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This paper describes the fragments obtained by digestion with RNAase T1 from rat liver serine-tRNA1 as well as from a mixture of very lipophilic serine-tRNAs. From the data obtained by these digest as well as from the digestion with pancreatic RNAase the nucleotide sequence of the anticodon of serine-tRNA1 can be constructed. Evidence from different nucleotide sequences in other serine-tRNAs is presented [ABSTRACT FROM AUTHOR]

1. Tryptic digest, s of the [14C]carboxymethylated derivatives of the t, hree isoenzymes EE, ES and SS of horse liver alcohol dehydrogenase have been compared in "fingerprint" experiments. 2. Eight peptide spots present in the digest of the carboxymethylated EE enzyme were not detected in the digest of the carboxymethylated SS enzyme; and seven spots found in the latter were not discovered in the former. No other differences were noticed. The ES derivative yielded both types of spots but in reduced amounts, It is concluded that the E- and S-types of subunits are very similar and that the ES isoenzyme is a hybrid molecule. 3. From the carboxymethylated SS and ES isoenzymes the S-chain peptides that, differ from their counterparts in the E-chain were prepared and their structures analysed. They were compared to the known structures [ 1 — 3] of the corresponding E-chain peptides. It is concluded that all the differences between the two sets of peptides are accounted for by amino acid changes at only six positions along the protein chains of the E- and S-types of subunits, and an ancestral geneduplication is suggested. The differences at five positions (17, 94, 101, 110 and 366) are amino acid exchanges compatible with one-base mutations, while the nature of the sixth difference (position 115) is not fully established. 4. The six differences make the S-chain more hydrophobic and three units of charge more positive than the E-chain. These properties fit the solubilities and electrophoretic mobilities of the three isoenzymes. The difference in substrate specificity between the E- and S-chains might be explained by a direct participation in the substrate binding site of some of the residues exchanged, but, other explanations cannot be excluded. [ABSTRACT FROM AUTHOR]

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

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

Debate continues over the physical characteristics and even the existence of arginase isoenzymes. This paper gives additional support for such multiplicity and reports differences in physical characteristics among the various forms. After 2500-5000-fold purification of rat liver arginase, three molecular forms were separated on carboxymethyl-cellulose columns and were purified 2500-5000-fold, 800-1000-fold and 600-1000-fold, respectively. The molecular forms have also been identified by chromatography in the supernatant of tissue extracts. The isolation of these molecular forms by affinity chromatography, using Sepharose-lysine as a competitive inhibitor of arginase, shows only one main form, however. Kinetic studies were done for two of the molecular forms isolated, specifically the activation energy (Ea, the energy of denaturatization (Ed), Km, pH and the effect of divalent cations were determined. Significant differences were found for the Ea between the two molecular forms. The isolated isoenzymes are cationic at pH 5.5 and pH 8.8. However, they show different mobilities in electrophoresis. The molecular weight determination by gel filtration yields a value of 110000 to 115000 for both forms. The use of thin-layer immunochromatography plates, a combination of molecular weight and immunodiffusion technique, gave only one peak with the same molecular weight as that determined by gel filtration. The immunological studies showed that the isoenzymes have similar antigenic determinants. [ABSTRACT FROM AUTHOR]

The object of this paper is to analyse the effects of the coenzymes NAD(P)+ and NAD(P)H, and the effectors ADP and GTP, on the polyhexameric structure of pig-liver dehydrogenase. The linear polymerisation model proposed by Eisenberg for the native quaternary structure of this protein is valid with any effector; the observed variations of the degree of polymerization are explained by the modification of the apparent association constant of the hexamers. The appendices I and II define the free and associated areas and give, the theoretical foundations of the variation of the association constant of hexamers in terms of the binding of the ligands to the protemers. The increase in the degree of polymerization of the glutamate dehydrogenase with the binding of NAD(P)H is explained by a higher affinity of the coenzymes for the protomers which have an associated area compared to the protomers which have a free area. No variation is observed with NAD(P)+, ADP, or GTP alone. The formation of the protein · GTP · NAD(P)H ternary complex leads to a complete depolymerization when the two ligands are in saturating concentrations. The systematic study of the variations of polymerization in terms of increasing concentration of NAD(P)H at constant concentration of GTP, or in terms of increasing concentration of GTP at constant concentration of NAD(P)H shows that the interaction between the two opposite protomers of two consecutive hexamers is responsible for the sigmoidal shape of the depolymerization curves. The reversibility of this effect by ADP is assigned to a competition between the binding of ADP and the binding of GTP. [ABSTRACT FROM AUTHOR]

An enzyme, exhibiting NAIDPH-dependent oxidoreductase activity towards alicyclic ketones has been extracted from pig liver and purified 122-fold with respect to the protein contained in the crude extract after centrifugation at 54000×g. General properties, ultraviolet spectrum, stability, kinetic constants (V and Km) for NADPH and for typical substrates are reported. The molecular weight of the enzyme was estimated at 500000 by gel-filtration. The enzyme is HS(HB)-specific with respect to coenzyme. Alicyclic ketones can be conveniently used to measure the activity at all stages of purification. The topography of the active site responsible for oxidoredutase activity has been investigated by use of rigid alicyclic ketones such as trans-decal-1-ones as probes. In the accompanying paper it is shown (1) that the biological function of the whole enzyme complex is that of a fatty acid synthetase and (2) that the oxidoreductase activity can be ascribed to its 3-oxoacyl-acyl-carrier protein reductase component. [ABSTRACT FROM AUTHOR]

This paper deals with investigations in isolated perfused rat livers on tryptophan-oxygenase a under various experimental conditions. Enzyme-activity Showed a linear rise with amounts of tryptophan (0; 125 and 250 mg of tryptophan/kg) in the perfusate. Adrenalectomized and sham-operated animals have been compared and activity in the adrenalectomized rats were significantly lower in all cases. A significant decrease in the substrate induced increase of tryptophan-oxygenase-activity occured 12 h after adrenaleetomy. Substrate-concentration was 250 mg/kg in these experiments. The influence of substrate and cortisol on tryptophan-oxygenase-activity has been investigated 7 days after operation, Combined application of both substrate and steroid resulted in no difference to sham-operated animals when compared to substrate only in the same concentration. On the other hand, in livers from adrenlectomized rats values were significantly higher with combined application than in livers of adrenalectomized animals which received substrate only. The substrate-dependent rise in trsyptophan-oxygenase-activity could not be influenced by actinomycin D in contrast to to he cortisol effect which was significantly inhibited under these conditions. Both "inducers" were inhibited by cycloheximid. These inhibition-experiments suggest and confirm two different mechanisms of the substrate-activation and steroid-induction of tryptophan-oxygenase. [ABSTRACT FROM AUTHOR]

This paper reports the partial purification and characterization of a new type of arginase from chicken liver. The enzyme can be demonstrated only in less than 10% of a fasting chicken population, and has never been found in fed animals. The new arginase differs with respect to several properties (chromatographic behaviour, sedimentation coefficient, Km for arginne) from the arginase normally found in chicken liver and is more like the ureotelic arginase isolated from rat liver. [ABSTRACT FROM AUTHOR]

1. Spectral changes following the addition of hydroperoxides to isolated hepatocytes and to perfused rat liver were observed. Cytochrome b5 is the major, if not the only, hemoprotein exhibiting redox changes under these conditions: cytochrome b5 is oxidized by added hydroperoxides, e.g. tert-butyl or cumene hydroperoxides. No spectral changes attributable to cytochrome b5 were obsewed with tert-butanol. 2. The effect is present also when the mitochondrial respiratory chain is inhibited by antimycin A, and it is not observable with isolated mitochondria. On the other hand, the oxidation of cytochrome b5 by hydroperoxides is readily demonstrable in microsomal fractions in presence of NADH. 3. Spectral evidence for a participation of the other microsomal hemoprotein, cytochrome P-450, in the hydroperoxide-linked effects was not obtained. Thus, in hepatocytes from phenobarbitalpretreated rats, no formation of cytochrome P-420, no displacement of a type I substrate, hexobarbital, and no major steady state redox change of cytochrome P-450 was detectable. However, when cytochrome P-450 was dithionite-reduced, an oxidation of this cytochrome occurred upon subsequent hydroperoxide addition. 4. Hydrogen peroxide addition to hepatocytes also leads to a lower steady-state degree of reduction of cytochrome b5. Evidence is provided with hepatocytes from rats pretreated with 3-amino1,2,4-triazole that H2O2 generated intracellularly, e.g. from added glycolate, also causes a detectable oxidation of cytochrome b5. 5. The mechanism of these hydroperoxide effects remains to be established, and it is not clear whether cytochrome b5 reacts directly or indirectly. However, it is suggested that these effects may be of significance for the further study of cytochrome-b5-linked metabolic pathways. [ABSTRACT FROM AUTHOR]

Six types of nuclease activities were found to be concentrated in the large granule fraction isolated from rat liver homogenates by differential centrifugation. Analysis by density equilibration shows that three nucleases are associated with mitochondria: an alkaline ribonuclease (pH optimum 8.8), an alkaline deoxyribonuclease (pH optimum 7.6) and an enzyme acting on polyriboadenylate (pH optimum 7.5). When the outer mitochondrial membrane is ruptured in hypotonic medium, the three mitochondrial nucleases are partially solubilized. Solubilization is however obtained by addition of KCl to the suspension medium. It is concluded that mitochondrial nucleases are localized in the intermembrane space but that an adsorption to the outer face of the inner mitochondrial membrane occurs in sucrose 0.25 M. The mitochondrial localization of alkaline ribonuclease, alkaline deoxyribonuclease and polyadenylate accounts for at least 80% of the activity of liver homogenate; nevertheless, an excess of these enzymes is present in the microsomal fraction. Although no definitive conclusion can be reached for the significance of this observation, it is shown by density equilibrium analysis that these nucleases are not associated either with ribosomes or with the membranes which are the major component of the microsomal fraction. [ABSTRACT FROM AUTHOR]

Smooth and rough endoplasmic reticulum and free ribosomes from rat liver each show cyclicAMP-stimulated protein kinase activity utilizing exogenous substrates. The protein kinases of smooth and rough endoplasmic reticulum can be divided into two classes, one which is extracted by ionic media, and one more firmly attached enzyme fraction which is solubilized by Triton X-100. The protein kinase of free ribosomes is extracted by ionic media. All the three microsomal fractions support an endogenous phosphorylation of proteins which is only slightly stimulated by cyclic AMP. The endogenous phosphorylation shows two pH optima at pH 6.5 and 8.5. The phosphate is incorporated into seryl and threonyl residues of several protein species. Two major phosphoproteins are present in both smooth and rough endoplasmic reticulum, while a third major phosphoprotein is present only in the smooth fraction. There are also several minor phosphoproteins in the two fractions. The endogenous phosphorylation is initially rapid, especially in smooth and rough endoplasmic reticulum where it reaches a maximum after 10—15 rain incubation. The endogenously phosphorylated microsomal fractions also support an endogenous dephosphorylation, which is rapid initially, but which leaves approximately 60% of the phosphoryl groups unhydrolyzed. Like the protein kinase the proteins of smooth and rough endoplasmic reticulum which can undergo endogenous phosphorylation can be divided into two classes, one which is extracted in ionic media and one more tightly bound which is solubilized by Triton X-100. Protein kinase, cyclic-AMP-binding material and protein substrates extracted from smooth and ruogh endoplasmic reticulum by salt or detergent all show recoveries substantially exceeding 100%, suggesting that these activities are partly masked while associated with membrane material. [ABSTRACT FROM AUTHOR]

The rates of synthesis and degradation of glucose-6-phosphate dehydrogenase in the liver of male C57BL mice are followed by a modified immunochemical method. Mice are given labeled amino acids for a predetermined period, and the labeled enzyme is isolated from liver homogenates with goat anti-enzyme, followed by rabbit anti-goat IgG serum. The precipitates are either counted directly or analyzed on acrylamide gels. The rate of glucose-6-phosphate dehydrogenase synthesis is found to be independent of the state of induction or repression of the animal. The synthesis of the enzyme constitutes 0.20-0.25% of the synthesis of all soluble liver proteins. This rate is maintained even during periods of most rapid formation or disappearance of enzyme activity. Quantitative immunoprecipitation shows that similar amounts of precipitable antigen are present in induced and non-induced liver homogenates. The results indicate that the large reversible increase in activity of glucose-6-phosphate dehydrogenase, observed when animals are transferred from a high-fat to a fatless diet, does not involve the synthesis of a new enzyme protein, and that mechanisms involving modulation of existing enzyme molecules ought to be considered for the adaptation of this enzyme in mouse liver.

A simple optical method for the assay of Δ4-3β-hydroxysteroid dehydrogenase activity in cell-free preparations of rate liver was designed. This test is based on the fact that under the chosen conditions no reduction of the Δ4-bond occurs. Thus the Δ4-3β-hydroxysteroids dehydrogenase activity can be directly measured by the production of Δ4-3-ketosteroids, the sum of which in turn may be quantitated by their isonicotinic acid hydrazone formation. Δ4-3β-Hydroxysteroid dehydrogenases are localized in both cytoplasmic and microsomal fractions, exhibiting higher activity with NAD than with NADP. Microsomal enzyme activities show a significant sex difference which is more pronounced with NADP as coenzyme than with NAD (male:female activity ratios 5.8 with NADP, 1.9 with NAD). No sex differences were observed in the cytoplasmic enzyme activity. These results indicate the existence of two NAD-dependent enzyme activities of which the microsomal activity shows sexual differences. On the basis of the male:female activity ratios a further separate NADP-dependent microsomal enzyme activity can also be distinguished. [ABSTRACT FROM AUTHOR]

This paper describes the fragments obtained by digestion with RNAase T1 from rat liver serine-tRNA1 as well as from a mixture of very lipophilic serine-tRNAs. From the data obtained by these digest as well as from the digestion with pancreatic RNAase the nucleotide sequence of the anticodon of serine-tRNA1 can be constructed. Evidence from different nucleotide sequences in other serine-tRNAs is presented [ABSTRACT FROM AUTHOR]

Beef liver 5-aminolevulinate dehydratase was purified 700-fold by salt fractionation, heat treatment, gel filtration and ion-exchange chromatography. The final product was homogeneous by electrophoretic, ultracentrifugal and immunological criteria and had a molecular weight, by the low-speed equilibrium method, of 260000. Treatment with sodium dodecylsutfate and 2-mercaptoethanol dissociated the enzyme into dimeric subunits which could be further dissociated into monomeric subunits by treatment with 7.3 M guanidine hydrochloride and dithioerythreitol. The molecular weight of the monomeric subunit and the results of amino acid analysis suggested the presence of 14 subunits per mole. Carboxymethylation and hydrolysis showed the presence of 56 cysteine residues per mole, approximately half of which were available for titration with p-ehloromereuribenzoate in the urea-denatured enzyme. The enzyme has a pH optimum of 6.3 and a Km of 0.46 mM in the crude state and 0.15 mM when pure. Thiol activation was required for catalytic activity and some evidence was obtained for a requirement for zinc as a co-factor. Heavy metals inhibited the enzyme in a complex manner. Zinc acted as a competitive inhibitor, lead acted as a non-competitive inhibitor, while cadmium acted in a unique manner, causing inhibition at low concentrations of subtrate and stimulation at high levels of substrate. [ABSTRACT FROM AUTHOR]

A method is described for the preparation of DNA polymerase purified about 800-fold from rat liver nuclei. The yield of enzyme is about 140-200 µg from 200 g liver. Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the enzyme in the final step, shows a main band corresponding to a polypeptide of molecular weight of 29 000 ± 3%. Sephadex G-100 column chromatography indicates the enzyme to have an apparent molecular weight of approximately 60 000 ± 2% at an ionic strength of 0.15, suggesting that the enzyme is a dimer as isolated. In 2 M NaCl, the apparent molecular weight is 42 000. The enzyme prefers double-stranded DNA templates but utilises most efficiently those activated by deoxyribonuclease I. It has the ability to carry out limited synthesis using only one deoxynucleoside-5'-triphosphate in the assay. The final preparation of DNA polymerase has nucleoside diphosphate kinase associated with it. [ABSTRACT FROM AUTHOR]

The activity of a highly purified nucleotidase from rat liver cytosol which splits certain 3'- anti 5'-nucleotides, Was studied in the presence of each of 36 (different nucleic acid nucleic acid constituents including14C, labeled and chemically modified nucleotides. It was found that the compounds either inhibited or had no effect, on dephosphorylation of thymidine 5'-phosphate, which was used as substrate for measuring 5'nucleotidase activity. On the other hand , the 3'-nucleotidase activity, which was measured with uridine 3'-phosphate as substrate, was stimulated 2.6 times by deoxy- guanosine and, furthermore, by dexyguanosine and, furthermore, by deoxyinosine, thymidine, deoxyuridine and inosine in decreasing order of effectiveness. Experiments with various phosphorylated derivatives of thymidine indicated that a 5'-phosphoryl group increase the stimulating effect of the nucleoside whereas a 3'-phosphoryl substitution reduces its ability to activate the enzyme. Di-and triphosphates were less stimulatory than the mono phosphate. The results are interpreted to indicate that the same catalytic site is responsible for the hydrolysis of the 3'-and 5'-nucleotides and that the enzyme possesses a regularly site, topographically different from the catalytic site, at which the deoxyribonucleic acid constituents act as stimulators of enzyme activity. [ABSTRACT FROM AUTHOR]

Tryptic peptide maps of human liver alcohol dehydrogenase show this protein to be homologous to the corresponding protein of the horse. A subunit molecular weight very close to 40000 is established for the human enzyme. Sequence analysis of about one quarter of the tryptic peptides of human alcohol dehydrogenase show identical residues to the horse enzyme at about 90% of the positions, which is considered fairly representative of the general resemblance between the two proteins. All amino acid exchanges found are compatible with one-base mutations in the genetic code. Two different types of subunits of human liver alcohol dehydrogenase are identified. They are essentially similar but differ at some positions, one of which is No 43 (valine in one subunit and alanine in the other). Still other subunit types may exist. The known occurrence of isoenzymes of human liver alcohol dehydrogenase may therefore be explained at least in part by subunits of different primary structures. The amino acid differences between the subunits of the human enzyme are not found at the same positions as those between the two types of subunit of the horse enzyme. The structural differences between subunits from the two species seem greater than between the subunits within either species. Isoenzyme differences may, therefore, have evolved independently in the two species. The suggestion that some of the amino acid exchanges between the horse subunits may be directly involved in the substrate binding is supported. The isoenzyme and species differences at position 43 is only three residues away from the reactive "active site" cysteine residue, The region around this important residue is thus not kept constant in liver alcohol dehydrogenase during evolution. [ABSTRACT FROM AUTHOR]

Mitochondrial ATPase from rat liver has been highly purified to a maximum specific activity of 110 in Tris-bicarbonate buffer at pH 8.0. Sedimentation velocity studies in the analytical ultracentrifuge show that the enzyme sediments as a single symmetrical peak with a sedimentation coefficient (sº20, w, w) of 12.9 S. Molecular weight determinations by both high-speed sedimentation equilibrium and polyacrylamide gel chromatography indicate a molecular weight of 360 000 ± 10 000. Polyacrylamide gel electrophoresis of the enzyme dissociated by sodium dodecyl sulfate reveals one major and three minor bands with molecular weights of 53 000, 28 000, 12 500, and 8000-9000. A calculated molecular weight of approximately 365 000 is obtained by assuming the presence of six subunits of molecular weight 53 000 and one of each of the lighter subunits. Like the beef-heart enzyme, rat-liver ATPase activity is lost on incubation at 5 °C and examination by high-speed sedimentation equilibrium at 5 °C indicates that the molecule is extensively dissociated to components of low molecular weight. The enzymatic activities of both rat-liver-mitochondrial and beef-heart-mitochondrial ATPases are sensitive to the anion of the buffer used at pH 8.0 with activity decreasing in the following series: HCO3- > maleate > chloride > acetate = sulfate. Sulfite, chromate and dinitrophenol stimulate the activity of either enzyme in Tris-sulfate buffer, but only sulfate increases the activity seen in Tris-bicarbonate buffer. Aurovertin decreases the enzymatic activity to approximately the same level, regardless of the anion(s) present. The enzyme is insensitive to oligomycin. [ABSTRACT FROM AUTHOR]

Rat liver serine-tRNA1 was partially cleaved with pancreatic RNAase and RNAase T1 or by digestion of a chemically modified tRNA with pancreatic RNAase. Fragments up to half molecules cleaved at the anticodon site were obtained. From the various fragments the structure of rat liver serine-tRNA1 has been constructed. [ABSTRACT FROM AUTHOR]

The action patterns of liver and muscle glycogen synthetases and of muscle phosphorylase b on glycogen samples of different molecular weight and on β-amylase limit dextrins were studied. For this purpose a method for measuring the number of newly added glucose residues that are at non-reducing ends was developed. It was found that glucose transfer to the non-reducing ends of glycogen catalyzed by liver glycogen synthetase and muscle phosphorylase followed a Poisson distribution. The number of outer chains in the glycogen molecules available to both enzymes appeared to be smaller than the actual number of outer chains in the polysaccharide. The number of such available chains diminished as the glycogen was heavier. For the same glycogen sample, the number of available chains to phosphorylase appeared to be equal or smaller than that, to glycogen synthetase. It was found that muscle phosphorylase transferred 1.2 to 1.4 glucose moieties successively per outer chain, independently of the molecular weight, of the glycogen. The number of glucose units added successively to the non-reducing ends of glycogen by liver glycogen synthetase increased from 1.7 to 6.8 with the molecular weight of the polysaccharide. Both phosphorylase and liver glycogen synthetase transferred more glucose units in a repetitive way to the same outer chain of the β-amylase limit dextrin than to the same outer chain of the undegraded glycogen. Muscle glycogen synthetase transferred a greater number of glucose units successively per outer chain of glycogen than the liver enzyme. [ABSTRACT FROM AUTHOR]

Within the first 30 sec after intravenous injection of [32P]Pi the P-flux was measured the mononucleotide pool of the normal mouse liver. It is only in this short time interval that the dynamic phase of tracer kinetics is met, permitting proper calculations of flux rates of the mononucleotides with high turnover. The simplified model applied for the calculation of the stated flux rates describes fairly adequately the main pathways within the complex network of possible mononucleotide interconversions. The P-flux from the β-P of ADP to the β-P of ATP, catalyzed preferentially by oxidative and substrate phosphorylation amounts to 33 µmoles per g fresh liver per min. 12, 0.7, 0.3 and 0.01 µmoles γ-P of ATP are transferred per g per min to the β-P of ADP, UDP, GDP and CDP, respectively, via the group nucleoside monophosphate kinases. These flux rates are 1-2 orders of magnitude less than expected from the kinetics behaviour of the involved enzymes in vitro. The reverse reaction of the nucleoside monophosphate kinases is probably small. 9, 4 and 0.5 µmoles of the γ-P of ATP are transferred per g per min to the γ-P of GTP, UTP and CTP via nucleoside diphosphate kinase. [ABSTRACT FROM AUTHOR]

Metabolism of phospholipids in post mortem tissues of liver, kidney, spleen, and heart of guinea pigs was studied. The per cent composition of the individual groups of phospholipids in the above tissues in the fresh state and after a certain period of the dying process taking place under the described conditions is shown. Generally, one can state that phosphatidyl choline is the most important component of phospholipids in these tissues, in liver and in heart representing about 70% of the total amount. They are metabolized relatively faster, while sphingomyelin and phosphatidyl serine are metabolized most slowly. On the basis of the lipid phosphorus determination it is possible to conclude that within 24 h about 20-30% of the phospholipids are decomposed. During the following phase of the dying process no further pronounced decrease of phospholipids takes place. After this period the enzyme system splitting phospholipids is practically inactive. During the dying process an apparent increase of inorganic phosphorus can be observed, which indicates the decomposition of other phosphorus compounds in the course of this process. The relative representation of the individual groups of phospholipids in liver mitochondria is different from that of the whole tissue. Liver mitochondria contain a relatively higher amount of phosphatidyl ethanolamine, a lesser amount of phosphatidyl choline, lysophosphatidyl choline being totally absent. [ABSTRACT FROM AUTHOR]

A rapid de novo synthesis of mitochondrial phospholipids can be shown following the incubation of different radioactively labeled precursors ([14C]choline, L-α-[14C]glycerophosphate, [14C]serine and [14C]ethanolamine) with isolated rate liver mitochondria. The data provide strong evidence that mitochondria are able to synthesize their own phospholipids in situ with little or no contribution from contaminating microsomes or bacteria. Some conditions for this incorporation are described which are relevant to the known pathways for phospholipid biosynthesis. [ABSTRACT FROM AUTHOR]

The isolated liver of a female pre-viable foetus was perfused with [4-14C]testosterone. 6β-Hydroxy-testosterone, 619-hydroxy-androstenedione, 2β-hydroxy-testosterone and androstenedione could be isolated from the perfusion medium. These compounds have been characterized by their gas-chromatographic behaviour on a SE-30 and XE-60 column, as free steroids, acetates and trimethylsilyl ethers. Furthermore 2β-hydroxy-testosterone as diacetate was crystallized to constant specific activity. [ABSTRACT FROM AUTHOR]

The ratio of the specific radioactivities of 3-hydroxybutyrate: citrate was determined in rat liver mitochondria which were incubated in the presence of [1-14C]palmitate, pyruvate, bicarbonate, ATP, phosphate and malonate. Without compartmentation this ratio would maximally be 2, however, under our conditions values of 2.5-3.7 were observed. In further experiments with mitochondria, the sensitivity of pyruvate carboxylase for acetyl-CoA produced from various precursors was tested. It was found that acetyl-CoA produced from L-acetylcarnitine or by oxidation from either pyruvate, octanoate or palmitylcarnitine but not from leucine led to a stimulation of pyruvate carboxylation. These results demonstrate a compartmentation of acetyl-CoA in liver mitochondria. The further finding that different mitochondrial fractions showed varying ratios of specific radioactivities of 3-hydroxybutyrate: citrate indicates that the observed compartmentation may be explained by the existence of different types of mitochondria with varying enzyme patterns and acetyl-CoA pools. [ABSTRACT FROM AUTHOR]

Dihydrofolate reductase from bovine liver has been purified 5000-fold employing conventional techniques and methotrexate/aminohexyl/Sepharose affinity chromatography. Electrophoresis of the isolated enzyme on polyacrylamide gels resulted in the separation of two enzymatically active protein components which were not interconvertible by treatment with dihydrofolate and/or the coenzyme. The two forms, present in a ratio of 20:1, were found by isoelectric focusing to have isoelectric points of 7.15 and 5.94. They had identical specific activities towards dihydrofoloate (26.1-27.0 U/mg) and folate (1.3 - 2.2 U/mg), and had identical molecular weights (23 5000) and amino acid compositions. Due to the small quantity of the acidic form and the similarity of the two forms, the amino-terminal sequence (19 residues) was determined on a mixture of carboxymethylated reductase. The single sulfhydryl group of the enzyme can be modified by several sulfhydryl reagents in the native enzyme without loss of activity. Modification of the same residue occurs in the denaturated state and partially inhibits renaturation to the fully active enzyme. One disulfide bridge was detected by reduction and alkylation. The cleavage of this bond did not effect the enzymatic activity. [ABSTRACT FROM AUTHOR]

Nucleoli isolated from rat liver were incubated for synthesis of RNA in vitro in the presence or absence of S-adenosyl[³H]methionine. The results obtained that neither the rate of RNA synthesis nor the processing of pre-ribosomal RNA was changed if methylation was allowed to take place. The methylation process acts on the RNA most recently synthesized, rather than on the bulk of the RNA already present in the nucleoli. The reaction seems to occur faithfully both quantitatively and qualitatively. It is calculated that 104 mol methyl groups were incorporated per mol of newly synthesized 45-S RNA. Methylation of the ribose rather than th bases predominated. The pattern of alkali-stable oligonucleotides of RNA methylated in vitro analyzed and found to correspond closely to that of ribosomal RNA labelled in vivo. [ABSTRACT FROM AUTHOR]

Poly(A) polymerase activities have been solubilized from rat liver nuclei and purified by chromatography on Bio-Gel A-1.5m, DEAE-Sephadex and CM-cellulose. Three distinct forms of nuclear poly(A) polymerase have been resolved by chromatography on CM-cellulose. According to their sequence of elution from CM-cellulose these enzyme activities have been termed A, B and C. Enzymes A and B are Mn2+-dependent, enzyme C requires Mg2+. With the same chromatographic step on CM-cellulose the Mn2+-dependent poly(A) polymerase activities were separated from a dependent enzyme system capable of synthesizing RNA a primed poly(U), poly(G) and poly(C). The effect of different nuclear and cytoplasmic RNA primers on the rate of poly(A) formation suggests enzyme A to be responsible for the elongation of preexisting poly(A) chains. The phosphorylated derivative of cordycepin, 3'-deoxyadenosine 5'-triphosphosphate (3'-dATP), which is known to inhibit nuclear poly(A) synthesis in vivo, also impairs poly(A) formation in vitro. It is shown that 3'-dATP very probably is not incorporated into poly(A) in vitro, suggesting that 3'-dATP primarily affects the catalytic activities of the poly(A) polymerase species rather than directly blocking chain elongation. [ABSTRACT FROM AUTHOR]

Horse liver alcohol dehydrogenase was reacted with glyoxal at different pH values ranging from 6.0 to 9.0. At pH 9.0 the enzyme undergoes a rapid activation over the first minutes of reaction, followed by a decline of activity, which reaches 10 % of that of the native enzyme. Chemical analysis of the inactivated enzyme after sodium borohydride reduction shows that 11 arginine and 11 lysine residues per mole are modified. At pH 7.7 the enzyme activity increases during the first hour of the reaction with glyoxal and then decreases slowly. Chemical analysis shows that 4 arginine and 3 lysine residues per mole are modified in the enzyme at the maximum of activation. At pH 7.0 the enzyme undergoes a 4-fold activation. Chemical analysis shows that in this activated enzyme 3 lysine and no arginine residues per mole have been modified. Steady-state kinetic analysis suggests that the activated enzyme is not subjected to substrate inhibition and that its Michaelis constant for ethanol is three times larger than that of the native enzyme. The possible role of arginine and lysine residues in the catalytic function of liver alcohol dehydrogenase is discussed. [ABSTRACT FROM AUTHOR]

Recent findings have confirmed the role of form A DNA-dependent RNA polymerase activity as that which is responsible for the transcription of the ribosomal RNA-coding genes. Unfortunately, the form A enzymes have proved to be very labile and difficult to work with, especially under high ionic strength conditions. We have, therefore, investigated a method fot the purification of the form Al and All enzymes from rat liver using mild low-ionic-strength conditions. Since preparations from whole nuclei were found to be grossly contaminated with protein having similar properties, the enzymes are extracted from nucleoli. Forms Al and Al! are separated on a phosphocellulose column, purified by further ion-exchange chromatography, and by sedimentation through a glycerol gradient. The purified enzymes each migrate as a single band on native polyacrylamide gels and have the expected characteristics of form A RNA polymerase. Sedimentation rates through glycerol gradients indicate that they both have a similar size to that of Escherichia coil RNA polymerase (Mr, about 500000). The purified enzymes are free of DNase and RNase. A method is also described for the purification of form B from the nucleoplasm remaining after isolation of nucleoli. The presence of form C activity was not detected. [ABSTRACT FROM AUTHOR]

1. The kinetics of the reaction catalysed by fructose bisphosphatase have been studied at pH 7.2 and at pH 9.5. The activity of the enzyme was shown to respond sigmoidally to increasing concentrations of free Mg2+ or Mn2+ ions at pH 7.2, whereas the dependence was hyperbolic at pH 9.5. At both pH values the enzyme responded hyperbolically to increasing concentrations of fructose 1,6-bisphosphate, although inhibition was observed at higher concentrations of this substrate. This high substrate inhibition was shown to be partial in nature and the enzyme was found to be more sensitive at pH 7.2 than at pH 9.5. 2. The kinetic results at pH 9.5, when taken together with other known properties of the enzyme, are consistent with the enzyme obeying either a random-order equilibrium mechanism or a compulsory-order steady-state mechanism in which fructose bisphosphate binds to the enzyme before the cation. 3. Reaction of the enzyme with a four-fold molar excess of p-chloromercuribenzoate caused activation of the enzyme when its activity was assayed in the presence of Mn2+ ions but inhibition when Mg2+ ions were used. Higher concentrations of p-chloromercuribenzoate caused inhibition. This activation at low p-chloromercuribenzoate concentrations, and the reaction of 5,5'-dithio-bis(2-nitrobenzoate) with the four thiol groups in the enzyme that reacted rapidly with this reagent, were prevented or slowed by the presence of inhibitory, but not non-inhibitory, concentrations of fructose bisphosphate. After reaction with a four-fold molar excess of p-chloromercuribenzoate the enzyme was no longer sensitive to high substrate inhibition by fructose bisphosphate. [ABSTRACT FROM AUTHOR]

1. The activity of beef liver fructose bisphosphatase has been shown to respond cooperatively to increasing concentrations of the activating cations Mg2+ and Mn2+. The allosteric inhibitor AMP caused an increase in this cooperativity and a decrease in the apparent affinity of the enzyme for the activating cation. 2. The cooperative response of the enzyme to AMP is similarly increased by increasing cation concentrations with a concomitant decrease in the apparent affinity. 3. Direct binding experiments indicated that in the absence of either Mg2+ or Mn2+ the enzyme bound AMP non-cooperatively up to a maximum of two molecules per molecule of enzyme, a result that is indicative of half-sites reactivity. The binding became increasingly cooperative as the concentration of the activating cation was increased. 4. The substrate fructose bisphosphate had no effect on any of these cooperative responses. 5. These results may be most simply interpreted in terms of a concerted model in which the activating cation functions both as an allosteric activator and as an essential cofactor for the reaction. [ABSTRACT FROM AUTHOR]

The halothane-dependent, calcium-induced loss of respiratory control in rat liver mitochondria [1,2] is Mg2+-dependent and is accompanied by an enhanced mitochondrial swelling. It is suggested that this swelling reflects an increase in calcium activity in the matrix space, due to a decrease in binding of the accumulated cation. This change in the partition of intramitochondrial calcium is correlated with an inhibition by halothane of energy-independent, calcium-induced swelling. The enhanced swelling associated with the active accumulation of calcium in the presence of halothane does not lead to a marked increase in permeability to other ions. Nevertheless, under conditions of energised calcium uptake, and in the presence of Mg2+, a halothane-dependent, ruthenium red-insensitive efflux of calcium is observed. This is consistent with the proposed halothane- dependent increase in the matrix activity of accumulated Ca2+. It is suggested that this mechanism accounts for the previously postulated [2] futile cycle of calcium uptake and release induced by halothane in rat liver mitochondria. [ABSTRACT FROM AUTHOR]

With a view to the study of the subcellular localization of nucleases, methods ensuring the specificity of enzyme determinations are presented for six types of nuclease activities in liver homogenates. The ribonuclease activity of rat liver is due to three enzymes with different pH optimum. For acid ribonuclease (pH optimum 5.3), it is possible to avoid interference from the other ribonucleases by performing the incubation at pH 5. Neutral ribonuclease (pH optimum 7.6) is differentiated by relying on its sensitivity to the natural inhibitor from the supernatant of liver homogenate. Comparison of activities before and after pretreatment at 50°C in acid medium permits the specific measurement of alkaline ribonuclease (pH optimum 8.8). The optimal conditions for the determination in liver homogenates of two deoxyribonucleases and of an enzyme acting on polyriboadenylate are also described. The activity of these various nucleases is compared and some of their properties are investigated. [ABSTRACT FROM AUTHOR]

The subunit structure of rat liver acetyl-coenzyme-A carboxylase has been studied by polycrylamide gel electrophoresis in the presence of dodecylsulfate. A number of individual preparations of the enzyme purified by the same procedures exhibited three different types of electrophoretic patterns as follows: first, a single slow-moving protein band (Mr 230 000); secondly two adjacent fast-moving protein bands (Mr 124 000 and 118 000); finally, all three protein bands. With the use of the [14C]biotin-labelled enzyme, the biotinyl prosthetic group was shown to be associated with the polypeptide of 230 000 Mr as well as with that of 124 000 Mr, but not with the polypeptide of 118 000 Mr. Studies were next made with the labelled enzyme to examine the possibility that the two light polypeptides might have been formed by proteolytic modification of the heavy polypeptide during the procedures used for the purification of the enzyme. Treatment of the enzyme with trypsin or chymotrypsin resulted in cleavage of the heavy polypeptide into two nonidentical polypeptides with molecular weights of approximately 120 000. Incubation of the enzyme with proteases derived from rat liver converted the heavy polypeptide into lighter polypeptides of 80 000-130 000 Mr. Acetyl-CoA carboxylase isolated from crude rat liver extracts by means of immunoprecipitation with specific antibody invariably showed only the heavy polypeptide. The biotin content of the enzyme was found to be 1 mil per 237 000 g protein. These results indicate that rat liver acytyl-CoA carboxylase, unlike bacterial and plant biotin enzymes, has only one kind of subunit, which has a molecular weight of 230 000 and contains one molecule of biotin. Thus, the mammalian enzyme exhibits a highly integrated subunit structure. [ABSTRACT FROM AUTHOR]

The matabolism of a mixture of [4-14C]- and [7&Beta-²H]testosterone by the hepatic microsomal fraction from adult female C57BL/6L mice has been investigated. The following metabolites were identified by their mass spectra and by their reteintion times on gas chromatography on one or two phases: 1ξ-, 2Β-, 6α-, 6Β-, 7α-, 15α-, 16α-, 16Β-hydroxytestosterone; 6α-, 6Β-, and 7α-hydroxy-4-androstene-3,17-dione; and 4-androstene-3,17-dione. A compound tentatively identified as 6- or 7-oxotestosterone was also isolated. 17Β-Hydroxy-4,6-androstadien-3-one, 17Β-hydroxy-1,4-androstadien-3-one and 4,6-androstadiene-3,17-dione were identified but are considered to arise non-enzymatically from 7α-hydroxytestosterone, 1ξ-hydroxytestosterone and 7α-hydroxy-4-androstene-3,17-dione, respectively. [ABSTRACT FROM AUTHOR]

Cytoplasmic and nuclear protein kinase activities from perfused rat liver have been studied in response to dibutyryl-adenosine cyclic 3′: 5′-monophosphate added at a concentration that stimulates hepatic gluconeogenesis (100 μM). Total nuclear protein kinase, as assayed using a mixed histone fraction as phosphate acceptor, is increased by 5-fold within 8 min of the addition of cyclic nucleotide to the perfusate. In contrast the total cytoplasmic protein kinase activity is decreased to 50% of the control value. The protein substrate specificity of the protein kinase that is present in the nucleus in response to dibutyryl-adenosine cyclic 3′:5′-monophosphate stimulation is similar to that of cytoplasmic, adenosine cyclic 3′:5′-monophosphate-dependent, protein kinase but is distinct from that of the enzyme(s) present in control nuclei. The predominant species of protein kinase from stimulated nuclei has a sedimentation constant of 3.9 S. This value is identical to that of the catalytic subunit of cytoplasmic adenosine 3′:5′-monophosphate-dependent protein kinase. These data suggest that some of the effects of adenosine 3′:5′-monophosphate on nuclear events may be mediated through its interaction with the inactive protein kinase holoenzyme in the cytoplasm and the subsequent redistribution of the active catalytic subunits generated by this interaction. [ABSTRACT FROM AUTHOR]

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

1. The activity of liver phosphorylase b from several mammalian species has been studied. The enzyme from rat or mouse has a higher activity than the rabbit enzyme, which is itself more active than pig liver phosphorylase b. 2. The activity of liver phosphorylase b is influenced by anions and by AMP, and these effects are influenced by pH. Fluoride, which is currently added to the assay mixture of phosphorylase a in crude preparations, is about as active as sulfate as a stimulator of phosphorylase b. 3. When assayed at pH 6.1 and in the presence of 0.15 M NaF, the activity of rat liver phosphorylase b reaches 25 % of that of the a enzyme; if I mM AMP is also present, this value rises to 50 %. 4. Methods are described that allow the determination of liver phosphorylase a without interference of b, and the determination of total phosphorylase (a + b) in rat liver. [ABSTRACT FROM AUTHOR]

The reduction, catalysed by liver alcohol dehydrogenase, of benzaldehyde in the presence and absence of pyrazole, and the oxidation of benzyl alcohol and cyclohexanol in the presence of isobutyramide, has been measured by the stopped-flow technique. In performing these experiments particular care was taken to purify the enzyme, coenzymes, substrates and inhibitors, and to minimise as much as possible the effects of a blank substrate reaction. The calculation of the amount of substrate converted to product during the various phases of the transient process was based on the absorption coefficients for the enzyme-coenzyme and enzyme-coenzyme-inhibitor complexes determined in the absence of substrate. The results show that the two active sites of liver alcohol dehydrogenase are kinetically equivalent and that the enzyme does not exhibit half-of-the-sites reactivity. [ABSTRACT FROM AUTHOR]

We have investigated the effect of the centrifugation speed on the behavior of rat-liver mitochondria during isopycnic centrifugation in an isoosmotic medium. The gradient was made with a macro-molecular compound, glycogen dissolved in 0.25 M aqueous sucrose. The distribution curves of several mitochondrial enzymes change when the centrifugation reaches a certain speed: they are shifted toward regions of lower density. The results are plausibly explained by supposing that the inner mitochondrial membrane becomes permeable to sucrose at high centrifugation speeds, and that the granules swell. The main causal agent of the phenomenon is the hydrostatic pressure the mitochondria are subjected to during centrifugation. Morphological observations show that mitochondria are markedly deteriorated when centrifuged at high speed in the glycogen gradient: they are swollen and the outer membrane is broken; also frequently, a large electron-dense granule is seen in the matrix near the inner membrane. [ABSTRACT FROM AUTHOR]

The distribution of oligonucleotides which are released from rat liver ribosomes by treatment with pancreatic ribonuclease has been studied. Rat liver monoribosomes lost from 15 to 17% of their nucleotides by treatment with pancreatic ribonuclease. This quantity was highly reproducible and did not depend significantly on the temperature (0–20 °C) and time (10–120 min) of incubation or on the concentration of enzyme (1:5000– 1 : 50). Whereas the amounts of oligonucleotides liberated was 16%, it was shown by column chromatography that they consisted of 71% mononucleotides, 16% dinucleotfdes, 6% trinucleotides, 4% tetranucleotides and 2% pentanucleotides and that these oligonucleotides were enriched in uridine, containing approximately half of the uridine residues present in the high-molecular-weight ribosomal RNA. The high molecular weight of the RNA from ribonuclease-treated ribosomes was preserved until it was heated; after heating, RNA fragments having sedimentation coefficients of 5 S and less were present. It is inferred that the oligonucleotides are derived from pyrimidine-rich clusters located in single-stranded ‘hairpin’ loops on the outside surface of the ribosome. [ABSTRACT FROM AUTHOR]

The binding of the diterpenoid drug 14-deoxy-14-[(2′-diethylamino-ethyl)-mercaptoacetoxy]-dihydromutilin hydrogen fumarate in the cell of rat liver is mainly to the microsomal fraction. Besides specific binding to cytochrome P-450, where the enzymic degradation of the drug occurs, we observed a very high number of identical sites (site A) with an affinity of approximately 4.2 × 10³ M-1 (25 °C, pH 7.4). Model investigations demonstrate that these interactions occur almost exclusively with the microsomal phospholipid moiety. Their capacity for the drug was determinated to be of the order of 0.2 mol/mol phospholipid. The specific interaction of the pleuromutilin derivative with cytochrome P-450 gives rise to different spectral changes of the protein. At low concentrations where weak cooperativity of the overall binding to microsomes (sites B) was found, the formation of a type I complex was observed. At increasing concentrations of the drug this interaction vanishes and a spectral change of a different type (modified type II) arises. The affinity for this complex is identical with that of the phospholipid binding sites. The interaction of the drug with the phospholipid moiety might give rise to dual effects, Firstly the very near neighbourhood of a multitude of relatively weak binding sites will facilitate a transport of the drug along the microsomal membranes. Secondly, the loading of the membranes with the drug at high concentrations might influence the binding to cytochrome P-450 so that a qualitatively different interaction takes place. [ABSTRACT FROM AUTHOR]

1. In order to demonstrate that triiodothyronine affects mitochondrial RNA synthesis by acting on the enzyme component of the DNA · RNA polymerase complex, mdtochondrial RNA polymerase from thyroidectomized and hormone-treated rats was purified up to a stage in which activity was dependent on the addition of exogenous template. In these conditions and using different DNAs as templates, the enzyme from hormone-treated animals displayed an activity about double that of the activity of thyroidectomized animals. 2. Measurements of stability of mitochondrial RNA synthesized in vitro suggest, however, that the hormone can act also at the template level in mitochondrial transcription: the RNA population synthesized in vitro from hormone-treated rats is indeed much more enriched in unstable, probably messenger, RNA species. 3. The turnover of mitochondrial messenger RNA is higher after hormone treatment. 4. Adenosine cyclic 3′: 5′-monophosphate (cAMP) and its dibutyryl derivative added in vitro to mitochondria from thyroidectomized animals do not affect the incorporation of labeled precursor into mitochondrial RNA, suggesting that the level of the cyclic nucleotide in mitochondria is probably not involved in the hormone action. 5. It is concluded from these and previous studies that the thyroid hormone affects more than one parameter in the mitochondrial transcription process. The interrelationship between these events at molecular level remains, however, to be clarified. [ABSTRACT FROM AUTHOR]

1.Alcohol dehydrogenase isoenzymes BB were isolated from human livers of both "normal" and "atypical" phenotypes [1]. Their pH-rate profiles, their behaviour on CM-cellulose and the number of hybrids formed with horse subunits A show that the "atypical" isoenzymes contain subunits B1 and B2, the "normal" one only subunits B1. These results indicate that most "atypical" individuals genetically represent heterozygotes. 2.A high degree of homology between horse subunit A human subunits B is found. However, the human isoenzymes have a molecular weight of 87000 and contain about 410 amino acid residues, 6 tyrosines and 3 tryptophans per subunits. 3.the sequence of one tryptic peptide from subunit B1 is Phe-Ala-Lys and is altered to Phe-Pro-Lys in the subunit B2. This substituted residue is located in a region which corresponds to the coenzyme-binding site of the horse enzyme. [ABSTRACT FROM AUTHOR]

1. The oxidation of benzyl alcohols by liver alcohol dehydrogenase under conditions [S]o » [E]o is a biphasic process with exponential rise to a steady state. The rate constants for the transient phase were determined by a curve-fitting procedure. 2. A large isotope effect was obtained for p-methoxybenzyl alcohol, indicating that hydrogen transfer is rate-determining for the transient phase. Rate constants for the hydrogen transfer step, k3, were obtained for the various alcohols and correlated with the Hammett σ constant by plotting log k3 against σ. The slope of this line (q = -0.76) is consistent with rate-limiting hydride transfer; a mechanism is proposed to account for the low magnitude of q. 3. The pre-steady-state rate constants, kb, for ali benzyl alcohols (<20 s -1) are lower than that for ethanol. 2-Methylpropan-l-ol and cyclohexylmethanol have pre-steady-state rate constants of about 150 s--1, indicating that the lower kb values for benzyl alcohols are due to the electronwithdrawing effect of the benzene ring, rather than to steric effects. [ABSTRACT FROM AUTHOR]

The association-dissociation equilibrium of beef-liver glutamate dehydrogenase was investigated in phosphate buffer of different ionic strengths and in the presence of the organic solvents ethylene glycol and dioxane. For some of the solvents the temperature dependence of the equilibrium constant was analyzed. The dependence of the apparent weight-average molecular weight on enzyme concentration shows always the same general shape as that found earlier in 0.067 M phosphate buffer at pH 7.6, Each curve can be fitted assuming an open association-dissociation equilibrium with one single equilibrium constant for all steps and with one single value for the second virial coefficient. Within the limit of the experimental error, the second virial coefficient is found to be the same for all systems (8-9 nmol × 1 × g-2), whereas the equilibrium constants vary by orders of magnitude. At low ionic strength a very small temperature dependence was found with a reduced association at higher temperatures. At high ionic strength a temperature maximum of the equilibrium constant is observed. Preferential phosphate binding is indicated by the high values of the refractive index increment. The organic solvents investigated here (ethylene glycol, dioxane) cause a dissociation into the unimers (oligomers). The equilibrium constant decreases from 9 × 105 M-1 in aqueous phosphate buffer to 5.2 × 10³ M-1 in 6 M ethylene glycol and to 2.4 × 105 M-1 in 2 M ethylene glycol. The effects obtained with 5% dioxane (0.57 M) are similar to that with 2 M ethylene glycol. In both cases a sharp decrease of the association with increasing temperature is observed. The reaction enthalpy and the reaction entropy of the association reaction decrease with decreasing temperature in all solvents, the enthalpy being positive in the absence and negative in the presence of organic solvents. The free enthalpy of reaction, however, increases with increasing temperature in buffers of high and low ionic strengths but decreases in the presence of organic solvents. The association reaction at high ionic strength appears mainly to be entropydriven whereas in the presence of the organic solvents the reaction seems to be energy-driven at all temperatures. It is suggested that the organic solvents interfere with the hydrophobic interactions between the unimers, The positive reaction enthalpy in the presence of high salt concentrations indicates that the attractive electrostatic forces are largely shielded, whereas shielding is imperfect for the repulsive forces which even seem to be enhanced. This could be due to a preferential binding of anions which would tend to increase the negative net charge and could explain the difference observed between the effects of phosphate and chloride solutions of identical ionic strength. [ABSTRACT FROM AUTHOR]

The effects of glucose and of AMP on several properties of purified dog liver phosphorylase a have been investigated. Glucose stimulated several-fold the conversion of phosphorylase a into b, catalyzed by purified phosphorylase phosphatase. The reaction was strongly inhibited by AMP in the absence of glucose, and much less in the presence of glucose. Conversely, the concentration of glucose that gave half-maximal stimulation was increased several-fold by the nucleotide. Similar effects were observed when the phosphorylase phosphatase was studied in crude liver preparations. 1,5-Anhydroglucitol, α-methylglucoside and 2-deoxyglucose, quoted in order of decreasing efficiency, also stimulated the phosphorylase phosphatase reaction. Similar results were obtained when the conversion of phosphorylase a into b′ by trypsin was studied. The effect of glucose on the kinetics of purified liver phosphorylase a was mostly to decrease affinity for glucose 1-phosphate and to increase cooperativity between glucose 1-phosphate binding sites. AMP had the opposite effect and each of the two ligands antagonized the binding of the other. Glucose derivatives, in the order of efficiency given above, mimicked the effects of glucose. Glucose and the same glucose analogues, as well as AMP, protected phosphorylase a against denaturation at 55 °C. These results indicate that the stimulation of the phosphorylase phosphatase reaction by glucose is secondary to the binding of the hexose to phosphorylase a, and that this binding is modulated by the concentration of AMP. The physiological implication of these findings is discussed. [ABSTRACT FROM AUTHOR]