Aging effects on the liver aldolase of rabbits (Short Communication)

A chemical method is used to determine the amount of aldolase sequence in rabbit livers. It is demonstrated that the aldolase sequences of the livers of young rabbits are more efficient catalytically than are those from the livers of old rabbits. The presence of altered residues in the aldolase sequences of old animals may account for these observations.     

Studies on the subunit structure of 4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating) from Pseudomonas acidovorans.

1. Homogeneous preparations of D-4-deoxy-5-oxoglutarate hydro lyase (decarboxylating)(EC4.2.1.41) were analysed in the ultracentrifuge by the high-speed sedimentation-equilibrium method of Yphantis (1964). The molecular weight in 0.1 M-potassium phosphate buffer, pH 7.2, in 6M-guanidine hydrochloride and in 0.1 M-beta-mercaptoethanol in 6M-guanidine hydrochloride was 113,000, 56,000 and 30,400 respectively. Polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate indicated a minimum molecular weight of 30,500. 2. Measurement of the thiol content of the enzyme, before and after reduction with NaBH4 or dithiothreitol under denaturing conditions, indicated the presence of eight thiol residues and two interchain disulphide bridges/enzyme molecule. 3. Amino acid analysis showed that the intact enzyme contains a total of approximately 100 arginine and lysine residues, but digestion of the enzyme with trypsin yielded about 49 peptides staining with ninhydrin in a peptide "map". 4. With the knowledge that the enzyme contains only two substrate-binding sites, it is suggested that the enzyme probably consists of four polypeptide chains arranged in an alpha2beta2 confirmation.     

Glucocerebrosidase: reconstitution of activity from macromolecular components. (Short Communication)

Glucocerebrosidase activity was reconstituted in vitro from a soluble glycoprotein factor (P) and a particle-bound factor (C). The physiological significance of the system is discussed.     

Haemoglobulin catabolism: the role of ferrihaems in studies of the degradation pathway (Short Communication)

The importance of ferrihaem aggregation in studies of haemoglobin catabolism is assessed in the light of recent work. Experimental evidence is put forward suggesting that monomeric ferrihaems are degraded much more readily than dimeric species. This may offer an alternative explanation for the apparent ;apoprotein catalysis' recently observed.     

Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine acetaldehyde-lyase (aldolase) in species of Pseudomonas

1. The route of l-threonine degradation was studied in four strains of the genus Pseudomonas able to grow on the amino acid and selected because of their high l-threonine aldolase activity. Growth and manometric results were consistent with the cleavage of l-threonine to acetaldehyde+glycine and their metabolism via acetate and serine respectively. 2. l-Threonine aldolases in these bacteria exhibited pH optima in the range 8.0–8.7 and K_m values for the substrate of 5–10mm. Extracts exhibited comparable allo-l-threonine aldolase activities, K_m values for this substrate being 14.5–38.5mm depending on the bacterium. Both activities were essentially constitutive. Similar activity ratios in extracts, independent of growth conditions, suggested a single enzyme. The isolate Pseudomonas D2 (N.C.I.B. 11097) represents the best source of the enzyme known. 3. Extracts of all the l-threonine-grown pseudomonads also possessed a CoA-independent aldehyde dehydrogenase, the synthesis of which was induced, and a reversible alcohol dehydrogenase. The high acetaldehyde reductase activity of most extracts possibly resulted in the underestimation of acetaldehyde dehydrogenase. 4. l-Serine dehydratase formation was induced by growth on l-threonine or acetate+glycine. Constitutively synthesized l-serine hydroxymethyltransferase was detected in extracts of Pseudomonas strains D2 and F10. The enzyme could not be detected in strains A1 and N3, probably because of a highly active `formaldehyde-utilizing' system. 5. Ion-exchange and molecular exclusion chromatography supported other evidence that l-threonine aldolase and allo-l-threonine aldolase activities were catalysed by the same enzyme but that l-serine hydroxymethyltransferase was distinct and different. These results contrast with the specificities of some analogous enzymes of mammalian origin.     

Assay for glutamine synthetase activity (Short Communication)

The use of phosphoenolpyruvate plus pyruvate kinase as an ATP-generating system in the assay for glutamine synthetase activity via the formation of gamma-glutamylhydroxamate from glutamate and hydroxylamine with crude tissue preparations is shown to give values far in excess of the true glutamine synthetase activity of the tissue. This is due to the generation of pyruvate, which reacts with hydroxylamine to give a compound that is chromogenic with the ferric chloride reagent used for measuring gamma-glutamylhydroxamate.     

Alternative routes of aromatic catabolism in Pseudomonas acidovorans and Pseudomonas putida: gallic acid as a substrate and inhibitor of dioxygenases.

When 3,4-dihydroxyphenylacetic acid (homoprotocatechuic acid) was added to Pseudomonase acidovorans growing at the expense of succinate, enzymes required for degrading homoprotocatechuate to pyruvate and succinate semialdehyde were strongly induced. These enzymes were effectively absent from cell extracts of the organism grown with 4-hydroxyphenylacetic acid, and this substrate was metabolized by the catabolic enzymes of the homogentisate pathway. Two separate ring-fission dioxygenases for 3,4,5-trihydroxybenzoic acid (gallic acid) were present in cell extracts of Pseudomonas putida when grown with syringic acid, and gallate was degraded by reactions associated with meta fission. One of the two gallate dioxygenases also attacked 3-O-methylgallic acid; the other, which did not, was induced when cells were exposed to gallate. This organism possessed ortho fission enzymes, including protocatechuate 3,4-dioxygenase (EC 1.13.11.3) and cis,cis-carboxymuconate-lactonizing enzyme (EC 5.5.1.2), after induction with 3,4-dihydroxybenzoic acid (protocatechuic acid). Gallate was a substrate for protocatechuate 3,4-dioxygenase, with a Vmax about 3% of that of protocatechuate and with an apparent Km slightly lower. Gallate was a powerful competitive inhibitor of protocatechuate oxidation.     

A temperature-jump study of the reaction between azurin and cytochrome c-551 from Pseudomonas aeruginosa (Short Communication)

Temperature-jump studies on the electron-transfer reaction between azurin and cytochrome c-551 clearly reveal two chemical relaxations. The amplitudes of these relaxation processes have identical spectral distributions, but the relaxation times show different dependences on the reactant concentrations. These findings are discussed in terms of possible models.     

Studies on the activity of aldolase from rabbit muscles in the presence of liposomes]

Electronmicroscopic studies have been made on the structure of liposomes obtained by the method of Bangham [13] with subsequent ultrasonic desintegration. The effect of muscle aldolase on the structure of liposomes was also investigated. Parallel studies were made on the effect of storage of liposomes upon the activity of aldolase. It was shown that liposomes obtained from chromatographically pure egg lecithin present discrete partially aggregated bodies, 1.000-3.000 A in size, composed by concentric layers, which have a dimension of approximately 40 A and periodicity of about 70 A. Interaction of these particles with the protein results into their desintegration and enlargement, this process being accompanied by the formation of a "fringe" at the edge of the particles. Aldolase activity in these systems in higher than in control. During storage of phosphatide-aqueous system, obtained by the metod of Bungenberg de Jong, activation of aldolase is gradually replaced by its inactivation.     

The reaction of Pseudomonas aeruginosa cytochrome c oxidase with sodium metabisulphite (Short Communication)

Spectrophotometric evidence is presented for the formation of a complex between metabisulphite and reduced Pseudomonas aeruginosa cytochrome c oxidase. The effects of metabisulphite on the recombination of CO with the reduced enzyme are discussed in terms of alternate binding sites for S(2)O(5) (2-) and CO.     

Dissimilation of the lignin model compound veratrylglycerol-beta-(o-methoxyphenyl) ether by Pseudomonas acidovorans: initial transformations.

The initial transformadomonas acidovorans are demethylation of the 4-methoxyl group of the 3,4-dimethoxyphenyl portion of the molecule, and a NAD+-linked dehydrogenation of the benzyl alcohol group. Based on these findings and those described before, an overall degradation scheme is postulated.     

Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine hydrolyase (deaminating) in a species of Corynebacterium

1. Three bacterial isolates capable of growth on l-threonine medium only when supplemented with branched-chain amino acids, and possessing high l-threonine dehydratase activity, were examined to elucidate the catabolic route for the amino acid. 2. Growth, manometric, radiotracer and enzymic experiments indicated that l-threonine was catabolized by initial deamination to 2-oxobutyrate and thence to propionate. No evidence was obtained for the involvement of l-threonine 3-dehydrogenase or l-threonine aldolase in threonine catabolism. 3. l-Threonine dehydratase of Corynebacterium sp. F5 (N.C.I.B. 11102) was partially purified and its kinetic properties were examined. The enzyme exhibited a sigmoid kinetic response to substrate concentration. The concentration of substrate giving half the maximum velocity, [S_0.5], was 40mm and the Hill coefficient (h) was 2.0. l-Isoleucine inhibited enzyme activity markedly, causing 50% inhibition at 60μm, but did not affect the Hill constant. At the fixed l-threonine concentration of 10mm, the effect of l-valine was biphasic, progressive activation occurring at concentrations up to 2mm-l-valine, but was abolished by higher concentrations. Substrate-saturation plots for the l-valine-activated enzyme exhibited normal Michaelis–Menten kinetics with a Hill coefficient (h) of 1.0. The kinetic properties of the enzyme were thus similar to those of the `biosynthetic' isoenzyme from Rhodopseudomonas spheroides rather than those of the enteric bacteria. 4. The synthesis of l-threonine dehydratase was constitutive and was not subject to multivalent repression by l-isoleucine or other branched-chain amino acids either singly or in combination. 5. The catabolism of l-threonine, apparently initiated by a `biosynthetic' l-threonine dehydratase in the isolates studied, depended on the concomitant catabolism of branched-chain amino acids. The biochemical basis of this dependence appeared to lie in the further catabolism of 2-oxobutyrate by enzymes which required branched-chain 2-oxo acids for their induction.     

A four-iron ferredoxin from Desulfovibrio desulfuricans (Short Communication)

Ferredoxin from Desulfovibrio desulfuricans was isolated, purified and crystallized. It contains four iron atoms and four sulphido or ;acid-labile' sulphur atoms for a molecule of 6000 daltons. The absorption spectrum in the u.v.-visible region and the electron-paramagnetic-resonance signals of the reduced protein are similar to those observed for other four-iron ferredoxins. The amino acid composition is different from that of Desulfovibrio gigas ferredoxin. The redox potential of -0.33V at pH7.0 was determined by dye techniques.     

Glucose metabolism in the rat small intestine: the effect of glucose analogues on hexokinase activity (Short Communication)

The effect of intubation of starved rats with solutions of glucose, 3-O-methylglucose, mannose, 2-deoxyglucose and sorbitol on the subcellular location of hexokinase in the mucosa of the small intestine was studied. Glucose, 3-O-methylglucose and mannose caused the soluble hexokinase activity to rise to a value similar to that found in fed animals, whereas sorbitol and 2-deoxyglucose caused smaller increases.     

Acceleration of gluconeogenesis from lactate by lysine (Short Communication)

l-Lysine (2mm) causes an increase (mean 60%) in the rate of gluconeogenesis from lactate in isolated liver cells. The effect is of a catalytic nature. No other amino acid has the same effect, though ornithine is slightly active. The effect is additional to the stimulatory effects of oleate and of dibutyryl cyclic AMP.     

Preparation of insoluble collagen from human cartilage (Short Communication)

A simple technique is described for the removal of proteoglycans from human cartilage by sequential treatments with H(2)O(2) followed by trypsin digestion. The resultant fibres contain no uronic acids and have the amino acid composition of highly purified collagen.     

Pathways of gluconeogenesis from l-serine in the neonatal rat (Short Communication)

Gluconeogenesis from amino acid precursors was increased in the perfused liver of neonatal rats compared with that of adults. Quinolinate (5mm) was less inhibitory to glucose formation from serine and hydroxyproline in the neonatal rat than in the adult, suggesting that the main route for glucose synthesis from these precursors in neonatal liver does not involve pyruvate as an intermediate.     

Anion effects on pyruvate kinase (Short Communication)

Rabbit muscle pyruvate kinase is shown not to be activated by several anions (acetate, chloride, lactate, nitrate and sulphate) when tested as their tetramethylammonium salts. This contradicts the conclusions of Focant & Watts (1973).     

Nuclear protein kinase activity in glucagon-stimulated perfused rat livers (Short Communication)

A simple method of purification of alpha-mannosidase from jack-bean meal is described which yields a product free of beta-N-acetylglucosaminidase activity.