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At least 19 recordsLinked to original sources
PubMed1998

Increased uridine kinase (ATP: uridine 5'-phosphotransferase; EC 2.7.1.48) activity in human and rat tumors.

The activity of uridine kinase (ATP: uridine 5'-phosphotransferase; EC 2.7.1.48), the rate-limiting enzyme of the UMP salvage pathway, was measured in human ovaries and ovarian carcinomas, in a spectrum of six rat hepatomas of different growth rates and in eleven normal rat tissues of high and low cell renewal rates. In a standard isotopic method developed for the 100,000 x g fraction, uridine kinase activity was linear for 20 min and proportional with protein concentration over a range of 0.1 to 0.8 mg per 0.1 ml reaction mixture. The apparent Kms for uridine, ATP and Mg++ in normal rat liver were 5.0, 3.4 and 1.5 mM and in the rapidly growing hepatoma 3924A, 0.8, 2.1 and 1.1 mM, respectively. In normal control ACl/N and Buffalo strain rat livers, kinase activity ranged from 159 to 180 nmol/h/mg protein. In hepatomas of slow and intermediate growth rates, kinase activity increased to 1.5- to 2.6-fold, and in hepatomas of rapid growth rates, to 5.1- to 5.8-fold over that of the relevant control, normal livers. When hepatoma 3924A tissue culture cells were plated and expressed their proliferative program, kinase activity increased to 2.1-fold in early log phase. To further clarify the linkage between uridine kinase and cell replicating capacity, the enzyme activity was measured in rat organs of high and low cell renewal. The kinase activity in liver of adult male Wistar rats was 176 +/- 6 nmol/h/mg protein. Activities in thymus, spleen and bone marrow were 4.7-, 2.1-, and 1.8-fold, respectively, of rat liver values; in adipose tissue, the activities were low. The decay rates of uridine kinase were examined in rats injected with a high dose of cycloheximide, which inhibits protein biosynthesis by 90%. The t(1/2) of the kinase in rat bone marrow was 0.64 h, in rat liver longer than 6 h. In human ovary and ovarian carcinoma, the apparent Kms for uridine were 11.5 and 0.5 mM, respectively. In human ovary (n = 3), kinase activity was 38 nmol/hr/mg protein; in ovarian carcinoma (n = 6), the activity increased to 5- to 13-fold over that in ovary. The positive linkage of uridine kinase activity with proliferation and transformation is apparent in human ovarian carcinomas and in rat hepatomas of different growth rates. Therefore, the increased uridine kinase activity should be an interesting target for anticancer chemotherapy.

Animals↗
PubMed1985

Inhibition of uridine kinase and the salvage of uridine by modified pyrimidine nucleosides.

Uridine kinase can play a crucial role in the provision of pyrimidine nucleotides for cellular nucleic acid synthesis, particularly when de novo synthesis is inhibited by chemotherapeutic agents. Therefore, uridine kinase is an attractive target for drug development. We examined a series of 29 analogs of uridine, most with modifications at the 5'-position, as inhibitors of uridine kinase in vitro and of uridine salvage by intact L1210 cells. Substitution at the 5'-position resulted in decreased efficacy as inhibitors of uridine kinase, particularly if the substituent was large. None of the analogs with 5'-position modifications effectively inhibited salvage of uridine by intact L1210 cells. Four carbocyclic pyrimidine nucleoside analogs (one series) were all effective competitive inhibitors of uridine kinase and of uridine salvage by intact L1210 cells. Cyclopentenyl uracil 19 shows promise for further development as it inhibits uridine salvage at nontoxic concentrations.

Animals↗
PubMed1982

Cytotoxicity of a new uridine analog, 4-hydroxy-1-(beta-D-ribofuranosyl)-pyridazine-6-one, and its interaction with uridine kinase.

A new uridine analog, 4-hydroxy-1-(beta-D-ribonfuranosyl)-pyridazin-6-one (3-deaza-6-azaUrd), inhibited the growth of L1210 cells in culture, with a concentration to reduce growth rate to 50% of control of 7 X 10(-5) M. After treatment for 24 or 48 h with 5 X 10(-4) M 3-deaza-6-azaUrd, 80% of the cells were unable to resume growth when the analog was removed from the cultures; also, 99% of the cells were killed, as determined by colony formation in soft agar. Studies on the prevention of the cytotoxic effects of 5 X 10(-4) M 3-deaza-6-azaUrd showed that uridine or cytidine gave complete protection. 2'-Deoxycytidine also gave partial protection, but orotic acid or thymidine had no effect on the growth inhibition by 3-deaza-6-azaUrd. These results suggested that growth inhibition by 3-deaza-6-azaUrd might be due to interference in pyrimidine biosynthesis. Activation of 3-deaza-6-azaUrd to its 5'-phosphate derivative appeared to be catalyzed by uridine kinase. 3-Deaza-6-azaUrd was shown to complete with uridine for phosphorylation (Ki = 4.7 mM) and, therefore, to be a possible alternative substrate for uridine kinase from mouse kidney (Km for uridine = 82 microM). The enzyme was partially purified by streptomycin sulfate precipitation, ammonium sulfate fractionation, and gel filtration. This preparation was found to be free of pyrimidine nucleoside phosphorylase and uridine monophosphate kinase.

Animals↗
PubMed1984

Uridine kinase molecular species and uridine uptake in some variants of rat hepatomas.

In a family of clonal lines derived from the Reuber H 35 rat hepatoma, four electrophoretically distinct molecular forms of uridine kinase (UK I, II, III, and IV) have been characterized. They are the same as those found in foetal rat liver. Different UK profiles occur in these cell lines, and no strict correlation could be established between the state of differentiation of the cells and the form of UK expressed. A clone of somatic hybrid cells between line p4 (form 1 only) and Fu5-5 (forms II, III, and IV) that does not express form I indicates that p4 cells may lack a factor controlling the polymerization of form I. This variety of clonal cell lines was used to study the uptake and phosphorylation of labeled uridine. The results suggest a relationship between the UK form present and the rate uridine phosphorylation by the intact cells.

Animals↗
PubMed1996

Cloning and expression of a cDNA encoding uridine kinase from mouse brain.

Uridine kinase is the rate-limiting enzyme in the pyrimidine salvage pathway of all mammalian cells. A cDNA for uridine kinase from mouse brain has been isolated, sequenced, and characterized. This is the first report of a complete nucleotide sequence for mammalian uridine kinase. The isolated cDNA is only 95% complete, missing the first 17 codons. The correct 5'-terminus sequence was obtained from high-stringency screening of a mouse liver genomic DNA library. The translated cDNA sequence encodes a protein of 277 amino acids (Mr 31,068). A truncated form of the cDNA was expressed in Escherichia coli. The expressed protein displayed uridine kinase activity and readily formed a tetramer, the most active form of the wild-type enzyme. Analysis of the amino acid sequence identified the three ATP-binding site consensus motifs. The predicted secondary structure for uridine kinase and the sequence comparison with three kinases having known crystal structures are consistent with uridine kinase having an alpha/beta core structure of the nucleotide-binding fold found in many kinases. We have also isolated and cloned a nonfunctional, processed pseudogene from mouse genomic DNA. This pseudogene sequence is 94% identical with the coding DNA.

Amino Acid Sequence↗
PubMed1981

[Isolation and properties or uridine kinase from Zajdela hepatoma cells].

Uridine kinase (ATP: uridine-5-phosphotransferase, EC 2.7.1.48) was isolated from cytosol of rat Zajdela ascite hepatoma cells by fractionation with ammonium sulfate and gel-filtration on Sephadex G-200. The enzyme has a pH optimum of 7.2 - 7.8; Km for uridine is 4.8 . 10(-5) M, that for ATP - 1.9 . 10(-4) M. The optimal ratio of ATP of Mg2+ is 2.6. The enzyme activity is inhibited by end products of pyrimidine biosynthesis with Ki for CTP of 6.0 . 10(-4) M and for UTP of 1.2 . 10(-3) M. The Ki values for uridine competitive analogs, i. e. 6-azauridine, 5-bromuridine and 5-azacytidine are equal to 4.0 . 10(-4) M, 1.5 . 10(-3) M and 2.5 . 10(-3) M, respectively. Further purification of the enzyme on Sepharose 4B allowed to obtain the most active, although heterogeneous fractions purified 86-fold, with specific activity of 11.2 mkmole/hour per mg of protein. Using electrofocusing, uridine kinase was found to consist of two major and one minor active fractions with pH of 6.2, 6.7 and 6.35, respectively. Chromatography on DEAE-cellulose DE-32 resulted in two major active fractions of the enzyme, differing in thermal stability and inhibition by CTP. It may be concluded that Zajdela ascite hepatoma cells contain at least two isoforms of uridine kinase.

Animals↗
PubMed1998

Uridine kinase: altered enzyme with decreased affinities for uridine and CTP.

Uridine kinase is the rate-limiting enzyme in the salvage pathway for uridine or cytidine of mammalian cells. Alignment of the uridine kinase sequence with other nucleoside and nucleotide kinases supports a common ancestor for all of these. Three polypeptide segments for the ATP site and three polypeptide segments for the acceptor nucleoside site have been identified. We report here the characterization of an altered form of the enzyme with a single amino acid change, Q146R, within or near the uridine-binding site. This single amino acid change leads to a 160-fold increase in Km for uridine (Km = 6.5 mM) and a decrease in kcat by more than 99%. This variant has normal affinity for ATP (Km = 130 microM), but shows substrate inhibition at ATP concentrations >3 mM. Mouse uridine kinase is normally an active tetramer that will dissociate to inactive monomers in response to CTP. In contrast, the altered protein is monomeric, but will associate to dimers and then to tetramers with increasing ATP. The Q146R enzyme has a 100-fold loss in affinity for the allosteric inhibitor CTP; this supports a model for CTP inhibition being caused by CTP binding backward at the catalytic site, as a bisubstrate analog.

Allosteric Regulation↗
PubMed1975

Non-allosteric regulation of the uridine kinase from seeds of Zea mays.

Uridine kinase (ATP: uridine 5'-phosphotransferase, EC 2.7.1.48) has been partially purified from ungerminated hybrid corn seed. It is associated with a soluble high molecular weight fraction from which it apparently cannot be dissociated without loss of activity. The stability of the enzyme is enhanced by the addition of dithiothreitol, glycerol and nucleotide substrate. The nucleoside specificity of the enzyme is limited to nucleosides containing pyrimidine and ribose moieties, such as uridine and cytidine. High concentrations of nucleosides cause substrate inhibition, however. The Km values for uridine and cytidine are 53 muM and 125 muM, respectively, and under subsaturating conditions uridine is phosphorylated about five times faster than cytidine. The reaction follows an ordered Bi Bi kinetic pattern, with ATP and ADP in competition for the free form of the enzyme. Purine, but not pyrimidine, nucleoside triphosphates serve as phosphate donors without regard to the sugar moiety. However, all of these triphosphates appear to compete for the same site on the enzyme. (Km ATP equals 590 muM, Km (app) GTP equals 61 muM, and CTP and UTP are linear competitive inhibitors against ATP, with Ki values of 60 muM and 240 muM, respectively.) Therefore, end product control of uridine kinase apparently does not involve allosteric sites, but instead is envisioned as simple competition between relatively effective or ineffective phosphate donors for a position on the enzyme.

Kinetics↗
PubMed1986

Regulation of uridine kinase. Evidence for a regulatory site.

Uridine kinase from mouse Ehrlich ascites tumor cells may exist at 4 degrees C in multiple aggregation states that only slowly equilibrate with one another. Increasing the temperature leads to dissociation, and the appearance of a single predominant species: at 22 degrees C the enzyme exists as a tetramer. There is also a break in the dependence of enzyme activity on temperature as measured in an Arrhenius plot. The feedback inhibitors CTP and UTP cause the enzyme to dissociate to the monomer, whereas the substrate ATP reverses this process. Kinetic studies show that the monomer has little or no activity. Studies of the reaction mechanism show that binding of substrates is ordered, leading to a ternary complex, and release of products is ordered: uridine is the first substrate bound, ADP the first product released. Except for the inhibitors UTP and CTP, all other nucleoside triphosphates, whether purine or pyrimidine, or containing ribose or deoxyribose, act as phosphate donor. Especially interesting are the opposite effects of CTP and dCTP on uridine kinase: unlike CTP, dCTP does not dissociate the enzyme and is competent as a phosphate donor. We propose that the various effects of different ligands are best explained by the existence of a regulatory site (with more stringent specificity than the catalytic site) that controls dissociation of uridine kinase to the inactive monomer.

Adenosine Triphosphate↗
PubMed1988

Cellular distribution, developmental changes and effects of cryptorchidism on uridine kinase in the rat testis.

High specific activity of uridine kinase was found in cultured peritubular cells (3.0 nmol/min per mg protein) which was more than 3-fold higher than that found in cultured Sertoli cells (0.79 nmol/min per mg protein). In the various classes of germ cells a decrease in specific uridine kinase activity was associated with increased maturity of the cells, primary spermatocytes, round spermatids and spermatozoa showing 1.3, 0.65 and 0.16 nmol/min per mg protein, respectively. A relationship between uridine kinase activity and the rate of RNA synthesis in these cells is suggested. A decrease in specific uridine kinase activity in testis with increasing age supports the finding of lower uridine kinase in mature germ cells than in earlier germ cells and somatic cells. This finding is further supported by the observation that cryptorchidism, which is associated with a time-dependent depletion of germ cells, resulted in an increase in specific uridine kinase activity. The results indicate that pyrimidine salvage is important in earlier germ cells, as well as in somatic cells in the testis, to produce substrates for nucleic acid synthesis.

Animals↗
PubMed1982

Regulation of uridine kinase quaternary structure. Dissociation by the inhibitor CTP.

Uridine kinase from mouse Ehrlich ascites cells can exist in a variety of different aggregation states, from monomer up to aggregates that may contain 32 or more subunits. With very crude enzyme preparations, uridine kinase activity is always associated with several different coexisting molecular weight species. Changes in the aggregation state are produced in the presence of normal effectors (orthophosphate, ATP and CTP) at physiological concentrations. With uridine kinase that has been purified 9,000-fold, enzyme activity is associated with only a single molecular weight species, but is still responsive to the same physiological effectors. In the presence of orthophosphate, uridine kinase has a molecular weight of 380,000 (appropriate for a dodecamer). In the presence of CTP, the enzyme dissociates with concomitant loss of activity. The dissociated enzyme can be reassociated to the native size. These results imply that alteration of the enzyme's quaternary structure by normal effectors constitutes a mechanism for regulating uridine kinase activity in vivo.

Animals↗
PubMed1980

Uridine kinase, adenylate kinase, and guanase in human lung tumors.

In pulmonary neoplasms, the uridine kinase concentration was higher (2- to 20-fold) than in the noninvolved lung portions of each of the 12 subjects studied. The extent of elevation of uridine kinase in the different tumors showed a significant positive correlation with the rises (1.5- to 30-fold) in thymidine kinase, suggesting that neoplastic transformation in human lung involved coordinated increases in the capacity for the reutilization of different nucleoside phosphates. Adenylate kinase was always at lower levels in neoplasms compared to noninvolved areas of the same lung, and the extent of this loss in the different tumors correlated inversely with the gain in uridine kinase and thymidine kinase. Normal fetal human lung was also deficient in adenylate kinase, while its uridine kinase and thymidine kinase (and also guanase) activities were above the adult levels. The guanase activities of the different neoplasms, unrelated to their uridine kinase or thymidine kinase content, correlated with the activities in the subjects' noninvolved lung. These individual differences were much more striking than those between the neoplastic and control samples. Variations in guanase activity thus appear to be "random," whereas observations on the three other enzymes attest to the orderly nature of biochemical differences among individual tumors and between normal and neoplastic lung.

Adenocarcinoma↗
PubMed1987

Uridine kinase: altered subunit size or enzyme expression as a function of cell type, growth stimulation, or mutagenesis.

Using antibody prepared against pure uridine kinase from Ehrlich ascites cells, we have measured the expression of enzyme protein by the Western blot technique. Variations were observed in the Mr of the enzyme subunit for uridine kinase from different species: 32,000 (mouse Ehrlich ascites cells), 30,000 (normal human lymphocytes), 28,000 (mouse tissues), 27,500 (rat tissues). For different normal tissues from the same species, there was no significant variation in the subunit size. Transformed human and mouse cell lines, selected for a deficiency of uridine kinase activity in the presence of inhibitors activated by this enzyme, expressed two cross-reacting proteins, one with a normal (30,000) and one with a smaller (21,000) subunit molecular weight than was found in the parental cell line (human lymphoma), or only a smaller protein of Mr 25,000 (mouse lymphoma). Our results show that selection protocols using metabolite inhibitors do not always repress the expression of the enzyme but instead may lead to selection of those cells that have a mutation in the uridine kinase gene, resulting in the expression of an inactive enzyme. The expression of uridine kinase protein changes when cells are stimulated to divide. For both mouse fibroblasts and human lymphocytes, expression of uridine kinase protein as well as activity clearly increased after cells were stimulated to grow. In fibroblasts, increases are seen by 3 hr after stimulation, and plateau after 9 hr at a sevenfold increase. In lymphocytes, no change is seen until 12 hr after stimulation, and a plateau is not reached until 72 hr, with a total increase of approximately 50-fold. There has been considerable interest in the possibility of uridine kinase isozymes. Except for cells that have been mutagenized, the present results show that, as judged by subunit molecular weight, there appears to be only one enzyme form in normal and neoplastic cells or in cells in which uridine kinase activity is induced.

Animals↗
PubMed1976

Stability of the insoluble form of uridine kinase coupled to zn2+ or pb2+ ions.

Partially purified calf brain uridine kinase precipitated by bivalent metal cations has been compared with the soluble enzyme fraction regarding its stability in the presence of inactivating factors. The freeze-dried preparations of uridine kinase precipitaated by Pb2+ or Zn2+ ions, althouth enzymatically highly active, are insoluble in aqueous solutions. The activity of metal-insolubilized enzymes disappears during their preincubation in acidic media or in the presence of silver ions. Also trypsin, chymotrypsin and cathepsin B1 caused decreases in enzyme activity. However, fractions which have been precipitated by metal ions and freeze-dried are stable at high temperatures, whereas the activity of soluble uridine kinase is completely lost. Both unheated metal-ion precipitated uridine kinase preparations and those heated at 100 degrees C are equally sensitive to the feedback inhibition by CTP.

Animals↗
PubMed1980

Effects of thyroid hormone on UTP content and uridine kinase activity of rat heart and skeletal muscle.

In rats made hyperthyroid by daily intramuscular injections of 250 microgram thyroxine (T4)/100 g body wt for 5 days, uridine kinase activity of extracts of psoas and cardiac muscle was markedly increased Vmax of the enzyme was elevated with no change in the apparent Km for uridine. In animals treated as above, significant increases in UTP and total uracil nucleotide contents were observed in heart and skeletal muscle. Twelve hours after a single intraperitoneal injection of 30 microgram/100 g body wt of 3,5,3'-triiodothyronine (T3), cardiac uridine kinase was significantly increased. Brain uridine kinase was unaffected by thyroid hormone treatment. In thyroidectomized rats, uridine kinase activity was lower than normal. The effect of thyroidectomy on uridine kinase activity was overcome by daily subcutaneous injections of 3 microgram T4/100 g body wt for 7 days. The rise in cardiac uridine kinase activity produced by T3 could be prevented by prior administration of actinomycin D.

Animals↗