Pyrimidine metabolism and sugar nucleotide synthesis in rat liver.

With radioactive precursors, the labelling kinetics of the soluble pyrimidine nucleotides and of RNA were measured in rat liver to determine the contribution of the metabolic flows through synthesis de novo and the salvage pathway. To separate and quantify all pyrimidine nucleotides, an h.p.l.c. technique was developed using anion-exchange chromatography and reversed-phase chromatography. The concentrations of cytidine nucleotides were in the range of 30-45 nmol/g wet weight, and the concentrations of the uridine phosphates and of the UDP-sugars were approx. 6 and 20 times higher respectively. After a single injection of [14C]orotic acid and of [3H]cytidine, the specific radioactivities were determined as a function of time. The 14C/3H ratio was calculated and gave a good indication of the involvement of the different flows. It could be concluded that UTP derived from synthesis de novo and from the salvage pathway is not completely mixed before being utilized. The flow of the salvage pathway is relatively more directed to RNA synthesis in the nucleus and that of synthesis de novo to cytoplasmic processes. For CTP it could also be concluded that the flow of the salvage pathway was relatively more directed to RNA synthesis in the nucleus. Because of the nuclear localization of the enzyme CMP-NeuAc (N-acetylneuraminate) synthase, special attention was paid to CMP-NeuAc. However, a conclusion about a location about the synthesis of CMP-NeuAc could not unequivocally be drawn, because of the small differences in 14C/3H ratio and the different values for the CDP-lipids.     

Acid inactivation of short-lived rat liver enzymes.

The stabilities of nine rat liver cytosol enzymes were compared at a variety of pH values. The cytosol enzymes studied were (a) those with half-lives in vivo of 3 days or longer: lactate dehydrogenase, arginase, glyceraldehyde phosphate dehydrogenase and alanine aminotransferase, (b) those with half-lives in vivo shorter than 2 days; glucokinase, dihydroorotase, serine dehydratase and tyrosine aminotransferase and (c) catalase, which has an intermediate half-life of 2.5 days for the protein protion. All the enzymes were stable in vitro at neurtal and alkaline pH values. However, at acidic pH values (pH 4): the long-lived enzymes (a) were stable; the short-lived enzymes (b) were completely inactivated with one exception; and catalase was partially inactivated. Tyrosine aminotransferase was the exception in that it is a short-lived enzyme in vivo but stable under all conditions tested in vitro. The finding that long-lived enzymes are stable in an acid milieu and short-lived enzymes are generally unstable was only observed if certain ligands (NAD+, pyridoxal 5'-phosphate, Mn2+, amino acids) were added to the invitro system. Lysosomal extracts did not accelerate the rate of inactivation of any cytosol enzyme in acidic solutions. These results indicate that if degradation of intracellular enzymes occurs in lysosomes, acid inactivation and denaturation of enzymes may be the initial event in determining the functional half-lives of the enzymes in vivo.     

Pyrimidine biosynthetic enzymes in Babesia hylomysci

Gero A. M. and Coombs G. H. 1982. Pyrimidine biosynthetic enzymes in Babesia hylomysci. International Journal for Parasitology12: 377–382. The enzymes that catalyse the conversion of carbamylaspartate to UMP have been demonstrated in the rodent piroplasm, Babesia hylomysci and partially characterised. They were shown to be similar to the corresponding mammalian enzymes in that dihydroorotase, orotate phosphoribosytransferase and orotidine-5'-phosphate decarboxylase were soluble, whilst dihydroorotate dehyrogenase was membrane bound and appeared to be associated with a respiratory chain. Dihydroorotate dehydrogenase was found to have a K_m (l-DHO) of 21 μm and a pH optimum of 7.8. It was inhibited by analogs of ubiquinone and several pyrimidines, dihydroazaorotate being the most effective ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 17 μ}\text{m}$). It is concluded that Babesia parasites contain a functional de novo biosynthetic pathway for pyrimidines which provides a potential target at which to direct putative chemotherapeutic agents.     

Effects of lithium on inducible enzymes of rat liver.

Chronic lithium administration to female rats results in elevation of serum corticoids, lowering of serum glucose and altered induction of liver enzymes. The cortisol induction of tyrosine transaminase is increased selectively over that of tryptophan oxygenase. The glucose induction of glucokinase following a fast is increased by chronic lithium treatment but diminished by acute treatment. These results indicate that lithium may alter the glucose metabolic set-point in rats.     

The development of oxidative enzymes in rat liver mitochondria.

During early postnatal development there was an increase in the specific activity of a number of oxidative enzymes localized on the outer and inner mitochondrial membrane. The succinic oxidase complex of the inner mitochondrial membrane, whose activity in 1-day-old rats was 50% of the value in adult animals, attained the maximum on about the 10th day after birth. Activity of the choline and the proline oxidase complex, both of which are also localized in the inner mitochondrial membrane, was minimal in 1-day-old rats and went on rising after the 10th day. Rotenone-insensitive NADH-cytochrome c reductase activity, which is localized on the outer mitochondrial membrane, remained stable up to the 10th day, and rose between the 10th and the 90th day. Developmental changes in monoaminooxidase activity, which is likewise localized on the outer mitochondrial membrane, followed a similar course to the choline and proline oxidase complexes. The amount of cytochromes a+alpha3 and cytochrome b in isolated mitochondria did not alter during development. The protein spectrum of the mitochondrial particles, determined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate, likewise displayed no marked changes during postnatal development. The above findings show that the metabolic functions of the mitochondria mature during development and that changes in the different enzymes have their own characteristic time course.     

Further purification and characterization of diacetyl reducing enzymes from beef liver

• 1.1. Two diacetyl reducing enzymes have been isolated from beef liver. One of them, a monomer of mol. wt 28-30,000 dalton and pI 6.2, corresponds to the low moleclular weight diacetyl reductase formerly accounted for using preparations of this organ; it has been now identified as an l-glycol dehydrogenase.
• 2.2. The other one, an oligomer of 78,000 dalton and pI 7.0, which matches the high molecular weight diacetyl reductase, is, in the authors' opinion, a new enzyme for which the systematic name L(+)-α-hydroxycarbonyl: NAD(P) oxidoreductase (EC 1.1.1...) and common name α-dicarbonyl reductase are proposed.

     

Acid inactivation of short-lived rat liver enzymes

The stabilities of nine rat liver cytosol enzymes were compared at a variety of pH values. The cytosol enzymes studied were (a) those with half-lives in vivo of 3 days or longer: lactate dehydrogenase, arginase, glyceraldehyde phosphate dehydrogenase and alanine aminotransferase, (b) those with half-lives in vivo shorter than 2 days; glucokinase, dihydroorotase, serine dehydratase and tyrosine aminotransferase and (c) catalase, which has an intermediate half-life of 2.5 days for the protein portion. All the enzymes were stable in vitro at neutral and alkaline pH values. However, at acidic pH values (pH 4): the long-lived enzymes (a) were stable; the short-lived enzymes (b) were completely inactivated with one exception; and catalase was partially inactivated. Tyrosine aminotransferase was the exception in that it is a short-lived enzyme in vivo but stable under all conditions tested in vitro. The finding that long-lived enzymes are stable in an acid milieu and short-lived enzymes are generally unstable was only observed if certain ligands (NAD⁺, pyridoxal 5′-phosphate, Mn²⁺, amino acids) were added to the iv vitro systems. Lysosomal extracts did not accelerate the rate of inactivation of any cytosol enzyme in acidic solutions. These results indicate that if degradation of intracellular enzymes occurs in lysosomes, acid inactivation and denaturation of enzymes may be the initial event in determining the functional half-lives of the enzymes in vivo.     

Postnatal development of peroxisomal and mitochondrial enzymes in rat liver.

Subcellular organellles from livers of rats three days prenatal to 50 weeks postnatal were separated on sucrose gradients. The peroxisomes had a constant density of 1.243 g/ml throughout the life of the animal. The density of the mitochondria changed from about 1.236 g/ml at birth to a constant value of 1.200 g/ml after two weeks. The peroxisomal and mitochondrial fatty acid beta-oxidation and the peroxisomal and supernatant activities of catalase and glycerol-3-phosphate dehydrogenase were measured at each age, as well as the peroxisomal core enzyme, urate oxidase, and the mitochondrial matrix enzyme, glutamate dehydrogenase. All of these activities were very low or undetectable before birth. Mitochondrial glutamate dehydrogenase and peroxisomal urate oxidase reached maximal activities per g of liver at two and five weeks of age, respectively. Fatty acid beta-oxidation in both peroxisomes and mitochondria and peroxisomal glycerol-3-phosphate dehydrogenase exhibited maximum activities per g of liver between one and two weeks of age before weaning and then decreased to steady state levels in the adult. Peroxisomal beta-oxidation accounted for at least 10% of the total beta-oxidation activity in the young rat liver, but became 30% of the total in the liver of the adult female and 20% in the adult male due to a decrease in mitochondrial beta-oxidation after two weeks of age. The greatest change in beta-oxidation was in the mitochondrial fraction rather than in the peroxisomes. At two weeks of age, four times as much beta-oxidation activity was in the mitochondria as in the peroxisomal fraction. Peroxisomal glycerol-3-phosphate dehydrogenase activity accounted for 5% to 7% of the total activity in animals younger than one week, but only 1% to 2% in animals older than one week. Up to three weeks of age, 85% to 90% of the liver catalase was recovered in the peroxisomes. The activity of peroxisomal catalase per g of rat liver remained constant after three weeks of age, but the total activity of catalase further increased 2.5- to 3-fold, and all of the increased activity was in the supernatant fraction.     

Repair of depurinated DNA with enzymes from rat liver chromatin.

DNA from T7 phage containing AP (apurinic/apyrimidinic) sites was repaired by the successive actions of three chromatin enzymes [AP endodeoxyribonuclease, DNAase IV (5'----3'-exodeoxyribonuclease) and DNA polymerase-beta] prepared from rat liver and T4-phage DNA ligase. Since DNA ligase is also found in rat liver chromatin, all the activities used for the successful repair in vitro are thus present in the chromatin of a eukaryotic cell. Our results show, in particular, that the chromatin DNAase IV is capable of excising the AP site from the DNA strand nicked by the chromatin AP endodeoxyribonuclease. We did not try to combine all the enzymes, since competition between some of them might have prevented the repair; we have, for instance, shown that DNA ligase can seal the incision 5' to the AP site made by the AP endodeoxyribonuclease. Changes in chromatin structure during repair might perhaps prevent this competition when nuclear DNA is repaired in the living cell.     

Distribution of lysosomal enzymes in different types of rat liver cells.

Eight lysosomal enzymes were measured in different types of rat liver cells. Hepatocytes were purified by low speed centrifugation of a cell suspension obtained by treating the perfused liver with collagenase. Nonparenchymal cells (NPC) were purified by centrifugation after treating the initial cell suspension with pronase, which selectively destroys the parenchymal cells (PC). Kupffer cells were found to attach selectively to tissue culture dishes after overnight culture of an NPC suspension. The specific activity of lysosomal enzymes was generally higher in NPC than in hepatocytes, but the different enzymes were concentrated to different degrees in the NPC. Specific activity of acid phosphatase was 1.7 times higher in NPC than in hepatocytes. Specific activity of acid DNAase, on the other hand, was 8 times higher in NPC than in hepatocytes. Other enzymes showed intermediate values. Assuming that 30% of the liver cells are nonparenchymal it may be calculated that from 7% (acid phosphatase) to 25% (acid DNAase) of the hepatic lysosomal enzymes are present in the NPC. The pattern of lysosomal enzymes in cultured Kupffer cells was similar to that of the NPC from which the Kupffer cells were derived. Cathepsin D and β-glucuronidase were, however, elevated in Kupffer cells as compared with NPC. The enzyme pattern in Kupffer cells was almost identical with that of rat peritoneal macrophages.     

On the presence of two non-specific nucleotide-sugar-hydrolysing enzymes in rat liver.

Evidence is presented for the occurrence of two different non-specific nucleotide-sugar hydrolases in rat liver and other rat tissues. These two enzymes (I and II) were separated by chromatography on a 5'-AMP-aminohexyl-Sepharose column. Enzyme I is most probably identical with phosphodiesterase I (EC 3.1.4.1). Enzyme II appeared to be identical with an enzyme described in literature as 'CMP-sialic acid hydrolase' [Kean & Bighouse (1974) J. Biol. Chem. 249, 7813-7823], since almost all activity with CMP-N-acetylneuraminate as substrate was recovered in this enzyme fraction. CMP-N-acetylneuraminate was a poor substrate for Enzyme I, whereas deoxythymidine-5'-p-nitrophenyl phosphate and all nucleoside-diphosphosugars tested were good substrates for both Enzyme I and II. Therefore it is suggested that CMP-N-acetylneuraminate is used as an additional substrate to discriminate between the activities of Enzyme I and II in homogenates or membrane preparations. The various substrates appeared to be competitive inhibitors of each other, suggesting that, in each enzyme preparation, only one enzyme is responsible for the hydrolysis of the various substrates. The dissimilar properties of the two enzymes are substantiated by studying the subunit molecular masses (Enzyme I, 125 kDa; Enzyme II, 50-55 kDa), the sensitivity towards Triton X-100, Sarkosyl and sodium dodecyl sulphate and towards trypsin treatment. It is discussed whether the alpha-N-acetylglucosamine phosphodiesterase described by Varki & Kornfeld [(1981) J. Biol. Chem. 256, 9937-9943] is identical with one of the nucleotide-sugar hydrolases described here.     

The nucleoids of rat liver cell microbodies. Fine structure and enzymes

The nucleoids of microbodies of rat liver cells were isolated in a highly homogeneous and pure state, by treating the microbody-rich fraction, prepared from 10% polyvinylpyrrolidone-0.25 M sucrose homogenate, with Triton X-100. Three treatments with 0.1% detergent were enough to render the nucleoids free from contamination with mitochondria, microsomes, lysosomes, and intact microbodies. Electron microscopically, the nucleoids were found to consist of parallel bundles of highly dense hollow tubules, the outer and inner diameters of which are approximately 150 and 50 A, respectively. Ten tubules are arranged around a longitudinal space 190 x 200 A in width. The nucleoids thus show a honeycomb appearance in the cross-plane and a parallel-packed structure in the longitudinal plane. Biochemically, the nucleoids were found to bear only urate oxidase among probably microbody-enzymes, and they might be the only cytoplasmic particles of rat liver cells in which the enzyme locates. Urate oxidase activity, on a unit protein basis, of the nucleoid preparation is approximately 380 times as high as that of the whole homogenate, and is almost comparable with that of a commercial type I enzyme preparation. No enzymes of mitochondrial, microsomal, and lysosomal origins were detected in the nucleoids. The fine structure of the nucleoids is described in detail, and a probable schematic diagram is presented.