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.     

Pyrimidine nucleotide synthesis in Pseudomonas citronellolis

Pyrimidine biosynthesis was active in Pseudomonas citronellolis ATCC 13674 and appeared to be regulated by pyrimidines. When wild-type cells were grown on succinate in the presence of uracil, the de novo enzyme activities were depressed while only four enzyme activities were depressed in the glucose-grown cells. On either carbon source, orotic acid-grown cells had diminished aspartate transcarbamoylase, dihydroorotase or OMP decarboxylase activity. Pyrimidine limitation of glucose-grown pyrimidine auxotrophic cells resulted in de novo enzyme activities, except for transcarbamoyolase activity, that were elevated by more than 5-fold compared to their activities in uracil-grown cells. Since pyrimidine limitation of succinate-grown mutant cells produced less enzyme derepression, catabolite repression appeared to be a factor. At the level of enzyme activity, aspartate transcarbamoylase activity in P. citronellolis was strongly inhibited by all effectors tested. Compared to the regulation of pyrimidine biosynthesis in taxonomically-related species, pyrimidine biosynthesis in P. citronellolis appeared more highly regulated.     

Pyrimidine nucleotide and nucleic acid synthesis in human monocytes and macrophages.

In most cell types, the production of deoxynucleotides is tightly coupled to the pace of cell division, and nearly all deoxynucleotides are used for semiconservative DNA synthesis. The capacity of peripheral blood monocytes and macrophages to proliferate is controversial. However, these cells have been reported to produce and release thymidine, which can serve as a precursor or regulator of DNA synthesis by lymphocytes and other cells. To determine to what extent de novo pyrimidine nucleotide synthesis is linked to cell division in peripheral blood monocytes and macrophages, compared to human U937 promonocytes and CEM lymphoblasts, we used a precise precursor-product labeling method. The results showed that in all three cell types, the pace of pyrimidine deoxynucleotide production, and of thymidylate synthesis, was in proportion to the rate of DNA synthesis. The human blood monocytes and macrophages, in contrast to U937 cells, had extraordinarily low deoxyribonucleotide pools (less than 1 pmol/10(6) cells) and synthesized neither thymidylate nor DNA de novo during 7 days culture. Colony-stimulating factors augmented RNA synthesis in monocyte-derived macrophages, and enhanced cell survival, without inducing either DNA or thymidylate synthesis. We conclude that the thymidine released by macrophages derives from dead or dying cells, and not from de novo synthesis.     

Localization of nucleotide pyrophosphatase in the rat kidney

Summary Hydrolysis of NAD by a nucleotide pyrophosphatase of renal membrane fractions has been reported previously. The aim of the present study was to localize this enzyme in the rat kidney. Nucleotide pyrophosphatase was assayed in glomeruli, in three parts of the proximal tubule and in four parts of the distal tubule dissected form freezedried sections. Nucleotide pyrophosphatase activity, expressed in mol·min^–1·mg protein^–1, ranged between 9.8 and 32.3 in the proximal tubular segments and between 1.1 and 2.7 in the distal tubular segments. It was 3.4 in the glomeruli. The enrichement of the activity during the purification of brush border vesicles was measured. A tenfold higher specific activity was found in the brush border vesicles as compared to the renal cortical homogenates. Thus, most of the renal nucleotide pyrophosphatase appears to be localized in the luminal membrane of the proximal tubule. A permeabilization of the membrane did not increase the activity of brush border vesicles. This indicates that all catalytic sites are accessible at the outer surface of the membrane.Supported by the Swiss National Foundation, grant nr. 3.813.084     

Localization of nucleotide pyrophosphatase in the rat kidney

Hydrolysis of NAD by a nucleotide pyrophosphatase of renal membrane fractions has been reported previously. The aim of the present study was to localize this enzyme in the rat kidney. Nucleotide pyrophosphatase was assayed in glomeruli, in three parts of the proximal tubule and in four parts of the distal tubule dissected form freeze-dried sections. Nucleotide pyrophosphatase activity, expressed in mumol X min-1 X mg protein-1, ranged between 9.8 and 32.3 in the proximal tubular segments and between 1.1 and 2.7 in the distal tubular segments. It was 3.4 in the glomeruli. The enrichment of the activity during the purification of brush border vesicles was measured. A ten-fold higher specific activity was found in the brush border vesicles as compared to the renal cortical homogenates. Thus, most of the renal nucleotide pyrophosphatase appears to be localized in the luminal membrane of the proximal tubule. A permeabilization of the membrane did not increase the activity of brush border vesicles. This indicates that all catalytic sites are accessible at the outer surface of the membrane.     

Pyrimidine nucleotide biosynthesis in Phaseolus aureus. Enzymic aspects of the control of carbamoyl phosphate synthesis and utilization

1. Aspartate transcarbamoylase from 4-day-old radicles of Phaseolus aureus was purified 190-fold by (NH(4))(2)SO(4) fractionation, DEAE-cellulose and DEAE-Sephadex chromatography and Sephadex-gel filtration. The partially purified enzyme, which required P(i) for maximum stability, had an apparent molecular weight of 83000+/-5000. 2. Uridine nucleotides were found to inhibit the activity; UMP was the most potent inhibitor, followed by UDP and UTP. No other nucleotide was found to affect the enzyme, nor could UMP inhibition be overcome by adding another nucleotide. Aspartate gives a hyperbolic substrate-saturation curve, both with and without UMP. The nucleotide inhibitor is non-competitive with respect to this substrate. Carbamoyl phosphate also yields a hyperbolic substrate-saturation curve in the absence of feedback inhibitor, but when UMP is added a sigmoidal pattern results, and the inhibition is competitive with carbamoyl phosphate. 3. The degree of inhibition by UMP is not affected by p-chloromercuribenzoate, urea, mild heat pretreatment or change in pH over the range 8.5-10.5, but is affected by temperature. 4. The aspartate analogue, succinate, both activates and inhibits the reaction, depending on the concentrations of aspartate and succinate used. 5. Kinetic studies with the partially purified enzyme showed that the K(m) for carbamoyl phosphate (0.091 mm) is much lower than that for aspartate (1.7mm). A sequential reaction mechanism was inferred from product-inhibition kinetics, with carbamoyl phosphate binding to the enzyme before aspartate, and the product, carbamoylaspartate, being released ahead of P(i). Initial-velocity studies gave a set of parallel reciprocal plots, compatible with an essentially irreversible step occurring before the binding of aspartate.     

Characterization of particulate cyclic nucleotide phosphodiesterases of rat kidney.

Particulate cyclic nucleotide phosphodiesterases of rat kidney display some distinct kinetic and regulatory properties. Only a small portion (5–10%) of the total homogenate low K_m cyclic AMP phosphodiesterase activity (measured with concentrations of cyclic AMP less than l μm) is tightly associated with kidney membranes. Cyclic GMP phosphodiesterase activity (measured with 0.25–200 μm cyclic GMP) is readily detectable in these fractionated and washed membranes. Low concentrations of cyclic GMP stimulated the hydrolysis of cyclic AMP (K_a ～- 0.5 μM), an effect not noted in most other membrane systems. High concentrations of cyclic GMP (K_i ～- 450 μM) and cyclic AMP (K_i ～- 150 μM) inhibited the hydrolysis of each other noncompetitively. Solubilization of membrane bound activities by sonication or Sarkosyl L markedly alters enzyme kinetic properties and the responses to cyclic nucleotides and sulfhydryl reagents. Incubation of membrane fractions with dithiothreitol (5 mm) or storage of the membranes at 4 °C results in a change in extrapolated kinetic constants for cyclic AMP hydrolysis and an increase in the rate of denaturation at 45 °C. Our findings raise the possibility that regulation of membrane-bound cyclic nucleotide phosphodiesterase activity involves interactions with cyclic nucleotides themselves, as well as oxidation and reduction of disulfide bonds and membrane-enzyme interactions.     

Nicotinamide nucleotide synthesis in regenerating rat liver

1. The concentrations and total content of the nicotinamide nucleotides were measured in the livers of rats at various times after partial hepatectomy and laparotomy (sham hepatectomy) and correlated with other events in the regeneration process. 2. The NAD content and concentration in rat liver were relatively unaffected by laparotomy, but fell to a minimum, 25 and 33% below control values respectively, 24h after partial hepatectomy. NADP content and concentration were affected similarly by both laparotomy and partial hepatectomy, falling rapidly and remaining depressed for up to 48h. 3. The effect of injecting various doses of nicotinamide on the liver DNA and NAD 18h after partial hepatectomy was studied and revealed an inverse correlation between NAD content and DNA content. 4. Injections of nicotinamide at various times after partial hepatectomy revealed that the ability to synthesize NAD from nicotinamide was impaired during the first 12h, rose to a peak at 26h and fell again by 48h after partial hepatectomy. 5. The total liver activity of NAD pyrophosphorylase (EC 2.7.7.1) remained at or slightly above the initial value for 12h after partial hepatectomy and then rose continuously until 48h after operation. The activity of NMN pyrophosphorylase (EC 2.4.2.12) showed a similar pattern of change after partial hepatectomy, but was at no time greater than 5% of the activity of NAD pyrophosphorylase. 6. The results are discussed with reference to the control of NAD synthesis in rapidly dividing tissue. It is suggested that the availability of cofactors and substrates for NAD synthesis is more important as a controlling factor than the maximum enzyme activities. It is concluded that the low concentrations of nicotinamide nucleotides in rapidly dividing tissues are the result of competition between NAD synthesis and nucleic acid synthesis for common precursor and cofactors.     

Pyrimidine synthesis in tissue culture

Abstract— Myelinated cerebellar tissue culture (organ culture) was used to assess the salvage and de novo pathways of pyrimidine synthesis in mammalian brain. Radioactive orotic acid and carbamyl aspartic acid were readily incorporated into UMP and into perchloric acid-insoluble RNA. The incorporation was effectively blocked by azauridine. Neither radioactive sodium bicarbonate or citrulline was incorporated into UMP or blocked by azauridine. [³H]Uridine, on the other hand, rapidly entered the cultures, was incorporated into UMP and perchloric acid-insoluble material, and was partially inhibited by azauridine. The failure to demonstrate activity of carbamyl phosphate synthetase suggests the potential importance of the salvage pathway and the likely dependence of the brain upon exogenous and endogenous pyrimidine precursors.     

Autoradiographic studies on the proliferation of glomerular and tubular cells of the rat kidney in early diabetes

Although Osterby and coworkers have shown that the onset of diabetes is associated with glomerular hypertrophy and enlargement of the filtration surface, the pathogenesis of this lesion and the behaviour of the glomerular tuft cells during the increase in glomerular filtration surface area remain unclear. Autoradiography and electron microscopic morphometry in kidneys of male Sprague-Dawley rats with streptozotocin-induced diabetes indicate that glomerular enlargement is combined with glomerular cell hyperplasia. 6 days after continuous 3H-thymidine infusion there is a significant increase in DNA-synthesis in tuft cells indicating cell proliferation. Since podocyte volume only increases in acute diabetes (as shown by Osterby and Gundersen in 1980), and the mean podocyte nuclear area remains unchanged, we conclude that the increased DNA-synthesis results in podocyte hypercellularity so that an increased glomerular filtration surface area can be covered by a greater number of cells.     

Pyrimidine reducing enzymes of rat liver.

Dihydrouracil dehydrogenase (NADP+) (EC 1.3.1.2) was partially purified from the cytosol fraction of rat liver and fractionated by disc gel electrophoresis. A major and minor band were visualized by staining for enzyme activity. The substrate specificity of these bands was investigated. It was found that both bands were two to three times more active with dihydrothymine as substrate than with dihydrouracil in the presence of NADP+ and the optimum pH of 7.4. Mitochondrial fractions containing most of the NADH-dependent uracil reductase of rat liver cells were fractionated by centrifugation in sucrose density gradients. Two procedures involving linear or discontinuous gradients were used. By both, good separation of NADH- and NADPH- dependent reductases was achieved. Marker enzyme studies supported the view that the NADH-dependent enzyme is located principally in mitochondria whereas the NADPH-dependent enzyme is mainly in plasma and endoplasmic reticulum membranes. For the NADH-dependent reductase the apparent Km for thymine at pH 7.4 was 1.39 times that found for uracil whereas for the NADPH-dependent enzyme the apparent Km values were similar for the two substrates at this pH. Dihydrouracil was the principal product isolated by paper chromatography from the reaction mixture containing a partially purified fraction of mitochondria, uracil and NADH at pH 7.4. This fraction also catalyzed the formation of radioactive carbon dioxide from [2-14C]uracil. The proportion of CO2 formed by the mitochondria was about 10% of that formed by the original homogenate.     

Morphological changes in the rat kidney following long-term diabetes

The morphological basis of diabetic nephropathy has been studied using light and electron microscopy. Kidneys of streptozotocin-induced diabetic rats were examined on the light microscope at 4 weeks and 8 months after induction of diabetes mellitus. In addition, the 8-month diabetic kidneys were examined with the electron microscope. Renal hypertrophy was evidenced by the increase in the weight of kidneys of diabetic rats. Whilst the diabetic kidneys were approximately twice as large after 4 weeks they were only 30% larger compared to age-matched controls after 8 months of induction of diabetes. After 4 weeks, light microscopy revealed dilated tubules within the cortex of the diabetic kidneys. Light microscopy showed a significant amount of destruction of the distal convoluted tubules while electron microscopy revealed a spectrum of damage that included basement membrane thickening, loss of podocytic foot processes, disruption of tubular basal infoldings and their related mitochondria and fibrosis of the tubules 8 months after induction of diabetes. It is concluded that renal hypertrophy persists after a prolonged occurrence of diabetes but the extensive damage and loss of renal tissue including the loss of the foot processes of podocytes might be partly responsible for the clinical presentation of diabetic nephropathy.     

Pyrimidine biosynthesis in rat brain

—Studies on the incorporation of [¹⁴C]NaHCO₃ into both orotic acid and RNA in tissue slices reveal the occurrence of the complete orotate pathway for the de novo biosynthesis of pyrimidines in the rat brain. A comparison of the rates of incorporation of bicarbonate into orotic acid and RNA in tissue slices of brain and liver indicate the brain to be one-fourth to one-half as active as the liver in the de novo biosynthesis of pyrimidines. The results of this study favor the proposal that the adult rat brain can meet its needs for pyrimidines through de novo synthesis and is not dependent upon salvage activity and an extraneural supply of pyrimidines.     

Molecular cloning and nucleotide sequence of cDNA encoding the rat kidney S-adenosylmethionine synthetase.

We previously reported the isolation of a cDNA encoding the liver-specific isozyme of rat S-adenosylmethionine synthetase from a lambda gt11 rat liver cDNA library. Using this cDNA as a probe, we have isolated and sequenced cDNA clones for the rat kidney S-adenosylmethionine synthetase (extrahepatic isoenzyme) from a lambda gt11 rat kidney cDNA library. The complete coding sequence of this enzyme mRNA was obtained from two overlapping cDNA clones. The amino acid sequence deduced from the cDNAs indicates that this enzyme contains 395 amino acids and has a molecular mass of 43,715 Da. The predicted amino acid sequence of this protein shares 85% similarity with that of rat liver S-adenosylmethionine synthetase. This result suggests that kidney and liver isoenzymes may have originated from a common ancestral gene. In addition, comparison of known S-adenosylmethionine synthetase sequences among different species also shows that these proteins have a high degree of similarity. The distribution of kidney- and liver-type S-adenosylmethionine synthetase mRNAs in kidney, liver, brain, and testis were examined by RNA blot hybridization analysis with probes specific for the respective mRNAs. A 3.4-kilobase (kb) mRNA species hybridizable with a probe for kidney S-adenosylmethionine synthetase was found in all tissues examined except for liver, while a 3.4-kb mRNA species hybridizable with a probe for liver S-adenosylmethionine synthetase was only present in the liver. The 3.4-kb kidney-type isozyme mRNA showed the same molecular size as the liver-type isozyme mRNA. Thus, kidney- and liver-type S-adenosylmethionine synthetase isozyme mRNAs were expressed in various tissues with different tissue specificities.     

Circadian variations in pyrimidine nucleotide synthesis in rat liver

The biosynthesis of pyrimidine components in rat liver varies with the time of the day. The concentrations of both the cytidine and the uridine components of the acid-soluble extract are lowest in the morning hours and highest around midnight. The utilization of [2-¹⁴C]orotic acid for the synthesis of the pyrimidine components of the acid-soluble extract, RNA, and DNA has a similar character. Analogous changes also are seen in the uptake of [U-¹⁴C]cytidine and its utilization for the synthesis of RNA cytosine.     

Perinatal changes in adenine nucleotide content of developing rat kidney

Adenine nucleotides have been measured in fetal and newborn kidneys of rat using the luciferine-luciferase method. In fetuses, between days 18 and 21 of gestation there is a drop of the relative amount of ATP in the renal nucleotide pool. Consequently, the kidneys of 21 days-old fetuses have lowered ATP/ADP ratio (3.6) and energy charge (0.80) compared with values found on day 18 (6.9 and 0.91, respectively); this relative energy deficit is heightened in progesterone induced postmaturity. One hour after delivery whether the gestational stage is 21, 22 or 23 days, there is a rise in ATP and a decrease in AMP content which restore a high energy level in kidney of the newborn and a 30% increase in the total adenine nucleotide pool.     

Influence of testosterone on purine nucleotide turnover in rat kidney

The influence of testosterone on purine nucleotide metabolism in rat kidney has been investigated in adult and in prepubertal castrated rats. Results have been evaluated through biomathematical model. Castration enhanced the turnover of purine nucleotides in adult rats and reduced it in young castrated rats. Treatment with testosterone in the castrated rats further enhanced nucleotide turnover both in the adult rats and also in the second group, with an oscillatory profile. A clear effect on the inosinic branch point was demonstrated, and specifically on GMP formation, which was opposite according to the age of the animal. The different behavior in the two groups after castration was partially ascribed to the action of other hormones in the absence of testosterone. The observed changes show that the action of the hormone is not limited to sexual organs; they might be at the basis of variations in cellular size and number which probably occur in the kidney after orchiectomy and following androgen administration.