STUDIES ON THE REGULATION OF GABA SYNTHESIS: SUBSTRATE-PROMOTED DISSOCIATION OF PYRIDOXAL-5''-PHOSPHATE FROM GAD

The association between glutamate decarboxylase (GAD) and its cofactor, pyridoxal-5′-phos-phate (pyridoxal-P), was studied using 20,0000 supernatant of rat brain. In this preparation GAD required added pyridoxal-P to maintain a linear reaction rate beyond 5 min of incubation. Following exhaustive dialysis the enzyme was more than 83% saturated with cofactor indicating that the cofactor was tightly bound to the enzyme. When incubations were performed in the presence of glutamate and without added pyridoxal-P there was a progressive inactivation of the enzyme which was dependent on the glutamate concentration. This lost activity was almost completely recovered by addition of pyridoxal-P to the dialyzed glutamate-inactivated enzyme. The results suggest that glutamate inactivates GAD by promoting the dissociation of pyridoxal-P from the enzyme thereby producing inactive apoen-zyme which can be reactivated by combining with available pyridoxal-P. This interpretation is supported by the finding that progress curves for the reaction were accurately described over a 30 min incubation period and 10-fold glutamate concentration range by an integrated rate equation which takes the glutamate-promoted dissociation of cofactor into account. The progressive inactivation could not be attributed to denaturation of the enzyme, impurities in the substrate, effects of pH, depletion of substrate, protein concentration, sulfhydryl reagents or product inhibition. The results presented here also show that certain precautions must be adopted to accurately measure GAD activity in the absence of added pyridoxal-P as has been widely done in studies of drug action. Specifically, measurements must be made at short times of incubation and low concentrations of glutamate to minimize the glutamate-promoted inactivation of the enzyme.     

Oxidative inactivation of an extramitochondrial acetyl-CoA hydrolase by autoxidation of L-ascorbic acid

The activity of acetyl-CoA hydrolase (dimeric form) purified from the supernatant fraction of rat liver was shown to have a half-life (t1/2) of 3 min at 0 degree C, but to stable at 37 degrees C (t1/2 = 34 h) [Isohashi, F., Nakanishi, Y. & Sakamoto, Y. (1983) Biochemistry 22, 584-590]. Incubation of the purified enzyme with L-ascorbic acid (AsA) at 37 degrees C resulted in inactivation of the enzyme (t1/2 = 90 min at 2 mM AsA). The extent of inactivation was greatly enhanced by addition of transition metal ions (Cu2+, Fe2+, and Fe3+). Thiol reducing agents, such as reduced glutathione and DL-dithiothreitol, protected the hydrolase from inactivation by AsA. However, these materials did not restore the catalytic activity of the enzyme inactivated by AsA. When AsA solution containing Cu2+ was preincubated under aerobic conditions at 37 degrees C for various times in the absence of enzyme, and then aliquots were incubated with the enzyme solution for 20 min, remaining activity was found to decrease with increase in the preincubation time, reaching a minimum at 60 min. However, further preincubation reduced the potential for inactivation. Catalase, a hydrogen peroxide (H2O2) scavenger, almost completely prevented inactivation of the enzyme by AsA plus Cu2+. Superoxide dismutase and tiron, which are both superoxide (O2-) scavengers, also prevented inactivation of the enzyme. A high concentration of mannitol, a hydroxyl radical (OH) scavenger, partially protected the enzyme from inactivation. These results suggest that inactivation of the enzyme by AsA in the presence of Cu2+ was due to the effect of active oxygen species (H2O2, O2-, OH) that are known to be autoxidation products of AsA. Valeryl-CoA, a competitive inhibitor of acetyl-CoA hydrolase, greatly protected the enzyme from inactivation by AsA plus Cu2+, but ATP and ADP, which are both effectors of this enzyme, had only slight protective effects. These results suggest that inactivation of this enzyme by addition of AsA plus Cu2+ was mainly due to attack on its active site.     

Characterization of the rhodobacter sphaeroides 5-aminolaevulinic acid synthase isoenzymes, HemA and HemT, isolated from recombinant Escherichia coli.

The hemA and hemT genes encoding 5-aminolaevulinic acid synthase (ALAS) from the photosynthetic bacterium Rhodobacter sphaeroides, were cloned to allow high expression in Escherichia coli. Both HemA and HemT appeared to be active in vivo as plasmids carrying the respective genes complemented an E. coli hemA strain (glutamyl-tRNA reductase deficient). The over-expressed isoenzymes were isolated and purified to homogeneity. Isolated HemA was soluble and catalytically active whereas HemT was largely insoluble and failed to show any activity ex vivo. Pure HemA was recovered in yields of 5-7 mg x L-1 of starting bacterial culture and pure HemT at 10 mg x L-1 x HemA has a final specific activity of 13 U x mg-1 with 1 unit defined as 1 micromol of 5-aminolaevulinic acid formed per hour at 37 degrees C. The Km values for HemA are 1.9 mM for glycine and 17 microM for succinyl-CoA, with the enzyme showing a turnover number of 430 h-1. In common with other ALASs the recombinant R. sphaeroides HemA requires pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor resulted in inactive apo-ALAS. Similarly, reduction of the HemA-PLP complex using sodium borohydride led to > 90% inactivation of the enzyme. Ultraviolet-visible spectroscopy with HemA suggested the presence of an aldimine linkage between the enzyme and pyridoxal 5'-phosphate that was not observed when HemT was incubated with the cofactor. HemA was found to be sensitive to reagents that modify histidine, arginine and cysteine amino acid residues and the enzyme was also highly sensitive to tryptic cleavage between Arg151 and Ser152 in the presence or absence of PLP and substrates. Antibodies were raised to both HemA and HemT but the respective antisera were not only found to bind both enzymes but also to cross-react with mouse ALAS, indicating that all of the proteins have conserved epitopes.     

Rapid Inactivation of Brain Glutamate Decarboxylase by Aspartate

In the absence of its cofactor, pyridoxal 5'-phosphate (pyridoxal-P), glutamate decarboxylase is rapidly inactivated by aspartate. Inactivation is a first-order process and the apparent rate constant is a simple saturation function of the concentration of aspartate. For the beta-form of the enzyme, the concentration of aspartate giving the half-maximal rate of inactivation is 6.1 +/- 1.3 mM and the maximal apparent rate constant is 1.02 +/- 0.09 min-1, which corresponds to a half-time of inactivation of 41 s. The rate of inactivation by aspartate is about 25 times faster than inactivation by glutamate or gamma-aminobutyric acid (GABA). Inactivation is accompanied by a rapid conversion of holoenzyme to apoenzyme and is opposed by pyridoxal-P, suggesting that inactivation results from an alternative transamination of aspartate catalyzed by the enzyme, as previously observed with glutamate and GABA. Consistent with this mechanism pyridoxamine 5'-phosphate, an expected transamination product, was formed when the enzyme was incubated with aspartate and pyridoxal-P. The rate of transamination relative to the rate of decarboxylation was much greater for aspartate than for glutamate. Apoenzyme formed by transamination of aspartate was reactivated with pyridoxal-P. In view of the high rate of inactivation, aspartate may affect the level of apoenzyme in brain.     

EFFECTS OF DEPOLARIZATION ON COFACTOR REGULATION OF GLUTAMIC ACID DECARBOXYLASE IN SUBSTANTIA NIGRA SYNAPTOSOMES

Abstract— The level of saturation of glutamate decarboxylase (GAD) by cofactor, pyridoxal-5'-phosphate (pyridoxal-P), determined in synaptosomes prepared from substantia nigra tissue, was reduced from 45 to 28%; when ATP was included in the homogenizing medium to prevent nonspecific activation of GAD by endogenous pyridoxal-P. When the synaptosomes were incubated for 5–20 min at 37°C in Krebs-Ringer phosphate buffer (KRP), the level of saturation of GAD by cofactor decreased further, from 28 to 20%. Depolarization of the nigral synaptosomes by either high K⁺ (55 mM) or veratridine resulted in a significant increase in the level of GAD saturation by cofactor, to 32 and 41%. respectively. Omitting Ca²⁺ from the incubation medium blocked the depolarization-induced rise in the level of saturation. Depolarization with high K⁺ and veratridine also caused a significant decrease in the ATP concentration in the synaptosomes. No difference in ATP concentration was observed when the samples were incubated at 37°C for 5–20min or incubated in the absence of added Ca²⁺ with high K⁺. Results provide further evidence that in vivo brain GAD is largely unsaturated by cofactor and support the possibility that increased release and utilization of GABA may be associated with increases in the amount of pyridoxal-P endogenously bound to GAD in nerve terminals.     

Modulation of testicular galactolipid sulfotransferase activity in vitro by ATP

Testicular galactolipid sulfotransferase activity is an early marker of differentiation during mammalian spermatogenesis. The enzyme will catalyze the sulfation of galactosylglycerol in the 3' position of the galactose moiety at 37 degrees C in vitro. However, sulfotransferase activity was found to be completely lost on preincubation of the solubilized enzyme preparation at 37 degrees C. This loss of activity was completely prevented by inclusion of ATP and Triton in the preincubation step. This protective effect was synergistic, pH dependent and correlated with an inhibition of endogenous phosphatase activity. These results are interpreted to suggest that the galactolipid sulfotransferase may be regulated by a phosphorylation mechanism.     

Factors affecting the cold inactivation of an acetyl-coenzyme-A hydrolase purified from the supernatant fraction of rat liver

An extramitochondrial acetyl-coenzyme-A hydrolase from rat liver is shown to be a cold-labile oligomeric enzyme that undergoes a reversible conformational transition between a dimeric and a tetrameric form in the presence of adenosine 5'-triphosphate or adenosine 5'-diphosphate at 25-37 degrees C, and between a dimeric and a monomeric form at low temperature. The enzymatically active dimer is fairly stable at 25-37 degrees C, but much less stable at low temperature, dissociating into monomer with no activity. At 37 degrees C and low concentrations of enzyme protein (less than or equal to 14 micrograms/ml), the activity decreased rapidly and only 10% of the initial activity remaining after 60 min. Addition of bovine serum albumin or immunoglobulin G to the medium completely prevented inactivation of the dimeric enzyme at low concentration at 37 degrees C, but had little effect on cold inactivation of the enzyme. Cold inactivation of the dimeric enzyme was partially prevented by the presence of various CoA derivatives. The order of potency was acetyl-CoA (substrate) greater than or equal to butyryl-CoA greater than octanoyl-CoA greater than CoA (product) greater than acetoacetyl-CoA. Another enzyme product, acetate, had little effect on cold inactivation. Polyols, such as sucrose, glycerol, and ethylene glycol, and high concentrations of NaCl, KCl, pyrophosphate and phosphate also greatly prevented cold inactivation. Cold inactivation was scarcely affected by pH within the pH range at which the enzyme was stable at 37 degrees C.     

STUDIES ON THE REGULATION OF GABA SYNTHESIS: THE INTERACTION OF ADENINE NUCLEOTIDES AND GLUTAMATE WITH BRAIN GLUTAMATE DECARBOXYLASE

The effects of adenine nucleotides and glutamate on glutamate decarboxylase were studied in a dialyzed, high-speed supernatant of rat brain. When incubated with 10 μm -pyridoxal-P the enzyme was strongly inhibited by ATP, ADP and their Mg²⁺ complexes at concentrations which were well below tissue levels. The enzyme was not significantly inhibited by 15 mm -AMP or by 100 μM-3′-5’cyclic AMP or 3′-5’cyclic GMP. Inhibition by the nucleotides cannot be described in conventional steady-state kinetic terms. Addition of ATP in the presence of pyridoxal-P resulted in a slow, progressive decrease in the reaction rate which was similar to the inactivation observed when the enzyme was incubated in the absence of pyridoxal-P. The progressive inactivation in the presence of ATP was minimal at concentrations of glutamate which were well below K_m and became much more pronounced at higher glutamate concentrations. Addition of suprasaturating amounts of pyridoxal-P late in the incubation when the enzyme was almost completely inactivated resulted in an immediate and complete reactivation of the enzyme. Inhibition by ATP could be prevented by addition of saturating amounts of pyridoxal-P at the start of the reaction and was also relieved by addition of potassium phosphate buffer. The results suggest that inhibition by the nucleotides involves the prior formation of the inactive apoenzyme which results from the glutamate-promoted dissociation of pyridoxal-P. In the absence of the nucleotides, the enzyme is normally reactivated by the added pyridoxal-P. The nucleotides act to block this reassociation of pyridoxal-P with the apoenzyme thereby producing a progressive inactivation of the enzyme. The implications of these results for the regulation of GABA synthesis are discussed.     

Effects of temperature and monovalent cations on activity and quaternary structure of tryptophanase

Effects of temperature and monovalent cations on the activity and the quaternary structure of tryptophanase of Escherichia coli were studied. The conversion of the apoenzyme into the active holoenzyme was attained at 30 degrees C in Tris-HCl buffer (pH 8.0) containing pyridoxal-P and K+, while no conversion occurred at 5 degrees C. The active holoenzyme thus formed was stable even at 5 degrees C, as long as the cation was present. When K+ was absent, however, the active enzyme gradually lost the activity upon chilling to 5 degrees C. The HPLC gel filtration analysis of the active holoenzyme and the low temperature-inactivated enzyme species revealed that the tetrameric holoenzyme dissociated into the dimeric apoenzyme concomitant with the low temperature-induced inactivation at 5 degrees C. The results of HPLC experiments together with other available evidence also suggest that the inactive tetrameric holoenzyme was first formed from the dimeric apoenzyme and pyridoxal-P prior to the formation of the active holoenzyme and that the cation promoted the conversion of the inactive holoenzyme into the active holoenzyme rather than being involved in the conversion of the apoenzyme and pyridoxal-P into the holoenzyme. Among various cations tested for the above effects, NH4+ exhibited the largest effect and K+ the second.     

Cytochemical model system for microsomal rat liver glucose-6-phosphate.

A method is described for the incorporation of a microsomal rat liver fraction into polyacrylamide films without significant loss of its glucose-6-phosphatase activity. The enzymatic activity was completely lost when the films were prepared with ammonium persulfate as initiator of the polymerization as previously described for alkaline phosphatase, but modification of this method showed that about 90% of the glucose-6-phosphatase activity could be retained. The enzyme in the films prepared with the new method was completely inhibited by alloxan, HgCl2, and preincubation in 0.05 M acetate buffer (pH 5.0) at 37 degrees C, as determined biochemically. Similar results were obtained for the enzyme in films determined histochemically according to the lead method of Wachstein and Meisel. In this respect the behavior of the incorporated enzyme is similar to that in suspension. Films fixed with 1.5% glutaraldehyde showed rapid inactivation of glucose-6-phosphatase. There was good correlation between the biochemical and histochemical activity determined after fixation. A method to embed polyacrylamide films in Epon for electron-microscopical investigation is also described. Dimethyl sulfoxide was used as the dehydrating agent instead of ethanol/acetone.     

Cloning, expression, and characterization of 5-aminolevulinic acid synthase from Rhodopseudomonas palustris KUGB306

The hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned from the genomic DNA of photosynthetic bacterium Rhodopseudomonas palustris KUGB306. The deduced protein (ALAS) of this gene contained 409 amino acids. The hemA gene was subcloned into an expression vector pGEX-KG and the encoded protein was overexpressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli BL21. The recombinant ALAS was purified and isolated free of the fusion partner (GST) by affinity purification on glutathione-Sepharose 4B resin and cleavage of the purified fusion protein by thrombin protease. The optimum pH and temperature of the recombinant ALAS was found to be at pH 7.5-8.0 and 35-40 degrees C, respectively. The Km value of the enzyme was 2.01 mM for glycine and 49.55 microM for succinyl-CoA. The enzyme activity was strongly inhibited by Pb2+, Fe2+, Co2+, Cu2+, and Zn2+ at 1 mM, but slightly affected by Mg2+ and K+. The recombinant ALAS required pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor led to complete loss of the activity. Ultraviolet-visible spectroscopy with the ALAS suggested the presence of an aldimine linkage between the enzyme and PLP.     

Characterization of the activity of purified recombinant human 5-lipoxygenase in the absence and presence of leukocyte factors.

Purified recombinant human 5-lipoxygenase was used to investigate the catalytic properties of the protein in the presence and absence of leukocyte stimulatory factors. Recombinant human 5-lipoxygenase was purified to apparent homogeneity (95-99%) from a high expression baculovirus system by chromatography on ATP-agarose with a yield of 0.6 mg of protein per 100 ml of culture (2 x 10(8) cells) and a specific activity of 3-6 mumol of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) per mg of protein in the presence of ATP, Ca2+, and phosphatidylcholine as the only factors. In the absence of leukocyte factors, the reaction catalyzed by the purified recombinant enzyme showed a half-time of maximal 5-HPETE formation of 0.5-0.7 min and was sensitive to the selective 5-lipoxygenase inhibitors BW755C (IC50 = 13 microM) and L-656,224 (IC50 = 0.8 microM). The reaction products of arachidonic acid oxidation were 5-HPETE and 6-trans- and 12-epi-6-trans-leukotriene B4, the nonenzymatic hydrolysis products of leukotriene A4 (LTA4), indicating that the purified protein expressed both the 5-oxygenase and leukotriene A4 synthase activities (ratio 6:1). The microsomal fraction and the 60-90% ammonium sulfate precipitate fraction from sonicated human leukocytes did not increase product formation by the isolated enzyme when assayed in the presence of ATP, Ca2+, and phosphatidylcholine. These factors were found to stabilize 5-lipoxygenase during preincubation of the enzyme at 37 degrees C with the assay mixture but they failed to stimulate enzymatic activity when added at the end of the preincubation period. The results demonstrate that human 5-lipoxygenase can be isolated in a catalytically active form and that protein factors from leukocytes protect against enzyme inactivation but are not essential for enzyme activity.     

Leishmanial phosphatase blocks neutrophil O-2 production

Leishmania donovani, the causative agent in kala-azar or visceral leishmaniasis, infects cells of the macrophage system. We show that a purified preparation of the tartrate-resistant acid phosphatase, isolated from the external surface of L. donovani promastigotes, inhibits superoxide anion production by human neutrophils. Preincubation of neutrophils for 15-30 min at 37 degrees C with 240 units (1 unit equals 1 nmol of 4-methylumbelliferyl phosphate cleaved per h) of the acid phosphatase decreases both the rate and extent of superoxide generation by 90% upon stimulation with the chemoattractant peptide fMet-Leu-Phe. The ability of the phosphatase to suppress superoxide anion production is abolished by heat inactivation of the enzyme or by incorporation of an acid phosphatase inhibitor in the preincubation medium, indicating that the effect is dependent on the catalytic activity of the enzyme. These results suggest a possible pathophysiologic role for the acid phosphatase of L. donovani promastigotes.     

Mutagenicity of methyl isocyanate in the modified test conditions of Ames Salmonella/microsome liquid-preincubation procedure

Methyl isocyanate (MIC) was tested for mutagenicity using the Ames Salmonella/microsome liquid-preincubation procedure with slight modification of test conditions. In the modification the preincubation mixture was incubated at 10 degrees C for 60 min. MIC was assayed both in the presence and absence of Aroclor-1254-induced S9, using 5 tester strains of Salmonella typhimurium, TA97a, TA98, TA100, TA102 and TA104. MIC induced mutagenic response in two base-pair substitution strains, TA100 and TA104, in the presence and absence of S9. However, mutagenic response in the presence of S9 was low as compared to that in the absence of S9. In the comparative mutagenic activity at 3 different preincubation test conditions (37 degrees C for 20 min, 20 degrees C for 40 min and 10 degrees C for 60 min), optimum mutagenic response was observed at 10 degrees C for the 60-min test condition. However, no mutagenic response was observed at 37 degrees C for the 20-min test condition.     

Stabilization of insulin receptor subunit structure by glutathione-insulin transhydrogenase

A partially purified insulin receptor preparation from rat liver was incubated at 37 degrees C with and without the protein-disulfide interchange enzyme, glutathione-insulin transhydrogenase (thiol: protein-disulfide oxidoreductase/isomerase, EC 1.8.4.2/5.3.4.1). Insulin-binding activity was then assessed by crosslinking receptor-125I-insulin complexes and subjecting them to electrophoresis on SDS-polyacrylamide gels in the absence and presence of reductant followed by autoradiography. Prior incubation of the receptor at 37 degrees C in the absence of the enzyme markedly decreased the subsequent binding of 125I-insulin to the holoreceptor (Mr 350 000) and to its subunits (Mr 180 000 and 130 000), while addition of the enzyme to the preincubation medium served to substantially prevent this decrease. The loss in binding at 37 degrees C was not restored by subsequent addition of the enzyme, nor was the loss prevented by any of the several known inhibitors of proteolysis. The apparent stabilization of receptor by transhydrogenase, as evidenced by the increase in binding above control levels, was proportional to both the enzyme concentration and the duration of incubation. These effects seem to be specific for transhydrogenase, since several other disulfide-containing proteins were found to be ineffective. These data suggest that the stabilization of the subunit structure of the insulin receptor at physiological temperatures may take place via a disulfide interchange reaction catalyzed by glutathione-insulin transhydrogenase.     

Mechanisms of heart adenylate cyclase activation by epinephrine and fluoride ions

The effects of epinephrine and NaF on the membrane preparations of adenylate cyclase from rabbit heart were studied. After preincubation with epinephrine or NaF at 37 degrees C and subsequent washing of the membranes at 4 degrees C from the effectors, adenylate cyclase passes into the activated state and loses its sensitivity to epinephrine and NaF. The effect may be "reversed" by preincubation of the membranes at 37 degrees C. The addition of ATP to the preincubation media does not affect the regulatory and catalytic properties of the enzyme. It is assumed that adenylate cyclase regulation by hormones and fluoride ions may occur without hypothetical processes of phosphorylation and dephosphorylation of the enzyme. The effect of preincubation is probably due to the temperature-dependent association and dissociation of the enzyme-receptor complex in the membrane. Epinephrine and NaF partially protect the cyclase against trypsin-induced inactivation, which is indicative of structural or conformational changes of the adenylate cyclase complex during its interaction with activators.     

Inactivation of bacteriophage PM2 during storage

The kinetics of bacteriophage inactivation in the medium that is optimal for its storage has been studied at temperatures from 4 to 55 degrees C. The plot of Arrhenius dependence of the constant of inactivation rate consists of the two linear parts with the energies of activation Ea = 25 kcal/mol for 4-37 degrees C and Ea = 91 kcal/mol for 37-55 degrees C. The DNA of inactivated bacteriophage remained mostly in superspiralized form and completely preserved its biological activity as tested by transfection in spheroplasts. The analysis of inactivation kinetics suggests ageing of virions cultivated at 4 degrees C. The addition of watersoluble antioxidant amoxipin did not change the inactivation kinetics. The addition of antioxidant ionol with twin-80 increased the inactivation that was paralleled by the bacteriophage DNA degradation.     

Co2+-mediated time- and temperature-dependent activation of neutral alpha-D-mannosidase from monkey brain.

Neutral alpha-D-mannosidase from monkey brain was purified by Co2+-chelate affinity chromatography and immunoadsorbent affinity chromatography. The purified enzyme, with subunit Mr 45,000, was essentially homogeneous with only traces of two contaminant proteins as revealed by SDS/polyacrylamide-gel electrophoresis and AgNO3 staining. The purified enzyme, on preincubation with Co2+ at 37 degrees C or 60 degrees C followed by assay, showed a time-dependent enhancement in activity. The enhanced activity of the enzyme persisted even after removal of the Co2+. Bacitracin could partially prevent the activation. An aminopeptidase activity that was stimulated by Co2+ both at 37 degrees C and at 60 degrees C was present in the purified enzyme. After preincubation of the enzyme with Co2+ there was evidence for the release of amino acids, as revealed by t.l.c., but the Mr determined by SDS/polyacrylamide-gel electrophoresis was not appreciably altered. It is suggested that a Co2+-stimulated thermostable aminopeptidase, inseparable from the neutral mannosidase, may be involved in the stimulation of neutral mannosidase activity during its preincubation with Co2+.     

Selective inactivation of the deoxyadenosine phosphorylating activity of pure human deoxycytidine kinase: stabilization of different forms of the enzyme by substrates and biological detergents

Deoxycytidine kinase, purified from human leukemic spleen to apparent homogeneity, is a multisubstrate enzyme that also phosphorylates purine deoxyribonucleosides [Bohman & Eriksson (1988) Biochemistry 27, 4258-4265]. In the present investigation we show that the stability and temperature dependence of dCyd kinase activity differed appreciably from the dAdo kinase activity of the same pure enzyme. Selective inactivation of dAdo activity was observed upon an incubation of the enzyme at both 4 and 37 degrees C. The half-life of dAdo activity at 4 degrees C increased from 36 to 84 h, when the protein concentration was increased by addition of bovine serum albumin. However, the half-life of dCyd activity increased from 72 h to more than 7 days under the same conditions. dCyd activity was stable for at least 6 h at 37 degrees C while the half-life of dAdo activity was 2 h. The presence of substrates like ATP, dTTP, or dAdo stabilized dAdo activity at both temperatures, and full maintenance of both activities at 37 degrees C was obtained by the addition of the zwitterionic detergent CHAPS. Furthermore, thermal inactivation of the dAdo activity occurred at a lower temperature (48 degrees C) as compared to the dCyd activity (54 degrees C). The presence of protease inhibitors had no effect on enzyme inactivation, nor was there a difference in the subunit structure of the selectively inactivated enzyme as compared to the fully active form, as revealed by size-exclusion chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)