Kinetic properties of a nucleoside phosphotransferase of chick embryo

1. A nonspecific nucleoside phosphotransferase (nucleotide : 3'-deoxynucleotide 5'-phosphotransferase, EC 2.7.1.77), purified from chick embryos, catalyzes the transfer of phosphate ester from a nucleotide donor to a nucleoside acceptor. 2. The enzyme exhibits sigmoidal kinetics with respect to nucleoside monophosphate donors, but with respect to nucleoside di- or triphosphate donors and nucleoside acceptors hyperbolic kinetics were obtained. 3. The nucleoside phosphotransferase of chick embryo is unstable to heat and is protected from inactivation by a large number of nucleosides. 4. Nucleoside di- and triphosphates lower both the concentration of nucleoside monophosphates required for half-maximal velocity and the kinetic order of reaction measured with these phosphate donors. On the contrary, nucleoside di- or triphosphate do not modify the kinetic parameters evaluated for nucleoside acceptors. 5. We suggest that the nucleoside phosphotransferase contains both substrate and regulatory sites. It seems that the free apoenzyme is converted, by means of cooperative interactions between regulatory sites, into an enzyme-nucleotide complex, which is particularly stable at 37 degrees C.     

Cytosolic 5′-nucleotidase/nucleoside phosphotransferase: A nucleoside analog activating enzyme?

Nucleoside phosphotransferase acting on inosine and deoxyinosine has been partially purified from cultured Chinese hamster lung fibroblasts (V79). The activity is associated with a cytosolic 5′-nucleotidase acting on IMP and deoxyIMP. The transfer of the phosphate group from IMP to inosine catalyzed by this enzyme was activated by ATP and 2,3-bisphosphoglycerate. Inosine, deoxyinosine, guanosine, deoxyguanosine, and the nucleoside analogs 2′,3′-dideoxyinosine and 8-azaguanosine are substrates, while adenosine and deoxyadenosine are not. IMP, deoxyIMP, GMP, and deoxyGMP are the best phosphate donors. The cytosolic 5′-nucleotidase/phosphotransferase substrate, 8-azaguanosine, was found to be very toxic for cultured fibroblasts (LD₅₀ = 0.32 μM). Mutants resistant to either 8-azaguanosine and the correspondent base 8-azaguanine were isolated and characterized. Our results indicated that the 8-azaguanosine-resistant cells were lacking both cytosolic 5′-nucleotidase and hypoxanthine-guanine phosphoribosyltransferase, while 8-azaguanine resistant cells were lacking only the latter enzyme. Despite this observation, both mutants displayed 8-azaguanosine resistance, thus indicating that cytosolic 5′-nucleotidase is not essential for the activation of this nucleoside analog.     

A novel non-radioactive method for detection of nucleoside analog phosphorylation by 5'-nucleotidase.

Cytosolic 5'-nucleotidase has been implicated in the phosphorylation of certain nucleosides of therapeutic interest. In vitro, IMP and GMP serve as the optimal phosphate donors for this nucleoside phosphotransferase reaction. Existing assays for nucleoside phosphorylation effected by 5'-nucleotidase require a radiolabeled nucleoside as the phosphate acceptor and separation of the substrate-nucleoside from product-nucleotide has been accomplished either by a filter binding method or HPLC. However, detection of the phosphorylation of unlabeled nucleoside by HPLC is difficult since the ultraviolet absorbance of the phosphate donor, IMP, frequently obscures the absorbance of newly formed nucleotide. The use of ribavirin 5'-phosphate (RMP, 1,2,4-triazole-3-carboxamide riboside 5-monophosphate) as the phosphate donor obviates this difficulty since this triazole heterocycle does not significantly absorb at the wavelengths used to detect most nucleoside analogs. Using this procedure, a 5'-nucleotidase activity from the 100,000 x g supernatant fraction of human T-lymphoblasts deficient in adenosine kinase, hypoxanthine-guanine phosphoribosyltransferase, and deoxycytidine kinase, was characterized with regard to structure-activity relationships for certain inosine and guanosine analogs.     

Nucleoside phosphotransferase activity of human colon carcinoma cytosolic 5'-nucleotidase.

A cytosolic 5'-nucleotidase, acting preferentially on IMP and GMP, has been isolated from human colon carcinoma extracts. This enzyme activity catalyzes also the transfer of the phosphate group of 5'-nucleoside monophosphates (mainly, 5'-IMP, 5'-GMP, and their deoxycounterparts) to nucleosides (preferentially inosine and deoxyinosine, but also nucleoside analogs, such as 8-azaguanosine and 2',3'-dideoxyinosine). It has been proposed that the enzyme mechanism involves the formation of a phosphorylated enzyme as an intermediate which can transfer the phosphate group either to water or to the nucleoside. The enzyme is activated by some effectors, such as ATP and 2,3-diphosphoglycerate. Results indicate that the effect of these activators is mainly to favor the transfer of the phosphate of the phosphorylated intermediate to the nucleoside (i.e., the nucleoside phosphotransferase activity). This finding is in accordance with previous suggestions that cytosolic 5'-nucleotidase cannot be considered a pure catabolic enzyme.     

The effect of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide on the rat liver microsomal glucose-6-phosphatase system

The effect of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC) on the reactions catalyzed by the glucose-6-phosphatase system of rat liver microsomes was studied. Modification of the intact microsomes by CMC leads to the inhibition of the glucose-6-phosphatase, pyrophosphate:glucose and carbamoyl-phosphate : glucose phosphotransferase activities of the system. The activities are restored by the disruption of the microsomal permeability barrier. The mannose-6-phosphate, pyrophosphate, and carbamoyl-phosphate phosphohydrolase activities of the intact as well as the disrupted microsomes were not affected by CMC. It follows from the results obtained that CMC inactivates the microsomal glucose-6-phosphate translocase, the inactivation is a result of the modification of a single sulfhydryl or amino group of the translocase; pyrophosphate, carbamoyl phosphate and inorganic phosphate are transported across the microsomal membrane without participation of the glucose-6-phosphate translocase; pyrophosphate and carbamoyl phosphate may act as the phosphate donors in the glucose phosphorylation reactions in vivo.     

Synthesis of nucleotides by phosphotransferase from human tissues

Several human tissues (prostate greater than liver greater than kidney greater than spleen) contain phosphotransferase capable of synthesizing nucleoside monophosphate by low-energy phosphate transfer to pyrimidines, purines as well as ribo- and deoxyribonucleosides. Phenyl phosphate, AMP, TMP, GMP and IMP were good phosphate donors while thymidine, deoxyadenosine and deoxyuridine are the most effectively utilized substrates. The phosphatransferase activity is present in all particulate fractions with most activity in microsomes. The enzyme is being purified from human tissues.     

Phosphotransferase-dependent glucose transport in Corynebacterium glutamicum

G.M. MALIN AND G.I. BOURD. 1991. The transport system for glucose and its non-metabolizable analogue methyl-α-D-glucoside (MG) has been described in Corynebacterium glutamicum. The initial product of the transport reaction was shown to be a phosphate ester of MG (MGP). Free MG appeared inside the cells as a result of MGP dephosphorylation. The bacteria transported MG with an apparent K_m of 0.08 ± 0.017 mmol/l and V_max of 21 ± 2.3 nmol/(min × mg dry wt). Toluenized cells and crude cell extracts catalysed phosphoenolpyruvate (PEP)-dependent phosphorylation of MG and glucose. Both the membrane and the cytoplasmic fractions of bacterial extracts were required for phosphotransferase reaction. Most of the spontaneous mutants resistant to 2-deoxyglucose (DG), xylitol and 5-thioglucose were defective both in transport and in PEP-dependent phosphorylation of MG. Some strains were defective only in glucose utilization and some were also unable to grow on a number of other sugars. The phosphotransferase activity in extracts from mutant cells was restored by the addition of either membrane or cytoplasmic fraction from wild type bacteria. It was concluded that Corynebacterium glutamicum accumulated glucose and MG by means of a PEP-dependent phosphotransferase system (PTS).     

The synthesis of 11-[(2-pyridyl)amino]- and 11-[(9-anthracenylcarbonyl)amino]undecyl phosphate and the study of their acceptor properties in the enzymatic reaction catalyzed by Salmonella galactosyl phosphotransferases

Undecyl phosphate derivatives with new fluorescent labels, 11-[(2-pyridyl)amino]undecyl phosphate and 11-[(9-anthracenylcarbonyl)amino]undecyl phosphate, were synthesized. These compounds were shown to be acceptor substrates of the galactosyl phosphate residue in the enzymatic reaction catalyzed by galactosyl phosphotransferase from Salmonella anatum or Salmonella newport membrane preparations.     

An adenosine 3':5'-monophosphate-dependent protein kinase from the human erythrocyte membrane. Purification and characterization.

An adenosine 3':5'-monophosphate-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) has been isolated from the human erythrocyte memebrane and the phosphotransferase activity exhibited by this enzyme has been purified 800-fold. In concentrated solutions, the membrane-derived protein kinase undergoes aggregation with a concomitant loss in observed phosphotransferase activity. This loss of activity can be restored by means of inducing deaggregation. The phosphotransferase activity of the protein kinase is virtually obliterated in the presence of high (300 mM) concentrations of sodium chloride. This effect is also reversible. The pH optimum for the phosphotransferase reaction that is catalyzed by the membrane-derived protein kinase is approximately 8. Micromolar concentrations of cAMP are optimal with respect to promoting the phosphotransferase reaction. Initial velocity and product inhibition studies were conducted on the cAMP-independent protein kinase derived from the cAMP-dependent enzyme. These studies indicate that the phosphotransferase reaction proceeds by a sequential kinetic mechanism.     

Purification and characterization of myo-inositol hexaphosphate-adenosine diphosphate phosphotransferase from Phaseolus aureus

myo-Inositol hexaphosphate adenosine diphosphate phosphotransferase transfers phosphate from myo-inositol hexaphosphate to adenosine diphosphate to synthesize adenosine triphosphate. This enzyme has been isolated and purified from ungerminated mungbean seeds and found to be different from guanosine diphosphate phosphotransferase. A purification of about 200-fold with 15% recovery has been obtained. The optimal pH of the reaction is 7.0 and is dependent on the presence of a divalent cation, i.e., Mg²⁺ and Mn²⁺. The K_m value for myo-inositol hexaphosphate has been found to be 0.41 × 10^?4m and V is 90.0 nmol of P_i transferred per milligram of protein per 20 min. K_m for ADP is 0.88 × 10^-4m and V is 83.3 nmol of phosphorus transferred to ADP per milligram of protein per 20 min. The ADP phosphotransferase reaction is reversible to the extent of about 50% of the forward reaction. dADP is partly effective as an acceptor but other ribonucleoside mono- and diphosphates cannot substitute for ADP. The products ATP and myo-inositol pentaphosphate have been confirmed by several criteria. It has also been shown that this enzyme transfers phosphate only from a specific phosphoryl group (C-2 position) of myo-inositol hexaphosphate for the synthesis of ATP and 1,3,4,5,6-myo-inositol pentaphosphate or pentakis (dihydrogen phosphate).     

Membrane-bound 5′-nucleotidase/nucleoside phosphotransferase from Bacillus cereus

• 1.1. A search for nucleoside phosphotransferase activity in Bacillus cereus led to the following results: (i) The phosphotransferase activity was associated with a membrane bound 5′-nucleotidase. (ii) The enzyme phosphorylates both purine and pyrimidine nucleosides as well as 2′,3′-dideoxyinosine. (iii) The enzyme was inhibited by adenylic nucleotide di- and triphosphates, and its nucleotidase activity was increased in the presence of inosine as phosphate acceptor.
• 2.2. Bacterial and vertebrate 5′-nucleotidases with phosphotransferase activity diner for several characteristics, such as cellular location, substrate specificity, magnesium requirement and regulation.

     

Multifunctional glucose-6-phosphatase: cellular biology.

Glucose-6-phosphatase is a multifunctional enzyme, displaying potent ability to synthesize as well as hydrolyze Glc-6-P. These multifunctional characteristics have been exploited in studies of the extended distribution of the enzyme, and their physiological significance has been examined. The enzyme is considerably more widely distributed than previously suspected. It has been found in pancreas, adrenals, lung, testes, spleen, and brain as well as in liver, kidney, and mucosa of small intestine. Approximately 15–20% of total hepatic glucose-6-phosphatase-phosphotransferase is present in nuclear membrane, 75–80% is found in endoplasmic reticulum, and small amounts have been detected also in plasma membrane and repeatedly-washed mitochondria. Both hydrolytic and synthetic functions, in constant proportions, have been found in livers of 21 species of birds, amphibia, reptiles, crustacea, fishes, and mammals (including man) studied. With 5 mM phosphoryl donor and 100 mM D-glucose as substrates, carbamyl-P:glucose phosphotransferase activity of glucose-6-phosphatase exceeded that of glucokinase by 5–50 fold. While latencies of activities of isolated microsomal preparations are extensive, those of nuclear membranes are not. Latencies of activities of intact endoplasmic reticulum of permeable hepatocytes are 28% for Glc-6-P phosphohydrolase and 56% for carbamyl-P:glucose phosphotransferase. Studies with isolated perfused livers from fasted rats suggest rather convincingly that such phosphotransferase activities may function as an hepatic glucose-phosphorylating system supplemental to glucokinase and hexokinase. This conclusion is based both on comparisons of rates of glucose uptake with hepatic enzyme levels (glucokinase, hexokinase, phosphotransferase), and on observed inhibitibility of glucose uptake by ornithine and 3-0-methyl-D-glucose. The question of availability of adequate concentrations of suitable phosphoryl donor(s) in cytosol of the liver cell constitutes a principal focus for continuing studies regarding physiological functions of this enzyme.     

Vinylglycolate resistance in Escherichia coli.

Escherichia coli K-12 vinylglycolate-resistant mutants have been isolated and characterized. Two of the mutants, JSH 150 and JSH 151, have been determined to be double mutants, lacking both membrane-bound L-and D-lactate dehydrogenases. The lactate transport system is intact in all strains; both radioactive lactate and vinylglycolate are actively taken up. Likewise, the phosphoenolypyruvate-dependent phosphotransferase system for hexose uptake is active. Vinylglycolate, previously shown to inhibit the phosphoenolpyruvate-dependent phosphotransferase system, has very little effect in the double mutants. The extent of vinylglycolate inhibition in other mutants seems directly related to the activity of the lactate dehydrogenases. This indicates that vinylglycolate is oxidized to 2-keto-3-butenoate before inactivating the phosphoenolpyruvate-dependent phosphotransferase system. These results were found in whole cells and confirmed in isolated membrane vesicles.     

Glucose-specific permease of the bacterial phosphotransferase system: phosphorylation and oligomeric structure of the glucose-specific IIGlc-IIIGlc complex of Salmonella typhimurium

The glucose-specific membrane permease (IIGlc) of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediates active transport and concomitant phosphorylation of glucose. The purified permease has been phosphorylated in vitro and has been isolated (P-IIGlc). A phosphate to protein stoichiometry of between 0.6 and 0.8 has been measured. Phosphoryl transfer from P-IIGlc to glucose has been demonstrated. This process is, however, slow and accompanied by hydrolysis of the phosphoprotein unless IIIGlc, the cytoplasmic phosphoryl carrier protein specific to the glucose permease (IIGlc) of the PTS, is added. Addition of unphosphorylated IIIGlc resulted in rapid formation of glucose 6-phosphate with almost no hydrolysis of P-IIGlc accompanying the process. A complex of IIGlc and IIIGlc could be precipitated from bacterial cell lysates with monoclonal anti-IIGlc immunoglobulin. The molar ratio of IIGlc:IIIGlc in the immunoprecipitate was approximately 1:2. Analytical equilibrium centrifugation as well as chemical cross-linking showed that purified IIGlc itself is a dimer (106 kDa), consisting of two identical subunits. These results suggest that the functional glucose-specific permease complex comprises a membrane-spanning homodimer of IIGlc to which four molecules of IIIGlc are bound on the cytoplasmic face.     

Properties of two sugar phosphate phosphatases from Streptococcus bovis and their potential involvement in inducer expulsion.

Streptococcus bovis possesses two sugar phosphate phosphatases (Pases). Pase I is a soluble enzyme that is inhibited by the membrane fractions from lactose-grown cells and is insensitive to activation by S46D HPr, an analog of HPr(ser-P) of the sugar phosphotransferase system. Pase II is a membrane-associated enzyme that can be activated 10-fold by S46D HPr, and it appears to play a role in inducer expulsion.     

The purification and properties of nucleoside phosphotransferase from mucosa of chicken intestine

Nucleoside phosphotransferase (nucleotide: 3'-deoxynucleoside 5'-phosphotransferase, EC 2.7.1.77) has been purified from chicken intestine mucosa to apparent homogeneity. The enzyme is represented by a multisubunit protein at different degrees of association. It can dissociate into a component with a marked fall in catalytic activity. The associated forms are similar to the enzyme previously purified from chick embryo as regards: substrate specificity both with respect to nucleoside monophosphate donors and to deoxyribonucleoside acceptors; sigmoidicity in the rate curve with a variable phosphate donor; instability to heat, dilution and lowering of pH; the activating and protecting effect of nucleotides, particularly the diphosphate forms. The dissociated form displays lower Vmax and higher S0.5 than the associated ones; and the Hill constants are always about 1. With this form, nucleotides show only a modest activating effect and do not protect. Mg2+, Mn2+ or Co2+ are required for catalytic activity, whereas the protective effect of nucleotides is independent of divalent metals. Inorganic phosphate stabilizes associated forms of the enzyme, but inhibits its activity by competing with nucleotide effectors. The enzyme behaves also as a phosphohydrolase, particularly with respect to deoxyribonucleoside monophosphates; deoxyuridine and deoxythymidine inhibit hydrolytic activity.     

Bacterial phosphoenolpyruvate-dependent phosphotransferase system. Mechanism of the transmembrane sugar translocation and phosphorylation

The phosphoryl-group transfer from PHPr to glucose or alpha-methylglucose and from glucose 6-phosphate to these same sugars catalyzed by membrane-bound EIIBGlc of the bacterial phosphoenolpyruvate-dependent phosphotransferase system has been studied in vitro. Kinetic measurements revealed that both the phosphorylation reaction and the exchange reaction proceed according to a ping-pong mechanism in which a phosphorylated membrane-bound enzyme II acts as an obligatory intermediate. The occurrence of a phospho-IIBGlc/IIIGlc has been physically demonstrated by the production of a glucose 6-phosphate burst from membranes phosphorylated by phosphoenolpyruvate, HPr, and EI. The observation of similar second-order rate constants for the production of sugar phosphate starting with different phosphoryl-group donors confirms the catalytic relevance of the phosphoenzyme IIBGlc intermediate. The in vitro results, together with data published by other investigators, have led to a model describing sugar phosphorylation and transport in vivo.     

Kinetic analyses of the sugar phosphate:sugar transphosphorylation reaction catalyzed by the glucose enzyme II complex of the bacterial phosphotransferase system.

The sugar phosphate:sugar transphosphorylation reaction catalyzed by the glucose Enzyme II complex of the phosphotransferase system has been analyzed kinetically. Initial rates of phosphoryl transfer from glucose-6-P to methyl alpha-glucopyranoside were determined with butanol/urea-extracted membranes from Salmonella typhimurium strains. The kinetic mechanism was shown to be Bi-Bi Sequential, indicating that the Enzyme II possesses nonoverlapping binding sites for sugar and sugar phosphate. Binding of the two substrates appears to occur in a positively cooperative fashion. A mutant with a defective glucose Enzyme II was isolated which transported methyl alpha-glucoside and glucose with reduced maximal velocities and higher Km values. In vitro kinetic studies of the transphosphorylation reaction catalyzed by the mutant enzyme showed a decrease in maximal velocity and increases in the Km values for both the sugar and sugar phosphate substrates. These results are consistent with the conclusion that a single Enzyme II complex catalyzes both transport and transphosphorylation of its sugar substrates.     

Effect of dietary phosphate intake on phosphate transport by isolated rat renal brush-border vesicles

Renal brush-border membrane vesicles isolated from rats kept for 6-8 weeks on a low-phosphate diet (0.15% of dry matter) showed a markedly faster Na(+)-dependent phosphate uptake than did membrane vesicles isolated from animals kept on a high-phosphate diet (2% of dry matter). Phosphate-uptake rate by brush-border membrane vesicles isolated from animals on a low-phosphate diet remained significantly increased after acute parathyroidectomy. Dietary adaptation was also observed in animals that had been parathyroidectomized before exposure to the different diets. In animals on the low-phosphate diet parathyrin administration inhibited phosphate uptake by brush-border vesicles only if the animals were repleted with P(i) (5ml of 20mm-NaH(2)PO(4)) 1h before being killed. After acute phosphate loading and parathyrin administration the difference in the transport rate between the two dietary groups remained statistically significant. The results suggest that the adaptation of proximal-tubule phosphate transport to dietary intake of phosphate is reflected in the Na(+)/phosphate co-transport system located in the luminal membrane of the proximal-tubule cell. Since the dietary effects on phosphate transport by brush-border membranes are only partially reversed by acute changes in parathyrin concentration and are also observed in chronically parathyroidectomized animals, the adaptation of the Na(+)/phosphate co-transport system to dietary phosphate intake seems to involve an additional mechanism independent of parathyrin.     

Properties of rat liver nuclear protein kinases.

Two cyclic nucleotide-independent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) have been purified to homogeneity from rat liver nuclei. While these enzymes have many similar catalytic properties (preference for acid rather than basic proteins), they differ in molecular weight and subunit composition. Protein kinase NII will utilize ATP and GTP as phosphate donors while protein kinase NI will only effectively use ATP. Both enzymes reveal an unusual activation by Fe2+.