sites on chicken and rat liver pyruvate carboxylase which are distinct from both the catalytic and activator sites. Interaction of chlorothricin with the two vertebrate liver pyruvate carboxylases differs from the effect exerted by this antibiotic on pyruvate carboxylase purified from Azotobacter vinelandii. A sigmoidal relationship between initial velocity and inhibitor concentration is observed for the vertebrate enzymes under most conditions whereas a hyperbolic profile characterizes the concentration dependence of inhibition of the Azotobacter vinelandii enzyme by chlorothricin. In the case of rat liver pyruvate carboxylase chlorothricin does not alter the extent of cooperativity in the relationship between initial rate and CoASAc concentration. However, a small but significant increase of the Hill coefficient from a value of 2.7 in the absence of antibiotic to that of 3.3 in the presence of 0.5 mM chlorothricin is observed for chicken liver pyruvate carboxylase. Chlorothricin decreases the rate of inactivation observed when rat liver pyruvate carboxylase is incubated with trinitrobenzenesulfonate and when chicken liver pyruvate carboxylase is incubated at 2 degrees C. The maximal decrease in inactivation observed in the presence of saturating concentrations of antibiotic is 50% for cold inactivation of the chicken liver enzyme and 60% for inactivation of the enzyme from rat liver by trinitrobenzenesulfonate. In both cases a sigmoidal relationship is observed between inactivation rate and chlorothricin concentration. These data as well as the initial rate studies suggest that multiple interacting sites for this antibiotic are present on the vertebrate liver pyruvate carboxylases. The occupancy of these sites appears to cause significant distortion of both the catalytic and the activator sites.


The distribution of J blood-group activity on lipoprotein and protein fractions of bovine serum.

There is a diversity of carriers of the J blood-group activity of bovine serum. The qualitative and quantitative distribution of the J activity on different carriers was studied, using various fractionation procedures. Approximately one third of J activity was found in the total lipids extracted from serum, two thirds in the lipid-free residue precipitated by lipid extraction. One third of the lipid J substance was found to be bound to the very low density lipoprotein, two thirds to the low density lipoprotein, while the high density lipoprotein was completely free of J activity. All non-lipidic J substance was present in the lipid-free protein. There was no J activity in the low molecular weight mucoproteins of serum and in the apoproteins of the lipoprotein fractions. The lipoprotein fractions were prepared by ultracentrifugation at different solvent densities. The lipoprotein fractions were characterized by chemical analyses and physical properties. The lower total cholesterol concentration of bovine serum, as compared to human serum, is reflected in a lower concentration of low density lipoprotein. The results obtained by ultracentrifugation coincide with the results obtained by precipitation of "beta-lipoproteins" with dextran sulfate and calcium chloride and with results obtained by gel filtration of bovine serum. The "beta-lipoprotein" fraction contains lipoproteins of very low and low density, and probably chylomicrons and a variety of other proteins, however no high density lipoprotein.


Control of hepatic protein synthesis. Differential effects of ATP levels on the initiation and elongation steps.

The effect of nucleotide energy levels in vivo on the different steps of protein synthesis has been studied. Hepatic anoxia was induced by interrupting the blood portal-vein flow. At 5 min of anoxia ATP fell to 59% of the control values and the amino acid incorporation into protein was inhibited by more than 70%. This strong inhibition was not paralleled by polyribosomal breakdown. On the contrary, when fasted rats were used, at 5 min of anoxia the ribosomal state of aggregation was found to increase. Longer periods of anoxia resulted in a further decrease in triphosphonucleoside content and polyribosomal breakdown. Based on these results and other reports from the literature it is concluded that the Km for the GTP of the peptide-chain-elongation mechanism must be higher than the Km of the initiation step. This finding implies that variations of nucleotide levels in vivo within the physiological range may control protein synthesis at the elongation step without apparent changes in the polyribosomal profiles.


Effect of substrate structure on the pre-steady-state kinetics of oxidation by liver alcohol dehydrogenase. A correlation with the Taft sigma parameter.

The oxidation of a series of primary alcohols by liver alcohol dehydrogenase has been studied under conditions of [S]o greater than [E]o using the stopped-flow method. A biphasic process, with exponential rise to a steady state, was observed for most of the alcohols and the rate constants for the transient phase were determined. No transient phase could be detected for 2-chloroethanol and 2-nitroethanol and steady-state measurements were made for these alcohols. The rate constants for the hydrogen transfer step were obtained from the pre-steady-state rate constants for the various alcohols and correlated with the Taft sigma constant. The (see article) value obtained (-1.8) is consistent with rate-limiting hydride transfer coupled with removal of the hydroxyl proton by a suitable basic group on the enzyme. A possible identity for this group is suggested.


Inhibition by carbon monoxide of the secondary-amine mono-oxygenase of Pseudomonas aminovorans and the photochemical action spectrum for its reversal.

The secondary-amine mono-oxygenase (EC 1.14.99.--) of Pseudomonas aminovorans is potently inhibited by carbon monoxide. The degree of inhibition of the purified enzyme was determined by the CO:O2 ratio rather than by the absolute concentration of carbon monoxide. The partition constant (the CO:O2 ratio causing 50% inhibition of activity) was 9.2 X 10(-4). The inhibition could be reversed by light, and the extent of reversal was proportional to the light intensity. With monochromatic light of wavelength 417 nm, the light sensitivity, L, was determined to be 2.5 X 10(8) cm2 min/mol quantum. The photochemical action spectrum for the light reversal of inhibition showed a single maximum of effectiveness at about 420 nm. The difference spectrum of the enzyme (reduced by NADH) on bubbling with CO (compared with an NADH-reduced reference sample) showed a peak at 426 nm. The preparations showed none of the spectral properties to cytochrome P-450 mono-oxygenase preparations, and was much more sensitive to carbon monoxide. The enzyme behaves as a typical o-type cytochrome (i.e. a carbon-monoxide-reactive b-type cytochrome), and its sensitivity to carbon monoxide as well as in its spectral properties, shows close resemblances to haemoglobin.


The properties of cytochrome f and P700 in chloroplasts suspended in fluid media at sub-zero temperatures.

1. The properties of P700 and cytochrome f have been studied at sub-zero temperatures in chloroplasts suspended in a medium containing 50% (v/v) ethylene glycol. The dark reduction of these components after a period of illumination provided information about the rate-limiting step of photosynthetic electron transport under these conditions. 2. The oxidation of P700 on illumination in the presence of methyl viologen and its subsequent dark reduction can be observed at -35 degrees C. This cycle of reactions could be repeated many times. The rate of reduction was increased by NH4Cl and reduction was inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea. 3. The oxidation and reduction of cytochrome f could also be observed under similar conditions. The activation energies for the reduction of cytochrome f and P700 are similar (about 75 kJ mol-1) and the reduction of cytochrome f is also inhibited by dichlorophenyldimethylurea and stimulated by NH4Cl. 4. The reduction of both cytochrome f and P700 seemed to follow first-order kinetics, but the t1/2 for the redcution of the cytochrome was at least three times that for the reduction of P700 at the same temperature. It was concluded that the results were only compatible with a model in which the main pathway of electrons from plastoquinone to P700 involved cytochrome f if the equilibrium constant between the cytochrome and P700 was very much less than that expected from their redox potentials.


Evidence for cooperative effects in the exchange reaction catalysed by the oxoglutarate translocator of rat-heart mitochondria.

The initial rates of the exchange external oxoglutarate/internal malate through the inner membrane of rat-heart mitochondria, for various concentrations of the two substrates, have been reinvestigated for an extended range of concentrations of the external oxoglutarate. This has been made possible by use of the inhibitor-stop technique that allows 100 times smaller incubation times than the centrifugation-stop technique used previously. Under the experimental conditions the uptake of the external-labelled oxoglutarate into the mitochondrial-matrix space is mediated by the oxoglutarate translocator performing a ono-to-one exchange of the anions oxoglutarate (external) and malate (internal). Two intermediary-plateau regions are observed in the kinetic saturation curve of the translocator by the external oxoglutarate, revealing a complex rate equation which is found to be the product of two one-substrate functions. Analysing these features it is shown that the model, proposed earlier, of a "double carrier" as catalyst in a rapid-equilibrium random bi-bi mechanism, is still applicable but that several external binding sites have to be considered. As already noticed the external and the internal substrates bind to their respective sites independently of each other. Furthermore, some additional requirements imposed by the observed kinetics suggest that the exchange reaction is performed by only one translocator species made of identical interacting subunits. The anion exchange is tentatively viewed as a rotation of a subunit around an axis situated in the plane of the membrane after two independent local configuration changes induced by the binding of the two substrates on this subunit.


Activity of polyribosomes from the muscle of normal and dystrophic mice in cell-free amino-acid incorporation.

Polyribosomes sedimenting in the manner characteristic of those from embryonic chick muscle, as described by Heywood et al. in 1967 (Proc. Natl Acad. Sci. U.S.A. 57,1002--1009) were reproducibly obtained from normal mouse muscle by homogenization of the muscle with a Dounce homogenizer. The polyribosome profiles of dystrophic muscle were qualitatively similar to those of normal muscle except that the relative amount of ribosomes in polyribosome complexes was smaller (44% +/- 3S.E.) in dystrophic muscle than in normal muscle (67% +/- 4S.E.). In spite of this difference, polyribosomes from dystrophic muscle incorporated amino acids in vitro at a faster rate and produced a larger amount of polypeptide at the end of the reaction than polyribosomes from normal muscle.


Antibodies to adenosine 5'-monophosphate: purification and specificity.

Antibodies to adenosine-5'-monophosphate were produced in rabbits by injecting a conjugate of the nucleotide (oxidized with periodate) with bovine serum albumin. Nucleotide-specific antibodies were isolated by affinity chromatography on oligoadenylic acids/agarose column. Pure immunoglobulin G antibodies were obtained by gel filtration on Sephadex G-200. These antibodies, as analyzed by double diffusion react with adenosine 5'-monophosphate--bovine serum albumin, slightly with inosine-5'-monophosphate conjugate and not at all with the other nucleotide conjugates. The association constants for adenosine-5'-monophosphate--antibody complex formation obtained by dialysis equilibrium and fluorescence measurements, are in good agreement. This latter technique was used to study on one hand the influence of temperture and salt on complex formation, on the other hand the interaction of the antibodies with AMP derivatives. The phosphate group, the ribose and the base are recognized by the antibody, but the C-8 atom of adenine residues is not directly involved in the binding.


A new mode of ring cleavage of 2,3-dihydroxybenzoic acid in Tecoma stans (L.). Partial purification and properties of 2,3-dihydroxybenzoate 2,3-oxygenase.

2,3-Dihydroxybenzoic acid has been shown to be oxidized via the 3-oxoadipate pathway in the leaves of Tecoma stans. The formation of 2-carboxy-cis,cis-muconic acid, a muconolactone, 3-oxoadipic acid and carbon dioxide during its metabolism has been demonstrated using an extract of Tecoma leaves. The first reaction of the pathway, viz., the conversion of 2,3-dihydroxybenzoate to 2-carboxy-cis,cis-muconic acid has been shown to be catalysed by an enzyme designated as 2,3-dihydroxybenzoate 2,3-oxygenase. The enzyme has been partially purified and a few of its properties studied. The enzyme is very labile with a half-life of 3--4 h. It is maximally active with 2,3-dihydroxybenzoate as the substrate and does not exhibit any activity with catechol, 4-methyl catechol, 3,4-dihydroxybenzoic acid, etc. However, 2,3-dihydroxy-p-toluate and 2,3-dihydroxy-p-cumate are also oxidized by the enzyme by about 38% and 28% respectively, compared to 2,3-dihydroxybenzoate. Sulfhydryl reagents inhibit the enzyme reaction and the inhibition can be prevented by preincubation of the enzyme with the substrate. Substrate also affords protection to the enzyme against thermal inactivation. Sulfhydryl compounds strongly inhibit the reaction and the inhibition cannot be prevented by preincubation of the enzyme with its substrates. Data on the effect of metal ions as well as metal chelating agents suggest that copper is the metal cofactor of the enzyme. Evidence is presented which suggests that iron may not be participating in the overall catalytic mechanism.


Isolation and identification of the urinary pigment uroerythrin.

The red pigment uroerythrin, a chromophore known to be adsorbed by the amorphous urate sediments (sedimentum lateritium), has been isolated from human urine and further purified as its trimethyl derivative. Urine was applied to a column of Amberlite XAD-2 resin on which uroerythrin and other pigments were adsorbed. The pigments were eluted with methanol and uroerythrin was further purified by extraction with ether at pH 4.0, repeated chromatography on lipophilic Sephadex LH-20 and thin-layer chromatography on silica gel. For optimal purification uroerythrin was converted into the trimethyl derivative and chromatographed on silical gel thin-layer plates. The structure of the pigment has been studied by chromate degradation followed by identification of the imide products by thin-layer chromatography. From these results and from infrared, mass spectral and nuclear magnetic resonance data a tripyrrole structure for uroerythrin is concluded. The proposed structure for the chromophore is related to that of the bile pigment biliverdin consisting, however, only of the rings A, B and C.


Isolation and identification of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid from the urine of a patient with sialuria.

N-Acetylneuraminic acid preparations from the urine of a patient with sialuria contain 1--2% of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. This new human sialic acid was isolated by ion-exchange and partition chromatography. The structure has been elucidated by mass spectrometry and confirmed by comparison with the synthetic compound. The properties of this unsaturated sialic acid in the orcinol/Fe3+/HCl and the periodic acid/thiobarbituric acid tests as well as in thin-layer and gas-liquid chromatography are described. It does not react with acylneuraminate pyruvatelyase. The origin of this new human sialic acid is discussed.


Two-dimensional polyacrylamide-gel electrophoresis of the proteins and glycoproteins of the human erythrocyte membrane.

A two-dimensional polyacrylamide gel electrophoresis technique has been developed, improving the analytical separation of some proteins and glycoproteins of the human erythrocyte membrane. Freshly prepared membranes are totally solubilized, subjected to dodecylsulfate--polyacrylamide gel electrophoresis in the first dimension, followed by electrophoresis in the second dimension, using a detergent-free polyacrylamide gradient gel. By this method the proteins of the human erythrocyte membrane could be resolved into a two-dimensional pattern, which has been shown to be highly reproducible with respect to various blood-groups and within one blood-group from specimen to specimen. The method enables especially the investigation of the hydrophobic and very likely integrated membrane proteins and glycoproteins. Thus, band III[Fairbanks, G., Steck, Th. & Wallach, D. F. H., Biochemistry, 10, 2606--2617 (1971)] could be shown to consist of five proteins, one of them being the major glycoprotein of the human erythrocyte membrand. The two spectrin bands differed considerably in their two-dimensional patterns. The value of the given method for the investigation of membrane defects, which may be linked with various diseases of human erythrocytes, could be demonstrated in the case of two patients suffering from congenital dyserythropoetic anaemia.


Labile sulfur in human Superoxide dismutase.

1. The nautre of the intense absorption band at 320 nm of the copper and zinc-containing enzyme superoxide dismutase, from human red blood cells, has been investigated. The band does not depend on the metal prosthetic groups of the enzyme, as it is still present in the apo protein. When, however, copper alone is removed from the enzyme with a treatment involving the use of cyanide, the band is also lost. Nevertheless the copper-free protein is able to recover both the enzyme activity and the native electron paramagnetic resonance spectrum as easily as the apo protein. 2. A number of other treatments are able to abolish the band. They include reaction with reducing agents such as dithiothreitol, sulfite, borohydride, exposure to denaturants such as guanidine HCl and sodium dodecyl sulfate, and exposure to pH values below pH 3 or above pH 13. 3. Four sulfur atoms per protein molecule were found to be associated to the 320-nm chromophore on the basis of quantitative determinations following reaction with cyanide or sodium borohydride. 4. A molar absorption coefficient of 1150 M-1 cm-1 was determined per each chromophoric group. In spite of this relatively high value and unusual stability, a persulfide group, R-S-SH, seems to be the most likely structure for this chromophore. 5. Bovine and equine superoxide dismutase do not show spectral or chemical evidence for such a group. This, and the recovery of activity and spectral properties of copper in the cyanide-treated human enzyme, indicate that labile sulfur is not associated with the superoxide dismutase activity of this protein.


Synthesis of apamin, a neurotoxic peptide from bee venom.

The apamin sequence has been synthesized by the solid-phase procedure. The synthetic peptide showed the same physiochemical and chemical properties as natural apamin in the following tests: paper electrophoresis, amino acid analyses after acid and enzymatic hydrolyses, sequence analysis, electrophoreses after tryptic and chymotryptic digestions. Synthetic apamin was as active as natural apamin in the neurotoxic assay in mice (LD50, after subcutaneous injection, for the 20-g mouse: 58 mug).


Flavocytochrome b2: simulation studies of the electron-transfer reactions among the prosthetic groups.

Simulation studies by digital computer were undertaken in order to test and clarify the interpretations deduced from experimental data concerning the electron transfer mechanism from L-lactate to flavocytochrome b2, which were presented in a preceding paper in this journal. The reaction scheme proposed as the "best" one is composed of 7 steps. It allows the best fitting of the time courses established for the oxidized flavin (Flox), the flavin semiquinone (Flsq), the fully reduced flavin (Flred), and the reduced haem (Hred); it can be extended to 1 s. This scheme also allows a good simulation of the general shape of preequilibrium titration curves obtained at a 200-ms reaction time for Hred and Flsq, and a valuable simulation of the reduction electron paramagnetic resonance time course established for Hred and Flsq at low lactate concentration. The agreement between experimental and simulated curves led to an estimation of some rate constants experimentally unknown, relative (in particular) to the electron exchange between flavin and haem and between couples of flavins. Another interest of these stimulation studies was to point out the obligatory involvement of a slow final step to perform the flavocytochrome b2 full reduction; this step could be controlled by some conformational change of the protein.


Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography.

Methods which use the scintillator PPO to record film images of 3H in chromatograms and polyacrylamide gels (fluorography) have been described elsewhere. This paper demonstrates that pre-exposure of the film to a brief flash of light greatly increases the sensitivity of fluorography. Pre-exposure also permits quantitative interpretation of the film image, because it corrects the non-linear relationship between radioactivity of the sample and absorbance of the film image. Therefore the distribution of radioactivity in the sample is accurately represented by microdensitometry of the image obtained on pre-exposed film. Using pre-exposed film 300 dis. 3H/min or 30 dis. 14C/min can be detected in a band in a gel in a 24-h exposure. The Appendix describes revisions and extensions of existing fluorographic procedures, including application to agarose gels and a rapid procedure for recovering PPO for re-use.


Metabolic activities of histones in rat liver and spleen.

Synthesis and turnover of histone I and II in normal rat liver and spleen were studied by Amberlite CG 50 column chromatography. Histone I was separated into three or four subfractions, each of which showed a different rate of incorporation of [3H]lysine. This was verified by a more shallow gradient chromatography developed by Kinkade and Cole [3] for very lysine-rich histone (F1), which showed tissue specific differences between liver and spleen in both the elution pattern and synthetic rates. These subfractions were distinguished from each other by dodecylsulphate electrophoresis. The turnover, or disassociation of histone I and II in chromatin was measured by double-labelling of normal rat liver with [3H] and [14C]lysine. A good correspondence was found between the synthesis and turnover patterns of individual histone I fractions, while the histone II synthesized was conserved for over a month. From consideration of the turnover in relation to the cell population of normal liver tissue, which consists of a very small fraction of growing cells and a very large fraction of resting ones, it was concluded that turnover of histone I must occur even in resting cells. When DNA synthesis in the spleen was completely inhibited by hydroxyurea, the synthesis of histone II was inhibited but that of histone I was only partially inhibited. The remaining synthesis seemed to occur in cells in the resting state. It was concluded tentatively, the continuous replacement of very lysine-rich histones of chromatin must occur even in resting cells in which DNA synthesis has ceased. The biological significance of disassociation of histones from chromatin was discussed.


Characterization of three new fucolipids from hog gastric mucosa.

Three new fucolioids have been isolated from the water-soluble glycolipid fraction of hog gastric mucosa. Fucolipid A and C exhibited blood-group-A activity, whereas Fucolipid B was not active in A-anti-A, B-anti-B and H-anti-H systems. The structures of these glycolipids were identified by partial acid hydrolysis and methylation analysis, as: (see article)


Glucagon and insulin control of gluconeogenesis in the perfused isolated rat liver. Effects on cellular metabolite distribution.

The metabolic effects of glucagon and glucagon plus insulin on the isolated rat livers perfused with 10 mM sodium L-lactate as substrate were studied. Glucagon stimulated gluconeogenesis, ketogenesis and ureogenesis at the concentration used of 2.1 nM. The addition of insulin to give a glucagon-to-insulin ratio of 0.2 reversed all the glucagon effects. The glucagon enhancement of gluconeogenesis was accompanied by a rise in cytosolic and mitochondrial state of reduction of the NAD system and a fall in the [ATP]/[ADP] ratio. The analysis of the intermediary metabolite concentrations suggested, as possible sites of glucagon action, the steps between pyruvate and phosphoenolpyruvate as well as the reactions catalyzed by phosphofructokinase and/or fructose bisphosphatase. All the changes in metabolite contents were abolished when insulin was present. Glucagon increased the intramitochondrial concentration of all the metabolites, whose intracellular distribution was calculated. The finding of a significant rise in the calculated intramitochondrial concentration of oxaloacetate points to pyruvate carboxylation as an important site of glucagon interaction with the gluconeogenic pathway. A primary event in the glucagon action redistributing intracellular metabolites seems to be the mitochondrial entry of malate. The possibility is discussed that the changes in metabolite cellular distribution were brought about by the increased cellular state of reduction caused by the hormone.


Studies on the fate of homologous DNA applied to seedlings of Matthiola incana.

Seedlings of Matthiola incana (crucifer) are able to take up exogenous homologous DNA by the roots. DNA homogenously labelled with [3H]adenine and 5-bromodeoxyuridine is incorporated into the plants in a macromolecular form. Intact donor DNA and a fraction with a buoyant density intermediate between that of the donor and the recipient DNA can be recovered. Analysis of this intermediate fraction by ultrasonication and alkali treatment allows the suggestion that homologous DNA is integrated as a double-stranded DNA which becomes covalently linked to the recipient DNA. Control experiments in which seedlings were incubated in a mixture simulating donor DNA degradation products in the presence and absence of unlabelled competitors suggest that these results are not due to the breakdown of donor DNA and reincorporation of the products during DNA synthesis in the recipient plants. When ultrasonicated or thermally denatured DNA is applied to the plants it may be degraded and reused for recipient DNA synthesis but it is not recovered in a macromolecular form. The possibility that the intermediate DNA fraction arises by bacterial contamination of the plants can be excluded by several arguments. Autoradiographic studies show that at least part of the radioactivity of the donor DNA taken up by the plants is associated with the cell nucleus.


Specific enzymic cleavage of polypeptides at cysteine residues.

A method has been developed for specific enzymic cleavage of polypeptides at the N-terminal side of modified cysteine residues. Lysine residues are blocked by trifluoroacetylation and cysteine residues subsequently converted to the 2-aminoethyl derivatives. Digestion of the modified polypeptide with the lysine-specific protease from Armillaria mellea (patented by Walton et al., 1972) occurs only at 2-aminoethylcysteine residues. With the beta chain of human haemoglobin, which contains 2 cysteine and 11 lysine residues, cleavage was observed at both modified cysteines but at none of the lysines. In the case of a polypeptide from bee venom which contains 4 half-cystine and 5 lysine residues, cleavage occurred at only 2 of the modified cysteines and also at 2 lysine residues. The pattern of cleavage in the latter case can be interpreted in terms of the amino acid sequence of the polypeptide.


On the biosynthesis of 5-methoxybenzimidazole. Precursor-function of 5-hydroxybenzimidazole, benzimidazole and riboflavin.

1. In Clostridium thermoaceticum 5-hydroxybenzimidazole is methylated to 5-methoxybenz-imidaxole and transformed to 5-methoxybenzimidazolylcobamide. 5-Hydroxybenzimidazolycobamide is also methylated to 5-methoxybenzimidazolylcobamide. These results indicate a possible precursor function of 5-hydroxybenzimidazole in the biosynthesis of 5-methoxybenzimidazole. 2. The same microorganism uses benzimidazole to form benzimidazolylcobamide. This or externally added benzimidazolylcobamide, although taken up by the cells, is not further transformed (i.e. hydroxylated and methylated to 5-methoxybenzimdazolylcobamidel). This excludes a precursor function of benzimidazole in the biosynthesis of 5-methoxybenzimidazole. 3. Contrary to the biosynthesis of 5,6-dimethylbenzimidazole, 5-methoxybenzimidazole is not formed from riboflavin, but riboflavin inhibits the growth and the production of 5-methoxybenzimidazolylcobamide in Clostridium thermoaceticum. A tentative scheme for the biosynthesis of 5-methoxybenzimidazole via a riboflavin analog is discussed.


The primary structure of a major polypeptide component from the venom of Naja melanoleuca.

The venom of the forest cobra, Naja melanoleuca, contains a number of homologous polypeptides containing between 60 and 71 amino acid resides. The primary structure of a major component (approx. 10% by weight of the crude venom) has been determined unambiguously. The molecule contians 61 amino acid residues and four disulphide bridges. It has not effect on neuromuscular transmission or the excitatory or inhibitory responses to acetylcholine of molluscan neurons. The molecule is similar to, but not identical with, the so-called cytotoxins VII2 and VII3 isolated, by others, from the same venom but reported to be minor components.


Conformational properties of pig-heart cytoplasmic aspartate aminotransferase. Circular-dichroism and absorption-spectroscopic study of dicarboxylate binding.

1. The interaction between aspartate aminotransferase and dicarboxylates of various chain lengths and geometries has been studied from pH 6.5 TO 8.5 by circular dichroism (CD) and absorption spectroscopy. Liganding causes protonation of the pyridoxal phosphate-enzyme Schiff's base complex; the consequent changes in optical properties deltaAlambda, deltaCDlambda at the coenzyme maxima (lambda = 363 or 430 nm) are analysed for binding constants and the degree of perturbation of the coenzyme protonic dissociation constant, pKa. 2. Aliphate dicarboxylates follow linear binding functions for all optical parameters; in contrast, m and p-phthalates follow non-linear binding functions for both deltaAlambda and deltaCDlambda, implying that successive phthalate ligands bind with decreasing affinity. The ratio detlaCDlambda is effectively constant for a given ligand and the characteristic values for aromatic ligands indicate a changed environment for the coenzyme. 3. Inspection of the non-linear process for phthalates suggests that initially, binding occurs with high affinity, but with characteristically small effects on pKa. It is inferred that alipathic and aromatic dicarboxylates bind at different subsites in the active site region, perturbing the coenzyme pKa by an indirect protein-mediated mechanism. 4. Non-linearity of binding could derive from multiple binding to an individual subunit. Alternatively, different single sites may exist on adjacent subunits of the dimer, implying non-equivalence between otherwise identical subunits, expressed in properties involving groups close to the active site.


Effect of myosin on conformational changes of F-actin in thin filament in vivo induced by calcium ions.

The measurements of ultraviolet linear dichroism has been carried out on glycerinated muscle fibers of rabbit psoas and crab leg having various sarcomere lengths and the effect of interaction of the thin filament with myosin on the dichroism has been investigated under various conditions. The dichroism spectrum of the muscle fiber has a large positive peak at 285 nm (smaller absorption of light polarized perpendicular to the fiber axis) and a small negative peak at 297.5 nm. The negative peak at 297.5 nm, which comes from the dichroism of F-actin in the thin filament, became smaller in the presence of Ca2+ than in its absence. The positive peak at 285 nm, where the thick filament has main contribution, showed no appreciable change by Ca2+. The dichroism at 292.5 nm, where the thin filament has no contribution, showed no change either. Thus, the decrease of negative dichroism at 297.5 nm by Ca2+ indicates a conformational change in F-actin in the thin filament. With decreasing sarcomere length or increasing overlapping between thin and thick filaments, the dichroism decrease at 297.5 nm by Ca2+ increased, and attained a maximum at maximum overlapping. That is the conformational change of F-actin by Ca2+ was amplified by interaction with myosin. With further decrease of the sarcomere length, the dichroism decrease by Ca2+ decreased. In the absence of Ca2+, the addition of ATP or pyrophosphate induced a large change of transmittance and transmittance anisotropy, and also a large increase of birefringence at long wavelengths.


Stabilization of promoter complexes with a single ribonucleoside triphosphate.

Under specific binding conditions RNA polymerase forms complexes at several sites of the replicative form DNA of bacteriophage fd. One of these complexes becomes stable to both high salt and low temperature after incubation with GTP. None of the complexes is stabilized by ATP. The stabilization by GTP results from the synthesis of an oligo(G) chain, which is bound in the complex. Size and pyrimidine fingerprints of the DNA segment protected by the enzyme against digestion with DNase are not changed upon initiation of oligo(G) synthesis. This result indicates that binding site and initiation site are identical parts of a promoter region.


Action of glycosyl transferases upon "Bombay" (Oh) erythrocytes. Conversion to cells showing blood-group H and A specificities.

Individuals of the rare "Bombay" (Oh) blood-group phenotype lacking, due to a genetic defect, the alpha(1-2)fucosyl transferase, which is responsible for converting blood-group H precursor substances to H-specific structures. Treatment with GDP-fucose and alpha(1-2)fucosyl transferase prepared from gastric mucosa of O individuals to transform native or ficin-treated "Bombay" erythrocytes into cells phenotypically resembling O cells. The transformation was achieved, however,  after prior incubation of the "Bombay" erythrocytes with neuraminidase, indicating that blood-group H precursor molecules on the surface of these cells are masked by sialyl residues. Blood-group A specificity was conferred upon neuraminidase-treated "Bombay" cells by enzymatic transfer of alpha-N-acetylgalactosamine residues, in addition to alpha-fucose residues.


The secondary structure and poly(A) content of globin messenger RNA as a pure RNA and in polyribosome-derived ribonucleoprotein complexes.

The conformation in solution of duck and rabbit globin mRNA, and of the duck mRNA in the mRNA - protein particle, has been investigated by optical methods and also by the use of the dye ethidium bromide which becomes highly fluorescent when intercalated into the double-stranded regions of a nucleic acid. On the basis of the properties of this dye and on the ability of homopolyribonucleotides to form double-stranded structures we have, in addition, developed a simple and sensitive assay for the detection and quantitisation of sequences rich in a particular residue that may be present in an RNA chain. In solution, 45 to 60% of the nucleotides of duck globin nRNA were found to be in bihelical regions. A similar degree of secondary structure was found in rabbit globin mRNA (this paper), as well as in calf lens mRNA and mRNAs from ewe mammary gland (other results). All samples of globin mRNA examined in this work containeda sequence of poly(A), which has poly(U) binding properties similar to that of synthetic poly(a): no specific interaction between the poly(A) sequence and the rest of the molecules can be detected. The fraction of adenosine residues within these poly(A) segments represents 4% in rabbit mRNA and 8 to 9% in duck mRNA. An additional adenosine-rich segment interspersed with guanosine and possibly other residues, was also detected in one duck mRNA sample. The RNA in the duck mRNA - protein particle is also highly structured. The melting profile in the range of 20 to 65 degrees C is quite similar to that of free mRNA and the ability of ethidium bromide to intercalate is reduced to the extent of 70%. Yet the dichroic spectra of free and bound mRNA are significantly distinct. These data suggest that free and protein-bound mRNA May have a very similar degree of secondary structure but with distinct detailed conformation in bihelical regions (change in base tilting for example). Direct evidence has been obtained that proteins stick to the poly(A) segment in the particle since the fraction of adenosine residues detectable by our poly(u) titration procedure is reduced to 50% of that observed in the free mRNA.


Synthesis of RNA molecules larger than 45 S by isolated rat-liver nucleoli.

Nucleoli, isolated from rat liver, synthesize in vitro high-molecular-weight RNA, the base composition and sedimentation pattern of which resembles that of ribosomal precursor RNA. In addition, RNA molecules larger than 45 S have been found. In this paper experiments are described which indicate that these large RNA molecules represent geniune transcription products and are not aggregates arising under the experimental conditions employed. This was established by comparing different extraction methods, by sedimentation analysis of the RNA after denaturation with formamide and by pulse-chase experiments. Hybridisation-competition studies showed that 45-S RNA competes with those rapidly molecules to about 80-90%, thus providing evidence for the presence of ribosomal precursor RNA sequences in those long transcription products. Intact nuclei are able to synthesize in the presence of Mg2+ and alpha-amanitin RNA molecules larger than 45 S too, provided that the RNAase activity is suppressed effectively by the addition of cytoplasmic RNAase inhibitor. The significance of these results is discussed with respect to the initial transcript of the rDNA genes in rat liver nucleoli.


Tertiary structure and function of closed-circular mitochondrial DNA and its transcription in vivo.

The conformation of the closed circular mitochondrial DNA in cultured human cells is changed by the addition of berenil to the culture medium in such a way that the 34-S mitochondrial DNA is converted into a 29-S DNA and finally into a 24-S DNA. The superhelix density of the covalently closed 29-S mitochondrial DNA is sigma O = -1.5 times 10(-2) (turns/10 base pairs) and consequently is intermediate between the superhelix density of the 34-S DNA (sigma0 = -2.8 times 10(-2)) and that of the closed circular 24-S DNA (sigmao appromiately 0). Removal of the drug reverses the transition, covalently closed circular 34-S DNA to covalently closed circular 29-S DNA to covalently closed circular 24-S DNA. The covalently closed circular 24-S mitochondrial DNA is not replicated. Transcription of this DNA is also inhibited as indicated by the fact that no synthesis of the mitochondrial ribosomal RNAs occurs as long as the mitochondrial DNA is in this conformation. The covalently closed circular 29-S mitochondrial DNA is replicated but not transcribed in vivo.


Studies on the control of ribosomal RNA synthesis in HeLa cells.

In many eucaryotic systems protein synthesis is coupled to ribosomal RNA synthesis such that shut-down of the former causes inhibition of the latter. We have investigated this stringency phenomenon in HeLa cells. The protein synthesis inhibitors cycloheximide and puromycin cause inactivation of both processes but valine starvation totally inhibits only the processing of 45-S RNA. DNA-dependent RNA polymerases from A, B and C (or I, II and III respectively) were extracted, separated partially by DEAE-cellulose chromatography and their activity levels determined. These do not decrease significantly during inhibition of protein synthesis. To find out whether or not form A is bound to its template under these conditions, proteins were removed from chromatin with the detergent sarkosyl. This does not affect bound RNA polymerase. Inhibition of protein synthesis caused up to 50% reduction in endogenous alpha-amanitin-insensitive chromatin-RNA-synthesising activity. This reduced level of activity was not affected by sarkosyl treatment. Levels in normal cells were stimulated. This result indicates that the form A RNA polymerase is not bound to its template when protein synthesis is inhibited.