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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]

To investigate in the human the mechanism by which BSP is excreted into the bile, various studies have been performed on T-tube bile samples following the intravenous injection of the dye. There is evidence to show that BSP under-goes alteration during the process of hepatic excretion. Certain fractions of bile-type BSP are probably excreted in the form of a conjugate. No evidence has been found that the formation of a glucuronide explains the bile-type BSP observed. [ABSTRACT FROM AUTHOR]

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In 30 diabetic inpatients the fatty acid pattern of triglycerides in parenchymal liver cells was studied by gas-liquid chromatography. With increasing size of fat droplets, a significant increase in the proportion of palmitic and oleic acid was observed as well as a significant fall of arachidonic (C 20: 4) and eicosapentaenoic acid (C 20: 5). [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]

1. The [14C]carboxymethylated protein of the ethanol-active isoenzyme of horse liver alcohol dehydrogenase has been treated with trypsin (with and without previous maleylation of the substrate), chymotrypsin, pepsin and cyanogen bromide, respectively. Peptide mixtures obtained have been fractionated and analysed. 2. Data are given for those peptides originating from a C-terminal region comprising about 60% of the protein chain, and the amino acid sequence of this segment, containing the last 234 residues, is deduced. The N-terminal part was known previously and the primary structure of the whole protein chain is therefore established. 3. The ethanol-active isoenzyme is found to be a dimer of two completely identical protein chains, each 374 residues long. The N-terminal part of the molecule contains the reactive cysteine residue, six out of the seven histidine residues in the whole chain, the majority of the mutations between the isoenzyme chains of different substrate specificities, and a region homologous to another dehydrogenase. 4. Serine residues that reacted during maleylation of the protein were found to undergo O &rarrow;N maleyl shift when they became N-terminal residues in peptides. Evidence was obtained suggesting that carboxymethylmethionyl peptide bonds are labile. [ABSTRACT FROM AUTHOR]

1. The ethanol-active isoenzyme of horse liver alcohol dehydrogenase has been carboxymethylated with iodo[14C]acetate and different samples of the labelled protein treated with trypsin (both with and without previous maleylation of the substrate), chymotrypsin, pepsin or cyanogen bromide. In each case most of the resultant peptides have been purified and characterized. 2. Data are given for those peptides originating from an N-terminal region comprising about 40% of the protein chain and the amino acid sequence of the first 140 residues is deduced. 3. It is established that over this region the two chains of the enzyme have an identical sequence. The N-terminus is an acetylated serine residue and the "active site" cysteine residue occupies position no. 46. One of the two tryptophans of the chain, but none of the four tyrosines, is in the sequence given. The region around position 100 is rich in cysteine residues. Some serine residues were found to have reacted during maleylation of the protein. 4. The amino acid sequence is compared to the previously known sequences of glyceraldehyde 3-phosphate dehydrogenase from two species. Similarities strongly suggest a true homology, at least of an N-terminal region, and support theories of a common ancestral origin for these enzymes. The reactive cysteine residues are, however, at widely different positions in the primary structures. [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]

Experiments were designed to determine whether renal excretion of uric acid is achieved by the same mechanism as for renal tubular excretion of hippurates and related organic acids. Surviving slices of rabbit kidney cortex were unable to accumulate C-urate by a concentrative mechanism. Further, entry of C-urate into renal slices was unaffected by acetate, probenecid or anoxia in accord with earlier observations from this laboratory with non-radioactive urate. Experience with isolated perfused rat liver supports the use of this experimental method as a model of the hippurate transport system. Unlike hippurate and a large number of related organic anions, neither urate nor C-activity derived from urate was concentrated in the bile from this preparation. Probenecid did not inhibit excretion of the small amounts of C-activity which did appear in the bile. Urate did not compete with indigo carmine, a nonmetabolizable substrate of the hippurate transport system, for excretion into the bile. From these findings, it is concluded that urate and organic acids such as hippurate do not behave similarly in kidney or in liver. The possibility that urate might be excreted by an independent active transport mechanism is not excluded. The demonstration that renal tissues can synthesize urate from hypoxanthine raises the possibility that urate synthesis might also occur in the intact animal and might contribute to the renal clearance of uric acid. [ABSTRACT FROM AUTHOR]

The surface ultrastructure of Kupffer cells in the rat liver has been studied by scanning electron microscopy (SEM). The results demonstrate that Kupffer cells are both significantly different and clearly distinct from endothelial cells. Kupffer cells have neither pores (and/or 'sieve plates') nor fenestrations, all of which are present in endothelial cells. They possess a stellate shape, and only indirectly, with slender and irregular evaginations, contribute to the lining of the sinusoidal wall. Furthermore, the luminal surface in some areas contains a large population of short microvilli, microplicae and invaginations. These elements form a kind of microlabyrinth which may correspond to the 'worm-like' structures described by transmission electron microscopy (TEM). In the present study, transition forms between endothelial and Kupffer cells were never found. On the contrary, considering the highly fenestrated nature of the endothelial cells, the Kupffer cells may, by ameboid movements, easily cross the overlapping barrier of the sinusoid and protrude into the lumen. Thus, acting as activated macrophages, the Kupffer cells might function to prevent the entrance of foreign material into the tissues of the liver through the fragile and highly fenestrated endothelium. Finally, the topographical reconstruction of the sinusoid by correlated SEM and TEM studies demonstrates that Kupffer cells, with their protruding cytoplasm and ability to extend into the lumen of the sinusoid, may actually change the caliber of the vessel, and thus function as a 'sphincter' which causes a temporary arrest of the blood flow when the diameter of the sinusoidal lumen is reduced. [ABSTRACT FROM AUTHOR]

Lysosomes in mouse liver parenchymal cells have been marked by intravenous injection of Thorotrast. They were subsequently followed in a time sequence from five hours up to sixteen weeks after injection. At two days after injection the majority of the lysosomes was heavily loaded with marker particles, while endocytosis was no longer observed. From six days after injection Thorotrast was partly accumulated in very large lysosomes (conglomerates) with mean diameters up to 2.5 μm. As the time after injection advanced the Thorotrast content of the cells was reduced while most of the remaining marker substance became concentrated in the conglomerates. Many Thorotrast conglomerates were shown to contain acid phosphatase and some of them were able to fuse with functionally younger lysosomes which were marked with colloidal gold. Morphometric analysis showed an increase in the volume density of the dense body population between 0 and 2 days after injection, followed by a decrease between 2 and 11 days. The observed decrease is probably caused by exocytosis of the contents of Thorotrast containing lysosomes in bile capillaries. [ABSTRACT FROM AUTHOR]

The case is reported of a 61-year-old man with primary diffuse alveolar septal pulmonary amyloidosis. Amyloid infiltration of the heart and other organs was also observed. The clinical findings and laboratory investigations reveal features characteristic of defective gas transfer with pulmonary oedema due to left ventricular failure from myocardial involvement. [ABSTRACT FROM PUBLISHER]

It is shown that alloxan inhibits binding of SITS to liver cells. This indicates the cell membrane as a site of alloxan action. Alloxan is found to react with tissues to form complexes that are detectable up to 3 hrs after alloxan treatment. On the basis of the present findings, an assumption is made that alloxan inhibits a cell membrane processes by blockade of functionally important groups. [ABSTRACT FROM AUTHOR]

In 40 diabetic inpatients the fatty acid pattern of triglycerides in liver fat and in subcutaneous adipose tissue was determined by gas liquid chromatography. With rising size of fat droplets in liver parenchymal cells there was a significant increase of palmitic acid (C∶16), oleic acid (C 18∶1) and linoleic acid (C 18∶2) as well as a decrease of myristoleic acid (C 14∶1), arachidonic (C 20∶4) and eicosapentaenoic acid (C 20∶5) in liver triglycerides, resulting in a fatty acid composition in big droplets similar to that of adipose tissue. Fatty acid pattern of subcutaneous depot fat was strikingly constant. [ABSTRACT FROM AUTHOR]

Normal male Sprague Dawley rats were infused under restrained conditions with hypertonic dextrose solution, with or without added protein, through a superior vena cava catheter delivering 130 to 341 Cal. per kg. body weight per day. Infusion of hypertonic dextrose solution for four days at high caloric level (> 300 Cal.) uniformly produced gross fatty infiltration in the liver. Addition of essential amino acids did not prevent the change. Protein hydrolysate seemed to minimize the change slightly at a lower caloric level (250 Cal.) but not at a high caloric level (315 Cal.) There was a positive correlation between the liver weight and caloric intake (r = 0.68). [ABSTRACT FROM AUTHOR]

In three species chronic treatment with the anorectic drug chlorphentermine causes a profound alteration of the phospholipid/lipid metabolism in the organism, resulting in an increase of the fractions of phospholipids and lipids, e.g. in lungs, livers and adrenals. The results are interpreted as drug-induced generalized phospholipidosis, which is caused by amphiphilic drugs, like chlorphentermine and others. Its extent depends on several factors, like content, pattern and turnover rate of phospholipids in different organs, and on the species. [ABSTRACT FROM AUTHOR]

A series of experiments indicated thatI-parathyroid hormone administered to the rat i.v. was deposited very rapidly in three organ systems only, bone, kidney and the liver. As indicated by increasing solubility in trichloracetic acid, the hormone seemed to be rapidly metabolized at the sites of deposition. The experiments were in part repeated employing quantitative extraction and gel filtration procedures. It was found that intact hormone predominated (82%) in the circulation but was cleared very rapidly: 91, 97, and 99% having left by 10, 30 and 60 minutes respectively after injection. The intact hormone was found at 10 minutes to be principally in kidney, bone, and liver. Although there was evidence of fragmentation preferentially to polypeptides of molecular weights of 6000, 4500 and 2500 these cannot be claimed to represent real molecular entities. By one hour, only a few per cent of the injected hormone remained intact, the bulk having been degraded to fragments of small molecular weight. [ABSTRACT FROM AUTHOR]

Autoradiographs were prepared from tissues of rats sacrificed 10 minutes after injection of biologically activeI-labeled parathyroid hormone. No radioactivity was seen in intestine and muscle. Deposition in liver was diffuse showing some sinusoidal concentrations. Deposition in kidney was high and, nearly all activity appeared in selected tubules (presumably proximal tubules) in the outer third of the cortex. Specific localization was also seen in bone particularly in the cellular layers of periosteum and endosteum adjacent to bony matrix and to some extent in osteocytes. [ABSTRACT FROM AUTHOR]

Biologically activeI-labeled parathyroid hormone (I-PTH) was used in a series of studies in dogs and chickens designed to confirm and augment earlier studies in rats. As in rats, a three exponential equation was required to describe disappearance ofI-PTH from the blood in the dog. The first two 'half-lives' (1.8 and 7 min) accounted for the bulk of the dose. Also as in rats, deposition of apparently intact hormone took place rapidly in kidney, liver and bone in both the dog and the chicken. Degradation occurred very rapidly in all three target organs. Three labeled hormones of different biological activities were compared in the rat. Inactive, oxidized hormone was rejected by the liver but showed markedly increased deposition in kidney and the higher the purity of the hormone the higher was its uptake by liver. Exploration of a wide range of dosages revealed few effects on distribution (smaller deposition in liver and kidney at highest dosages, 65 μg/rat). Fresh sera did not degrade hormone rapidly or extensively. There was no deposition of hormone in intestinal mucosa, marrow, and red cells. Nephrectomy increased deposition in liver and bone. Finally, the perfused liver was capable of extensive degradation of the hormone. [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]