Magnetic resonance studies of three forms of creatine kinase. Comparison of the properties of native, CH-S-blocked, and H2NCOCH-blocked enzymes.

Magnetic resonance methods are applied in a comparative study of native creatine kinase from rabbit muscle with two sulfhydryl-modified forms of the enzyme--one inactive form obtained by reaction of the enzyme with iodoacetamide and one form with reduced activity obtained by reaction of the iodoacetamide-sensitive sulfhydryl group with methyl methanethiolsulfonate, which blocks the sulfhydryl with a CH3S-group. Water proton relaxation rate (PRR) titrations with the CH3S-blocked enzyme show that the modification does not alter appreciably the affinities of the enzyme for MnADP and for creatine in the presence of MnADP. Similar measurements for the H2NCOCH2-blocked enzyme indicate that this modification weakens the affinity of the enzyme for MnADP. In agreement with previous findings, there is no observable change in the PRR enhancement upon additions of creatine to solutions of the ternary complex, enzyme-MnADP, for the H2NCOCH2-blocked enzyme. PRR titrations enabled the measurement of binding of creatine to the ternary CH3S-enzyme-MnADP complex and show that specific anions such as nitrate, formate, and thiocyanate decrease the apparent dissociation constant for creatine in its complex with the CH3S-blocked enzyme and MnADP, as is observed with native creatine kinase. However, the change in the PRR enhancement for the CH3S-enzyme-MnADP upon binding of creatine in the presence or absence of anions was appreciably smaller than for the native enzyme. For the H2NCOCH2-blocked enzyme, these anions failed to bring about any influence of creatine on the PRR enhancement. Consistent with the diminished influence of these anions on the PRR enhancement of the CH3S-enzyme-MnADP-creatine complex, EPR spectra of bound Mn(II) show that the CH3S-blocking group interferes with the pronounced anion-induced spectral changes which are observed with the native enzyme. EPR spectra for the H2NCOCH2-enzyme-MnADP complex were not influenced upon additions of creatine, even in the presence of anions. These results suggest that the altered catalytic properties of the CH3S-blocked enzyme arise from structural perturbations at the active site which are also reflected in the PRR enhancement factors and EPR spectral features of the Mn(II) complexes. Moreover, the results clearly indicate that the H2NCOCH2-blocking group, which completely inactivates the enzyme, also eliminates the ability of the MnADP site to sense the presence of the second substrate, creatine, alone and in combination with anions which are structural analogs of the migrating phosphoryl group.


Properties of a CH3-blocked creatine kinase with altered catalytic activity. Kinetic consequences of the presence of the blocking group.

Steady state kinetic parameters for rabbit muscle creatine kinase (EC 2.7.3.2) and this enzyme stoichiometrically blocked at the iodoacetamide-sensitive cysteinyl residue with a CH3S-group have been measured at 30+/-0.1 degrees, pH 9.00, using Mg(II) as the required metal ion cofactor. The double reciprocal plots for the CH3S-blocked enzyme with MgATP as the variable substrate are biphasic, each curve showing a break at approximately 1.9 mM MgATP, and suggest the possibility of negative cooperativity in metal-nucleotide binding. Furthermore, extrapolated lines at high MgATP concentrations intersect on the abscissa, indicating loss of synergism in binding of substrates. In contrast, observed Michaelis constants for creatine are, within experimental error, the same for both native and blocked enzymes. The maximal velocity of the CH3S-blocked enzyme is found to be 28.1% of the value of the native enzyme. Product inhibition patterns for both native and blocked enzyme are also compared. Again, these patterns indicate that the CH3S-blocking group alters the nucleotide binding site more than the guanidino substrate binding site. Calculations using the methods of Chou and Fasman (1970) Biochemistry 13, 211-222) lead to the prediction that the active cysteinyl residue occurs at the beginning of a beta turn which separates two portions of beta sheet structure of the enzyme, and so may be in a position to mediate conformational changes in the protein.


Nature of interaction of dextran sulfate with lecithin dispersions and lysolecithin micelles.

Lecithin and lecithin/cholesterol dispersions as well as lysolecithin micelles were used to provide basic information on the mechanism of the interaction of zwitterionic phospholipids with dextran sulfate. The addition of dextran sulfate to lecithin dispersions or lysolecithin micelles in the presence of Ca2+ produced insoluble complexes. At each Ca2+ concentration, the amount of insoluble complex formed was maximal at the equivalence dextran sulfate/phospholipid weight ratio. An increase in CaCl2 concentration up to 10 mM progressively increased the equivalence ratios for the phospholipids. Further increase in Ca2+ concentration did not influence the equivalence ratio for maximal complex formation. The conversion of lecithin dispersions into insoluble complexes was very effective even at low Ca2+ concentrations. Approximately 70% of the lecithin was converted to the insoluble complex at CaCl2 concentrations as low as 0.5 mM and the complete conversion was observed at CaCl2 concentrations above 2.5 mM. In the presence of cholesterol, the precipitation curve for insoluble complex formation became broader than that of lecithin alone, indicating the enhancing effect of cholesterol on the insoluble complex formation both below and above the equivalence ratio. The stoichiometry of the interaction of the zwitterionic phospholipids with dextran sulfate inthe presence of calcium was determined using lysolecithin micelles. At the CaCl2 concentrations above 20 mM, the insoluble complex possessed a ratio of lysolecithin/calcium/sulfate group of approximately 2/1/3 (mol/mol/mol). It appears that the formation of the insoluble complex of lecithin or lysolecithin with dextran sulfate represents the mutually enhancing interactions involving both positive and negative charges of the zwitterionic phospholipids. These are the direct electro static interaction between the phospholipid choline nitrogen and the sulfate groups of dextran sulfate and the calcium cross-linking of the phosphate groups to the sulfate groups or to the phosphate groups of neighboring phospholipids.


Novel enzymic machinery for the metabolism of oxalacetate, phosphoenolpyruvate, and pyruvate in Pseudomonas citronellolis.

The metabolic pathways for the interconversion of oxalacetate, phosphoenolpyruvate, and pyruvate in Pseudomonas citronellolis form an interlocking system (Scheme 1) that would appear to require complex regulatory mechanisms to permit a proper flow of metabolites through the pathways and to prevent futile cycling. Oxalacetate decarboxylase (I in Scheme 1), P-enolpyruvate synthase (II), P-enolpyruvate carboxylase (III), and pyruvate kinase (V) are constitutive enzymes in this organism. Pyruvate carboxylase (VI) is inducible and has its highest activity in cells grown on glucose or lactate, moderate activity in cells grown on acetate, citrate, or glutamate, and virtually no activity in aspartate-grown cells. P-enolpyruvate carboxykinase (IV) was not detected. The presence of these five enzymes in a single cell has not been previously reported. In Scheme 1, three futile cycles are possible: the simultaneous operation of Reactions I and VI; of Reactions II and V; or of I, II, and III. An examination of the regulatory properties of the individual enzymes after partial purification offers support for the hypothesis of an intricate regulatory system. Oxalacetate decarboxylase (I) is inhibited by acetyl-CoA; phosphoenolpyruvate carboxylase (III) is activated by acetyl-CoA and ADP and inhibited by aspartate; phosphoenolpyruvate synthase (II) is inhibited by 5'-AMP and phosphoenolpyruvate; and pyruvate kinase (V) is activated by 5'-AMP and 2 keto, 3-deoxy,6-phosphogluconate and inhibited by ATP. The presence of metabolites with reciprocal but reinforcing functions is noteworthy. As an example, acetyl-CoA both inhibits the breakdown of oxalacetate and stimulates its formation. Only pyruvate carboxylase appears to be regulated by the carbon substrates of the growth medium.


Binding of concanavalin A to the surface of sea urchin eggs and its alteration upon fertilization.

The binding of concanavalin A (Con A) to Strongylocentrotus purpuratus eggs has been investigated using 125I-concanavalin A (125I-Con A). The lectin binds specifically to the egg surface and does not produce agglutination of the eggs. High 125I-Con A concentrations are necessary to saturate all of the surface binding sites. Scatchard plots of the binding data are biphasic and may be interpreted as showing two main classes of sites. Unfertilized eggs have 4 X 10(8) high affinity sites/egg (Ka=8 X 10(-7) M) and 4.5 X 10(8) istes of lower affinity (Kb=4 X 10(-6) M). The sites may be assigned to different layers of the egg surface by studying the effects of removal of egg coats on the binding reaction. Removal of the jelly coat by washing eggs at pH 4.5 has no effect on binding. However, disruption of the vitelline layer with dithiothreitol leads to a decrease in the number of high affinity sites. After fertilization, the high affinity sites are found upon the fertilization membrane produced from the vitelline layer. Low affinity sites predominate in the plasma membrane, and are not affected by treatments which alter the vitellinelayer. The number of low affinity sites double upon fertilization, with the insertion of new membrane into the egg surface, as a result of cortical granule exocytosis. The doubling of sites is not due to hyaline material released from the cortical vesicles at fertilization, and thus these sites appear to reside upon the new membrane that is inserted from the cortical vesicles. If eggs are activated with ammonia, bypassing the cortical reaction, no change in the binding of ConA occurs. Con A inhibits fertilization at concentrations higher than 0.1 mg/ml, where less than 50% of the high affinity (viteline layer) binding sites are occupied, and there is little binding to the low affinity (plasma membrane) sites. Thus, the interaction of sperm with vitelline layer components may be an obligatory step in the fertilization process. A fraction of the 125I-Con A binding sites is cleaved from the egg surface upon fertilization or after activation by the calcium ionophore A23187. This release of Con A binding sites occurs during the limited proteolysis of surface components that accompanies the cortical reaction, and does not occur with ammonia activation of the egg, where the cortical reaction does not occur. Thus, the changes in Con A binding at fertilization are caused by the massive cortical granule exocytosis that occurs within minutes of sperm penetration, and not by activation of the egg per se.


Pyruvate metabolism in bovine epididymal spermatozoa.

Treating bovine epididymal spermatozoa with rutamycin or rotenone inhibited both respiration and motility supported by endogenous substrates. When oxidative phosphorylation had been blocked with various inhibitors, pyruvate was metabolized to yield ATP and restored motility. Fructose, which is metabolized via glycolysis to yield ATP, was also able to resuscitate the cells. Other substrates tested (lactate, acetate, alpha-ketoglutarate, or glyoxylate) were unable to restore motility in rutamycin-treated cells. In the presence of pyruvate, the phosphorylation uncoupler, carbonylcyanide-p-trifluoromethyoxphenylhydrazone, reduced motility and ATP to common levels in untreated cells or cells treated with rutamycin or rotenone. Pyruvate is thus metabolized to produce ATP by a pathway independent of oxidative phosphorylation associated with the electron transport chain. 5-Methoxyindole-2-carboxylic acid, an inhibitor of lipoyldehydrogenase, prevented the increase of motility and ATP in rutamycin-treated cells, indicating that alpha-keto acid oxidation is involved in the production of ATP from pyruvate when rutamycin is present. With pyruvate present, bongkrekic acid, antimycin A, and anaerobiosis eliminated motility, reduced ATP to low levels, and also significantly reduced the rate of pyruvate metabolism. Acetate was produced from pyruvate only when cellular ATP concentrations were low. Decreases in free carnitine concentrations showed that pyruvate initially used was converted to acetylcarnitine. The results indicate that the intramitochondrial lactate dehydrogenase X, which is unique to spermatozoa, allows the NADH resulting from pyruvate oxidation to reduce other pyruvate molecules to lactate. Pyruvate thus competes with, and can substitute for, the NADH dehydrogenase of the electron transport chain. Pyruvate rapidly repletes the acetylcarnitine pool under a variety of conditions.


Mitochondrial metabolism of pyruvate in bovine spermatozoa.

Treatment with the polyene antibiotic, filipin, renders the spermatozoan cell membrane permeable to small molecules, but not to the intracellular enzymes aldolase and lactate dehydrogenase. Pyruvate (10 mM) as the sole substrate was metabolized very slowly. L-Carnitine increased pyruvate metabolism 3- to 4-fold and allowed limited rates of oxidative phosphorylation. When spermatozoa treated with filipin were supplemented with malate, there was a rapid, almost linear rate of pyruvate metabolism which was slightly increased by L-carnitine. In the absence of malate, 20 to 30% of the pyruvate used was reduced to lactate; this increased to 57% in the presence of malate. Without malate, about 90% of the pyruvate metabolized was converted to lactate and acetate or L-acetylcarnitine. Rutamycin or rotenone increased both the rate of pyruvate use and the delta lactate/deltapyruvate ratio. Under all treatments, L-carnitine consistently reduced the percentage of pyruvate converted to lactate by about 10%; part of the pyruvate was preferentially shunted into L-acetylcarnitine rather than lactate. The mitochondrial inhibitors, rotenone or rutamycin, did not change the amount of pyruvate that was converted to metabolites other than lactate, or L-acetylcarnitine, or both. Pyruvate-supported State 3 respiration was linear only if L-carnitine, or malate, or both, were added to the incubation medium. Added malate was necessary to produce a rapid State 3 respiratory rate and was also required for significant respiratory activity in the presence of rotenone or rutamycin. From cells metabolizing [2-14C]pyruvate (1.4 mM), 14C-labeled acid-extractable metabolites were separated by ion exchange column chromatography. All of the [2-14C]pyruvate (+/-5%) used was recovered in 14C-labeled metabolites and 14CO2. In the presence of malate, citrate accumulation was significant, and was always large in comparison to flux through the citric acid cycle. Glutamate, beta-hydroxybutyrate, acetoacetate, fumarate, aspartate, and alpha-ketoglutarate did not accumulate in significant amounts. Some 14C-labeled succinate was produced but only in the presence of malate. Alkaline hydrolysis of a fraction containing carnitine esters yielded acetate and a compound tentatively identified as beta-hydroxybutyrate or lactate. As in intact cells, intramitochondrial lactate dehydrogenase competes successfully with the electron transport system for the NADH generated by pyruvate metabolism. The role of lactate and L-carnitine, and conclusions suggested by the accumulation of certain metabolites are discussed in relation to control of citric acid cycle activity.


Estrogen binding proteins of calf uterus. Molecular and functional characterization of the receptor transforming factor: A Ca2+-activated protease.

It was previously shown that calf uterus cytosol contains a Ca2+-activated receptor transforming factor (RTF) which irreversibly converts the larger molecular states of estrogen receptor (5.3 to 8.6 S, depending on ionic strength) into a smaller, salt-stable form (4.5 S, independent of ionic strength). We now describe a method for rapid and reliable separation of precursor and RTF-transformed receptor forms, which takes advantage of a difference in isoelectric point between the two: the more acidic precursor (isoelectric point, 6.2) is still retained by DEAE-cellulose under conditions (0.12 M KCl, pH 8.3) which produce release from cellulose of the less acidic transformed form (isoelectric point, 6.6 to 6.8). Based on this method of separation, RTF activity can be assayed easily and we could thus progress in the purification and physical and functional characterization of this factor, RTF has been purified about 100-fold. Molecular properties, as assayed by methods suited to partially purified preparations, are as follows: sedimentation coefficient, 6.4 S; Stokes radius, 45 A; molecular weight, 115,000; isoelectric point, 4.9. The Michaelis constant, expressed as moles/liter of estradiol binding sites, is 1.25 X 10(8), at pH 7.5 and 4 degrees, pH 8.5 is optimum for activity. RTF attacks native casein (Km, 1.25 X 10(-5) mol/liter at pH 7.5 and 22 degrees) but not hemoglobin, ovalbumin, or albumin. N-Benzoylarginine methyl ester is a competitive inhibitor of RTF-induced receptor transformation, while L-leucylglycylglycine and N-benzoyltyrosinamide are not. RTF activity is protected by -SH compounds. RTF activity is Ca2+-dependent. Ca2+ starts an activation-inactivation cycle of RTF, with permanent loss of transforming activity which proceeds at a particularly fast rate in the absence of substrate. Mg2+ is inactive, while Sr2+ and Mn2+ may in part substitute for Ca2+. RTF is present in both endometrium and myometrium. RTF is not a lysosomal hydrolase, as shown by its alkaline pH optimum (8.5) and exclusive location in cytosol, nor is it trypsin or a protease of the trypsin group. Also, it is distinct from known proteases of human uterus. The functional significance of this Ca2+-activated protease of cytosol with alkaline pH optimum and high affinity for the larger native form of receptor is still unknown.


Sequence analysis of protamine mRNA from the rainbow trout. Depurination and nearest neighbor analysis of protamine cDNA.

Protamine cDNA, which was a full length copy of protamine mRNA was labeled during its synthesis by using deoxynucleoside [alpha-32P]triphosphates. Depurination analysis showed that there were 19 different pyrimidine oligonucleotides in protamine cDNA, some of which contained isomeric sequences. The stoichiometry of the pyrimidine oligonucleotides indicated that, while some sequences probably occur in each of the protamine mRNA components, other sequences are clearly absent from one or more of the components. Several of the pyrimidine oligonucleotides had sequences consistent with the amino acid sequences of the rainbow trout protamines. The longest oligopyrimidine tract, C7T4, had a complementary RNA sequence of AGGAGAGGAGG, a stoichiometry of close to 1, and fitted the amino acid sequence Arg-Arg-Gly-Gly which occurs near the COOH terminus of each of three major protamine components. Other pyrimidine oligonucleotides analyzed were complementary to RNA sequences from the noncoding region of protamine mRNA. There appears to be no preferential use of one particular arginine codon or set of codons. Of the 21 to 22 arginine codons in protamine mRNA no less than 7 and no more than 12 are of the CGX series. The other two codons, AGA and AGG, both occur but not in a series of more than two together. This indicates that the RNA sequences coding for the arginine tracts tend to contain a mixture of arginine codons. Nearest neighbor frequency analysis of protamine cDNA gives a low value for the frequency of the CpG doublet, despite its occurrence in four out of the six arginine codons. This is in accordance with the observation that the sequence CpG is surprisingly rare in vertebrate DNA and in the RNA transcribed from it.


Kinetic properties and substrate specificities of two cellulases from auxin-treated pea epicotyls.

Two cellulases purified from growing regions of auxin-treated peas (buffer-soluble and buffer-insoluble) hydrolyze cellulose powder, partially substituted carboxymethylcellulose (CM-cellulose), higher cellodextrins, and certain mixed linkage glucans (e.g. barley beta-glucan), at rates comparable to these reported for the most active fungal cellulases, and with kinetics and product formation characteristic of endohydrolase action. They are unable to cleave 1,3-linkages in beta-glucans, or 1,4-linkages in dextrins containing excessive substitution at C6, alpha configuration, alternating beta-1,3- and 1,4-linkages, or residues other than anhydroglucose. They are not active towards cellobiose or the 1,4-linkage adjacent to the reducing end of cellodextrin chains. It is concluded that buffer-soluble and buffer-insoluble cellulases are true beta-1,4-glucan 4-glucanohydrolases (EC 3.2.1.4). On a molar basis, Vmax values for buffer-insoluble are higher than buffer-soluble cellulase acting towards any of the substrates tested, but Km values towards CM-cellulose and cellohexaose are essentially identical. Both cellulases were inhibited by C12+, Hg2+, and sulfhydryl-binding reagents. Buffer-insoluble, but not buffer-soluble, cellulose was inactivated by reagents that bind serine and threonine, which reflects differences in their amino acid composition. No major qualitative differences have been detected in the mode of action of the two enzymes. Despite marked differences in their physical and immunological properties, close similarities between buffer-soluble and buffer-insoluble enzymic properties suggest that their active sites are the same.


The 24-hydroxylation of 1,25-dihydroxyvitamin D3.

The production of 1,24,25-trihydroxyvitamin D3 in vivo in vitamin D-deficient rats has been demonstrated from either 25-hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3. The stereochemical configuration of the hydroxyl on the 24 position of 1,24,25-trihydroxyvitamin D3 has also been unambiguously established to be R. Nephrectomy failed to eliminate the conversion of 1,25-dihydroxyvitamin D3 to the 1,24,25-trihydroxyvitamin D3, establishing that the 24-hydroxylase can be demonstrated in at least one organ in addition to kidney in contrast to the 1-hydroxylase. 1,24,25-Trihydroxyvitamin D3 can also be produced in vivo from 1alpha-hydroxyvitamin D3 or from 24-hydroxyvitamin D3, analogs of vitamin D3 which are not naturally occurring. Using chick kidney mitochondrial preparations it has been demonstrated that the 24-hydroxylase is able to utilize 1,25-dihydroxyvitamin D3 as a substrate whereas it is unable to utilize 1alpha-hydroxyvitamin D3. In addition, the chicke kidney 1-hydroxylase is known to convert 24,25-dihydroxyvitamin D3 to the 1,24,25-trihydroxyvitamin D3, but this hydroxylase is unable to act on the 24-hydroxyvitamin D3. These results demonstrate that the renal vitamin D hydroxylases require that a hydroxyl be on the 25 carbon of the vitamin D molecule before it can be 1- or 24-hydroxylated.


P-enolpyruvate carboxykinase ferroactivator. Purification and some properties.

P-enolpyruvate carboxykianse ferroactivator was purified from rat liver cytosol to apparent homogeneity. This protein has an S20 of 4.69. High speed sedimentation equilibrium indicated a molecular weight of 82,000; that obtained by gel filtration was 126,000. this discrepancy in molecular weight by the two methods may indicate that this protein has a high axial ratio. A subunit molecular weight of 23,600 was obtained by sodium dodecyl sulfate electrophoresis. Synthesis of P-enolpyruvate is stimulated 3-fold when P-enolpyruvate carboxykinase is incubated in the presence of ferroactivator plus Fe2+; activity with Fe3+, Mn2+, Co2+, Cd2+, Mg2+, or Ca2+ was not affected by the ferroactivator. P-enolpyruvate synthesis by carboxykinase was activated by Mn2+ in the absence of ferroactivator. Quinolinic acid strongly inhibits carboxykinase activated by ferroactivator and Fe2+, but does not inhibit the enzyme activated by Mn2+. In the reverse reaction, ferroactivator and Fe2+ stimulated oxalacetate synthesis only briefly and carboxykinase activity rapidly returned to control rates. Increased oxalacetate synthesis by carboxykinase that had been incubated with ferroactivator and Co2+ was sustained. Stimulation of P-enolpyruvate synthesis by enzyme incubated in the presence of sulfate and Fe2+ rapidly diminished as a function of incubation time. Under similar conditions, P-enolpyruvate carboxykinase ferroactivator maintained the enzyme in the Fe2+-activated state for at least 4 h. The amounts of purified ferroactivator required to stimulate P-enolpyruvate carboxykinase maximally and the amount present in liver cytosol are sufficient to activate the enzyme maximally in vivo if adequate Fe2+ were present. It is possible that the rate of P-enolpyruvate synthesis in gluconeogenic tissues may be regulated by the availability of intracellular Fe2+ to the ferroactivator and P-enolpyruvate carboxykinase.


Regulation by insulin of amino acid release and protein turnover in the perfused rat hemicorpus.

Net changes in the concentrations of 18 amino acids in perfusate and skeletal muscle were followed during perfusion of hemicorpus preparations from fed rat. Perfusate levels of 16 amino acids showed little change from their initial concentrations during the 1st h, but increased dramatically during the 2nd and 3rd h. Aspartate and glutamate levels decreased continuously throughout the perfusion. Release of alanine and glutamine accounted for approximately 50% of the total change in perfusate amino acids. The increase in perfusate amino acids was derived from net breakdown of muscle proteins and not from leakage from the intracellular pool as evidenced by elevated concentrations of intracellular amino acids in perfused muscle. Addition of insulin to the perfusate did not change the pattern of amino acid release during the 1st h of perfusion. However, during the 2nd and 3rd h the hormone completely prevented the net release of most amino acids and maintained intracellular concentrations of most amino acids at levels found in upperfused tissue. Effects of time of perfusion and insulin on amino acid release were accounted for by changes in the rate of protein turnover. Protein synthesis in gastrocemius and psoas muscles in control perfusions decreased after 1 h to approximately 50% of the initial rate. This decrease was accompanied by a 2-fold increase in the level of ribosomal subunits, indicating development of a block in peptide chain initiation. Addition of insulin maintained the initial rate of synthesis and the in vivo level of ribosomal subunits, demonstrating that the hormone prevented the block in peptide chain initiation from forming. Addition of insulin after 2 h reversed the perfusion-induced block in initiation. Synthesis of the specific muscle protein myosin was increased 45% over the control rate in the presence of insulin. Insulin also produced a 50% decrease in the rate of protein degradation during the 2nd and 3rd h of perfusion. A similar effect was noted when protein synthesis was inhibited by addition of cycloheximide. Higher concentrations of insulin were required to maximally inhibit protein degradation than to increase protein synthesis. Involvement of lysosomal proteases in the effect of insulin on protein degradation was evaluated by measuring cathepsin D activity in psoas muscle homogenates. "Free" enzyme activity increased as a result of perfusion while addition of insulin maintained this activity at the unperfused level. Neither perfusion nor insulin had any effect on total cathepsin D activity. Alterations in protein degradation and lysosomal enzyme activity were not due to changes in levels of adenine nucleotides, GTP, or creatine phosphate.


Electron microscopy of the large form of transcarboxylase with six attached subunits.

In this paper we show that the native form of transcarboxylase may be a species which has six rather than three subunits attached to the central subunit. We have designated this form as the 26 S enzyme. Electron micrographs support the view that the six subunits are attached in sets of three at the opposite faces of the central subunit, in contrast to the 18 S form in which all three subunits appear to be attached only at one face. In addition, evidence is presented that the dissociation of the 26 S to the 18 S form of transcarboxylase occurs with the loss of three subunits exclusively from one face of the central subunit. This result may indicate that the two faces of the central subunit differ structurally or there is negative cooperativity in the dissociation of subunits. The 26 S transcarboxylase, which was made by attachment of subunits to the 18 S enzyme or trypsin-treated 18 S enzyme was shown to have subunits on both faces of the central subunit. Treatment of the 26 S enzyme with carbodimide to cross-link the subunits to the central subunit and thus stabilize the structure resulted in improved electron micrographs. A model of the 26 S form of the enzyme is presented.


Dissociation of prelycopersene pyrophosphate synthetase from phytoene synthetase complex of tomato fruit plastids.

The partially purified phytoene synthetase enzyme complex obtained from tomato fruit plastids dissociates into two or more subunit species on chromatography in low ionic strength buffer on DEAE-cellulose. One of these subunits prelycopersene pyrophosphate synthetase, has a molecular weight of approximately 40,000, whereas the phytoene synthetase complex has a molecular weight of 200,000. The prelycopersene pyrophosphate synthetase catalyzes the conversion of isopentenyl pyrophosphate to geranylgeranyl and prelycopersene pyrophosphates. The identities of these substances were established by thin layer chromatography in several solvent systems. The formation of both geranylgeranyl and prelycopersene pyrophosphates by this enzyme supports earlier results with cruder enzyme systems which suggested that these compounds are intermediates in the synthesis of phytoene.


Studies on inactivation of pyruvate dehydrogenase by palmitoylcarnitine oxidation in isolated rat heart mitochondria.

The oxidation of an optimal concentration of palmitoyl-carnitine, buffered with bovine serum albumin, by isolated rat heart mitochondria was found to give rise to an inactivation of pyruvate dehydrogenase, provided that the concentration of pyruvate present in the mitochondrial incubation was less than 250 muM. The greatest degree of inactivation was found at the lowest pyruvate concentration used, 50 muM, and this concentration was adopted for further studies in which the rate of mitochondrial respiration was varied. This was done by varying the activity of added hexokinase, in the presence of ATP, MgCl2, and glucose, and thus the availability of ADP to the mitochondrion. The pyruvate concentration in the incubation was approximately stabilized by adding pyruvate on the basis of oxygen consumption, with the ratio of pyruvate consumed:O2 consumed determined by trial and error. This device allowed the maintenance of essentially steady pyruvate concentrations and ATP/ADP ratios for at least 5 min, and allowed the pyruvate dehydrogenase interconversion time to approach a steady state. Activities of pyruvate dehydrogenase after 5 or 6 min of respiration were as follows, with values given in nanomoles/min/mg of protein for incubations containing pyruvate as sole substrate, and values for incubations containing pyruvate plus palmitoylcarnitine given in parentheses: State 4, 27 (9); 55% of State 3, 54 (14); 85% of State 3, 73 (28); State 3, 90 (93). Respiratory states are defined by Chance and Williams (1955) J. Biol. Chem. 217, 409-427). Values at earlier time points are also presented so that some idea may be formed of the time course of pyruvate dehydrogenase inactivation. CoASH/acetyl-CoA, NAD+/NADH, and ATP/ADP ratios were measured at the same time points in precisely scaled up incubations. The presence of palmitoylcarnitine in State 4 was found to give essentially no change in NAD+/NADH and ATP/ADP ratios and thus the inactivation of pyruvate dehydrogenase in that state may be attributed to a decreased CoASH/acetyl-CoA ratio. At a respiratory rate of 85% of State 3, palmitoylcarnitine did not change the ATP/ADP ratio, but lowered both CoASH/acetyl-CoA and NAD+/NADH ratios, both of which may contribute to pyruvate dehydrogenase inactivation. In State 3 there was no pyruvate dehydrogenase inactivation, despite a lowered CoASH/acetyl-CoA ratio in the presence of palmitoylcarnitine. It is concluded that ATP/ADP ratio has a pronounced effect on the interconversion of active and inactive pyruvate dehydrogenase, in according with previous work. Moreover, at a given ATP/ADP ratio, the effects of palmitoylcarnitine oxidation on enzyme interconversion are consistent with a mechanism involving the modulation of the interconversion by NAD+/NADH and CoASH/acetyl-CoA ratios...


Evidence for the presence of a natural inactivating factor of 3-hydroxy-3-methylglutaryl coenzyme A reductase in soluble extracts from rat liver microsomes.

The temperature sensitivity of crude solubilized 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, prepared from rat liver microsomes by different procedures, varies widely. When enzyme was solubilized by several techniques, HMG-CoA reductase activity in these extracts was rapidly and irreversibly inactivated at 4 degrees (Brown, M.S. Dana, S.E. Dietschy, J.M. and Siperstein, M.D. (1973) J. Biol. Chem 248, 4731-4738). In contrast, HMG-CoA reductase, solubilized from microsomes by a slow freeze-thaw method, was reversibility inactivated at 4 degrees over an interval of 2 h (Heller, R.A. and Gould, R.G. (1974) J. Biol. Chem 249, 5254-5260). In the present article irreversible inactivation at 4 degrees of crude solubilized HMG-CoA reductase, prepared from liver microsomes by a slow freeze-thaw technique, was investigated. In the absence of preincubation at 37 degrees, HMG-CoA reductase activity in the crude soluble extract had a half-life at 4 degrees of 4 h. Enzyme activity was more rapidly inactivated (t1/2=4 h) in extracts from younger (150 to 250 g) rats than in extracts from older (500 g) rats (t1/2=37 h). In contrast, partially purified HMG-CoA reductase was far more stable at 4 degrees (t1/2=312 h). However, when partially purified enzyme was treated with crude soluble extract, the enzyme was inactivated much more rapidly (t1/2=26 h). It is concluded that rapid irreversible inactivation of HMG-CoA reductase at 4 degrees is not an intrinsic property of this enzyme, but instead, this inactivation is caused by a factor or factors present in the crude soluble extract. While HMG-CoA reductase activity was rapidly inactivated in crude extracts from animals killed at the midpoint of the dark cycle, enzyme activity was inactivated much more slowly in extracts obtained from animals killed at the midpoint of the light cycle. These findings suggest that the concentration of the inactivating factor may vary, depending upon the physiological state of the animal. The nature of the inactivating factor is not known at the present time; however, it may be a protein since it is nondialyzable and destroyed by heat.


Effect of membrane environment on succinate dehydrogenase activity.

The turnover number of succinate dehydrogenase from mammalian heart determined by the spectrophotometric phenazine methosulfate assay, after complete activation, is approximately 21,000 mol of succinate oxidized/min/mol of histidyl flavin at 38 degrees in relatively intact inner membrane preparations and mitochondria. Reconstitutively active soluble preparations, extracted anaerobically in the presence of succinate from inner membrane preparations show turnover numbers of 11,500 to 14,500 and a significantly lower apparent Km for phenazine methosulfate than the parent particles. The decline of both the turnover number and of the Km occurs during the brief period when the enzyme is detached from the membrane. The observed values represent the activities in the soluble extract of both the reconstitutively active and reconstitutively inactive enzyme. The latter may be from 10 to 40% even in the most carefully prepared enzyme; it has a lower turnover number in the phenazine methosulfate assay than the average for the solution and is devoid of catalytic activity in the "low Km" ferricyanide assay (Vinogradov, A. D., Ackrell, B.A.C., and Singer, T.P. (1975) Biochem. Biophys. Res. Commun. 67, 803-809). The reconstitutively active form of the soluble enzyme has a turnover number of at least 15,000 and an equal activity in the low Km ferricyamide assay. When recombined with the membrane the total activity of the enzyme is increased by over 60% and it regains the original turnover number, Km for phenazine methosulfate, and sensitivity of the phenazine methosulfate reductase activity to thenoyltrifluoroacetone, carboxamides, and cyanide. It appears, therefore, that the membrane environment or some component of it exerts a positive modulating influence on the enzyme even in the fully activated state. In certain particulate sources (Keilin-Hartree preparations, Complex II) the enzyme shows lower turnover numbers (11,000 to 12,500) than in more intact inner membranes. This seems to be due to inactivation in the course of preparation and, in the case of Complex II, in part also to loss of the normal membrane environment or of a membrane component, possibly Q-10, during isolation.


Rat muscle 5'-adenylic acid aminohydrolase. Role of K+ and adenylate energy charge in expression of kinetic and regulatory properties.

The kinetic and regulatory properties of homogeneous AMP deaminase from rat skeletal muscle have ben examined with particular emphasis on (a) the role of potassium ions in the expression of these properties and (b) the role of the adenylate energy charge in the regulation of AMP deaminase activity. Although the enzyme has an absolute requirement for K+, the concentration required for maximum activation is dependent on the concentration of substrate. At saturating levels of AMP (greater than or equal 2.0 mM) maximum activation is observed with 25 mM KCl, whereas at lower substrate concentrations (0.2 mM) approximately 50 mM KCl is needed for maximum activation. Conversely, the response of enzyme activity ot increasing levels of substrate is dependent on the level of potassium ions. At substrating concentrations of K+, the saturation curve for AMP is highly sigmoidal (nh=3.2) whereas at higher KCl concentrations, the apparent cooperativity between AMP sites is almost completely abolished (nh=1.5). The inhibition by a number of phosphorylated metabolites, including ATP, GTP, creatine-P, and P1, is likewise sensitive to the concentration of K+. These results suggest that a significant amount of interaction between K+ sites and both substrate and effector sites is required for the expression of the catalytic and regulatory properties of the enzyme. The specific effects of ATP, creatine-P, and P1 on the parameters of Km and Vmax indicate that each of these profile of AMP deaminase activity generated in response to variations in the adenylate energy charge shows that within the physiological range of energy charge (0.75 to 0.95), the activity increases linearly with decreasesing energy charge and is insensitive to both the total adenylate pool size and the presence of P1 and creatine-P. These data suggest that the most important factor in the regulation of AMP deaminase activity is the state of the energy charge rather than the absolute concentrations of the individual effectors.


Rat liver proteins capable of transferring phosphatidylethanolamine. Purification and transfer activity for other phospholipids and cholesterol.

Two proteins, one in a highly purified form, have been isolated from the soluble fraction of rat liver homogenate. These proteins accelerate the transfer of labeled phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, sphingomyelin, and cholesterol from liposomes to mitochondria or erythrocyte ghosts. The fraction obtained after ammonium sulfate precipitation, gel filtration on Sephadex G-75, ion-exchange chromatography on CM-cellulose, ampholyte displacement chromatography, and heat treatment exhibited an 876-fold increase in its phosphatidylethanolamine transfer activity as compared with the postmitochondrial supernatant adjusted to pH 5.1. Isoelectric focusing on polyacrylamide gels shows a single band between pH 8.6 and 9.0. The transfer activity is abolished by trypsin, but withstands 5-min heating at 90 degrees. After heat treatment, a single major band is seen on polyacrylamide gel electrophoresis followed by two minor ones. The molecular weight of the major band is 12,500, as determined by electrophoresis on 15% polyacrylamide gels in the presence of sodium dodecyl sulfate. A molecular weight of 13,500 was calculated from molecular filtration through Sephadex G-50. The relative transfer activities toward the different phospholipids remain constant throughout the last three steps of the purification procedure in spite of the extensive change in the electrophoretic profile of the protein mixture. The cholesterol transfer activity remains unchanged after the final heat treatment as well. This indicates that all of the transfer activities are present in a single protein.


Mutants of mouse fibroblasts altered in the synthesis of cell surface glycoproteins. Preliminary evidence for a defect in the acetylation of glucosamine 6-phosphate.

We hve reported the isolation and preliminary biochemical characterization of two mutants of mouse fibroblasts selected for a decrease incell-to-substrate adhesion (Pouysségur, J., and Pastan, I. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 544-548). We attributed the adhesive defect of these mutants (AD6 and AD8) to the absence of iodinatable cell surface proteins. This study demonstrates that a defect in glycoprotein synthesis is the biochemical basis for the reduction in proteins exposed at the outer surface of the mutant cells. When D-glucosamine or L-fucose was used as radioactive precursor, analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a decrease in the labeling of all the glycoproteins of both clones. This decrease in glycoprotein labeling was not due to a defect in D-glucosamine uptake, since this precursor was taken up at a higher rate in the mutants than in the wild type. In spite of this high uptake, the rate of D-glucosamine incorporation into macromolecules was decreased by 60% and the carbohydrate content of membranes (mannose, galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid) from clone AD6 was diminished by 40 to 60%. When the various cell lines were labeled for 1 to 3 h with glucosamine and the acid-soluble pool analyzed, the wild type cells were found to accumulate UDP-N-acetylhexosamine as the major component. In contrast, clones AD6 and AD8 accumulated glucosamine 6-phosphate as the major component. This last finding suggests that there is a block in the N-acetylation of glucosamine 6-phosphate in both instants. This suggestion is supported by the finding that feeding the mutant cells 10 mM N-acetylglucosamine reverts them to the wild type phenotype (Pouysségur, J., Willingham, M. and Pastan, I. (1977) Proc. Natl. Acad. Sci. U.S.A., in pressy. In wild type cells all of the iodinatable proteins of the cell surface have the same mobility as the glycoproteins when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the early block in glycoprotein synthesis accounts for the reduction in iodinatable surface proteins in the mutant cells.


1-Alkyl-sn-glycero-3-phosphate: acyl-CoA acyltransferase in rat brain microsomes.

1-Alkyl-sn-glycero-3-phosphate:acyl-CoA acyltransferase activity was found in six rat tissues: heart, spleen, brain, kidney, liver, and lung. The enzyme in rat brain showed highest specific activity in the microsomal fraction and its properties were studied in detail. Triton X-100 and bovine serum albumin were found to stimulate the activity of the enzyme. It was found that Triton X-100 affects the acyltransferase activity differentially depending on the acyl-CoA donor. Albumin reversed the inhibition caused by a high concentration of 1-alkyl-sn-glycero-3-phosphate. The enzyme was shown to be stereospecific in acylating the alkylglycerophosphate substrate. Only the 1-alkyl-sn-glycero-3-phosphate isomer, not the other optical enantiomer, was active with brain acyltransferase. Evidence is presented to show that this enzyme is different from 1-acyl-sn-glycero-3-phosphate:acyl-CoA acyltransferase. Investigations of the specificity of the brain acyltransferase activity for different acyl-CoA species (16:0, 18:0, 18:2, 20:4, 22:4, 22:6) showed a selectivity which was dependent on the alkylglycerophosphate concentration. At low 1-alkyl-sn-glycero-3-phosphate concentration the enzyme selected polyunsaturated acyl-CoA species over saturated species. Providing a low but continuous amount of 1-alkyl-sn-glycero-3-phosphate to the acyltransferase was accomplished by the in situ enzymatic reduction of alkyldihydroxyacetone phosphate with NADPH in the incubation mixtures. The acyl composition of ether-containing phosphoglycerides in rat brain microsomes, as well as the free fatty acid composition, was determined. Alkenylacylglycerophosphorylethanolamine contains a high percentage of 22:6 ester, and alkenylacylglycerophosphorylcholine contains a large amount of 16:0 ester. Comparison of the acyl-CoA specificity of 1-alkyl-sn-glycero-3-phosphate acyltransferase and ether/glycerolipid acyl composition suggests that this acyltransferase could partially determine the composition of acyl groups in ether lipids.


Complete covalent structure of statherin, a tyrosine-rich acidic peptide which inhibits calcium phosphate precipitation from human parotid saliva.

The complete amino acid sequence of human salivary statherin, a peptide which strongly inhibits precipitation from supersaturated calcium phosphate solutions, and therefore stabilizes supersaturated saliva, has been determined. The NH2-terminal half of this Mr=5380 (43 amino acids) polypeptide was determined by automated Edman degradations (liquid phase) on native statherin. The peptide was digested separately with trypsin, chymotrypsin, and Staphylococcus aureus protease, and the resulting peptides were purified by gel filtration. Manual Edman degradations on purified peptide fragments yielded peptides that completed the amino acid sequence through the penultimate COOH-terminal residue. These analyses, together with carboxypeptidase digestion of native statherin and of peptide fragments of statherin, established the complete sequence of the molecule. The 2 serine residues (positions 2 and 3) in statherin were identified as phosphoserine. The amino acid sequence of human salivary statherin is striking in a number of ways. The NH2-terminal one-third is highly polar and includes three polar dipeptides: H2PO3-Ser-Ser-H2PO3-Arg-Arg-, and Glu-Glu-. The COOH-terminal two-thirds of the molecule is hydrophobic, containing several repeating dipeptides: four of -Gn-Pro-, three of -Tyr-Gln-, two of -Gly-Tyr-, two of-Gln-Tyr-, and two of the tetrapeptide sequence -Pro-Tyr-Gln-Pro-. Unusual cleavage sites in the statherin sequence obtained with chymotrypsin and S. aureus protease were also noted.


Metabolic properties of the products of mitochondrial protein synthesis in HeLa cells.

The metabolic behavior of the mitochondrial protein synthesis products has been investigated in HeLa cells. Particular attention was given to the four major electrophoretic components (designated as Nos. 2, 3, 5, and 8) of the 10 previously identified as organelle-specific products. Inhibition of cytoplasmic protein synthesis with emetine or cycloheximide causes a rapid decline in the rate of mitochondrial protein synthesis, with an estimated half-life of 1 to 2 h, affecting in a parallel way all the discrete components. About 30% of the original synthetic activity appears to be resistant to emetine treatment for at least 24 h; however, all the polypeptides synthesized after the first 4 h of cell exposure to emetine are metabolically unstable, possibly because of lack of integration into the inner mitochondrial membrane. An analysis of the stability of newly synthesized products of mitochondrial protein synthesis pulse-labeled in the presence of cycloheximide and then chased in the absence of the drug (i.e. under conditions of resumed cytoplasmic protein synthesis) has revealed marked differences among the various discrete components. In particular, about three-fourths of the radioactivity associated with components 3 and 5 decays within 4 h of chase, the remainder being substantailly stable afterwards; by contrast, the radioactivity in components 2 and 8 shows only a slow decline during a 3-day chase. If the chase is carried out under conditions of a persistent block of cytoplasmic protein synthesis, as is the situation after a pulse labeling in the presence of emetine, all newly synthesized components appear to be destablized in various degrees, with the exception of component 5, which is to a great extent stabilized. Inhibition of mitochondrial protein synthesis with chloramphenicol has a progressive stabilizing effect on most of the discrete components newly synthesized after removal of the drug; this effect is especially striking in the case of component 5 which, in experiments of continuous labeling in the presence of emetine after prolonged chloramphenicol treatment, becomes, after 24 h of labeling or more, the only recognizable peak in the electrophoretic pattern of the sodium dodecyl sulfate-lysed mitochondrial fraction. The results of the kinetic experiments described here are interpreted in terms of two roles of cytoplasmically synthesized proteins, one required for the synthesis of polypeptides within the organelles, the other necessary for the stabilization of the mitochondrial products.


Formation of adenosine 5'-phosphoroglycerol from ATP and glycerol by rat liver plasma membranes.

Rat liver plasma membranes are shown to catalyze the formation of adenosine 5'-phosphoroglycerol and adenosine 5'-phosphoromethanol from ATP and glycerol or methanol, respectively. In the presence of 2.7 M glycerol and 1 mM ATP, 30 nmol of adenosine 5'-phosphoroglycerol were formed in 10 min per mg of rat liver plasma membranes. The structures of these phosphodiesters were determined from the following evidence. Radioactivity was incorporated into the nucleotide from [alpha-32P]ATP, [2,8-3H]ATP, or [2-3H]glycerol. Treatment with snake venom phosphodiesterase I converted the nucleotides to AMP. The compound formed from glycerol and ATP co-migrated with adenosine 5'-phosphoroglycerol synthesized from glycerol and adenosine 5'-phosphoromorpholidate in five thin layer chromatography systems. The methyl derivative co-migrated with adenosine 5'-phosphoromethanol synthesized from methanol and adenosine 5'-phosphormorpholidate in several thin layer chromatography systems. The synthesis of these phosphodiesters was also catalyzed by chicken embryo fibroblast membranes and solubilized rat liver plasma membranes but not by rat heart plasma membrane preparations. Formation of significant amounts of these phosphodiesters required relatively high concentrations of the alcohols (greater than 1 M). The alcohol concentration dependence did not exhibit substrate saturation at physiologically meaningful concentrations of glycerol or methacol. It is proposed that either the alcohols examined were not the natural substrates for this enzyme or that the alcohol/AMP phosphodiesters were formed as a result of trapping of an enzyme/nucleotide intermediate. Adenosine 5'-phosphoroglycerol formation was inhibited approximately 50% by 15 mM NaF. Epinephrine, norepinephrine, glucagon, and prostaglandin E1 were without effect. Alloxan, an inhibitor of adenylate cyclase did not inhibit formation of adenosine 5'-phosphoroglycerol. It is concluded that adenylate cyclase was not responsible for formation of these phosphodiesters. The physiological significance of this reaction remains undefined.


Penicillinase-releasing protease of Bacillus licheniformis 749 Specificity for hydroxyamino acids.

The membrane penicillinase of Bacillus licheniformis 749/C differs from the exopenicillinase in that it has an additional 24 amino acid residues and a phosphatidylserine at the NH2 terminus (Yamamoto, S., and Lampen, J.O. (1976) J. Biol. Chem. 251, 4095-4101). The conversion of the membrane penicillinase to the exo form is probably carried out by a specific penicillinase-releasing protease (PR-protease) whose properties are generally consistent with the properties of penicillinase secretion. The substrate specificity of the PR-protease was determined by identifying the NH2 and COOH termini of the peptides produced by hydrolysis of ribonuclease B and beef insulin. The enzyme hydrolyzed only peptide bonds involving the carboxyl groups of serine or thrombine. Similar bonds in synthetic di- or tripeptides of L-serine were not cleaved. The existence of seryl-lysine and threonyl-glucamic acid bonds in the protease-susceptible (phospholipopeptide) region of the membrane penicillinase and the presence of only lysine or glutamic acid at the NH2 terminus of the exoenzyme released in vivo are consistent with the specificity of PR-protease; hence, we propose that this enzyme has an essential role in the formation of exopenicillinase. The PR-protease is a potential tool for protein sequence determination because of its narrow and novel substrate specificity.


Hydrogen peroxide lowers ATP levels in platelets without altering adenyalte energy charge and platelet function.

H2O2 irreversibly reduced metabolic platelet ATP levels with a corresponding accumulation of hypoxanthine. This process was enhanced by sodium azide or potassium cyanide and by increasing H2O2 concentrations. The adenylate energy charge was unaltered when less than two thirds of the metabolic ATP had disappeared but decreased markedly when more ATP disappeared. Platelet shape change, primary aggregation, dense granule and alpha-granule secretion were unaffected by H2O2-induced lowering of ATP provided that the adenylate energy charge did not fall by more than 5%; at greater adenylate energy charge reduction, platelet functions were inhibited. These results indicate that cell functions depend more on adenyalte energy charge than on the ATP level and expands the applicability of this view from bacterial systems to a mammalian cell, the human platelet.


The 5'-terminal structures of murine alpha- and beta-globin messenger RNA.

The 5'-terminal structures of murine alpha- and beta-globin mRNA were determined after incubating cells of the erythropoietic spleens of mice with [methyl-3H]methionine. Globin mRNA was obtained from total cellular RNA by oligo(dT)-cellulose chromatography followed by elution of mRNA from formamide gels after electrophoresis. The globin mRNA was then hydrolyzed with KOH or digested with a combination of RNase T2 and bacterial alkaline phosphatase, and 5'-terminal structures were isolated by DEAE-cellulose chromatography. The methylated nucleotides of these 5'-structures were determined following digestion with specific ribonucleases and bacterial alkaline phosphatase. Analyses of mRNA fractions enriched for either alpha- or beta-mRNA gave similar results. Our data indicate that murine alpha- and beta-globin mRNAs are identical through the first three nucleotides and that partial dimethylation exists at the second position: m7G(5')ppp(5') [m6Am/Am]pCmpNp.


Methylated nucleosides in globin mRNA from mouse nucleated erythroid cells.

Poly(A)-containing mRNAs labeled with [methyl-3H]methionine were isolated from nucleated erythroid cells obtained from the spleens of anemic mice. The RNAs were further separated into non-globin poly(A)-containing RNAs and highly purified globin mRNA by globin cDNA-cellulose affinity chromatography. DEAE-Sephadex column chromatography of the T2 ribonuclease digestion products of the cDNA-purified globin mRNA fraction yielded methylated resistant fragments with charges of -4.7 (Cap 1) and -5.3 (Cap 2). Digestion of the non-globin RNA fraction revealed a similar pattern with the addition of a methylated mononucleotide identified as 6-methyladenosine at -2 charges. Alkaline phosphatase treatment of the T2 resistant fragments reduced their charges by approximately 2, which is consistent with the removal of one terminal phosphate. Treatment of the globin T2 and alkaline phosphatase-resistant fragments withpenicillium P1 nuclease and alkaline phosphatase yielded a P1-resistant core structure in both fragments. In addition to the core, 2'-O-methylcytidine (Cm) was released from the more negatively charged globin fragment. The P1-resistant cores of the cap structures eluted from DEAE-Sephadex with the known standard m2G5'ppp5'Am and were found to be pyrophosphatase-sensitive establishing a 5'-5'-triphosphate linkage. The pyrophosphatase and alkaline phosphatase digestion products of the globin Cap 1 and Cap 2 core structures were analyzed by high voltage electrophoresis and paper chromatography and found to be 7-methyiguanosine (m7G) and the dimethylated nucleoside 6-methyl-2'-O-methyladenosine (N6mAm). A small amount of the singularly methylated adenosine, 2'-O-methyladenosine (Am) was also observed. The predominant sequences of the methylated nucleosides in the globin cap structures are therefore m7G5'ppp5'N6mAm and m7G5'ppp5'N6mAmpCm.


Effects of UDP-glucose addition on the synthesis of mannosyl lipid-linked oligosaccharides by cell-free fibroblast preparations.

The pattern of mannosyl lipid-linked oligosaccharides synthesized by cell-free enzyme preparations from cultured fibroblasts is altered substantially when 0.2 muM UDP-glucose is added to the incubation medium. Inclusion of UDP-glucose results in the appearance of a new labeled oligosaccharide, which is 1 or 2 glycose units larger than the lipid-linked oligosaccharide synthesized in the presence of only GDP-mannose (2 muM) and UDP-N-acetylglucosamine (20 muM). Label from UDP-[3H]glucose is incorporated into the same larger oligosaccharide size class. The results can be explained most easily by assuming that the new mannosyl lipid-linked oligosaccharide contains 1 or 2 glucose residues in addition to 5 to 6 mannose residues. The results are compatible with the recent finding by Spiro et al. (Spiro, M.J., Spiro, R.G., and Bhoyroo, V.D. (1976) Fed Proc. 35, 1375; Spiro M.J. Spiro, R.G., and Bhoyroo, V.D. (1976) J. Biol. Chem. 251, 6400-6408; Spiro, R.G., and Spiro, M.J. AND Bhoyroo, V.D. (1976) J. Biol. Chem. 251, 6409-6419; Spiro, M.J., Spiro R.G., and Bhoyroo, V.D. (1976) J. Biol. Chem. 251, 6420-6425) that naturally occurring mannosyl lipid-linked oligosaccharides contain glucose. In addition to being incorporated into lipid oligosaccharides, glucose residues are also incorporated into endogenous glycoproteins. Incorporation of glucose into glycoproteins that give rise to pronase glycopeptides of the typical asparagine-linked size classes is almost completely dependent on the presence of GDP-mannose.


Requirement of the delta subunit of chloroplast coupling factor 1 for photophosphorylation.

1. Chloroplast coupling factor (CF1) can be prepared by a rapid and simple procedure after release of the protein from the membrane by brief exposure to chloroform. It is suitable for large scale preparation of the coupling factor. 2. The protein contains five subunits (alpha, beta, gamma, delta, and epsilon in order of decreasing molecular weight) and serves as a coupling factor for photophosphorylation. However, when this preparation of CF1 was passed through a DEAE-Sephadex A-50 column, a protein with four subunits, missing the delta subunit, was obtained. The four-subunit protein did not serve as a coupling factor. 3. Photophosphorylation was restored to CF4-depleted chloroplast by addition of the four-subunit protein together with a partially purified preparation of the delta subunit. The latter subunit, when added alone, not only did not stimulate photophosphorylation but consistently diminished the residual activity. 4. The delta subunit is required for the binding of CF1 and may represent the stalk seen in electron micrographs as a link between the protein and the membrane.


Synthesis of tubulin and actin by neuronal and glial nuclear preparations from devloping rat brain.

A system was established in which nuclear preparations from rat brains were capable of protein synthesis under cell-free conditions. The electrophoretic pattern of the synthesized proteins was similar to that found in vivo provided that the reaction mixture contained pH 5 precipitated factors derived from the high speed supernatant fraction of brain. In the absence of the pH 5 factors, using nuclear preparations from brains of 2-day-old rats, approximately 1.5% and 2% of the newly synthesized proteins were identified as tubulin and actin, respectively. In the presence of pH 5 factors, protein synthesis was stimulated and the proportion of the newly synthesized tubulin and actin increased to 26% and 11%, respectively. In contrast to nuclear fractions from 2-day-old rats, when nuclei from brains of 1-month-old rats were tested in the presence of pH 5 factors, the proportion of tubulin and actin synthesized was lower and amounted to 10% and 4%, respectively. The age-dependent change in the relative amount of the tubulin and actin synthesized is in good agreement with the translational pattern shown by brain polyribosomes in a brain cell-free system as well as with the pattern obtained with brain mRNA translated in a wheat germ cell-free system. Nuclei enriched for either neuronal or glial populations synthesized similar proportions of tubulin and actin in vitro. We conclude that the reduction in the synthesis of tubulin and actin during the postnatal development of the rat brain occurs in both neuronal and glial cells.


Chemical evidence for common genetic ancestry of complement components C3 and C5.

Sequence studies reveal a marked similarity between the partial primary structures of human C3a and C5a anaphylatoxins. Eight of the first 25 residues are identical when threonine at position 1 in C5a is aligned with glutamine at position 3 in C3a and one gap is inserted in the C3a sequence. A unique cysteinyl-cysteine sequence is conserved in the 2 molecules, which further suggests a genetic relatedness. Computer-generated alignment scores from comparison of the NH2-terminal portions of human C3a and C5a exceeded by 4 standard deviations the score from random permutations of these same sequences. This sequence similarity between the two anaphylatoxins suggests that the respective precursors, C3 and C5, had a common genetic ancestry. The proposed genetic relationship between C3 and C5 may express itself functionally through a similar molecular behavior in complement-dependent cytolysis.


Stability of glycolytic enzymes of human erythrocytes.

The stability of various glycolytic enzymes of human erythrocytes has been studied by the mechanical shaking method. The rate of denaturation apparently followed first order kinetics. The t1/2, the shaking time required to denature 50% of the original activity, for glucose-6-phosphate dehydrogenase, phosphofructokinase, and pyruvate kinase was less than 1 min; that for hexokinase, 6-phosphogluconate dehydrogenase, and monophosphoglyceromutase was between 2 and 13 min; that for all the other enzymes was more than 30 min. Since the t1/2 value for each enzyme is highly reproducible if the shaking conditions are kept constant, these parameters may be used as an indicator of protein stability in solution. The mechanical denaturation method may also be used to remove unstable components from a mixture of proteins with different stabilities.


Pseudosequestration.

Three children with radiologic findings consistent with right-sided pulmonary sequestration were found at surgery to have a defect in the right hemidiaphragm which permitted a portion of normal liver to herniate into the chest and was associated with an anomalous systemic circulation to the right lower lobe. We refer to this abnormality as "pseudosequestration" and feel it is related to true pulmonary sequestration within the sequestration spectrum.


Radiology services for one million people (1974 Diagnostic Radiology Services in the Province of Manitoba).

For the year 1974, in Manitoba, 905,586 x-ray examinations were performed. The services were provided in 154 facilities, using 332 x-ray units, conducted by 566.6 full-time equivalent individuals, at a cost of $11,117,183. The examinations per capita have increased from 0.52 to 0.86 in the past decade while the cost per examination rose only from $11.00 to $12.63.


[Atypical achondrogenesis. Review of the literature apropos of 2 cases].

The authors describe two cases of a minor form of achondrogenesis. This form has not been published previously. A discussion of the different possibilities of differential diagnosis follows.


Ureteropelvic junction obstruction in the adult.

Cases of ureteropelvic (UP) obstruction presenting for the first time in the adult were analyzed and etiologic factors considered. Besides idiopathic causes, calculi and neoplasm were found in our series. Comparison with other reports of UP obstruction in pediatric patients revealed differences pertaining to mode of clinical presentation, sex predominance and incidence of neoplasm occuring at the UP junction in the adult.


Lymphocyte transformation in lung  cancer patients.

Lymphocyte mitogen transformation has been studied before and after treatment with sugery and radiation in lung cancer patients, in surgically treated patients with non-malignant lesions, and in healthy persons. We have shown a fall in lymphocyte response following operation, even in patients with benign lesions. Patients with cancer have a lymphocyte response which is depressed following operation more than the patients with benign lesions. Following cobalt irradiation there is a further fall in response. We were not able to identify a factor in the serum of patient with depressed lymphocyte transfromation which affected lymphocyte response to Phytohemagglutinin. A recovery in response towards pre-treatment levels occurs earliest in patients with benign lesions. In the patients who had not received irradiation, lymphocyte transformation did not have any prognostic value. In patients with advancing cancer, who ultimately die following irradiation, there is no recovery of response. This lack of recovery signals a very poor prognosis.


Telephone line transmission of rectilinear radionuclide scans. Report of a pilot project.

The General Electric-Elscint system for telephone line transmission of rectillinear scans has been tested and with certain limitations found to be acceptable in terms of diagnostic accuracy for many scan procedures. The system tested had some logistic disadvantage requiring batch processing of the scans, and making quality control of the scans difficult with the one video data processor available at the reference centre.


Diagnosis of rotator cuff tear on routine radiographs.

Routine shoulder radiographs were reviewed in 42 patients with proven rotator cuff tears and in 38 patients in whom a rotator cuff tear had been excluded by shoulder arthrography. Five radiographic findings were identified which were commonly associated with a rotator cuff tear. One or more of these findings were seen in 90.4% of those patients with a rotator cuff tear. These findings were not specific and were also seen in 64.3% of those patients with a frozen shoulder.


Precocious onset of Paget's disease--a report of three cases and review of the literature.

Paget's disease generally has its onset in mid-adult life, however cases are known to occur in young adults, albeit rare. Three patients with precocious onset of Paget's disease are reported and the literature is reviewed. The classical radiographic appearance of Paget's disease of bone is described.


"Brown tumor" of hyperparathyroidism induced with anticonvulsant medication.

A case history is presented of a 50-year-old epileptic receiving massive dose of phenobarbital and small doses of dilantin. Following a minor injury to her forearm a "brown tumor" of hyperparathyroidism and osteomalacia, both of which responded to vitamin D.A review of the literature is given; this is the second case reported which documents hyperparathyroidism under such circumstances, and the first to show radiographs of such changes. Radiologists should be aware of the possibility of these changes in patients on long-term anticonvulsants.


[Pyelocalyceal submucosal hemorrhage in a child treated with oral anticoagulants].

A 15 1/2-year-old girl with disseminated lupus erythematosus presented with acute flank pain and hematuria during oral anticoagulant therapy for thrombophlebitis of the lower extremities. The intravenous pyelogram demonstrated multiple filling defects of the renal collecting system interpreted as pyelocalyceal submocosal hemorrhage. This benign complication disappeared following adjustment of the anticoagulant therapy. An identical appearance has been described in a case of aplastic anemia, Henoch-Schonlein purpupa, and in renal truma. Submocosal hemorrhage of the renal collecting system is to be differentiated from pyeloureteritis cystca, uroepithelial tumor, vascular impressions from collateral circulation and submucosal edema.


Bilateral intrathoracic meningoceles.

Bilateral intrathoracic meningoceles are reported in a 22-year-old man with a family history of neurofibromatosis. Clinical evaluation and conventional radiography are inadequate to differentiate intrathoracic meningocele from other posterior mediastinal masses, especially dumbbell neurofibroma. Myelography is the diagnostic procedure of choice.


A simple device for testing the accuracy of x-ray timers.

A simple device for testing the accuracy of x-ray timers uses a portable record player whose turntable spins the film cassette beneath the lid on which lead shielding protects the film except for a tiny pin-hole through which the exposures  are made. Multiple exposures of different times of the reproducibility of one time setting can all be made on one film.


Ultrastructural and histochemical differences in cell surface properties of strain-specific and nonstrain-specific TA3 adenocarcinoma cells.

Transmission and scanning electron microscopy and histochemical and biochemical methods were used to investigate differences in cell structure and cell surface properties between the strain-specific TA3-St and nonstrain-specific TA3-Ha ascites sublines of the TA3 murine mammary adenocarcinoma. The TA3-St subline is lethal only to the syngeneic strain A mouse (the strain of origin), whereas the TA3-Ha subline is lethal even to foreign species. In contrast to the TA3-St cell surface, which has numerous folds and irregular microprojections, the TA3-Ha cell has abundant long microvilli of uniform dimensions. An extensive cell surface coat which resembles the "fuzz" coat found on microvilli of normal epithelium was present on the TA3-Ha, but not on the TA3-St cells. After routine fixation, the surface coat of the TA3-Ha cell usually appeared as a filamentous network extending 30-50 nm from the plasmalemma; occasionally, longer filamentous or rod-like structures were found extending 200-400 nm from the plasmalemma. The cell coat material was more extensive on the microvilli than on the intermicrovillous membranes. Free virus-like particles associated with TA3-Ha cells have a similar-appearing surface coat on their outer membranes. The density of surface anionic sites, determined with polycationic ferritin, was greater on the TA3-Ha than on the TA3-St cell surface, consistent with the presence at the TA3-Ha cell surface of several-fold more neuraminidase-susceptible sialic acid groups. The observed surface features of the nonstrain-specific TA3-Ha cell, in comparison to the strain-specific TA3-St cell, are consistent with the suggestion that sialic acid-rich glycoproteins at the TA3-Ha cell surface mask histocompatibility antigens and enhance the ability of malignant cells to invade foreign species.


Mobility of ribosomes bound to microsomal membranes. A freeze-etch and thin-section electron microscope study of the structure and fluidity of the rough endoplasmic reticulum.

The lateral mobility of ribosomes bound to rough endoplasmic reticulum (RER) membranes was demonstrated under experimental conditions. High-salt-washed rough microsomes were treated with pancreatic ribonuclease (RNase) to cleave the mRNA of bound polyribosomes and allow the movement of individual bound ribosomesmfreeze-etch and thin-section electron microscopy demonstrated that, when rough microsomes were treated with RNase at 4 degrees C and then maintained at this temperature until fixation, the bound ribosomes retained their homogeneous distribution on the microsomal surface. However, when RNase-treated rough microsomes were brought to 24 degrees C, a temperature above the thermotropic phase transition of the microsomal phospholipids, bound ribosomes were no longer distributed homogeneously but, instead, formed large, tightly packed aggregates on the microsomal surface. Bound polyribosomes could also be aggregated by treating rough microsomes with antibodies raised against large ribosomal subunit proteins. In these experiments, extensive cross-linking of ribosomes from adjacent microsomes also occurred, and large ribosome-free membrane areas were produced. Sedimentation analysis in sucrose density gradients demonstrated that the RNase treatment did not release bound ribosomes from the membranes; however, the aggregated ribosomes remain capable of peptide bond synthesis and were released by puromycin. It is proposed that the formation of ribosomal aggregates on the microsomal surface results from the lateral displacement of ribosomes along with their attached binding sites, nascent polypeptide chains, and other associated membrane proteins; The inhibition of ribosome mobility after maintaining rough microsomes at 4 degrees C after RNase, or antibody, treatment suggests that the ribosome binding sites are integral membrane proteins and that their mobility is controlled by the fluidity of the RER membrane. Examination of the hydrophobic interior of microsomal membranes by the freeze-fracture technique revealed the presence of homogeneously distributed 105-A intramembrane particles in control rough microsomes. However, aggregation of ribosomes by RNase, or their removal by treatment with puromycin, led to a redistribution of the particles into large aggregates on the cytoplasmic fracture face, leaving large particle-free regions.


Ultrastructure of blebbing and phagocytosis of blebs by hyperplastic thyroid epithelial cells in vivo.

In addition to pseudopods, somewhat pleomorphic blebs were consistently found protruding from the apical surfaces of hyperplastic rat thyroid epithelial cells into the follicular lumens in vivo. Many blebs were knobby, roughly hemispherical protrusions, with a diameter of 2-3 mum. Such blebs had a densely packed microfilamentous core and contained numerous apparent ribosomes. They were morphologically similar to blebs that have been observed in a variety of cultured cells. Other blebs were larger, more elongate, and less knobby, but had a similar ultrastructural organization. Blebs of all sizes appeared to be phagocytosed on some occasions by nearby epithelial cells. The phagocytic process involved partial engulfment of the bleb by a typical epithelial pseudopod, followed by an apparent pinching-off process, presumably resulting in the separation of the bleb from its cells or origin. The pinching-off process was associated with a band of approx. 6-nm diameter microfilaments that developed within the pseudopod cytoplasm surrounding the base of the bleb and is postulated to function as a contractile ring. The finding of blebbing is an intact tissue in vivo indicates that this phenomenon is not restricted to cultured cells, and thus tends to extend the significance of in vitro observations of the process. In relation to their occurrence in the hyperplastic thyroid gland in vivo, possible interconversions are considered between different types of blebs, and between blebs and pseudopods.


Daughter 3T3 cells. Are they mirror images of each other?

Using a new technique to visualize the tracks of moving 3T3 cells and combining it with the visualization of actin-containing microfilament bundles by indirect immunofluorescence (Lazarides, E. and K. Weber. 1974, Proc. Natl. Acad. Sci. U.S.A. 71:2268-2272), I present experiments which suggest that: (a) 30-40%  of the pairs of daughter 3T3 mouse fibroblasts in noncloned cultures have mirror symmetrical actin-bundle patterns. (b) The angle between separating daughter cells is approx. 90 degrees or 180 degrees and seems related to the directions of certain actin-containing bundles. (c) Approximately 40% of separately moving daughter cells which did not collide with any other cell in the culture performed directional changes in a mirror symmetrical way. Both daughter cells entered the next mitosis at approximately the same time. I suggest that the actin-bundle pattern, the angle of separation, major directional changes during interphase, and the time of the next mitosis are predetermined by the parental cell.


Gap junctions. Structural changes after uncoupling procedures.

The freeze-fracture appearance of rat stomach and liver gap junctions changes after uncoupling procedures such as inhibition of the metabolism of perfusion with hypertonic sucrose. In control stomach, either fixed immediately or kept for 1 h in a well-oxygenated Tyrode's solution at 37 degrees C, most gap junctions between mucous cells contain particles irregularly packed at an average center-to-center spacing of 10.3-10.5 nm. After 1-h treatment with 2,4-dinitrophenol (DNP), at the same temperature and oxygenation, most particles aggregate hexagonally at an average spacing of approximately 8.5 nm. Similar changes are seen in hypoxic specimens. In control liver, fixed by perfusion, most junctional particles are irregularly packed at an average center-to-center spacing of approximately 10 mm. Small areas of fairly regular hexagonal packing are occasionally seen, where the average particle spacing is 9.2-9.5 nm. In hypoxic liver, the junctional particles form regular hexagonal packings in which the average center-to-center particle spacing is approximately 8.5 nm. In liver perfused with hypertonic sucrose-calcium solutions, following EDTA solutions, most junctions are pulled apart. The separated junctional membranes, expected to be highly impermeable, contain particles regularly and tightly packed as in hypoxic or DNP-treated junctions. Preliminary measurements indicate also a possible change in particle diameter, from approximately 8.6 nm (control) to approximately 7.7 nm (treated). The structural changes are similar to those previously reported in crayfish and may reflect conformational changes in particle subunits resulting in functional uncoupling.


Freeze-fracture studies of the developing cell surface. I. The plasmalemma of the corneal fibroblast.

The freeze-fracture technique was used to study changes in the corneal fibroblast cell membrane during morphogenesis in chick embryos. Fibroblasts migrate into the acellular primary corneal stroma on day 6 of embryogenesis, moving between the orthogonal layers of collagen fibrils which serve as their substratum. Morphometric analysis of the intramembrane particles (IMP) reveals their concentration on the P face to decrease from 756 to 534/mum2 from day 6 to day 14. After day 14, fibroblast migration and cell division cease and the stroma condenses due to dehydration, so that by day 18 all of the layers of fibroblasts are extremely flattened and the cornea has taken on its mature, transparent form. The cell membranes of the terminally differentiated, highly compacted fibroblasts are rich in IMP (1,300/MUM2, P face). In seeking to relate the particle increase to cell differentiation, we analyzed synthetic events taking place at this time, but no correlation, we analyzed synthetic events taking place at this time, but no correlation with 25SO4 or proline-3H incorporation was found. The event which seems best correlated with the doubling of P face particles between days 15 and 18 is the dehydration and condensation of the stroma, an event which is associated with cessation of both cell division and migration. Thyroxine stimulates premature condensation of the stroma, whereas thiouracil delays condensation, but neither of these treatments affects IMP concentration. Interestingly, IMP concentration on the filopodia of migrating fibroblasts is similar to that on the cell bodies, suggesting that the new membrane has the same composition as the pre-existing membrane. Observations are also presented on tight and gap junctions between fibroblasts and on the relation of extracellular matrix to the outer etched surface of the fibroblast plasmalemma.


Two fractions of rough endoplasmic reticulum from rat liver. I. Recovery of rapidly sedimenting endoplasmic reticulum in association with mitochondria.

Low-speed centrifugation (640 g) of rat liver homogenates, prepared with a standard ionic medium, yielded a pellet from which a rapidly sedimenting fraction of rough endoplasmic reticulum (RSER) was recovered free of nuclei. This fraction contained 20-25% of cellular RNA and approximately 30% of total glucose-6-phosphatase (ER marker) activity. A major portion of total cytochrome c oxidase (mitochondrial marker) activity was also recovered in this fraction, with the remainder sedimenting between 640 and 6,000 g. Evidence is provided which indicates that RSER may be intimately associated with mitochondria. Complete dissociation of ER from mitochondria in the RSER fraction required very harsh conditions. Sucrose density gradient centrifugation analysis revealed that 95% dissociation could be achieved when the RSER fraction was first resuspended in buffer containing 500 mM KCl and 20 mM EDTA, and subjected to shearing. Excluding KCl, EDTA, or shearing from the procedure resulted in incomplete separation. Both electron microscopy and marker enzyme analysis of mitochondria purified by this procedure indicated that some structural damage and leakage of proteins from matrix and intermembrane compartments had occurred. Nevertheless, when mitochondria from RSER and postnuclear 6,000-g pellet fractions were purified in this way fromanimals injected with [35S]methionine +/- cycloheximide, mitochondria from the postnuclear 6,000-g pellet were found to incorporate approximately two times more cytoplasmically synthesized radioactive protein per milligram mitochondrial protein (or per unit cytochrome c oxidase activity) than did mitochondria from the RSER fraction. Mitochondria-RSER associations, therefore, do not appear to facilitate enhanced incorporation of mitochondrial proteins which are newly synthesized in the cytoplasm.


Two fractions of rough endoplasmic reticulum from rat liver. II. Cytoplasmic messenger RNA's which code for albumin and mitochondrial proteins are distributed differently between the two fractions.

Subcellular fractions were obtained from rat liver homogenates under conditions which prevented degradation of polysomes (pH 8.5 and high ionic strength). Rough endoplasmic reticulum (RER) was recovered in high yields from a low-speed nuclear pellet (rapidly sedimenting endoplasmic reticulum, RSER) and from a postmitochondrial supernate (rough microsomes). The polysomal RNA content of these two fractions was very similar. When polyA+-RNA's were translated inthe mRNA-dependent wheat embryo cell-free system, both fractions yielded polypeptide products which had similar electrophoretic patterns on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Activities of messenger RNA's which code for albumin and for polypeptides destined for transport to the inner membrane and matrix of mitochondria (i.e. 'mitoplasts') were assayed by translating in the more active rabbit reticulocyte cell-free system followed by immunoprecipitation of radioactive products and coelectrophoresis with immunoprecipitated marker proteins on SDS-polyacrylamide gels. These tests indicated that albumin mRNA is about equally distributed between the two fractions of RER, or slightly enriched in the RSER fraction when activity is expressed as a percentage of total polypeptide synthesis. Activities of cytoplasmic mRNA's which code for at least some mitoplast proteins could be detected in both fractions, but all were enriched in the rough microsome fraction, not the RSER (two- to threefold when corrected for differences in total polypeptide synthesis in the lysate). Comparisons of mRNA's from free vs. membrane-bound polysomes indicated that most of the albumin mRNA activity (86-91%) and mitoplast protein mRNA activities (75%) were present in the bound fraction. Assuming that RSER and rough microsomes do not derive exclusively from different cells types, the evidence suggests that, compared to albumin and most other membrane-bound mRNA's, cytoplasmic mRNA's coding for mitoplast proteins may be preferentially segregated or compartmentalized within the cell on the microsomal class of RER.


Freeze-fracture of monolayer cultures.

This paper describes a simple method for the freeze-fracturing of cells in monolayers or multi-layer tissue cultures. The method produces high quality replicas and is applicable to the study of virtually any tissue culture or organ culture system. It uses standard materials and equipment for both tissue culture and freeze-fracturing.


Secretion of nerve growth factor by central nervous system glioma cells in culture.

Bacteriophage immunoassays, radioimmunoassays, and biological assays have been used to measure levels of NGF in media conditioned by rat C-6 glioma cells in culture. By all three criteria, these cells secrete a macromolecule which is indistinguishable from mouse submandibular gland NGF.


Rapid decline thymidine kinase activity during differentiation of Naegleria.

A logarithmically growing population of Naegleria gruberi differentiate into flagellates synchronously in about 85 to 90 minutes after the stimulation. Using this system, two separate enzymes that phosphorylate thymidine were assayed. Thymidine kinase activity decreases rapidly after the stimulation, while nucleoside phosphotransferase activity remains approximately constant. The possibility that this observed decrease in thymidine kinase activity is due to an expanded thymidine pool or to an increased phosphatase activity has been ruled out. Mixing experiments indicated that there is neither an activator of thymidine kinase in growing cells nor an inhibitor in differentiating cells. Thus, the observed decrease is likely to be due to an inactivation of the enzyme. Addition of actinomycin D or cycloheximide does not prevent the decrease in thymidine kinase activity suggesting that neither transcription nor translation is involved in the putative inactivation of thymidine kinase enzyme. There is very good correlation between the degree (or rate) of decrease in thymidine kinase activity and the percent of cells differentiating into flagellates. Thus, it appears certain that the residual thymidine kinase activity is primarily due to some portion of cells which did not respond to the stimulation and did not differentiate. Since there is an early termination of nuclear DNA synthesis in differentiating Naegeria, the decrease in thymidine activity (but no change in nucleoside phosphotransferase) suggests a correlation between thymidine kinase and DNA synthesis in Naegleria.


Messenger RNA regulation in humam diploid fibroblasts.

In resting, non-growing human diploid fibroblasts the amount of rRNA is reduced 1.8-fold, cytoplasmic polysomes are disaggregated, and the level of poly-A RNA (mRNA) is reduced 1.8-fold in relation to growing cells. The distribution of poly-A RNA is altered in resting, non-growing cells so that an average of 64% of the total cytoplasmic poly-A RNA sediments along with particles  lighter than 80S (prepolysomal) in sucrose density gradients. By camparison, in growing cells only 30% of the cytoplasmic poly-A RNA sediments in the prepolysomal region. In SDS sucrose gradients, the sedimentation profile of the prepolysomal poly-A RNA from resting cells resembles that of polysomal poly-A RNA from those cells. In contrast, the average size of prepolysomal poly-A RNA from growing cells is much smaller than that of the polysomal poly-A RNA from those cells. These data are compatible with the possibility that resting cell prepolysomal poly-A is untranslated mRNA. Also consistent with this interpretation are experiments which demonstrate that one-quarter to one-third of the prepolysomal poly-A RNA of resting cells is recruited into polysomes in the presence of of cyoloheximide.


The effects of erythropoietin in vitro on spleen colony-forming cells.

Erythropoietin (epo) added to liquid cultures of mouse bone marrow cells effected both the numbers of spleen colony-forming cells (CFU) in the cultures and the types of spleen colonies formed from these cells in irradiated hosts. Epo caused an increase in the number of CFU detected in cultures on the second day; this effect persisted through day 10, with the maximal increase occurring on the seventh day. The magnitude of the rise on day 7 was proportional to the amount of epo added. The increase in spleen colonies found with cells cultured in the presence of epo was due solely to erythroid colonies. After seven days in culture without epo, there was a peak of cells that formed non-erythroid colonies. This peak did not appear when the cells were cultured in the presence of epo.


Quantitation of mitochondrial DNA, RNA, and protein in starved and starved-refed rat liver.

The purpose of this study was to investigate the problem of mitochondrial biogenesis in rat liver. The approach consisted of isolating mitochondria from control, 6 day starved and 6 day starved-5 day refed rats and comparing their DNA, RNA and protein content. This was performed by isolating the mitochondria by reorienting rate zonal centrifugation in sucrose gradients. It was found that six days of starvation resulted in a loss of 30% of the body weight, 55% of the liver weight, 40% of the mitochondrial protein, 60% of the mitochondrial RNA, but only 20% of the mitrochondrial DNA. It was also shown that refeeding of the rats for five days resulted in a restoration to normal or near normal levels in all the parameters measured. Further experiments employing the incorporation of 3H-TTP into into isolated mitochondria indicated that the maintenance of mitochondrial DNA was not the result of continuous DNA sythesis.


Accumulation of polyploid cells and G2-phase cells during ascites tumor growth.

Ehrlich ascites tumor cells from the plateau phase of growth were transplanted into new hosts, pulse-labeled with tritiated thymidine and blocked with repeated injections of vinblastine. When unlabeled cells were analyzed for their cellular DNA content utilizing a cytophotometric technique it was found that in relation to the total number of cells (labeled plus unlabeled), 13% had a 2C DNA content, 36% a 4C DNA content and 5% and 8C DNA content at 0.5 hours after tranplantation. By 24 hours the distributions changed dramatically: the initally unlabeled 2C cells were now 4C, the 36% of the cells that were initially 4C partitioned into 24% that were still 4C and 12% that progressed to 8C, and the initial 8C cells remained 8C. These studies indicate that the accumulation of 4C cells during the plateau phase of growth is due to a combination of G2 diploid and G1 tetraploid cells.


Gas-liquid chromatographic studies of reactions and structural relationships of steroids. V. Concurrent substitution in the pregnane side-chain and position II.

Qualitative and quantitative effects of classical reactions on steroids observed by gas-liquid chromatography (GLC) under standardized conditions, including the double internal standard technique, are reported. Simple procedures applicable to nanogram amounts of reactants are described. Reactions studied include the conversion of keto groups to hydroxyl groups by NaBH4, and the removal of the pregnane side-chain with NaBiO3. GLC chromatograms readily provide information on effects on functional groups at positions 3, 11, 17, 20, and 21 and the retention times of many steroids unavailable from commercial sources. GLC data analysis provides relationships between steroid structure and retention time from which methods for the computation of retention times and steroid identification are designed. The accuracy of these methods is demonstrated.


Liquid chromatography of nucleic acid components and their analogues on hydroxyethyl methacrylate gels.

Spheron hydroxyethyl methacrylate gels are advantageous sorbents for the high-performance liquid chromatography of various nucleic acid components and their analogues. In aqueous media these compounds are reversible sorbed on the surface of the gel. Differences in the sorption characteristics of particular derivatives enable good separations of even relatively complicated mixtures to be achieved.


Gas chromatography of methyl glycosides as their tri-methylsilyl ethers. The methanolysis and re-N-acetylation steps.

Gas chromatographic procedures for the analysis of carbohydrates in glyco-proteins frequently include methanolysis and re-N-acetylation steps. The usual methanolysis conditions were found to result in almost complete conversion of the methanolic HCl into methyl chloride. A neutral solution of methyl chloride in methanol was found capable of releasing fucose, sialic acid, and a portion of the galactose from alpha 1-acid glycoprotein. During the re-N-acetylation of the amino sugars O-acetylation of the alditols commonly employed as internal standards occurs. Addition of the standards after the re-N-acetylation step avoided the problem. The complete procedure for the determination of both neutral and amino sugars has been simplified to involve only one transfer step prior to final gas chromatography.


Gas chromatographic separation of diastereoisomeric amino acid derivatives on glass capillaries. THE USE OF PENTAFLUOROPROPIONYL-AMINO ACID (+)-3-METHYL-2-BUTYL ESTERS.

The separation of diastereoisomeric pentafluoropropionyl-D, L-amino acid (+)-3-methyl-2-butyl esters, including those derived from histidine, arginine and tryptophan, is achieved on glass capillaries. Separation factors of between 1.03 and 1.09 are observed for the derivatives of protein amino acids. The gas chromatographic separation of a mixture of common amino acids usually takes less than 1 h.


Determination of (+)-(3R), (4S)-3-[(4methoxyphenoxy)methyl]-1-methyl-4-phenylpiperidine hydrochloride (FG 4963) in biological fluids using competition for adsorption to glass.

A procedure has been developed for the gas chromatographic determination of a structurally new 5HT-uptake inhibitor with antidepressant properties (FG 4963) in plasma and urine. The method involves an extraction from alkaline solution with n-pentane, and a quantitative determination by gas chromatography using an internal standard and a nitrogen-sensitive detector. The binding of FG 4963 to glass during storage and evaporation necessitates the addition of a structurally similar compound to the vials used for collection of the blood and urine, in order to compete for the binding sites of the glass. The method is suitable for pharmacokinetic and clinical studies, the sensitivity being ca. 5 ng/ml in plasma and 1 ng/ml in urine and the precision being 10-15% for concentrations greater than 10 ng/ml.


Determination of papaverine in blood samples by gas-liquid chromatography and mass fragmentography.

The measurement of papaverine in blood samples by using either a glass capillary column with a flame-ionization detector or a packed column with mass fragmentographic detection is described. The two methods permit the determination of the normal range of concentrations of papaverine in blood (2-500 ng/ml). Owing to its high specificity, mass fragmentography is greatly superior to capillary chromatography, which is sometimes subject to interferences by solvent impurities.


Determination of debrisoquine and its 4-hydroxy metabolite in biological fluids by gas chromatography with flame-ionization and nitrogen-selective detection.

Specific methods have been devised for the simultaneous determination of the guanidino-type antihypertensive agent debrisoquine and its 4-hydroxy metabolite in human urine, involving in situ derivatisation with acetylacetone, extraction of the resulting pyrimidines, and gas chromatography using a flame-ionisation detector or a nitrogen-specific detector. Using the latter, a similar procedure was also developed to measure debrisoquine in human plasma and whole blood following prior extraction at pH 13.5 with chloroform. Concentrations down to 3 ng/ml can be measured accurately using a 1.0-ml sample. The methods were applied to the analysis of samples collected after a single 20-mg oral dose of debrisoquine hemisulphate.


A nitrosamide-specific detector for use with high-pressure liquid chromatography.

A highly specific automated method for identification of nitrosamides has been developed for use in conjuction with reversed-phase high-pressure liquid chromatography. The method is based on the well-known Griess reagent, and is capable of sensitivities of 0.5 nmoles of injected nitrosamide in most cases. The method may be used for analysis of nitrite, nitrosamides, nitrosocarbamates, and alkylnitrites.


Rapid method for the simultaneous determination of 1,4-dioxan and its major metabolite, beta-hydroxyethoxyacetic acid, concentrations in plasma and urine.

1,4-Dioxan and its principle metabolite, beta-hydroxyethoxyacetic acid (HEAA), are determined by gas chromatography-mass spectrometry (GC-MS) on a 3% OV-17 column using selected ion monitoring, following the methylation of HEAA directly in plasma or urine without extraction. The recoveries of dioxan from plasma and urine are 98 and 94%, respectively, and the recoveries of HEAA from plasma and urine are 86 and 94%, respectively. The detection limits of 1,4-dioxan in plasma and urine are 0.07 ppm, and the detection limits of HEAA in plasma and urine are 0.5 and 0.1 ppm, respectively. Separate simultaneous measurements of 1,4-dioxan and HEAA methyl ester concentrations in urine and plasma are obtained after the methylation via GC-MS without additional preparation of the samples.