Induction of phosphodiesterase by cyclic adenosine 3':5'-monophosphate in differentiating Dictyostelium discoideum amoebae.

Cyclic adenosine 3':5'-monophosphate added to the starvation media of Dictyostelium discoideum amoebae induces both intracellular and extracellular phosphodiesterase activities of these cells. The induced enzyme activity appears several hours earlier than that in starved cells which have not been induced with cyclic nucleotide. In both cases, the appearance of enzyme is inhibited by cycloheximide, and actinomycin D, and daunomycin. The KmS for the extracellular enzyme(s) of nucleotide-induced and uninduced control cells are identical. The induction of enzyme activity seems specific for cyclic adenosine 3':5'-monophosphate since cyclic guanosine 3':5'-monophosphate, as well as other nucleotides, have no effect. No differences in the activity or excretion of either N-acetylglucosaminidase or the inhibitory of the extracellular phosphodiesterase are observed between cyclic adenosine 3':5'-monophosphate-induced and control cells. A direct activation of phosphodiesterase by cyclic adenosine 3':5'-monophosphate can be excluded, since the addition of this nucleotide to cell lysates has no effect on the enzyme activity.     

The effect of substrate concentration and pH on the enzymic sulphation of l-tyrosyl derivatives

1. The kinetics of the enzymic transfer of sulphate from adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate to derivatives of l-tyrosine were investigated with a partially purified enzyme preparation from rat liver. 2. At pH7.5 and 37 degrees C the K(m) values for l-tyrosine methyl ester and adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate are 0.3mm and 8nm respectively. The K(m) value for either substrate is independent of the concentration of the other. The available data are consistent with the sulphation reaction proceeding according to a rapid-equilibrium random Bi Bi mechanism. 3. From the effect of pH on the K(m) and V(max.) values for l-tyrosine methyl ester, tyramine and N-acetyl-l-tyrosine ethyl ester it is concluded that the enzyme is specific for substrate molecules with a free and unprotonated amino group and an un-ionized hydroxyl group. 4. The only ionizing group that can be positively attributed to the enzyme appears to influence the binding of adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate and has an apparent pK value of approx. 9.5. It is suggested that this group may be an essential thiol. 5. The enzyme is inhibited by iodoacetamide at pH7.5 and 30 degrees C and this inhibition is prevented by the presence of adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate but not by l-tyrosine methyl ester.     

Effects of 8-substituted adenosine 3',5'-monophosphate derivatives on high Km phosphodiesterase activity.

Most of twenty-one 8-substitued adenosine 3',5'-monophosphate derivatives were found to inhibit competitively the hydrolysis of adenosine 3'5'-monophosphate by partially purified high Km (Michaelis-Menten constant) phosphodiesterase from hog brain cortex, which had one active site at high concentration of adenosine 3',5'-monophosphate (0.3 to 4.0 mM). The Ki value for the 8-substituted alkylaminoadenosine 3'5'-monophosphate derivative was found to decrease with increasing unbranched carbon chain of the substituent, and a minimum value was obtained in the case of 8-octylaminoadenosine 3',5'-monophosphate. The Ki value, however, increased gradually as the substituent of derivative became longer than that of 8-octylminoadenosine 3'5'-monophosphate. The similar phenomenon was observed in the 8-substituted alkylthioadenosine 3',5'-monophosphate. The standard affinity for adenosine 3,5'-monophosphate of the high Km phosphodiesterase was 5.0 kcal/mol, which was calculated from Km. The standard affinity for 8-hexylthioadenosine 3',5'-monophosphate, which inhibited most strongly the enzyme activity, was 7.2 kcal/mol. The difference (2.2 kcal/736) between the standard affinity for adenosine 3',5'-monphosphate and that for 8-hexylthioadenosine 3',5'-monophosphate seems to be based on the partial affinity for the substituent (hexylthio group) of the active site on the enzyme or its neighborhood. A characteristic similar interrelation between substituent length of derivatives and their inhibitory effect on the enzyme activity was observed similarly in two different series of derivatives, 8-substituted alkylaminoadenosine 3',5'-monophosphate and alkylthioadenosine 3',5'-monophosphate. The results may indicate the characteristic structure of the active site of the enzyme or its neighborhood.     

Irreversible inactivation of adenylyl cyclase by the "P"-site agonist 2',5'-dideoxy-,3'-p-fluorosulfonylbenzoyl adenosine

2',5'-Dideoxy,3'-p-fluorosulfonylbenzoyl Adenosine (2',5'-dd3'-FSBA) was synthesized and found to be an agonist and affinity label for the "P"-site of adenylyl cyclase. This compound irreversibly inactivated both a crude detergent-dispersed adenylyl cyclase from rat brain and the partially purified enzyme from bovine brain. The irreversible inactivation by 100 to 200 microM 2',5'-dd3'-FSBA was blocked in a concentration-dependent manner by several established P-site inhibitors of adenylyl cyclase, 2',5'-dideoxyadenosine, 2'-d3'-AMP, adenosine, and 2'-deoxyadenosine, but not by inosine, N6-(phenylisopropyl)adenosine, adenine, 2'-d3':5'-cAMP, or 5'-AMP, agents known not to act at the P-site. Moreover, irreversible inactivation by 2',5'-dd3'-FSBA occurred in the presence of ATP at concentrations up to 3 mM, making it unlikely that inactivation was due to an effect on the enzyme's catalytic site. Adenylyl cyclase was also irreversibly inactivated by 5'-FSBA, although modestly (less than 20%) and apparently nonspecifically. Dithiothreitol protected the enzyme from irreversible inactivation by 2',5'-dd3'-FSBA, but reversible inhibition of the enzyme was still observed, although with reduced potency. When 2 mM dithiothreitol was added after a 30-min preincubation with 2',5'-dd3'-FSBA, the rat brain enzyme was partially (approximately 80%) reactivated. The data suggest that 2',5'-dd3'-FSBA may irreversibly inactivate adenylyl cyclase by reacting with a cysteinyl moiety in proximity to the P-site domain of the enzyme. These data together with results of studies of P-site inhibition kinetics published elsewhere (Johnson, R. A., and Shoshani, I. (1990) J. Biol. Chem. 265, 11595-11600) strongly suggest that the P-site and catalytic site are distinct domains on the enzyme. 2',5'-dd3'-FSBA, and especially its radiolabeled analog, should prove to be a useful probe for structural studies of adenylyl cyclase, particularly with regard to the P-site.     

S-Adenosylhomocysteine hydrolase from human placenta. Affinity purification and characterization.

S-Adenosylhomocysteine hydrolase (EC 3.3.1.1) was purified to homogeneity from human placenta by using S-adenosylhomocysteine-agarose affinity chromatography. The enzyme is a tetramer with a native Mr of 189 000 and subunit Mr of 47 000-48 000; there were nine cysteine residues per subunit and no disulphide bonds. The pI was 5.7. H.p.l.c. analysis revealed that the enzyme contained four molecules of tightly bound cofactor (NAD) per tetramer, of which 10-50% was in the reduced form. The enzyme had four binding sites per tetramer for adenosine, of which 10-35% were found to be occupied. Two types of adenosine-binding sites could be distinguished on the basis of differences in rates of dissociation of the enzyme-adenosine complex, and by examining binding of adenosine at 0 degree C and 37 degrees C. The enzyme catalysed the interconversion of adenosine and 4',5'-dehydroadenosine; the equilibrium constant for this reaction was 2.1 and favoured 4',5'-dehydroadenosine formation. Variability in the specific activity of preparations of S-adenosylhomocysteine hydrolase was related to the NAD+/NADH ratio of the preparation. The capacity to bind radioactively labelled adenosine depended on the adenosine content of the purified enzyme. The rate of adenosine binding and the sensitivity of S-adenosylhomocysteine hydrolase to inactivation by adenosine were both diminished in the absence of dithiothreitol.     

Properties of enzyme fraction A from Chlorella and copurification of 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase, adenosine 5'phosphosulfate sulfohydrolase and adenosine-5'-phosphosulfate cyclase activities.

Enzyme fraction A from Chlorella which catalyzes the formation of adenosine 5'-phosphosulfate from adenosine 3'-phosphate 5'-phosphosulfate is further characterized. Fraction A is found to contain an Mg2+ -activated and Ca2+ -inhibited 3' (2')-nucleotidase specific for 3' (2'), 5'-biphosphonucleosides. This activity has been named 3' (2), 5'-biphosphonucleoside 3' (2')-phosphohydrolase. The A fraction is also found to contain an activity which catalyzes the formation of adenosine 3':5'-monophosphate (cyclic AMP) from adenosine 5'-phosphosulfate (adenosine 5'-phosphosulfate cyclase). Under the same conditions of assay, 5'-ATP and 5'-ADP are not substrated for cyclic AMP formation. Unlike the 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase activity, the adenosine 5'-phosphosulfate cyclase activity does not require Mg2+, requires NH+4 or Na+, and is not inhibited by Ca2+. The A fraction also contains an adenosine 5'-phospho sulfate sulfohydrolase activity which forms 5'-AMP and sulfate. The three activities remain together during purification and acrylamide gel electrophoresis of the purified preparation yields a pattern where only one protein band has all three activities. The phosphohydrolase can be separated from the other two activities by affinity chromatography on agarose-hexyl-adenosine 3'n5'-bisphosphate yielding a phosphohydrolase preparation showing a single band on gel electrophoresis. The adenosine 5'-phosphosulfate cyclase may provide an alternate route of cyclic AMP formation from sulfate via ATP sulfurylase, but its regulatory significance in Chlorella, if any, remains to be demonstrated. In sulfate reduction, the phosphohydrolase may serve to provide a readily utilized pool of adenosine 5'-phosphosulfate as needed by the adenosine 5'-phosphosulfate sulfotransferase. The cyclase and sulfohydrolase activities would be regarded as side reactions incidental to this pathway, but may be of importance in other metabolic and regulatory reactions.     

2-[(4-Bromo-2,3-dioxobutyl)thio]- and 2-[(3-bromo-2-oxopropyl)thio]adenosine 2'5'-bisphosphate: new nucleotide analogues that act as affinity labels of nicotinamide adenine dinucleotide phosphate specific isocitrate dehydrogenase

Two new reactive adenine nucleotide analogues have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 2',5'-bisphosphate (2-BDB-TA-2',5'-DP) and 2-[(3-bromo-2-oxopropyl)thio]adenosine 2',5'-bisphosphate (2-BOP-TA-2',5'-DP). Starting with NADP+, 2'-phospho-adenosine 5'-(diphosphoribose) (PADPR) was generated enzymatically and was converted to PADPR 1-oxide by reaction with m-chloroperoxybenzoic acid. Treatment with NaOH followed by reaction with carbon disulfide yielded 2-thioadenosine 2',5'-bisphosphate (TA-2',5'-DP). Condensation of TA-2',5'-DP with 1,4-dibromobutanedione or 1,3-dibromo-2-propanone gave the final products 2-BDB-TA-2',5'-DP and 2-BOP-TA-2',5'-DP, respectively. The structure of these new reagents was determined by UV, 1H NMR, 31P NMR, and 13C NMR spectroscopy as well as by bromide and phosphorus analysis. Both of these reagents exhibit properties expected for an affinity label of the coenzyme site of NADP+-dependent isocitrate dehydrogenase. With both reagents, biphasic kinetics of inactivation are observed that can be described in terms of a fast initial phase of inactivation resulting in partially active enzyme of 6-7% residual activity, followed by a slower phase leading to total inactivation. The inactivation rate constants for both reagents exhibit a nonlinear dependence on reagent concentration, consistent with the formation of a reversible complex with the enzyme prior to irreversible modification. The enzyme incorporates both reagents to a limited extent and is protected against inactivation by NADP+ and NADPH. The reaction of these new nucleotide analogues with isocitrate dehydrogenase is compared to the much slower inactivation caused by bromoacetone, indicating the importance of the nucleotide moiety in the functioning of the affinity labels. It is likely that 2-BDB-TA-2',5'-DP and 2-BOP-TA-2',5'-DP will have general applicability as affinity labels for other NADP+ binding enzymes.     

Biosynthesis of 9-beta-D-arabinofuranosyladenine: hydrogen exchange at C-2' and oxygen exchange at C-3' of adenosine

The data presented here describe new findings related to the bioconversion of adenosine to 9-beta-D-arabinofuranosyladenine (ara-A) by Streptomyces antibioticus by in vivo investigations and with a partially purified enzyme. First, in double label in vivo experiments with [2'-18O]- and [U-14C]adenosine, the 18O:14C ratio of the ara-A isolated does not change appreciably, indicating a stereospecific inversion of the C-2' hydroxyl of adenosine to ara-A with retention of the 18O at C-2'. In experiments with [3'-18O]- and [U-14C]-adenosine, [U-14C]ara-A was isolated; however, the 18O at C-3' is below detection. The adenosine isolated from the RNA from both double label experiments has essentially the same ratio of 18O:14C. Second, an enzyme has been isolated and partially purified from extracts of S. antibioticus that catalyzes the conversion of adenosine, but not AMP, ADP, ATP, inosine, guanosine, or D-ribose, to ara-A. In a single label enzyme-catalyzed experiment with [U-14C]adenosine, there was a 9.9% conversion to [U-14C]ara-A; with [2'-3H]-adenosine, there was a 8.9% release of the C-2' tritium from [2'-3H]adenosine which was recovered as 3H2O. Third, the release of 3H as 3H2O from [2'-3H]adenosine was confirmed by incubations of the enzyme with 3H2O and adenosine. Ninety percent of the tritium incorporated into the D-arabinose of the isolated ara-A was in C-2 and 8% was in C-3. The enzyme-catalyzed conversion of adenosine to ara-A occurs without added cofactors, displays saturation kinetics, a pH optimum of 6.8, a Km of 8 X 10(-4) M, and an inhibition by heavy metal cations. The enzyme also catalyzes the stereospecific inversion of the C-2' hydroxyl of the nucleoside antibiotic, tubercidin to form 7-beta-D-arabinofuranosyl-4-aminopyrrolo[2,3-d]pyrimidine. The nucleoside antibiotic, sangivamycin, in which the C-5 hydrogen is replaced with a carboxamide group, is not a substrate. On the basis of the single and double label experiments in vivo and the in vitro enzyme-catalyzed experiments, two mechanisms involving either a 3'-ketonucleoside intermediate or a radical cation are proposed to explain the observed data.     

Purification and properties of one component of acid phosphatase produced by Aspergillus niger.

One component, the i form, of acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) produced by Aspergillus niger was purified from the mycelial extract. The purified enzyme was homogenous on Sephadex G-200 gel filtration, disc electrophoresis and heat inactivation. The purified enzyme was studied and the following results were obtained: 1. The enzyme catalyzed the hydrolysis of a wide variety of phosphomonoesters, but not that of bis(p-nitrophenyl)phosphate, adenosine 3',5'-cyclic monophosphate, fructose 1,6-diphosphate, adenosine 5'-diphosphate or adenosine 5'-triphosphate. 2. Fluoride, orthophosphate, arsenate, borate, molybdate and (+)-tartrate acted as inhibitors. This enzyme was inactivated by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide, and was not affected by p-chloromercuribenzoate, N-acetylimidazole, p-diazobenzenesulfonic acid and tetranitromethane. From these results, tryptophan was estimated to play an important role in the enzyme activity. 3. The apparent molecular weight was 310000 by Sephadex G-200 gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate suggested that the molecular weight of the subunit was approximately 89000. 4. The purified enzyme contained 29% carbohydrate consisting of glucosamine, mannose and galactose. The amino acid composition of this enzyme was not specific compared with other known acid phosphatases.     

Thermostable Pyrococcus furiosus DNA ligase catalyzes the synthesis of (di)nucleoside polyphosphates

DNA ligase from the hyperthermophilic marine archaeon Pyrococcus furiosus (Pfu DNA ligase) synthesizes adenosine 5'-tetraphosphate (p4A) and dinucleoside polyphosphates by displacement of the adenosine 5'-monophosphate (AMP) from the Pfu DNA ligase-AMP (E-AMP) complex with tripolyphosphate (P3), nucleoside triphosphates (NTP), or nucleoside diphosphates (NDP). The experiments were performed in the presence of 1-2 microM [alpha-32P]ATP and millimolar concentrations of NTP or NDP. Relative rates of synthesis (%) of the following adenosine(5')tetraphospho(5')nucleosides (Ap4N) were observed: Ap4guanosine (Ap4G) (from GTP, 100); Ap4deoxythymidine (Ap4dT) (from dTTP, 95); Ap4xanthosine (Ap4X) (from XTP, 94); Ap4deoxycytidine (Ap4dC) (from dCTP, 64); Ap4cytidine (Ap4C) (from CTP, 60); Ap4deoxyguanosine (Ap4dG) (from dGTP, 58); Ap4uridine (Ap4U) (from UTP, <3). The relative rate of synthesis (%) of adenosine(5')triphospho(5')nucleosides (Ap3N) were: Ap3guanosine (Ap3G) (from GDP, 100); Ap3xanthosine (Ap3X) (from XDP, 110); Ap3cytidine (Ap3C) (from CDP, 42); Ap3adenosine (Ap3A) (from ADP, <1). In general, the rate of synthesis of Ap4N was double that of the corresponding Ap3N. The enzyme presented optimum activity at a pH value of 7.2-7.5, in the presence of 4 mM Mg2+, and at 70 degrees C. The apparent Km values for ATP and GTP in the synthesis of Ap4G were about 0.001 and 0.4mM, respectively, lower values than those described for other DNA or RNA ligases. Pfu DNA ligase is used in the ligase chain reaction (LCR) and some of the reactions here reported [in particular the synthesis of Ap4adenosine (Ap4A)] could take place during the course of that reaction.     

A stereochemical investigation of the hydrolysis of cyclic AMP and the (Sp)-and (Rp)-diastereoisomers of adenosine cyclic 3'':5''-phosphorothioate by bovine heart and baker''s-yeast cyclic AMP phosphodiesterases.

Bovine heart cyclic AMP phosphodiesterase, which has a requirement for Mg2+, hydrolyses cyclic AMP with inversion of configuration at the phosphorus atom, but only the (Sp)-diastereoisomer of adenosine cyclic 3':5'-phosphorothioate is hydrolysed by this enzyme. By contrast, the low-affinity yeast cyclic AMP phosphodiesterase, which contains tightly bound Zn2+, hydrolyses both the (Sp)- and the (Rp)-diastereoisomers of adenosine cyclic 3':5'-phosphorothioate, the (Rp)-diastereoisomer being the preferred substrate under V max. conditions. Both of the diastereoisomers of adenosine cyclic 3':5'-phosphorothioate, as well as cyclic AMP, are hydrolysed with inversion of configuration at the phosphorus atom by the yeast enzyme. It is proposed that, with both enzymes, the bivalent metal ion co-ordinates with the phosphate residue of the substrate, and that hydrolysis is catalysed by a direct "in-line' mechanism.     

A slowly dissociating form of the cell surface cyclic adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum.

Experiments using a phosphodiesterase-minus mutant of Dictyostelium discoideum indicate that ligand-induced loss of cell surface cyclic adenosine 3':5'-monophosphate binding sites (down regulation) can be evoked with concentrations of cyclic adenosine 3':5'-monophosphate as low as 10(-8) M. The loss of receptor sites is observed after 5 min of cell preincubation with cyclic adenosine 3':5'-monophosphate and can be as extensive as 75 to 80%. This decrease in binding sites is correlated with the appearance of a slowly dissociating cyclic adenosine 3':5'-monophosphate binding component. Radioactive cyclic adenosine 3':5'-monophosphate bound to this form of receptor cannot be competed for by nonradioactive cyclic adenosine 3':5'-monophosphate or adenosine 5'-monophosphate and is not accessible to hydrolysis by cyclic adenosine 3':5'-monophosphate phosphodiesterase. The extent of appearance of this binding component is dependent upon the concentration of cyclic adenosine 3':5'-monophosphate used to elicit the down regulation response and the temperature of the incubation medium.     

The binding site for the adenosyl group of coenzyme B12 in diol dehydrase

The binding of cob(II)alamin (CblII) and 5'-deoxyadenosine to diol dehydrase was studied spectroscopically and with [U-14C]5'-deoxyadenosine. CblII was bound to this enzyme forming a tight 1:1 complex which was resistant to oxidation by O2 even in the presence of CN-. An irreversible 1:1:1 ternary complex was formed between enzyme, CblII, and 5'-deoxyadenosine, when the enzyme was incubated first with the nucleoside and then with CblII. When this order of addition of the constituents was reversed, no 5'-deoxyadenosine was bound to the enzyme-CblII complex. Hydroxocobalamin could also bind to the enzyme together with the nucleoside, while other cob(III)alamins bearing a bulkier Co beta ligand displaced the nucleoside upon binding to the enzyme. The binding of [U-14C]5'-deoxyadenosine was strongly inhibited by unlabeled 5'-deoxy-ara-adenosine, 4',5'-anhydroadenosine, adenosine, adenine, and 5',8-cyclic adenosine, in this order, but not by 5'-deoxyuridine. These results constitute direct evidence for the presence of the binding site for the adenosyl group of adenosylcobalamin, which is spatially limited to and highly specific for adenine nucleosides. The binding of 5'-deoxyadenosine to the apoenzyme was reversible.     

Adenine nucleoside dialdehydes: potent inhibitors of bovine liver S-adenosylhomocysteine hydrolase

Various ribonucleoside 2',3'-dialdehydes, including adenosine dialdehyde, S-adenosylhomocysteine (AdoHcy) dialdehyde, and 5-(methylthio)-5'-deoxyadenosine (MTA) dialdehyde, were shown to be potent inhibitors of bovine liver AdoHcy hydrolase (EC 3.3.1.1). These ribonucleoside 2',3'-dialdehydes produce both time-dependent and concentration-dependent inactivation of the AdoHcy hydrolase. The inactivation appears to be irreversible since the enzyme activity cannot be recovered after prolonged dialysis against phosphate buffer. However, a substantial percentage of the enzyme activity could be recovered when the inactivated enzyme was dialyzed against a nitrogen buffer [e.g., tris(hydroxymethyl)aminomethane (Tris)]. This reversal of inhibition could be prevented, however, by pretreatment of the ligand-enzyme complex with sodium borohydride prior to dialysis in Tris buffer. Inclusion of substrates (e.g., adenosine or AdoHcy) afforded protection of the enzyme from the inactivation induced by the ribonucleoside 2',3'-dialdehydes. These data suggest that the bond formed between the enzyme and the inhibitor is probably a Schiff base linkage between the aldehydic functionality of the inhibitor and a protein lysinyl residue in or around the adenosine-AdoHcy binding site. When [2,8-3H]adenosine dialdehyde was used, a stoichiometry of 1.73 nmol of inhibitor bound per nmol of AdoHcy hydrolase was determined. Analysis of the kinetics of enzyme inactivation using the Ackermann-Potter approach indicates that adenosine dialdehyde is a tight-binding inhibitor, exhibiting a stoichiometry of one to two molecules of inhibitor bound to one molecule (tetramer) of enzyme and a Ki = 2.39 nM.     

3',5'-Cyclic nucleotide phosphodiesterase activity of the sulphatase A of ox liver

The sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) of ox liver hydrolyses adenosine 3',5'-monophosphate (cyclic AMP) to adenosine 5'-phosphate at an optimum pH of approx. 4.3, close that for the hydrolysis of cerebroside sulphate, a physiological substrate for sulphatase A. The Km is 11.6 mM for cyclic AMP. On polyacrylamide gel electrophoresis sulphatase A migrates as a single protein band which coincides with both the arylsulphatase and phosphodiesterase activities, suggesting that these are due to a single protein. Cyclic AMP competitively inhibits the arylsulphatase activity of sulphatase A, showing that both activities are associated with a single active site on the enzyme. sulphatase A also hydrolyses guanosine 3',5'-monophosphate, but not uridine 3',5'-monophosphate nor adenosine 2',3'-monophosphate.     

Fluorescence studies of ATP-diphosphohydrolase from Solanum tuberosum var. Desirée

Chemical modification of potato apyrase suggests that tryptophan residues are close to the nucleotide binding site. Kd values (+/- Ca2+) for the complexes of apyrase with the non-hydrolysable phosphonate adenine nucleotide analogues, adenosine 5'-(beta,gamma-methylene) triphosphate and adenosine 5'-(alpha,beta-methylene) diphosphate, were obtained from quenching of the intrinsic enzyme fluorescence. Other fluorescent nucleotide analogues (2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate, 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-diphosphate. 1,N6-ethenoadenosine triphosphate and 1,N6-ethenoadenosine diphosphate) were hydrolysed by apyrase in the presence of Ca2+, indicating binding to the active site. The dissociation constants for the binding of these analogues were calculated from both the decrease of the protein (tryptophan) fluorescence and enhancement of the nucleotide fluorescence. Using the sensitised acceptor (nucleotide analogue) fluorescence method, energy transfer was observed between enzyme tryptophans and ethene-derivatives. These results support the view that tryptophan residues are present in the nucleotide-binding region of the protein, appropriately oriented to allow the energy transfer process to occur.     

Affinity chromatography and binding studies on immobilized 5'-monophosphate and adenosine 2',5'-bisphosphate of nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa.

1. Nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa was purified to apparent homogeneity with an improved method employing affinity chromatography on N6-(6aminohexyl)-adenosine 2', 5'-bisphosphate-Sepharose 4B. 2. Polyacrylamide gel electrophoresis of the purified transhydrogenase carried out in the presence of sodium dodecyl sulphate, indicated a minimal molecular weight of 55000 +/- 2000. 3. The kinetic and regulatory properties of the purified transhydrogenase resembled those of the crude enzyme, i.e., NADPH, adenosine 2'-monophosphate and Ca2+ were activators whereas NADP+ was inhibitory. 4. Nicotinamide nucleotide-specific release of binding of the transhydrogenase to N6-(6-aminohexyl)-adenosine-2',5'-bisphosphate-Sepharose and N6-(-aminohexyl)-adenosine-5'-monophosphate-Sepharose suggests the presence of at least two separate binding sites for nicotinamide nucleotides, one that is specific for NADP(H) and one that binds both NAD(H) and NADP(H). 5. Binding of transhydrogenase to N6-)6-aminohexyl)-adenosine-2',5'-bisphosphate-Sepharose and activation of the enzyme by adenosine-2',5'-bisphophate showed a marked pH dependence. In contrast, inhibition of the Ca2+-activated enzyme by adenosine 2',5'-bisphosphate was virtually constant at various pH values. This descrepancy was interpreted to indicate the existence of separate nucleotide-binding effector and active sites.     

Kinetics and mechanism of the rat brain phenol sulphotransferase reaction.

Some properties of rat brain phenol sulphotransferase were investigated in in vitro at pH7.4. The enzyme was purified 10-fold by chromatography on DEAE-Sephadex -50. It can be assayed with 4-hydroxy-3-methoxyphenylethylene glycol or 4-methylumbelliferone as the sulphate acceptor. The partially purified enzyme is stable for at least 1 week when stored at 4 degrees C. It is, however, additionally activated (10--20%) and stabilized by 1 mM-dithiothreitol. The activity of the enzyme does not depend on the addition of exogenous Mg2+. The pH optima for the sulphation of 4-hydroxy-3-methoxyphenylethylene glycol and 4-methylumbelliferone are 7.8 and 7.4 respectively. Substrate inhibition by the sulphate acceptor is apparent at concentrations over 0.05mM. Initial-velocity studies in the absence and presence of product and dead-end inhibitors suggested that the mechanism of the rat brain sulphotransferase reaction is sequential ordered Bi Bi with a dead-end complex of enzyme with adenosine 3',5'-biphosphate and sulphate acceptor. The sulphate donor adenosine 3'-phosphate 5'-sulphatophosphate is the first substrate that adds to the enzyme, and the sulphate acceptor is the second substrate. The dissociation constant for the complex of enzyme with sulphate donor is 21 micron. The sulphated substrate is the first product and adenosine 3',5'-biphosphate is the second product that leaves the enzyme.     

Changes in cyclic AMP-dependent protein dinase activity in Tetrahymena pyriformis during the growth cycle.

An adenosine 3':5'-monophosphate-dependent protein kinase II (ATP:protein phosphotransferase, EC 2.7.1.37) was partially purified from the cytosol fraction of an exponentially growing culture of Tetrahymena pyriformis. Protein kinase II represented approximately 90% of the cytosolic protein kinase activity. The enzyme had a high degree of substrate specificity for calf thymus and Tetrahymena histones as compared to casein, protamine and phosvitin. The enzyme incorporated the terminal phosphate of ATP into serine and threonine residues of all the histone fractions. The apparent Km of the enzyme for adenosine 3':5'-monophosphate (cyclic AMP) was 1-10-minus 8 M. Protein kinase II was also activated by other cyclic nucleotides with apparent Km values in the range 2.k-10-minus 6 M. Ther specific activity of the cyclic AMP-dependent protein kinase of Tetrahymena decreases markedly from initial high values during the transition from the lag to early log phase of growth. This is followed by a shrp increase in the activity of the enzyme as the log phase of growth progresses. The specific activity of the enzyme increases rapidly during the heat-induced synchronization of Tetrahymena cells. The capacity for rapid phosphorylation of multiple classed of organelle-specific phosphoproteins and the level of cyclic AMP were maximal in Tetrahymena during the earliest phase of growth. These results demonstrate that the cell cycle of Tetrahymena may be coordinated by marked variations in the level of cyclic AMP which in turn regulate the cyclic AMP-dependent protein kinase.     

Mechanism of ribonucleic acid chain initiation. 1. A non-steady-state study of ribonucleic acid synthesis without enzyme turnover

A non-steady-state kinetic method has been developed to observe the initiation of long RNA chains by Escherichia coli RNA polymerase without the enzyme turnover. This method was used to determine the order of binding of the first two nucleotides to the enzyme in RNA synthesis with the first two nucleotides to the enzyme in RNA synthesis with poly(dA-dT) as the template. It was shown that initiator [ATP, uridyly(3'-5')adenosine, or adenyly(3'-5')uridylyl-(3'-5')adenosine] binds first to the enzyme-template complex, followed by UTP binding. The concentration dependence of UTP incorporation into the initiation complex suggests that more than one UTP molecule may bind to the enzyme-DNA complex during the initiation process. Comparison of the kinetic parameters derived from these studies with those obtained under steady-state conditions indicates that the steps involving binding of initiator or UTP during initiation cannot be rate limiting in the poly(dA-dT)-directed RNA synthesis. The non-steady-state technique also provides a method for active-site titration of RNA polymerase. The results show that only 36 +/- 9% of the enzyme molecules are active in a RNA polymerase preparation of high purity and specific activity. In addition, the minimal length of poly(dA-dT) involved in RNA synthesis by one RNA polymerase molecule was estimated to be approximately 500 base pairs.