Preparation of analogues of ATP, ADP and AMP suitable for binding to matrices and the enzymic interconversion of ATP and ADP in solid phase.

Alkylation of ATP with iodoacetic acid at pH 6.5 yielded 1-carboxymethyl-ATP which, after alkaline rearrangement, gave N-6-carboxymethyl-ATP. Condensation of this analogue with 1,6-diaminohexane in the presence of a water-soluble carbodiimide generated N-6-[(6-aminohexyl)carbamoylmethyl]-ATP in an overall yield of 40% based on the parent nucleotide ATP. The coenzymic activities of both N-6-adenine-substituted derivatives of ATP were tested with three kinases. Both derivatives showed coenzymic function against hexokinase with the "long" derivative having highest activity (95%) relative to unsubstituted ATP. Their activities towards the other two kinases tested was negligible except with the "long" analogue against glycerokinase (20%). The latter ATP analogue, when bound to Sepharose through its terminal amino group, could be dephosphorylated to the corresponding ADP analogue with soluble hexokinase yielding glucose 6-phosphate in an enzymic "solidphase" fashion. The Sepharose-bound ADP formed could subsequently be phosphorylated back to ATP using soluble acetate kinase. Sepharose-ATP preparations were also used in preliminary affinity chromatography studies using citrate synthase. Alkylation of ADP following the above procedure yielded the corresponding ADP analogue, N-6-[(6-aminohexyl)carbamoylmethyl]-ADP in an overall yield of 40%. Alkylation of AMP yielded the corresponding N-6-[(6-aminohexyl)carbamoylmethyl]-AMP in an overall yield of 45%.     

A new method of chemical modification of N6-amino group in adenine nucleotides with formaldehyde and a thiol and its application to preparing immobilized ADP and ATP.

Reaction of AMP with formaldehyde and 3-mercaptopropionic acid at pH 11.7 gave a new AMP derivative, N6-[(2-carboxyethyl)thiomethyl]-AMP (I) in 91% yield and reaction at pH 3.1 gave another new derivative, N6,N6-bis[(2-carboxyethyl)thiomethyl]-AMP (II) in 57% yield. The structures were determined by their 13C and 1H nuclear magnetic resonance spectra coupled with those of the simple analogues, N6-[(2-carboxyethyl)thiomethyl]-9-methyladenine (III) and N6,N6-bis[(2-carboxyethyl)thiomethyl]-9-methyladenine (IV) which were synthesized from 9-methyladenine in the same way as for derivatives I and II. ADP and ATP were treated in the same way as AMP to afford the corresponding carboxyl derivatives, N6-[(2-carboxyethyl)thiomethyl]-ADP (V), N6-[(2-carboxyethyl)thiomethyl]-ATP (VI), N6,N6-bis[(2-carboxyethyl)thiomethyl]-ADP (X) and N6,N6-bis[(2-carboxyethyl)thiomethyl]-ATP (XI) in 71%, 75%, 53% and 40% yield, respectively. These compounds were coupled to 1,3-diaminopropane with a water-soluble carbodiimide to give the corresponding amino derivatives, N6-([N-3-aminopropyl)carbamoylethyl]thiomethyl)-ADP (VIII), N6-(N-(3-aminopropyl)carbamoylethyl]thiomethyl)-ATP (IX), N6,N6-bis([N-(3-aminopropyl)carbamoylethyl]thiomethyl)-ADP (XIII), and N6,N6-bis([N-(3-aminopropyl)carbamoylethyl]thiomethyl)-ATP (XIV), which were further bound to CNBr-activated dextran to give new polymer-bound derivatives of ADP and ATP. These free and bo-nd derivatives were tested for their coenzymic activities against several kinases. The activities of the ADP derivatives, V, VIII, X, XIII, dextran-bound VIII, and dextran-bound XIII against acetate kinase were 82%, 81%, 68%, 55%, 35%, and 15%, respectively, relative to ADP and those of the ATP derivatives, VI, IX, XI, XIV, dextran-bound IX, and dextran-bound XIV against hexokinase were 88%, 94%, 60%, 81%, 58%, and 49%, respectively, relative to ATP.     

Preparation of N6-[N-(6-aminohexyl) carbamoyl]-adenine nucleotides and their application to coenzymically active immobilized ADP and ATP, and affinity adsorbents.

Reaction of ADP with hexamethylene diisocyanate in hexamethylphosphoramide followed by treatment in an acidic medium afforded three new adenine nucleotide analogues, N6-[N-(6-aminohexyl)carbamoyl]-ADP, N6-[N-(6-aminohexyl)carbamoyl]-ATP, and N6-[N-(6-aminohexyl)carbamoyl]-AMP in yields of 13%, 12% and 17%, respectively. The occurrence of the ATP analogue may be interpreted in terms of the equilibrium, 2ADP = ATP + AMP. Coenzymic activities of the ADP analogue against acetate kinase and pyruvate kinase were 82% and 20%, respectively, relative to ADP and those of the ATP analogue against hexokinase and glycerokinase were 63% and 87%, respectively, relative to ATP. These analogues were bound to CNBr-activated soluble dextran through their terminal amino group to give an immobilized ADP and an immobilized ATP, each of which was recycled in a system comprising acetate kinase and hexokinase, and when placed in a membrane reactor together with the enzymes, functioned as an immobilized coenzyme continuously yielding glucose 6-phosphate. A series of chemically defined affinity adsorbents were obtained by coupling these analogues to CNBr-activated Sepharose, and were used to separate the enzymes in a mixture of hexokinase, pyruvate kinase, phosphoglycerate kinase, lactate dehydrogenase, and alcohol dehydrogenase.     

The Synthesis of New Polymer Derivatives of ATP by Radical Copolymerization and Their Coenzymic Activity

Three polymerizable ATP derivatives, N⁶-[N-(6-methacrylamidohexyl)carbamoylmethyl]-, N⁶-[N -[2-[N -(2-methacrylamidoethyl)carbamoyl]ethyl]carbamoylmethyl]-, and N⁶ -[N -[N -(2-hydroxy- 3-methacrylamidopropyl)carbamoylmethyl]carbamoylmethyl]-ATP, were synthesized and radically copolymerized with comonomers [acrylamide, N -(2-hydroxyethyl)-, N -ethyl-, N, N - diethylacrylamide, acrylic acid, and 6-methacrylamidohexylammonium chloride] to obtain 18 new polymer derivatives of ATP. The molecular weight distributions were controlled by appropriate initiator concentrations. The monomeric and polymeric ATP derivatives were all coenzymically active against both hexokinase and glycerol kinase. The observed coenzymic activities (Km and V_max) are discussed in connection with the structures of the derivatives.     

High-performance liquid chromatography separation of some NAD+ derivatives suitable for covalent binding

Separation of NAD⁺, N¹-carboxymethyl-NAD⁺, N⁶-carboxymethyl-NAD⁺, and N⁶-[N-(6-aminohexyl)carbamoylmethyl]-NAD⁺ by high performance liquid chromatography is described. Reversed-phase chromatography with the acidic mobile phase (phosphate buffer pH 2.0–3.6) proved to be the most suitable method, particularly for the separation of impurities. The proposed method can be used for monitoring the course of the synthesis of N⁶-[N-(6-aminohexyl)carbamoylmethyl]-NAD⁺ and for the separation of the intermediates. Identification of the peaks was performed by means of spectroscopic measurement as well as a specific coenzyme activity test. Performance of the described method is greater in comparison with thin-layer chromatography.     

Preparation of an alcohol-dehydrogenase--NAD(H)--sepharose complex showing no requirement of soluble coenzyme for its activity.

1. Horse liver alcohol dehydrogenase and an NADH analogue, N6-[(6-aminohexyl)carbamoylmethyl]-NADH, have been co-immobilized to Sepharose 4B under conditions permitting binary complex formation between the enzyme and the cofactor. 2. The enzyme-coenzyme-matrix preparations were assayed with a coupled oxidoreduction reaction and showed activities, prior to addition of coenzyme, that were up to 40% of that obtained in excess of free coenzyme. 3. A molar ratio of 1:1 between the amount of bound enzyme was sufficient to obtain high activities in the absence of free coenzyme. 4. The highest recycling rate obtained for the immobilized nucleotide was 3400 cycles per hour. 5. Both thermal and storage stability of alcohol dehydrogenase was increased when the enzyme was co-immobilized with the NADH analogue. 6. The efficiency of the immobilized preparations (measured as product formation per minute and per assay volume) was higher (1.4 to 5 times in our assays) than the corresponding systems of free enzyme (in total enzyme units) and nucleotide in an identical assay volume.     

Production of macrophage-derived cytotoxic factor by N-[3-[(carbamoylmethyl)thio]propionylated] neoglycoproteins

6-Phosphomannosylated bovine serum albumin (Man6P-BSA), a neoglycoprotein endocytosed by macrophages, bearing either 3-(2-pyridyldithio)propionyl or 3-[(carbamoylmethyl)thio]propionyl residues coming from alkylation of thiol residues by iodoacetamide were prepared and tested for their immunomodulator properties. The supernatants of mouse peritoneal macrophages incubated with Man6P-BSA bearing 3-[(carbamoylmethyl)thio]propionyl groups, and by a lesser extent 3-(2-pyridyldithio)propionyl groups, were cytotoxic to L929 cells, suggesting the presence of a tumor necrosis factor like compound. This macrophage-activation process is linked to the capacity of Man6P-BSA to be endocytosed via membrane lectins of macrophages, because the supernatants of macrophages incubated with unglycosylated conjugates were not cytotoxic. The cytotoxic activity induced by 3-[(carbamoylmethyl)thio]propionyl groups bound onto Man6P-BSA was similar to that induced by Man6P-BSA bearing muramyl dipeptide, indicating that endocytosed neoglycoproteins bearing 3-[(carbamoylmethyl)thio]propionyl residues are potent macrophage activators.     

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.     

Biospecific affinity chromatography of an adenosine 3′:5′-cyclic monophosphate-stimulated protein kinase (protamine kinase from trout testis) by using immobilized adenine nucleotides

1. Two adenine nucleotides, 8-(6-aminohexyl)aminoadenosine 3':5'-cyclic monophosphate and 8-(6-aminohexyl)amino-AMP, were synthesized. Their structures were established in particular by using mass spectroscopy. 2. Free cyclic AMP and 8-(6-aminohexyl)amino cyclic AMP both stimulate protamine kinase activity at low concentrations, but are inhibitory at concentrations above 0.1mm. AMP is an inhibitor of enzymic activity, whereas neither 8-(6-aminohexyl)amino-AMP nor the earlier synthesized N(6)-(6-aminohexyl)-AMP is inhibitory. 3. The nucleotides were coupled to Sepharose 4B and used for biospecific chromatography of partially purified protamine kinase. Enzyme applied at high buffer concentrations to the cyclic AMP-Sepharose material was retarded and thereby purified tenfold. At low buffer concentrations the enzyme was adsorbed to the affinity material, and was subsequently released by a pulse of the inhibitor AMP, yielding a 50-100-fold purification. Enzyme applied to immobilized 8-(6-aminohexyl)amino-AMP or N(6)-(6-aminohexyl)-AMP was eluted together with the main protein peak in the void volume. 4. Protamine kinase eluted from 8-(6-aminohexyl)amino cyclic AMP-Sepharose was no longer activated by cyclic AMP. Results from sucrose gradient centrifugation suggest that a dissociation of the enzyme took place on the immobilized nucleotide. 5. Further information on the mass spectroscopy has been deposited as Supplementary Publication SUP 50026 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.     

Antagonists of calcium fluxes and calmodulin block activation of the p21-activated protein kinases in neutrophils

Neutrophils stimulated with fMLP or a variety of other chemoattractants that bind to serpentine receptors coupled to heterotrimeric G proteins exhibit rapid activation of two p21-activated protein kinases (Paks) with molecular masses of approximately 63 and 69 kDa (gamma- and alpha-Pak). Previous studies have shown that products of phosphatidylinositol 3-kinase and tyrosine kinases are required for the activation of Paks. We now report that a variety of structurally distinct compounds which interrupt different stages in calcium/calmodulin (CaM) signaling block activation of the 63- and 69-kDa Paks in fMLP-stimulated neutrophils. These antagonists included selective inhibitors of phospholipase C (1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione), the intracellular Ca(2+) channel (8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate), CaM (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide; trifluoperazine), and CaM-activated protein kinases (N-[2-(N-(chlorocinnamyl)-N:-methylaminomethyl)phenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide). This inhibition was dose-dependent with IC(50) values very similar to those that interrupt CaM-dependent reactions in vitro. In contrast, less active analogues of these compounds (1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione; N-(6-aminohexyl)-1-naphthalenesulfonamide; N-(4-aminobutyl)-1-naphthalenesulfonamide; promethazine; 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine]) did not affect activation of Paks in these cells. CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; trifluoperazine), but not their less-active analogues (N-(6-aminohexyl)-1-naphthalenesulfonamide; promethazine), were also found to block activation of the small GTPases Ras and Rac in stimulated neutrophils along with the extracellular signal-regulated kinases. These data strongly suggest that the Ca(2+)/CaM complex plays a major role in the activation of a number of enzyme systems in neutrophils that are regulated by small GTPases.     

Covalent binding of an NAD analogue to liver alcohol dehydrogenase resulting in an enzyme-coenzyme complex not requiring exogenous coenzyme for activity.

1. The NAD analogue, N6-[N-(6-aminohexyl)carbamoylmethyl]-NAD, was covalently bound to horse liver alcohol dehydrogenase in a carbodiimide-mediated reaction and in such a way that it was active with the very same enzyme molecule to which it was coupled. 2. The degree of substitution, i.e. the number of NAD analogues per enzyme subunit, could be varied (0.3-1.6). In one preparation 1.6 coenzyme molecules were bound per subunit; the alcohol dehydrogenase activity of this preparation was 40% of the activity obtained after addition of free NAD in excess. 3. It was calculated that every fourth active site of this preparation was provided with a covalently bound functioning coenzyme analogue, and that this analogue had a cycling rate of about 40 000 cycles/h in a coupled substrate assay. 4. The presence of the covalently bound coenzyme made the active sites difficult to inhibit with a competitive inhibitor. For example, 10 mM AMP inhibited the activity of the preparation by 50% whereas a reference system containing native alcohol dehydrogenase was inhibited by 80% in spite of the fact that the reference system contained about 20 000 times as high a concentration of coenzyme.     

Covalent binding of an NAD+ analogue to horse liver alcohol dehydrogenase in a ternary complex with pyrazole

Examination of the model of the fixation site of the adenosine phosphate part of NAD+ on horse liver alcohol dehydrogenase led us to synthesize a NAD+ analogue N6-[N-(8-amino-3,6-dioxaoctyl)carbamoylmethyl]-NAD+ in order to alkylate the carboxylic acid group of Asp-273 and to convert the normally dissociable coenzyme into a permanently bound prosthetic group. This NAD+ analogue is coupled to the horse liver alcohol dehydrogenase in the ternary complex formed with pyrazole. In these conditions the degree of fixation varies between 0.4 and 0.58 coenzyme molecule/enzyme subunit molecule. The N6-[N-(8-amino-3,6-dioxaoctyl)carbamoylmethyl]NAD+ acts as a true prosthetic group which can be reduced and reoxidized by a coupled substrate reaction and the internal activity of this holoenzyme corresponds to the amount of analogue incorporated.     

A kinetic study on the binding of monomeric and polymeric derivatives of NAD+ to yeast alcohol dehydrogenase

The binding to yeast alcohol dehydrogenase of NAD+ and its five derivatives (N6-[2-[N-[2-[N-(2-methacrylamidoethyl)carbamoyl]ethyl] carbamoyl]ethyl]-NAD (I), N6-[N-[2-[N-(2-methacrylamidoethyl) carbamoyl]ethyl]carbamoylmethyl]-NAD (II), copolymer of I with acrylamide (PA-I), copolymer of II with acrylamide (PA-II), and copolymer of I with N,N-dimethylacrylamide (PDMA-I] were studied statically and kinetically by the stopped-flow method by using the quenching of the enzyme fluorescence in the presence of pyrazole. Apparent dissociation constants and apparent rate constants were determined therefrom. It was concluded that (1) the N6-CH2CH2CO group (of I) is effective in making the derivative bind more strongly as well as faster than NAD+, while the N6-CH2CO group (of II) is not; and (2) the binding of the polymer derivatives of NAD+ to the enzyme is not essentially weaker and slower than that of native NAD+, but is even faster in some cases. The coenzymic activities of the above compounds were also determined with yeast alcohol dehydrogenase, pig heart malate dehydrogenase, and rabbit muscle lactate dehydrogenase.     

Phosphorothioate analogues of 2',5'-oligoadenylate. Enzymatic synthesis, properties, and biological activities of 2',5'-phosphorothioates from adenosine 5'-O-(2-thiotriphosphate) and adenosine 5'-O-(3-thiotriphosphate)

The chiral and achiral phosphorothioate analogues of 2',5'-oligoadenylates (2-5A) have been enzymatically synthesized from the Sp and Rp isomers of adenosine 5'-O-(2-thiotriphosphate) [(Sp)-ATP beta S and (Rp)-ATP beta S, respectively] and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) by 2-5A synthetase from L929 cells and lysed rabbit reticulocytes. These 2',5'-phosphorothioate analogues were separated, purified, and structurally characterized. While ATP gamma S and (Sp)-ATP beta S were as efficient substrates for the 2-5A synthetase as was ATP, (Rp)-ATP beta S was more than 50-fold less efficient a substrate. The beta- and gamma-phosphorothioates were more resistant to enzymatic hydrolysis than was authentic 2-5A. Compared to 2-5A, there were marked differences in the biological activities of the 2',5'-phosphorothioates as determined by (i) binding to 2-5A-dependent endoribonuclease (RNase L), (ii) activation of RNase L to hydrolyze RNA, and (iii) inhibition of protein synthesis in intact L929 cells. These studies extend previous reports on the elucidation of the stereochemical requirements of 2-5A synthetase and RNase L [Karikó, K., Sobol, R. W., Jr., Suhadolnik, L., Li, S. W., Reichenbach, N. L., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (first of three papers in this issue); Karikó, K., Li, S. W., Sobol, R. W., Jr., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (second of three papers in this issue)] with the phosphorothioate analogues of 2-5A.(ABSTRACT TRUNCATED AT 250 WORDS)     

The human SH-SY5Y neuroblastoma cell-line expresses a functional P2X7 purinoceptor that modulates voltage-dependent Ca2+ channel function

Fura-2 imaging of purinergic stimulation of non-differentiated neuronal human SH-SY5Y cells resulted in a rapid elevation in intracellular Ca2+ ([Ca2+]i) that was dependent on extracellular Ca2+. The rank order of agonists (200 micro m) was as follows: 2',3'-O-(4-benzoyl-benzoyl)-ATP (BzATP) > ATP4- > ATP; whereas 2-(methylthio)-ATP, ADP, UTP and alpha,beta-methylene-ATP and beta,gamma-methylene-ATP were ineffective. The response to BzATP was inhibited by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic-acid (PPADS, 1 micro m), 1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-l-tyrosyl)-4-phenylpiperazine (KN-62, 100 nm) and 8-(3-benzamido-4-4-methylbenzamido)-naphthalene-1,3,5-trisulfonic-acid (suramin, 200 micro m). The presence of a P2X7 receptor was confirmed by western blot studies using anti-P2X7. EC50 for BzATP was 212 +/- 6 micro m. BzATP > 30 micro m induced an initial, transient increase in [Ca2+]i before a plateau level was reached. BzATP < 30 micro m only produced a monophasic increase to the plateau level. The transient phase was reduced by the introduction of nimodipine (3 micro m) and to a smaller degree by omega-conotoxin GVIA (1 micro m) despite an almost equal presence of L and N-type Ca2+-channels. In whole-cell voltage-clamp studies at - 90 mV, BzATP (300 micro m) produced a fast activating inward current with a similar pharmacology as observed with Fura-2 imaging. Current clamp studies showed a dose-dependent depolarization to BzATP and ATP4-. BzATP also triggered transmitter release. Thus, the human neuronal SH-SY5Y cell line expresses a functional P2X7 receptor coupled to activation of Ca2+-channels.     

Evidence for a Mg2+-induced conformational change at the ATP-binding site of (Na+ + K+)-ATPase demonstrated with a photoreactive ATP-analogue

1. The 3'-ribosyl ester of ATP with 2-nitro-4-azidophenyl propionic acid has been prepared and its ability to act as a photoaffinity label of (Na+ + K+)-ATPase has been tested. 2. In the dark 3'-O-[3-(2-nitro-4-azidophenyl)-propionyl]adenosine triphosphate (N3-ATP) is a substrate of (Na+ + K+)-ATPase and a competitive inhibitor of ATP hydrolysis. 3. Upon irradiation by ultraviolet light, N3-ATP photolabels the high-affinity ATP-binding site and is covalently attached to the alpha-subunit and an approximately 12000-Mr component. 4. Photolabeling of the alpha-subunit by N3-ATP irreversibly inactivates (Na+ + K+)-ATPase. 5. Photoinactivation is strictly Mg2+-dependent. Na+ enhances the inactivation. ATP or ADP and K+ protect the enzyme against inactivation. 6. Mg2+, in concentrations required for photoinactivation, protects (Na+ + K+)-ATPase against inactivation by tryptic digestion under controlled conditions. 7. It is assumed that a conformational change of the ATP-binding site of (Na+ + K+)-ATPase occurs upon binding of Mg2+ to a low-affinity site.     

ADP receptor-induced activation of guanine-nucleotide-binding proteins in human platelet membranes.

ADP receptor-regulated binding of the labeled GTP analog, guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTP[gamma S]), to guanine-nucleotide-binding proteins (G proteins) was studied in human platelet membranes. The potent ADP receptor agonist, 2-methyl-thio-adenosine 5'-diphosphate (2MeSADP), a non-hydrolyzable analog of ADP, increased the binding of [35S]GTP[gamma S] without apparent lag phase. Under optimal conditions, i.e. in the presence of GDP (1-10 microM), 2MeSADP increased the binding up to about threefold, with half-maximal and maximal increase observed at 10 nM and 1 microM 2MeSADP, respectively. ADP itself increased the binding of [35S]GTP[gamma S] by maximally about twofold, with half-maximal increase occurring at 0.1 microM ADP. The agonist-induced stimulation was competitively antagonized by the ADP receptor(s) antagonist, (1S)-adenosine 5'-O-(1-thiotriphosphate) [(Sp)-ATP[alpha S]]. Other platelet receptor agonists known to act through receptors coupled to G proteins also increased binding of [35S]GTP[gamma S] in human platelet membranes, but without being inhibited by (Sp)-ATP[alpha S]. The data presented indicate that the platelet ADP receptor(s) can interact with and efficiently activate G proteins, the nature of which remains to be identified.     

The Co-immobilization of NAD and Dehydrogenases and Its Application to Bioreactors for Synthesis and Analysis

A polymerizable NAD derivative, N⁶-[N-[N-(2-hydroxy-3-methacrylamidopropyl)carbamoylmethyl]carbamoylmethyl]-NAD, formate dehydrogenase, and malate dehydrogenase were entrapped all together in polyacrylamide gels. The entrapment was carried out by radical copolymerization, and consequently NAD was bound on the matrix which enclosed the enzymes. These gels had the function of producing l-malate from oxalacetate and formate. The l-malate production was also continuously done in a column reactor for 3 days. Another gel was similarly prepared with N⁶-[N-(6-methacrylamidohexyl)carbamoylmethyl]-NAD, horse liver alcohol dehydrogenase, and diaphorase. This gel was shown to catalyze the formation of resorufin from resazurin and ethanol. This gel was applicable to ethanol analysis using a fluorescence spectrophotometer to determine resorufin. The analyzer was usable for one week.