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Presents forthcoming papers for the "European Journal of Biochemistry". "A guanyloribonuclease mouse liver cytosol," by A. Pantazaki and colleagues; "Revised nonmenclature for eukaryotic DNA polymerases," by P.M.J. Burgers and colleagues; "The folding and solution conformation of penicillin G acylase," by C.D. Lindsay and colleagues.

Lists papers due for publication in the "European Journal of Biochemistry" after the January 16, 1984 issue. "Labelling and cross-linking of Escherichia coli penicillin-binding proteins with bis-Beta-lactam antibiotics"; "Sorbitol dehydrogenase. The primary structure of the sheep-liver enzyme"; "Extensive variations and basic features in the alcohol dehydrogenase-sorbitol dehydrogenase family".

The article presents information on various papers to be published in the "European Journal of Biochemistry." Some of them are, "Lipid Domains in Plasma Membranes From Rat Liver," by F. Schroeder, "Calcium Modulates Fatty Acid Dynamics in Rat Liver Plasma Membranes," by F. Schroeder, and C.S. Argilaga, and others.

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]

The colon and lung amiloride-binding proteins were cloned from rat tissues. Two sizes of transcripts were identified. The 2.7-kb transcript codes for an 85-kDa protein, whereas the 1.2-kb transcript codes for a 25-kDa polypeptide. The 2.7-kb transcript was detected in the proximal and distal colon and in duodenum, liver, placenta and thymus. The 1.2-kb transcript was the only form present in lung and spleen, and it was also detected in placenta and colon. The short form corresponds to the 3′ terminus of the longer one. It is formed by alternative transcription under the control of an internal promoter. Cells stably transfected with cDNAs encoding these two proteins were used for binding studies using [3H]phenamil, a potent blocker of the epithelial Na+ channel, derived from amiloride. Both the long and short forms of the protein bind amiloride and some of its derivatives, but they have distinct pharmacologies. The order of potency of the different amiloride derivatives to inhibit [3H]phenamil binding was phenamil (K0.5 =10 nM) > benzamil (K0.5 = 43 nM) > amiloride (K0.5 = 1.4 μM) ≈ ethylisopropylamiloride (K0.5 = 1.6 μM) for the long form, whereas it was phenamil (K0.5 = 68 nM) > amiloride (K0.5 = 3.2 μM) ≈ ethylisopropylamiloride (K0.5 = 4 μM) ≈ benzamil (K0.5 = 6.3 μM) for the short form. Although the binding proteins described here are distinct from the pore-forming protein of the epithelial Na+ channel, the pharmacological profile of the long form of the ABP is identical to that described previously in pig and human kidney, and similar to that expected for an epithelial Na+ channel. The pharmacological profile of the short form resembles that previously described for an amiloride-binding protein in pneumocytes. Results presented in this paper suggest that previously purified preparations showing Na+ channel activity contain different forms of the amiloride-binding protein, possibly associated with other proteins. The similarity between amiloride-binding proteins and a protein identified in seminal vesicles suggests that amiloride-binding proteins are the best members of a new family of epithelial-specific proteins. [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]

Exposure of perfused rat livers to zymosan, arachidonic acid and phenylephrine, but not to latex particles, induces pronounced oxygen uptake, glycogenolysis and Ca2+ mobilization. The oxygen uptake induced by arachidonic acid and by zymosan remains elevated even after the agents have been removed. NaN3 was found to be much more effective in inhibiting the oxygen uptake induced by phenylephrine than that induced by zymosan or arachidonic acid. Glucose release induced by zymosan and by arachidonic acid reaches a maximum after about 2 min and then declines very rapidly even while the agents are still being infused. In contrast, glucose release induced by phenylephrine remains elevated for the duration of the infusion. Ca2+ fluxes induced by arachidonic acid are similar to those induced by phenylephrine in that efflux occurs when the agent is administered and influx occurs only when the agent is removed. This contrasts to the Ca2+ flux changes induced by zymosan where both Ca2+ effiux and Ca2+ influx occur even while zymosan is still being infused. Glucose release induced by zymosan is inhibited by bromophenacylbromide and nordihydroguaiaretic acid, but not by indomethacin. Indomethacin, however inhibits the arachidonic-acid-induced glucose release which is also inhibited by nordihydroguaiaretic acid but not by bromophenacylbromide. Indomethacin inhibits also the arachidonic-acid-induced Ca2+ flux changes whereas the zymosan- and the phenylephrine-induced Ca2+ flux changes are not inhibited by the cyclooxygenase inhibitor. The data presented in tbs paper suggest that in the perfused rat liver the zymosan-induced glycogenolysis, as well as the Ca2+ flux changes and glycogenolysis induced by arachidonic acid, are mediated by eicosanoids. [ABSTRACT FROM AUTHOR]

Nuclear RNP complexes, cytoplasmic mRNP particles and free and membrane-bound polysomes were prepared from rat liver and their tow-molecular-muss RNA components were analyzed on polyacrylamide/formamide gels. The separated small RNAs transferred to diazophenylthioether paper were hybridized to the nick-translated recombinant plasmid pA6 containing cDNA sequences for the low-Mr RNA called 7S(L) RNA. Nuclear RNP particles and free and membrane-bound polysomes were found to contain 7S(L) RNA. In the cytoplasm 7S(L) RNA could be identified as the major small RNA in 20-S cmRNP particles. [ABSTRACT FROM AUTHOR]

Apolipoprotein A-I, the major protein in mammalian high-density lipoprotein, acts as a cofactor for lecithin—cholesterol acyltransferase during the formation of cholesterol ester and as such, is thought to promote cholesterol efflux from peripheral cells to the liver. In this paper, we report the partial purification of rat liver apolipoprotein A-I mRNA by a polysome immunoadsorption technique, and its cDNA cloning. Isolation of two overlapping cDNA clones enabled us to derive the whole rate apolipoprotein A-I cDNA coding sequence. Comparison of the deduced protein sequence with its human counterpart reveals a striking homology between the prepropeptide precursors. Both mature protein amino-terminal regions are very homologous, suggesting that this particular domain could be involved in lipid/protein binding or lecithin—cholesterol acyltransferase activation. [ABSTRACT FROM AUTHOR]

As previously reported [Favilla, R. & Cavatorta, P. (1975) FEBS Lett. 50, 324–329], the enzyme horse liver alcohol dehydrogenase catalyzes a reaction between NAD+ and H2O2. The final isolated product was then called NADX because of its unknown structure. In this paper the results of spectroscopic investigations on this compound are described. They indicate that only the nicotinamide moiety of the original NAD+ molecule was modified by the action of hydrogen peroxide. From the ¹H and 13C nuclear magnetic resonance spectra of NADX the following structure was deduced: adenosine(5′)diphospho(5)-β-D-ribose-NH-CH=C(CHO)-CONH2. This structure is consistent with both ultraviolet and reactivity measurements performed on NADX. A tentative mechanism for the whole peroxidatic reaction pathway leading to NADX is finally proposed. [ABSTRACT FROM AUTHOR]

Liver and muscle aldolase display similar reaction mechanisms. Both the enzymes, by reacting with dihydroxyacetone phosphate, form an acid-labile intermediate which is in rapid equilibrium with an eneamine intermediate. Differences are found in the equilibrium concentration of the acid-labile intermediate, which represents approximately 25% of the total intermediates in the liver (this paper) and 60% in the muscle enzyme [E. Grazi and G. Trombetta, Biochem. J. 175, 361 (1978)] and in the rate of formation of the eneamine intermediate which is much slower in the liver enzyme. Furthermore, with liver aldolase, the rate by which the C-3H bond of dihydroxyacetone phosphate is cleaved is increased by 60 times in the presence of glyceraldehyde 3-phosphate. This, mechanistically, indicates that glyceraldehyde 3-phosphate is bound to the enzyme before the formation of the eneamine from dihydroxyacetone phosphate, and, physiologically, that in liver aldolase the gluconeogenetic activity is favoured over the glycolytic activity. [ABSTRACT FROM AUTHOR]

The mechanism of action of 3-hydroxy-3-methylglutaryl-CoA reductase solubilized from rat liver microsomes has been investigated. In the reduction of 3-hydroxy-3-methylglutaryl-CoA to mevalonate, an overall initial velocity study gave a linear intersecting pattern when both 3-hydroxy-3- methylglutaryi-CoA and NADPH were variable substrates. Adenosine 2′-monophospho-5′-diphos- phoribose, which was found to be a reversible inhibitor of reductase, inhibited the enzyme competitively with respect to NADPH, and uncompetitively with respect to 3-hydroxy-3-methylglutaryl-CoA. On the other hand, the inhibition of reductase by ML-236B (sodium salt), a specific reversible inhibitor of the enzyme isolated from the culture of a Penicillium (whose structure is given in the paper) is competitive with respect to 3-hydroxy-3-methylglutaryl-CoA and noncompetitive with respect to NADPH [Endo et al. (1976) FEBS Lett. 72, 323-326]. The reduction of 3-hydroxy-3-methylglutaryl-CoA to mevalonate was subject to the substrate inhibition by NADPH attributed to the formation of a productive enzyme-NADPH complex. These results indicate that the two substrates bind to the enzyme effectively in an ordered manner; reductase first interacts with 3-hydroxy-3-methylglutaryl-CoA to make a binary complex, which, in turn, forms a ternary complex with one molecule of NADPH. Considered together with the results of product inhibition study, and assuming a hemithioacetal of mevaldate and CoA is an intermediate of the reductase reaction, a bi-uni-uni-ter-ping- pong mechanism is proposed as a model of the overall reaction. [ABSTRACT FROM AUTHOR]

A product of mitochondrial protein synthesis in rat liver mitochondria, characterized by a low molecular weight (Mr < 10000) and an unusually high hydrophobicity, has been identified as the dicyclohexylcarbodiimide-binding protein and as a peptide of the hydrophobic sector of the mitochondrial ATPase complex. The purified protein still possesses the ability to bind dicyclohexylcarbodiimide. [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]

The present paper reviews recent advances which have been made in studies, predominantly in rat liver, on the nutritional and hormonal regulation of gene expression of lipogenic enzymes (acetylCoA carboxylase, fatty acid synthase, malic enzyme and glucose-6-phosphate dehydrogenase). [ABSTRACT FROM AUTHOR]

We have identified a mannosidase in rat liver that releases α1 → 2, α1 → 3 and α1 → 6 linked manose residues from oligosaccharide substrates, MannGlcNAc where n = 4-9. The end product of the reaction is Manα1 → 3[Manα1 → 6]Manβ1 → 4GlcNAc. The mannosidase has been purified to homogeneity from a rat liver microsomal fraction, after solubilization into the aqueous phase of Triton X-114, by anion-exchange, hydrophobic and hydroxyapatite chromatography followed by chromatofocusing. The purified enzyme is a dimer of a 110-kDa subunit, has a pH optimum between 6.1 and 6.5 and a Km of 65 μM and 110 μM for the Man5GlcNAc-oligosaccharide or Man9GlcNAc-oligosaccharide substrates, respectively. Enzyme activity is inhibited by EDTA, by Zn2+ and Cu2+, and to lesser extent by Fe2+ and is stabilized by Co2+. The pattern of release of mannose residues from a Man6GlcNAc substrate shows an ordered hydrolysis of the α1 → 2 linked residue followed by hydrolysis of α1 → 3 and α1 → 6 linked residues. The purified enzyme shows no activity against p-nitrophenyl-α-mannoside nor the hybrid GlcNAc Man5GlcNAc oligosaccharide. The enzyme activity is inhibited by swainsonine and 1-deoxymannojirimycin at concentrations 50-500-fold higher than required for complete inhibition of Golgi-mannosidase II and mannosidase I, respectively. The data indicate strongly that the enzyme has novel activity and is distinct from previously described mannosidases. [ABSTRACT FROM AUTHOR]

A major inducible form of heine oxygenase (EC 1.14.99.3) was purified from liver microsomes of chicks pre-treated with cadmium chloride. The purification involved solubilization of microsomes with Emulgen 913 and sodium cholate, followed by DEAE-Sephacel, carboxymethyl-cellulose (CM-52) and hydroxyapatite chromatography, and FPLC through Superose 6 and 12 columns operating in series. The final product gave a single band on silver-stained SDS/polyacrylamide gels (Mr = 33000). Optimal conditions for measurement of activity of solubilized heme oxygenase were studied. In a reconstituted system containing purified heme oxygenase. NADPH -cytochrome reductase, biliverdin reductase and NADPH, the Km for free heme was 3.8 ± 0.5 μM; for heme in the presence of bovine serum albumin (5 mol heme/3 mol albumin) the Km was 5.0 ± 0.8 μM ; and the Km for NADPH was 6.1 ± 0.4 μM (all values mean ± SD, n = 3). Oxygen concentration as low as 15 μM, with saturating concentrations of heme and NADPH, did not affect the reaction rate, indicating that the supply of oxygen is not involved in the physiological regulation of activity of the enzyme. The pH optimum of the reaction was 7.4: at 37 C, the apparent Vmax was 580 ± 44 nmol biliverdin · (mg protein)-1 · min-1 and the molecular activity was 19.2 min-1. Biliverdin IXa was the sole biliverdin isomer formed. In the presence of purified biliverdin reductase, biliverdin was converted quantitatively to bilirubin. Addition of catalase to the reconstituted system decreased the breakdown of heine to non-biliverdin products and led to nearly stoichiometric conversion of heine to biliverdin. Activity of the enzyme in the reconstituted system was inhibited by metalloporphyrins in the following order of decreasing potency: tin mesoporphyrin > tin protoporphyrin > zinc protoporphyrin > manganese protoporphyrin > cobalt protoporphyrin. Protoporphyrin (3.3 or 6.6 μM) (and several other porphyrins) and metallic ions (100 μM) alone had little if any inhibitory effect, except for Hg2+ which inhibited by 67% at 10 μM and totally at 15 μM. Following partial cleavage, fragments of the purified enzyme were sequenced. Comparison of sequences to those derived from eDNA sequences for the major inducible rat and human heme oxygenase showed 69% and 76% similarities, respectively. The histidine residue at position 132 of rat heme oxygenase-1 and the residues (Lys128-Arg136) flanking His132 were conserved in all three enzymes, as well as in the corresponding portion of a fourth less highly similar rat enzyme, heme oxygenase-2. It is suggested that this region plays a critical role in heme binding and in catalyzing the heme oxygenase reaction. [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]

Embryonic and adult chicken liver galactosyltransferases behave differently on DEAE-Sepharose chromatography. After solubilization, two embryonic activities (one transfers galactose in a β1&-4 linkage to asialo-agalacto-α1-acid glycoprotein and to N-acetylglucosamine; the other transfers galactose in a β-3 linkage to asialo-ovine submaxillary mucin) elute after the bulk of the protein, and after free glucose. The same two enzymes in adults elute more rapidly, almost coincident with the bulk of the protein, and before free glucose. The difference in elution patterns occurs when the column buffer contains 0.1 M NaCl. Without salt, both embryonic and adult transferases bind to the column, but with 0.5 M NaCl, the embryonic and adult transferases elute identically, and with the bulk of the protein. After treatment with neuraminidase, the embryonic transferase activities elute significantly earlier on a DEAE-Sepharose column in the presence of 0.1 M NaCl. The embryonic forms migrate more rapidly than do the adult forms on cellulose acetate electrophoresis, but neuraminidase treatment renders both enzyme forms immobile in this system. Neuraminidase treatment also inhibits the binding of the embryonic transferases to a wheat-germ-agglutinin-Sepharose column. Kinetically, the embryonic and adult transferases are indistinguishable. [ABSTRACT FROM AUTHOR]

Possible mechanisms are evaluated for the acute regulation of the hamster brown-fat mitochondrial proton conductance pathway which is active during non-shivering thermogenesis. Isolated mitochondria are incubated under conditions designed to approximate to the non-thermogenic state, and the effect of the steady infusion of fatty acids or acyl derivatives upon respiration, membrane potential and membrane proton conductance is monitored continuously. Fatty acids increase the proton conductance with no detectable threshold concentration, allowing the generated acyl carnitine to be rapidly oxidized. The extent of depolarization and of respiratory increase is a function of the rate of infusion. Immediately infusion is terminated the conductance decreases, the mitochondria repolarize and respiration returns to the initial rate. Infusion of acyl-CoA and acylcarnitine cause only a slight depolarization or respiratory increase after high concentrations of these derivatives have accumulated. Any factor which decreases the rate of conversion of fatty acid to acyl-CoA potentiates the conductance increase. An effect of acyl-CoA upon chloride permeability is not specific to brown-fat mitochondria. Fatty acids infused into rat liver mitochondrial incubations produced a small conductance increase, comparable to that of acyl-CoA or acylcarnitine. It is concluded that fatty acids are the most plausible acute regulators of the proton conductance. The relation to the brown-fat-specific 32000-Mr protein is discussed. [ABSTRACT FROM AUTHOR]

Addition of ammonium ions to isolated rat hepatocytes stimulated the rate of synthesis of pyrimidines. Isolation and quantification of pyrimidine nucleotides, orotic acid an the acid-hydrolyzed product of carbamoylaspartic acid by non-exchange chromatography and high-pressure liquid chromatography show a marked simulation in the incorporation of [14C]bicarbonate in incubations with added ammonium ions. The incorporation into total uridine nucleotides (σUMP) was increased twofold in the presence of 5 mM ammonium ion, and approximately eightyfold into orotic acid. There was a parallel increase in labelling a carbamoylaspartic acid from undetectable to a level similar to that of orotic acid. The specific activity of urea formed during the incubations did not change during incubations or in the presence of ammonium ions confirming that the change in labelling of pyramidine was not due to a change in the specific activity of precursor. Despite the stimulation in incorporation of label into pyrimidines, there was no increase in the hepatcyte content of σUMP, which was 11.5 µmol/g dry weight, although the orotic acid content increased from 0.09 µmol/g dry weight in the absence of added ammonium ions (but in the presence of 2 mM L-glutamine) to 8.6 µmol/g dry weight with 5 mM ammonium ion. The stimulated incorporation of [14C]becarbonate in the presence of 5 mM and 10 mM ammonium ion was shown to be due to a stimulated synthesis of carbamoyl phosphate, since greater than 80% of label in the uracil ring was present at position C-2. Incubation of hepatocytes in basal medium (Eagles) containing 2.5% foetal calf serum and 20 mM bicarbonate showed that there was a significant stimulation of pyrimidine synthesis with 1 mM ammonium completely reversed by 5 mM L-ornithine and was partially reversed by 1 mM L-ornithine. Evidence for a contribution of the urea cycle carbamoyl phosphate synthetase to pyrimidine synthesis is discussed. [ABSTRACT FROM AUTHOR]

Characterization of polysomes from estrogenized chicken liver revealed that very-low-density lipoprotein II (VLDL&subII, serum albumin and vitellogenin mRNAs are differently packed with ribosornes during translation in vivo. The ribosome density per number of nucleotides is high for VLDL&subII mRNA. intermediate for serum albumin mRNA and low for vitellogenin mRNA. This difference in ribosomal load is maintained throughout the period of hormone effect. The differential utilization observed for vitellogenin and VLDL&subII mRNAs partly explains the large difference in molar production rate between these yolk protein precursors. Translation properties and efficiency of the three hepatic mRNAs were also compared in the mRNA-depleted reticulocyte lysate. Elongation of the nascent chains for vitellogenin and serum albumin proceeded in a discontinuous fashion. Initiation in vitro was studied at varying ionic strengths., in the presence of aurintricarboxylic acid, and at suboptimal hemin concentrations. VLDL&subIImRNA expression is by far the most resistant to 7-methylguanosine 5'- triphosphate (m[sup7]GTP) and high salt concentrations, vitellogenin mRNA the least. This behavior resembles the differential utilization of the mRNAs in vivo. The putative structural basis of these differences is discussed. [ABSTRACT FROM AUTHOR]

The results of steady-state kinetic measurements on the initial rate of the peroxidatic reaction between β-NAD+ and hydrogen peroxide, catalyzed by horse liver alcohol dehydrogenase, at pH 7 are described. A simple sequential mechanism is deduced from graphical analysis of the data plotted according to Eadie-Augustinsson-Hofstee primary plots and the values of the true kinetic parameters KmNAD, KmH2O2 and V are estimated from the corresponding secondary plots. Ethanol has been found to compete with hydrogen peroxide for the same enzyme active site. During the catalytic process a progressive inactivation of the enzyme occurs caused by H2O2. The rate law of this process is quantitatively described at pH 7 both in the absence and in the presence of NAD+. The coenzyme has been found to protect the enzyme against inactivation by H2O2, which oxidized essential cysteine residues. The results obtained from the study of both catalytic and inactivating processes are finally rationalized on the basis of a general mechanistic scheme. [ABSTRACT FROM AUTHOR]

The kinetic properties of two of the partially separated isoenzymes I and V of pig liver esterase were studied. The cholinesterase-like isoenzyme I hydrolyses butyrylcholine as well as various other esters and aromatic amides. This isoenzyme is sensitive to 0.01 mM physostigmine and to fluoride. The second type (isoenzyme V) has the features of the so-called aliesterase: it acts preferentially on short-chain aliphatic esters, does not hydrolyse butyrylcholine and has only low activity towards amides. Further differences exist with regard to sensitivity towards organophosphorous inhibitors, to the influence of organic solvents and to the pH optimum. Transacylation reactions with methanol as an acceptor are mainly catalyzed by the isoenzyme of the aliesterase type. The esterase forms III and IV, which are located between isoenzymes I and V on the isoelectric focussins column, show kinetic features similar to those of a mixture of I and V. A stepwise increase or decrease, respectively, of certain kinetic properties, e.g. specific activities, is observed for the sequence I, III, IV, V, A quantitative comparison of the kinetic properties supports our proposed subunit model [1], according to which the main components of these four esterase fractions are the trimers γγγ, αγγ, ααγ, and ααα. These results offer explanations for many of the very complex and often controversial results formerly obtained with the heterogeneous pig liver esterase. [ABSTRACT FROM AUTHOR]

Three different subunits of highly purified pig liver esterase (EC 3.1.1.1) can be separated by analytical dodecyl sulfate electrophoresis, though their relative mobilities are very similar. The same subunit bands are obtained with microsomes, in which the esterases have been labeled with the specific active-site-directed inhibitor bis( 4-nitro-[14C] phenyl)phosphate. The heterogeneity of the native trimeric enzyme is much more complex, as is demonstrated by isoelectric focussing and polyacrylamide gel electrophoresis. Fractions of esterase which were partially separated by preparative isoelectric focussing show differences in their subunit composition, their amino acid analyses, their tryptic peptide maps, and their C-terminal amino acids. From these experiments various features of the differing esterase subunits can be deduced. Based on the chemical results and on various experiments which did not indicate any secondary modification of the protein side-chains, the molecular basis of the esterase heterogeneity is discussed. We conclude that the native trimeric esterase is a mixture of numerous hybrids of at least three protein subunits with differing but closely related primary sequences. A comparison of the relative specificity of various preparations of pig liver microsomes indicates that genetic differences concerning the composition of liver esterase exist between individuals. [ABSTRACT FROM AUTHOR]

The prostaglandin-synthesizing system in rat liver was studied. When rat liver microsomes were incubated with radioactive arachidonic acid, two radioactive metabolites were detected which were exclusively identified with prostaglandin F2α and E2 by the double isotope method and gas chromatography-mass spectrometry (both electron impact and chemical ionization). Rat liver was found to produce always more prostaglandin F2α than prostaglandin E2. Thromboxane B2 and other prostaglandins such as prostaglandin D2 and 6-ketoprostaglandin F1α were not detected. When rat liver homogenates, instead of microsomes, were incubated with radioactive arachidonic acid, most of the arachidonic acid was found to be incorporated into a phospholipid fraction instead of being transformed into prostaglandin F2α, and E2. When rat liver cytosol was added to the incubation mixture of microsomes prostaglandin synthesis was inhibited, while the incorporation of arachidonic acid into phospholipids was enhanced. The effect of the cytosol was lost when the cytosol was boiled beforehand. These facts suggest that some enzymes or heat labile co-factors which facilitate the incorporation of arachidonic acid into phospholipids are present in rat liver cytosol. The prostaglandin synthetase was sensitive to small changes in pH, the optimum pH for the enzyme system being 9.7 for prostaglandin F2α and 8.9 for prostaglandin E2. Reduced glutathione, hydroquinone and EDTA were essentially inactive for the prostaglandin synthesis in liver. Under optimum conditions, rat liver microsomes were able to transform radioactive arachidonic acid into prostaglandin F2α and E2 with a conversion rate as high as 20%. [ABSTRACT FROM AUTHOR]

Horse-liver alcohol dehydrogenase requires Zn2+ for enzymatic activity. Reactivation experiments after dissociation and denaturation of the enzyme in 6 M guanidinium hydrochloride and subsequent separation of zinc prove that the effect of the metal on the rate and yield of reconstitution is complex. In the absence of Zn2+ no reactivation is detectable, while excess of Zn2+ leads to inactive aggregates. Optimum reactivation yields are obtained at 10 μM Zn2+ after short incubation in the denaturant; increasing zinc concentration causes a decrease of the rate of reactivation. The refolding of the zinc-free enzyme is characterized by consecutive first-order processes which may be separated from second-order dimer formation. Addition of 10 μM Zn2+ during refolding may be used to block side reactions competing with the reconslitution. The transition from sigmoidal kinetics to second-order profiles by adding Zn2+ after completion of the aforementioned first-order process corroborates the proposed uni-bimolecular reactivation mechanism which implies the involvement of inactive monomers. These gain their enzymatic function as a consequence of dimerization. The effect of Zn2+ may be explained by a side reaction in the overall reaction scheme of reactivation and renaturation which allows the kinetic measurements to be quantitatively described. [ABSTRACT FROM AUTHOR]

A new isotopic method, based upon the stereospecific replacement of a proton (³H) by a hydroxyl group has been developed for the measurement of rat liver testosterone and dehydroepiandrosterone 16α-hydroxylase activity. Specifically 16-tritiated substrates were prepared by microbiological (Cylindrocarpon radicicola) transformation of the [16-sup3;H]progesterone and [16-³H]pregnenolone. The incubation medium consists of a phosphate buffer (pH 7:150 mM), NADPH (0.1 mM), nicotinamide (10 mM) and magnesium chloride (4 mM). Tween 80 (1 mg/ml) is used to solubilize saturating concentrations of [16-³H]testosterone (50 µM) or [16-³H]dehydroepiandrosterone (100 µM). The enzymatically released tritium is recovered in the incubation medium as tritiated water which is distilled under reduced pressure and counted by liquid scintillation. The method is easy to perform. very sensitive (50 pmol of 16α-hydroxylated metabolites) and is independent of any further metabolism of the 16α-hydroxylated products. [ABSTRACT FROM AUTHOR]

The metabolites of 2-deoxy-D-galactose were quantitatively determined in liver and in various tissues of the rat. After intraperitoneal administration of 2-deoxy-D-[1-14C]galactose in a dose of 1 mmol/kg the liver was the predominant organ metabolizing this galactose analog. 2-Deoxy-Dgalactose 1-phosphate and UDP-2-deoxy-D-galactose were the major hepatic metabolites and after 3 h comprised 78% and 20% of the total radioactivity in liver, respectively; UDP-2-deoxy-Dglucose amounted to 0.7%. Liver was the only tissue in which substantial formation of UDP-2deoxy-D-hexoses was observed. The synthesis of UDP-2-deoxy-D-galactose was shown to be catalyzed by UDP-glucose: galactose1-phosphate uridylyltransferase. The low affinity of this enzyme for 2-deoxy-D-galactose 1-phosphate explained the accumulation of this substrate analog to levels as high as 14 mmol/kg liver. Enzymatic analysis of alkaline ethanol extracts showed a marked fall of the hepatic P1 content during the period of rapid 2-deoxy-D-galactose 1-phosphate formation. The trapping of Pi was associated with a loss of ATP and a lowering of the adenylate energy charge. The formation of UDP-2-deoxy-D-galactose occurred only after high levels of 2-deoxy-D-galactose 1-phosphate had been attained. The limited epimerization of UDP-2-deoxy-D-galactose to UDP-2deoxy-D-glucose led to an accumulation of the former deoxy-sugar nucleotide. The trapping of uridylate in the form of UDP-2-deoxy-D-galactose was accompanied by a transient decrease in the hepatic contents of UTP, UDP-glucose, and UDP-galactose. The administration of 2-deoxy-Dgalactose together with uridine or orotate completely prevented the depression of UTP. The accumulation of 2-deoxy-D-galactose 1-phosphate and the associated depression of Pi, in the absence of UTP deficiency, represent a state analogous to that induced by galactose in uridylyltransferase-deficient cells and tissues and may serve as a biochemical model in the study of the cellular injury in galactosemia. [ABSTRACT FROM AUTHOR]

1. A factor, which amplifies the inductions of several liver enzymes by glucocorticoid, was partially purified from Proteus mirabilis from rat intestine. The factor (amplifier) was completely inactivated by α-glucosidase, but not by other glycoside hydrolases, proteases, nucleases or phosphatases tested; it was also hydrolysed by HCl with liberation of reducing sugars. Thus the oligosaccharide in this factor seems to be essential for the amplification. 2. In adrenalectomized rats the amplifier increased the inductions of several liver enzymes, such as tyrosine aminotransferase and leucine aminotransferase, by glucocorticoid. But it did not amplify the induction of tyrosine aminotransferase by glucagon or insulin or the activities of enzymes that are not induced by glucocorticoid. The amplifier by itself did not have any glucocorticoid-like action in adrenalectomized rat. These results show that the amplifier specifically increases the inductions of liver enzymes by glucocorticoid. 3. Since a similar amplification was also observed in isolated perfused liver and cultured hepatoma cells in vitro, the amplifier seems to act directly on the target organ or cells. [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]