A new enzymatic method for the determination of inorganic phosphate and its application to the nucleoside diphosphatase assay

A new enzymatic method has been developed for the determination of inorganic phosphate, in which purine nucleoside phosphorylase and xanthine oxidase are used as indicator enzymes. This method has been applied to the assay of nucleoside diphosphatase. Incidental to this work, the apparent Michaelis constant of phosphate for calf spleen purine nucleoside phosphorylase was determined to be 0.25 mm, and the extinction coefficient of uric acid at 293 nm and pH 7.4 was found to be 13.0 × 10³m^?1 cm^?1.     

Identity of aliphatic L- -hydroxyacid oxidase and glycolate oxidase from rat livers

l-α-Hydroxyacid oxidase and glycolate oxidase have been partially purified from rat livers and found to be identical, judging by substrate specificities, K_m values for certain substrates and coenzyme (FMN), activation energy, inhibition rates by various reagents and pH optimum. K_m values are as follows; glycolate, 2.4 × 10^?4m; l-α-hydroxyisocaproate, 1.26 × 10^?3; glyoxylate, 1.41 × 10^?4m; and FMN, 1.13 × 10^?6m. K_m values for glycolate and FMN are one-tenth and one-twentieth the literature values for hepatic glycolate oxidase. Sucrose density gradient centrifugation establishes that this enzyme is located in hepatic peroxisomes.     

Purine nucleoside phosphorylases purified from rat liver and Novikoff hepatoma cells.

Purine nucleoside phosphorylase (PNP) was purified from rat hepatoma cells and normal liver tissue utilizing the techniques of ammonium sulfate fractionation, heat treatment, ion-exchange and molecular exclusion chromatography, and polyacrylamide gel electrophoresis. Homogeneity was established by disc gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Purified rat hepatoma and liver PNPs appeared to be identical with respect to subunit and native molecular weight, substrate specificity, heat stability, kinetics and antigenic identity. A native molecular weight of 84,000 was determined by gel filtration. A subunit molecular weight of 29,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point was observed at pH 5.8, and the pH optimum was 7.5. Inosine, guanosine, xanthosine, and 6-mercaptopurine riboside were substrates for the enzymes. The apparent K_m for both inosine and guanosine was about 1.0 × 10^?4m and for phosphate was 4.2 × 10^?4m. Hepatoma and liver PNP showed complete cross-reactivity using antiserum prepared against the liver enzyme.     

MEMBRANE THIAMINE TRTPHOSPHATASE FROM RAT BRAIN: INHIBITION BY ATP AND ADP

—The hydrolysis of ThTP by rat brain membrane-bound ThTPase is inhibited by nucleoside diphosphates and triphosphates. ATP and ADP are most effective, reducing hydrolysis by 50% at concentrations of 2 × 10^?5m and 7·5 × 10^?5m respectively. Nucleoside monophosphates and free nuclcosides as well as P_i have no effect on enzyme activity. ThMP and ThDP also fail to inhibit hydrolysis in concentrations up to 5 × 10^?3m . Non-hydrolysable methylene phosphate analogs of ATP and ADP were used in further kinetic studies with the ThTPase. The mechanism of inhibition by these analogs is shown to be of mixed non-competitive nature for both compounds. An observed K_i, of 4 × 10^?5m for the ATP analog adenosine-PPCP and 9 × 10^?5m for the ADP analog adenosine-PCP is calculated at pH 6·5. Formation of the true enzyme substrate, the [Mg²⁺. ThTP] complex, is not significantly affected by concentrations of analogs producing maximal (>95%) inhibition of enzyme activity. Likewise the relationships between pH and observed K_m and pH and V_max are not shifted by the presence of similar concentrations of inhibitor.     

Deoxyguanosine toxicity on lymphoid cells as a cause for immunosuppression in purine nucleoside phosphorylase deficiency.

To delineate the pathogenesis of the immunodeficiency disease associated with purine nucleoside phosphorylase deficiency, the effects of guanosine, inosine, deoxyguanosine and deoxyinosine on the growth of a mouse T cell lymphoma line in culture were studied. Of these four purine nucleosides, deoxyguanosine was the most toxic. At 5 x 10^?6 to 10^?5 M, deoxyguanosine inhibits growth of the lymphoma cells; higher concentrations result in complete killing. The cytotoxic effects of this deoxynucleoside can be prevented by simultaneous addition to culture medium of deoxycytidine and hypoxanthine. Determination of nucleotide pools in deoxyguanosine-treated cells shows a marked reduction of the deoxycytidine triphosphate and the adenine ribonucleotide pools, accompanied by a sharp rise in the guanosine deoxyribonucleotide and a smaller increase in the corresponding ribonucleotide pools.Deoxyguanosine as well as guanosine, inosine and deoxyinosine were known to accumulate to relatively high levels in the plasma of a patient with T cell immunodeficiency disease associated with purine nucleoside phosphorylase deficiency. The other three purine nucleosides are much less toxic than deoxyguanosine. Thus it is very probable that the patient's clinical manifestations of T lymphocytopenia are the consequence of deoxyguanosine inhibition of lymphoid cell proliferation, resulting from depletion of deoxycytidine triphosphate and adenine nucleotides.     

Irreversible inhibition of phosphorylase phosphatase by fluorophosphate.

The inactivation of phosphorylase phosphatase by fluorophosphate is described. The inactivation is dependent upon time and concentration of fluorophosphate and cannot be reversed by removal of fluorophosphate from the enzyme. Acid hydrolysis of fluorophosphate destroys the capacity for inhibition. The inactivation exhibits saturation kinetics. A dissociation constant for the enzyme-fluorophosphate complex and a rate constant for the reaction were calculated to be 5.5 × 10^?3 M and 0.22 min^?1, respectively. A competitive inhibitor, phosphate, protects the enzyme against inactivation. The data are consistent with an irreversible covalent modification of the active site of phosphorylase phosphatase by fluorophosphate.     

Cloning, purification and characterisation of a recombinant purine nucleoside phosphorylase from Bacillus halodurans Alk36

A purine nucleoside phosphorylase from the alkaliphile Bacillus halodurans Alk36 was cloned and overexpressed in Escherichia coli. The enzyme was purified fivefold by membrane filtration and ion exchange. The purified enzyme had a V _max of 2.03 × 10⁻⁹ s ⁻¹ and a K _m of 206 μM on guanosine. The optimal pH range was between 5.7 and 8.4 with a maximum at pH 7.0. The optimal temperature for activity was 70°C and the enzyme had a half life at 60°C of 20.8 h.     

Purification of the chloroplastic valyl-tRNA synthetase from Euglena gracilis.

Euglena gracilis chloroplast valyl-tRNA synthetase was purified 990 fold to a specific activity of about 1100 units/mg protein, by a series of steps including ammonium sulfate precipitation and chromatography on hydroxyapatite, DEAE-cellulose, Blue Dextran — Sepharose and Sephadex G200. The enzyme gives a single band upon polyacrylamide gel electrophoresis, appears to be a monomer with a molecular weight of 126,000 daltons and has Km values of 1.5 × 10^?5 M for L-valine, 5 × 10^?5 M for ATP, and 6 × 10^?8 for tRNA^Val.     

S-adenosylmethionine: Protein-carboxyl methyltransferase from erythrocyte

Protein methylase II (S-adenosylmethionine:protein—carboxyl methyltrans-ferase), which modifies free carboxyl residues of protein, was purified from both rat and human blood, and properties of the enzymes were studied. The pH optima for the reaction were dependent on the substrate proteins used; pH 7.0 was found with endogenous substrate, 6.1 with plasma, 6.5 with γ-globulin, and 6.0 with fibrinogen. The molecular weight of the enzymes from both rat and human erythrocytes were identical (25,000 daltons) determined by Sephadex G-75 chromatography. Partially purified enzyme from rat erythrocytes showed three peaks on electrofocusing column at pH 4.9, 5.5 and 6.0. The K_m values of the enzymes from rat and human erythrocytes showed 3.1 × 10^?6m and 1.92 × 10^?6m at pH 6.0, 1.96 × 10^?6m and 1.78 × 10^?6m at pH 7.2, respectively, for S-adenosyl-l-methionine. It is also found that S-adenosyl-l-homocysteine is a competitive inhibitor for protein methylase II with K_i value of 1.6 × 10^?6m.     

Purification and properties of a D-fructose 1,6-bisphosphatase from Saccharomyces cerevisiae.

Fructose 1,6-bisphosphatase (EC 3.1.3.11) from Saccharomyces cerevisiae has been purified to homogeneity. A molecular weight of 115,000 has been obtained by gel filtration. The enzyme appears to be a dimer with identical subunits. The apparent K_m for fructose bisphosphatase varies with the Mg²⁺ concentration of the enzyme, being 1 × 10^?6m at 10 mm Mg²⁺ and 1 × 10^?5m at 2 mm Mg²⁺. Other phosphorylated compounds are not significantly hydrolyzed by the enzyme. An optimum pH of 8.0 is exhibited by the enzyme. This optimum is not changed by addition of EDTA. AMP inhibits the enzyme with a K_i of 8.0 × 10^?5m at 25 °C. The inhibition is temperature dependent, the value of K_i increasing with raising temperature. 2-Deoxy-AMP is also inhibitory with a K_i value at 25 °C of 1.6 × 10^?4m. An ordered uni-bi mechanism has been deduced for the reaction with phosphate leaving the enzyme as the first product and the fructose 6-phosphate as the second one.     

Purification and characterization of uricase,a nitrogen-regulated enzyme,from Neurospora crassa

Uricase, a purine catabolic enzyme, is controlled by induction and by nitrogen catabolite repression in Neurospora. Uricase was purified nearly 1000-fold to homogeneity. Only a single protein band could be detected in analytical gels of the pure enzyme, and the protein band in each case corresponded exactly to the position of in situ staining for enzyme activity. The molecular weight of native uricase was estimated to be 123,000 ± 7000. The enzyme is a tetramer composed of identical or similar-sized subunits. The K_m value of uricase for uric acid was estimated to be 4.2 × 10^?5, m. Oxonic acid was shown to be a competitive inhibitor of uricase, with a K_i value of 6.7 × 10^?7, m. Uricase is a stable enzyme and is not subject to feedback inhibition by ammonia, glutamate, or glutamine in Neurospora. The regulation of uricase appears to occur primarily at the biosynthesis level. Uricase appears to be a metalloenzyme with no essential sulfhydryl groups.     

A critical reexamination of the continous spectrophotometric assay for adenosine deaminase

The continuous spectrophotometric assay for adenosine deaminase has been reinvestigated, using both adenosine and 9-β-d-arabinofuranosyladenine as substrates. This assay is based on the reported decrease in absorbance at or near 265 nm between the adenine nucleoside substrate and the hypoxanthine nucleoside product. In the substrate concentration range 1,5 – 8.0 × 10^?4m, the progress of the reaction is associated with an anomalous sigmoidal dependence of absorbance on time, and the overall change in absorbance decreases with increasing substrate concentration. Near 8 × 10^?4m substrate, the deamination proceeds with no change in absorbance, while at higher concentrations, small increases in absorbance are observed. These effects, if ignored, generate initial “rate” data exhibiting an apparent substrate inhibition whieh, however, is completely an artifact induced by the spectral anomalies. Over the entire concentration range 5 × 10^?6–1 × 10^?3m, alternative assay methods (e.g., discontinuous detection of the product, ammonia) yeld normal Michaelis-Menten kineties. The anomalous behavior manifested in the continuous spectrophotometric assay is due to large negative deviations from Beer's law. These deviations are observed for all four of the nucleosides tested, viz., adenosine, 9-β-d-arabinofuranosyladenine, inosine, and 9-β-d-arabinofuranosylhypoxanthine. The departure from Beer's law is detectable anywhere in the concentration range 5 × 10^?6–1 × 10^?3m, but is most marked at concentrations above 1 × 10^?4m. Thus, the continuous spectrophotometric assay for adenosine deaminase should be utilized withextreme caution, and should not be employed at concentrations exceeding 1 × 10^?4m, irrespective of the K_m value for the substrate. Specific recommendations are given for future assays.     

A second purine nucleoside phosphorylase in Escherichia coli K-12

Summary The presence of a second purine nucleoside phosphorylase in wild-type strains of E. coli K-12 after growth on xanthosine has been demonstrated. Like other purine nucleoside phosphorylases it is able to carry out both phosphorylosis and synthesis of purine deoxy- and ribonucleosides whilst pyrimidine nucleosides cannot act as substrates. In contrast to the well characterised purine nucleoside phosphorylase of E. coli K-12 (encoded by the deoD gene) this new enzyme could act on xanthosine and is hence called xanthosine phosphorylase. Studies of its substrate specificity showed that xanthosine phosphorylase, like the mammalian purine nucleoside phosphorylases, has no activity towards adenine and the corresponding nucleosides. Determinations of K _m and gel filtration behaviour was carried out on crude dialysed extracts. The presence of xanthosine phosphorylase enables E. coli to grow on xanthosine as carbon source. Xanthosine was the only compound found which induced xanthosine phosphorylase. No other known nucleoside catabolising enzyme was induced by xanthosine. The implications of non-linear induction kinetics of xanthosine phosphorylase is discussed.     

Ethanolamine kinase from Culex pipiens fatigans

Ethanolamine kinase was partially purified from the larvae of Culex pipiens fatigans and its properties were studied. The enzyme was separated from choline kinase by acetic acid precipitation at pH 5.0 of a 13,000g supernatant of the larval homogenate. Alkaline phosphatase activity was removed from the enzyme preparation by the acid treatment followed by ammonium sulfate fractionation. The enzyme was localized in the cytosolic fraction and had a requirement for Mg²⁺ as a cofactor. The K_m values for ethanolamine and ATP were 4 × 10^?4 and 1.54 × 10^?4m, respectively. The affinity of the enzyme for nucleotide triphosphates was in the order, ATP > ITP > GTP while UTP and CTP were poorly utilized. p-Chloromercuribenzoate and N-ethylmaleimide inhibited the enzyme activity and reduced glutathione protected the enzyme from their inhibition. Choline and serine had no effect on the enzyme activity. The enzyme had a molecular weight of 44, 000 daltons as determined by gel filtration chromatography. Eggs contained the highest specific activity of the enzyme while adult insects had the highest total enzyme activity.     

The purine and pyrimidine metabolism of Tetrahymena pyriformis

The metabolism of purines and pyrimidines by the ciliated protozoan Tetrahymena was investigated with the use of enzymatic assays and radioactive tracers. A survey of enzymes involved in purine metabolism revealed that the activities of inosine and guanosine phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, E.C. 2.4.2.1) were high, but adenosine phosphorylase activity could not be demonstrated. The apparent K_m for guanosine in the system catalyzing its phosphorolysis was 4.1 ± 0.6 × 10^?3 M. Pyrophosphorylase activities for IMP and GMP (GMP: pyrophosphate phosphoribosyltransferase, E.C. 2.4.2.8), AMP (AMP: pyrophosphate phosphoribosyltransferase, E.C. 2.4.2.7), and 6-mercaptopurine ribonucleotide were also found in this organism; but a number of purine and pyrimidine analogs did not function as substrates for these enzymes. The metabolism of labeled guanine and hypoxanthine by intact cells was consistent with the presence of the phosphorylases and pyrophosphorylases of purine metabolism found by enzymatic studies. Assays for adenosine kinase (ATP: adenosine 5'-phosphotransferase, E.C. 2.7.1.20) inosine kinase, guanosine kinase, xanthine oxidase (xanthine: O₂ oxidoreductase, E.C. 1.2.3.2), and GMP reductase (reduced-NADP: GMP oxidoreductase [deaminating], E.C. 1.6.6.8) were all negative. In pyrimidine metabolism, cytidine-deoxycytidine deaminase (cytidine aminohydrolase, E.C. 3.5.4.5), thymidine phosphorylase (thymidine: orthophosphate ribosyltransferase, E.C. 2.4.2.4), and uridine-deoxyuridine phosphorylase (uridine: orthophosphate ribosyltransferase, E.C. 2.4.2.3) were active; but cytidine kinase, uridine kinase (ATP: uridine 5'-phosphotransferase, E.C. 2.7.1.48), and CMP pyrophosphorylase could not be demonstrated.     

Studies on inhibitory effect of phosphoramidon and its analogs on thermolysin.

Phosphoramidon, N-(α-l-rhamnopyranosyloxyhydroxyphosphinyl)-l-leucyl-l-tryptophan, and its analog, N-phosphoryl-l-leucyl-l-tryptophan, inhibited thermolysin in a competitive manner and K_i values were calculated to be 2.8 × 10^?8 and 2.0 × 10^?9m, respectively. The l-rhamnose moiety in phosphoramidon was suggested to be not involved in inhibition of thermolysin. A phosphoramidon analog containing histidine instead of tryptophan showed weaker inhibition. Spectrophotometric titration based on difference ultraviolet absorption spectra of the enzyme-inhibitor complex showed equimolar binding of the inhibitor to the enzyme.     

Uridine-cytidine kinase. Kinetic studies and reaction mechanism.

The initial velocity pattern has been determined for uridine-cytidine kinase purified from the murine mast cell neoplasm P815. With either uridine or cytidine as phosphate acceptor, and ATP as phosphate donor, the pattern observed was one of intersecting lines, ruling out a ping-pong reaction mechanism, and suggesting that the reaction probably proceeds by the sequential addition of both substrates to the enzyme to form a ternary complex, followed by the sequential release of the two products. This pattern was obtained whether the reaction was run in 0.01 m potassium phosphate buffer, pH 7.5, or in 0.1 m Tris-HCl, pH 7.2. When analyzed by the Sequen computer program, the data indicated an apparent K_m of the enzyme for uridine of 1.5 × 10^?4m, an apparent K_m for cytidine of 4.5 × 10^?5m, and a K_m for ATP, with uridine or cytidine as phosphate acceptor, of 3.6 × 10^?3m or 2.1 × 10^?3m, respectively. The V was 1.83 μmol phosphorylated/min/mg enzyme protein for the uridine kinase reaction and 0.91 μmol for the cytidine kinase reaction.     

Characterization of the subunits of purine nucleoside phosphorylase from cultured normal human fibroblasts

In previous communications we have demonstrated that the subunits of normal human erythrocyte purine nucleoside phosphorylase can be resolved into four major (1–4) and two minor (1p and 2p) components with the same molecular weight but different apparent isoelectric points (and net ionic charge). The existence of subunits with different charge results in a complex isoelectric focusing pattern of the native erythrocytic enzyme. In contrast, the isoelectric focusing pattern of the native enzyme obtained from cultured human fibroblasts is simpler. The multiple native isoenzymes obtained from human erythrocytes and human brain have isoelectric points ranging from 5.0 to 6.4 and from 5.2 to 5.8, respectively, whereas cultured human fibroblasts have two major native isoenzymes with apparent isoelectric points of 5.1 and 5.6.Purine nucleoside phosphorylase has been purified at least a hundredfold from ³⁵S-labeled cultured human fibroblasts. A two-dimensional electrophoretic analysis of the denatured purified normal fibroblast enzyme revealed that it consists mainly of subunit 1 (90%) with small amounts of subunits 2 (10%) and 3 (1%). This accounts for the observed differences between the native isoelectric focusing and the electrophoretic patterns of the erythrocyte and fibroblast enzymes. The purine nucleoside phosphorylase subunit 1 is detectable in the autoradiogram from a two-dimensional electrophoretic analysis of a crude, unpurified extract of ³⁵S-labeled cultured normal human fibroblasts. The fibroblast phosphorylase coincides with the erythrocytic subunit 1 of the same enzyme, and the cultured fibroblasts of a purine nucleoside phosphorylase deficient patient (patient I) lack this protein component, genetically confirming the identity of the purine nucleoside phosphorylase subunit in cultured fibroblasts.This work was supported by a grant from the National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, United States Public Health Service. L. J. G. is supported by a fellowship from the National Institute of Child Health and Human Development. D. W. M. is an Investigator, Howard Hughes Medical Institute.     

Esteroproteolytic enzymes from the submaxillary gland. Kinetics and other physicochemical properties.

Two esteroproteolytic enzymes (A and D) have been isolated from the mouse submaxillary gland and shown to be pure by ultracentrifugation, immunoelectrophoresis, acrylamide-gel electrophoresis, and amino acid analyses. The enzymes have molecular weights of approximately 30,000 and are structurally and antigenically related. Narrow pH optima between 7.5 and 8.0 are exhibited by both enzymes. The “pK₁'s” are between 6.0 and 6.5 and the “pK₂'s” are near 9.0. A marked preference for arginine-containing esters is shown by both enzymes. The maximum specific activity of enzyme A on p-tosylarginine methyl ester (TAME) at pH 8 was 2500–3000 μm min^?1 mg^?1 and for enzyme D, 400–600 μm min^?1 mg^?1. With TAME as substrate, the K_m for enzyme A was 8 × 10^?4m at 25 °C and 6 × 10^?4m at 37 °C. For D, K_m was 3 × 10^?4 at 25 °C and 2 × 10^?4m at 37 °C.An apparent activation of enzyme D by tosylarginine (TA), a product of TAME hydrolysis, and all α-amino acids examined was due to removal of an inhibitor by chelation. This effect could be duplicated by 8-hydroxyquinoline and diethyldithiocarbamate but not by EDTA. Enzyme A was not affected by these substances to any remarkable extent. Several divalent ions proved to be potent inhibitors of enzyme D. Both enzymes are inactivated by the active site reagents diisopropyl phosphofluoridate and tosyllysine chloromethylketone but much less rapidly than is trypsin. Nitrophenyl-4-guanidionobenzoate reacts with a burst of nitrophenol liberation but with a rapid continuing hydrolysis. One active site per molecule is indicated. Enzyme D is inactivated by urea, reversibly at 10 m and with maximal permanent losses at 6 m. Autolysis of the unfolded form by the native enzyme when they coexist at intermediate urea concentrations appears to occur.Identity of enzyme D and the epithelial growth factor binding protein is demonstrated.     

Some kinetic studies on ribonucleosides degradation by extracts of penicillium citrinum

Cell-free extracts of 3–4 days old mats of nitrate-grown Penicillium citrinum catalyze the hydrolytic cleavage of the N-glycosidic bonds of inosine, guanosine and adenosine optimally at pH 4, 0.1 M citrate buffer. The same extracts catalyze the hydrolytic deamination of cytidine at a maximum rate in 0.08 M Tris-acetate buffer pH 6.5, 40°C and 50°C were the most suitable degrees for purine nucleoside hydrolysis and cytidine deamination, respectively. The incubation of the extracts at 60°C, in the absence of cytidine caused a loss in the deaminating activity, while freezing and thawing had no effect on both activities. The deaminating activity seems to be cytidine specific as neither cytosine, adenine, adenosine nor guanosine could be deaminated. Uridine competively inhibited this activity, while ammonia had no effect. The apparent K_m value of this enzyme for cytidine was 1.57×10^?3M and its K_i value for uridine was 7.8×10^?3M. The apparent K_m values of the N-glycosidic bond cleaving enzyme for inosine, guanosine and adenosine were 13.3, 14.2 and 20×10^?3 M, respectively.