Showing papers on "Nucleotidase published in 1972"

CYTOCHEMICAL LOCALIZATION OF 5'-NUCLEOTIDASE IN SUBCELLULAR FRACTIONS ISOLATED FROM RAT LIVER : I. The Origin of 5'-Nucleotidase Activity in Microsomes

Abstract: A procedure has been developed for the cytochemical localization of 5'-nucleotidase in isolated, unfixed, rat liver microsomes. Membranes were incubated with adenosine 5'-phosphate (5'-AMP) and Pb(NO3)2 and then isolated on sucrose density gradients: all the phosphate released was recovered with the membranes by this procedure. Adenosine 2'-phosphate (2'-AMP) and adenosine 3', 5'-cyclic phosphate (3',5'-AMP) were shown to be competitive inhibitors, but not substrates, for purified 5'-nucleotidase and were employed to determine the specificity of the cytochemical reaction. It was found that the incubation conditions for the cytochemical assay did not affect the specificity of 5'-nucleotidase. Microsomes incubated as controls with Pb2+, or Pb2+ and 2'-AMP or 3',5'-AMP were of the same density, although slightly denser than microsomes incubated without Pb2+, and were unassociated with lead precipitate when examined by electron microscopy; microsomes incubated with Pb2+ and 5'-AMP were much denser and were stained heterogeneously with lead phosphate when examined by electron microscopy. Precipitates formed artificially from Pb2+ and inorganic phosphate did not resemble the reaction product. Microsomes were, therefore, separated on sucrose gradients and the subfractions were examined cytochemically. Lead precipitates were associated with the majority of rough-surfaced vesicles, and the reaction product was distributed heterogeneously in all fractions. Vesicles which stained like the membranes of the bile canaliculi in isolated plasma membranes were observed in the lightest subfraction. The reaction product was localized on the outside surface of the microsomal membranes, and was solubilized by low concentrations of ethylenediaminetetraacetic acid. It is concluded that 5'-nucleotidase is present in the endoplasmic reticulum and that the microsome fraction contains, in addition, vesicles derived from the plasma membrane.

Phosphodiesterase in Dictyostelium discoideum and the chemotactic response to cyclic adenosine monophosphate.

Abstract: A phosphodiesterase with a low Michaelis constant for cyclic adenosine monophosphate was found in the membrane fraction of the cellular slime mold. This activity was highest during the aggregation stage. Enzyme with similar properties was also secreted by the cells. Dithiothreitol inhibited both enzymes and potentiated the cellular response to cyclic adenosine monophosphate.

The structure and function of ribonuclease T1. 18. Gel filtration studies on the interaction of ribonuclease T1 with substrate analogs.

Abstract: 1. The interaction of ribonuclease Tj [EC 2. 7. 7. 26] with its substrate analogs has been investigated by a gel filtration method. The enzyme bound maximally one molecule of 3'-GMP per molecule of protein, indicating the presence of one specific binding site in the enzyme molecule. 2. At pH5.5 and 25°C, the apparent binding strengths of the substrate analogs were in the order: 2'-GMP>3'-GMP>3', 5'-GDP>9-(2'-hydroxyethyl)guanine 2'-phosphate> 5'-GMP>guanosine>8-bromoguanosine>3'-AMP> deoxyguanosine> 5'-l-MeGMP> 3'CMP, 3'-UMP, guanine and the other eight nucleosides examined. The results show the importance of the integrity of the guanine, ribose, and phosphate portions for the binding. The importance of the N-l and N-7 positions and the 2-amino and 6-oxo (or hydroxy) groups in the guanine portion for the specific binding was also suggested. 3. The optimum pH for the binding of 3'-GMP and guanosine to the enzyme was around pH 5. The pH dependence of the binding of these analogs to the enzyme indicated the implication in the binding of at least two groups in the enzyme with pKa values of about 3.5 and 7, respectively, presumably a carboxyl and an imidazole groups. The significant loss of the binding ability toward 3'-GMP at temperatures above 40°C or in the presence of urea demonstrates the importance of the native conformation of the enzyme for the binding. The effects of some added substances on the binding were also investigated. 4. The Glu-58-carboxymethylated, inactive enzyme retained a considerable binding ability toward 3'-GMP and moreover it possessed almost the same binding ability toward guanosine over a wide pH range as that of the native enzyme. These results appear to indicate that Glu-58 is part of the catalytic site rather than part of the binding site, although the latter possibility cannot be excluded. The binding ability toward 3'-GMP was lowered significantly by the modification of Arg-77 with phenylglyoxal and also by the cleavage of the disulfide bonds with /9-mercaptoethanol. Arginine-77 may be the phosphate binding site or may be situated near the active site. Abbreviations used are: GMP, guanosine monophosphate ; GDP, guanosine diphosphate ; GpA, guanylyl(3'—»5>adenosine; AMP, adenosine monophosphate; CMP, cytidine monophosphate; UMP, uridine monophosphate ; 9-GEtP, 9-{2'-hydroxyethyl)guanine 2'-phosphate ; 1-MeGMP, 1-methylguanosine monophosphate.

Ribonuclease T1 Spectrophotometric Investigations of the Interaction of the Enzyme with Substrate Analogues

Abstract: Spectral changes on interaction of ribonuclease T1 with a number of substrate analogues have been measured. For guanosine and guanosine monophosphates these changes are the same as those produced by protonation of the guanine chromophore. It is concluded that the guanine ring of the substrate analogues is protonated by ribonuclease T1 in position N-7. Evidence is presented for a similar interaction of the enzyme with the purine ring of adenosine monophosphate. From the magnitude of the spectral changes binding constants have been determined. They decrease in the order guanosine 2′-phosphate > guanosine 3′-phosphate > guanosine 5′-phosphate > guanosine. The temperature dependence of the binding constants has been measured and the changes in enthalpy, free energy and entropy, which occur upon the RNAase T1-ligand interaction, have been calculated. The pH dependencies of the different ligand · enzyme complex formations revealed several pKa values (2.5,3.7,6.5,8.5). The pKa values of 2.5 and 6.5 have been assigned to protonations of the ligand. The pKa values of 3.7 and 8.5 have been assigned to specific groups in the active site of ribonuclease T1, which are involved in the recognition of the guanine base. From the pH dependencies of the guanosine monophosphate/enzyme interaction it is concluded that only the monoanionic form of guanosine monophosphate can interact with the enzyme. The nature of binding of guanosine monophosphate to ribonuclease T1 is discussed. It is proposed that the positions N-1 and N-7 of the guanine base are recognized by a basic and an acid group in the active site of ribonuclease T1 and that the monoanionic phosphate moiety interacts with a basic group of the enzyme.

An Improved Kinetic 5′Nucleotidase Assay

Abstract: 5′nucleotidase activity was measured by a coupled optical assay in which adenosine, liberated by action of the primary enzyme, released ammonia which in turn formed L-glutamate from 2-oxoglutarate and NADH. Oxidation of the latter is monitored at 340 nm. Greater activity was obtained when triethanolamine buffer, pH 7.2, and Mn++ were substituted for tris buffer, pH 7.9, and Mg++. The concentration of substrate, 5′AMP, was altered to 1 mmole/liter and that of β-glycerophosphate to 50 mmoles/liter to achieve optimal activity of true nucleotidase and suppression of 5′AMP-hydrolysis by non-specific phosphatases.

Regulation of rat-liver nucleotidase activity involving deoxyribonucleic-acid components as allosteric effectors.

Abstract: The activity of a highly purified nucleotidase from rat liver cytosol which splits certain 3′- and 5′-nucleotides, was studied in the presence of each of 36 different nucleic acid constituents including 14C-labeled and chemically modified nucleotides. It was found that the compounds either inhibited or had no effect on dephosphorylation of thymidine 5′-phosphate, which was used as substrate for measuring 5′-nucleotidase activity. On the other hand, the 3′-nucleotidase activity, which was measured with uridine 3′-phosphate as substrate, was stimulated 2.6 times by deoxyguanosine and, furthermore, by deoxyinosine, thymidine, deoxyuridine and inosine in decreasing order of effectiveness. Experiments with various phosphorylated derivatives of thymidine indicated that a 5′-phosphoryl group increases the stimulating effect of the nucleoside whereas a 3′-phosphoryl substitution reduces its ability to activate the enzyme. Di- and triphosphates were less stimulatory than the monophosphate. The results are interpreted to indicate that the same catalytic site is responsible for the hydrolysis of the 3′- and 5′-nucleotides and that the enzyme possesses a regulatory site, topographically different from the catalytic site, at which the deoxyribonucleic acid constituents act as stimulators of enzyme activity.

A simple competitive protein-binding assay for cyclic adenosine 3':5'-monophosphate.

Abstract: A cyclic adenosine 3': 5'-monophosphate (Ado-3':5'-P) assay is proposed basing on the principle of competitive protein-binding. Free and bound Ado-3':5'-P is separatedjby coated (Dextran T 70, bovine serum albumine) charcoal. The Ado-3':5'-P binding protein is obtained from bull adrenals. The detection limit is 0.25 pmol in the incubation tube. The affinity constant of the protein-Ado-3': 5'-P complex is 2.8 · 10l/mol. The free enthalpy is approximate lOkcal/mol. The high specificity is tested with other similar nucleotides. There is a great difference between the N-monoand N-2'O-dibutyryl derivatives of Ado-3':5'-P with respect to their affinities for the binding protein, or protein kinase. The criteria of reliability are given by precision, detection limit, specificity and accuracy data. The method is suitable for serial determinations. One person is able to measure 100 samples (including the standard curve) per day. For linearisation of the dose response curve a new method is described. The proposed plot has the advantages of simplicity and ease of curve calculation. In addition, the plot yields the affinity constant and binding capacity of the binding protein. Moreover, this method allows the estimation of the affinity constant of any competitor of a labeled ligand. This method for linearisation is discussed regarding its limitations and advantages.

Adenosine 3,5-cyclic monophosphate palmitates

Abstract: THE TWO NEW COMPOUNDS 2''-O-PALMITOYLADENOSINE 3''5''-CYCLIC MONOPHOSPHATE AND N6,2''-O-DIPALMITOYLADENOSINE-3''-5''-CYCLIC MONOPHOSPHATE ARE POTENT INHIBITORS OF ADENYLATE CRYLASE AND ARE USEFUL FOR THE SUPPRESSION OF HYPERHORMONAL STATES SUCH AS HYPETHYROIDISM AND HYPERADRENALISM.