Characterization of intestinal brush border guanylate cyclase activation by Escherichia coli heat-stable enterotoxin

Intestinal brush border guanylate cyclase was previously reported to be activated by the Escherichia coli enterotoxin (STa). This system was reexamined in order to develop a hypothesis for the mechanism of activation. The extent of activation was previously underestimated, since by using sodium azide to inhibit competing reactions and ethylene glycol bis(beta-aminoethyl ether) N,N-tetraacetic acid to chelate Ca2+, which is inhibitory, maximal activations of 30- to 50-fold were obtained. Ca2+ inhibition was only partially relieved by the calmodulin inhibitor calmidazolium. Inhibitors of the O2-dependent activation of soluble guanylate cyclase had no effect on STa activation; hence, it was concluded that STa activation did not involve arachidonate release and oxidation. STa was able to further increase activity already elevated by the nonionic detergent Lubrol PX. The membrane-active agent filipin, which was previously reported to inhibit both basal and agonist-stimulated adenylate cyclase, did not inhibit STa activation of guanylate cyclase. Digitonin, another cholesterol binder, inhibited STa activation at low concentrations, which disappeared at higher concentrations. Both of these agents stimulated basal activity. Dimethyl sulfoxide produced a concentration-dependent inhibition of STa activation, while increasing basal activity 7-fold. Ethanol inhibited both basal and STa-stimulated activity, with the former being more affected. Benzyl alcohol, like ethanol, a "fluidizer" of cell membranes, also inhibited both basal and activated enzymes. We concluded that STa directly activates this guanylate cyclase and, because of the differential effects of inhibitors on basal and STa-stimulated activity, propose a receptor-mediated mechanism.     

Regulation of ciliary adenylate cyclase by Ca2+ in Paramecium.

In the ciliated protozoan Paramecium, Ca2+ and cyclic nucleotides are believed to act as second messengers in the regulation of the ciliary beat. Ciliary adenylate cyclase was activated 20-30-fold (half-maximal at 0.8 microM) and inhibited by higher concentrations (10-20 microM) of free Ca2+ ion. Ca2+ activation was the result of an increase in Vmax., not a change in Km for ATP. The activation by Ca2+ was seen only with Mg2+ATP as substrate; with Mn2+ATP the basal adenylate cyclase activity was 10-20-fold above that with Mg2+ATP, and there was no further activation by Ca2+. The stimulation by Ca2+ of the enzyme in cilia and ciliary membranes was blocked by the calmodulin antagonists calmidazolium (half-inhibition at 5 microM), trifluoperazine (70 microM) and W-7 (50-100 microM). When ciliary membranes (which contained most of the ciliary adenylate cyclase) were prepared in the presence of Ca2+, their adenylate cyclase was insensitive to Ca2+ in the assay. However, the inclusion of EGTA in buffers used for fractionation of cilia resulted in full retention of Ca2+-sensitivity by the ciliary membrane adenylate cyclase. The membrane-active agent saponin specifically suppressed the Ca2+-dependent adenylate cyclase without inhibiting basal activity with Mg2+ATP or Mn2+ATP. The ciliary adenylate cyclase was shown to be distinct from the Ca2+-dependent guanylate cyclase; the two activities had different kinetic parameters and different responses to added calmodulin and calmodulin antagonists. Our results suggest that Ca2+ influx through the voltage-sensitive Ca2+ channels in the ciliary membrane may influence intraciliary cyclic AMP concentrations by regulating adenylate cyclase.     

Proton pump-linked Mg2+-ATPase activity in isolated rat liver lysosomes

ATPase activity in highly purified rat liver lysosome preparations was evaluated in the presence of other membrane cellular ATPase inhibitors, and compared with lysosome ATP-driven proton translocating activity. Replacement of 5 mM Mg2+ with equimolar Ca2+ brought about a 50% inhibition in divalent cation-dependent ATPase activity, and an 80% inactivation of ATP-linked lysosomal H+ pump activity. In the presence of optimal concentrations of Ca2+ and Mg2+, ATPase activity was similar to that seen in an Mg2+ medium. Mg2+-dependent ATPase activity was greatly inhibited (from 70 to 80%) by the platinum complexes; cis-didimethylsulfoxide dichloroplatinum(II) (CDDP) at approximately 90 microM and cis-diaminedichloroplatinum(II) at twofold higher concentrations. Less inhibition, about 30 and 45%, was obtained with N,N'-dicyclohexylcarbodiimide and N-ethylmaleimide, and the maximal effect occurred in the 50-100 microM and 0.1-1.5 mM ranges, respectively. The concentration dependence of inhibition by the above drugs was determined for both proton pumping and ATPase activities, and half-maximal inhibition concentration of each activity was found at nearly similar values. A micromolar concentration of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) prevented ATP from setting up a pH gradient across the lysosomal membranes, but stimulated Mg2+-ATPase activity significantly. ATPase activity in Ca2+ medium was also inhibited by CDDP and stimulated by FCCP, but both effects were two- to threefold less than those observed in Mg2+ medium. FCCP failed to stimulate ATPase activity in a CDDP-supplemented medium, thus suggesting that the same ATPase activity fraction was sensitive to both CDDP and FCCP. Mg2+-ATPase activity, like the proton pump, was anion dependent. The lowest activity was recorded in a F-medium, and increased in the order of F- less than SO2-4 less than Cl- approximately equal to Br-. The CDDP-sensitive ATPase activity observed, supported by Mg2+ and less so by Ca2+, may be related to lysosome proton pump activity.     

Adenylate cyclase in silkworm. Effects of adenosine 3'-phosphate and 2'-deoxyadenosine 3'-phosphate on the enzyme system in fat body

Adenosine 3'-phosphate and 2'-deoxyadenosine 3'-phosphate inhibit silkworm fat body adenylate cyclase. The inhibition has a rapid onset, and is dependent on the concentration of Mn2+ or Mg2+. The concentrations of 2'-deoxy-3'-AMP required for 50% inhibition (Ki) are 13 microM with 2 mM Mn2+ and 32 microM with 10 mM Mg2+. These Ki values are 7-30 times lower than that for 2'-deoxyadenosine. Stimulation of adenylate cyclase by NaF renders the activity more sensitive to the nucleotide inhibition, reducing the Ki value to 4 microM in the presence of Mn2+. The inhibitory activity is specific for adenine 3'-nucleotide; Ki for 2'-AMP and 5'-AMP are ten times or more higher than that for 3'-AMP, and the other 3'-nucleotides including 8-bromo-3'-AMP, 3'-IMP and 3'-GMP have little or no inhibitory activity.     

Purification and characterization of phosphatidylinositol kinase from porcine liver microsomes

Phosphatidylinositol kinase was solubilized and purified from porcine liver microsomes to apparent homogeneity. The purification procedure includes: solubilization of microsomes by 2% Triton X-100, ammonium sulfate precipitation (20-35% saturation), Reactive blue agarose chromatography, DEAE-Sephacel chromatography and two consecutive hydroxyapatite chromatographies. A total of 4900-fold purification with 8% recovery of enzyme activity was achieved. The molecular weight of the enzyme as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 55000. The enzyme is stimulated in a decreasing order by Mg2+, Fe2+, Mn2+, Fe3+ and Co2+. Ca2+ inhibited Mg2+-stimulated activity with an I50 of 0.4 mM. Apparent Km values for phosphatidylinositol and ATP are 120 and 60 microM, respectively. The enzyme is inhibited by adenosine (I50 = 70 microM), ADP (I50 = 120 microM) and quercetin (I50 = 100 microM). The enzyme is also sensitive to sulfhydryl inhibitors. Using the purified enzyme as an immunogen, we have successfully prepared antibodies for phosphatidylinositol kinase in rabbits. The antibodies appear to recognize an antigen of Mr 55000 on SDS-polyacrylamide gel electrophoresis from various porcine tissues in Western blot analysis.     

Purification and properties of magnesium- and manganese-dependent ribonucleases H from chick embryo

Two RNases H, Mg2+- and Mn2+-dependent RNases H, are present in extracts of chick embryo. These RNases H can be separated by phosphocellulose column chromatography. Mg2+-dependent RNase H was purified over 900-fold and Mn2+-dependent RNase H over 1,700-fold from chick embryo extracts. The molecular weight of the purified Mg2+-dependent RNase H was about 40,000 and of the Mn2+-dependent RNase H about 120,000, when estimated by gel filtration. Mg2+-dependent RNase H exhibits maximal activity at pH 9.5, and requires 15 to 20 mM Mg2+ for maximal activity, whereas Mn2+-dependent RNase H is most active at pH 8.5, and is maximally active at the concentration of 0.4 mM Mn2+, and has some activity with Mg2+. Both enzymes require a sulfhydryl reagent for maximal activity. Mn2+-dependent RNase H was inhibited by o-phenanthroline, pyrophosphate, and those polyamines tested, whereas Mg2+-dependent enzyme was not, although it was inhibited by NaF. Both RNases H liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini endonucleolytically.     

Selective activation of particulate guanylate cyclase by a specific class of porphyrins

Guanylate cyclase was activated 3- to 10-fold by hemin in a dose-dependent manner in membranes prepared from homogenates of rat lung, C6 rat glioma cells, or B103 rat neuroblastoma cells. Maximum activation was observed with 50 to 100 microM hemin with higher concentrations being inhibitory. Activation was observed when Mg2+-GTP but not when Mn2+-GTP was used as the substrate. Increased enzyme activity reflected selective activation of the particulate form of guanylate cyclase; hemin inhibited the soluble form of guanylate cyclase 70 to 90% over a wide range of concentrations. Activation was not secondary to proteolysis since a variety of protease inhibitors failed to alter stimulation by hemin. Protophorphyrin IX had little effect on particulate guanylate cyclase activity and sodium borohydride almost completely abolished hemin-dependent activation. These data suggest a requirement for the ferric form of the porphyrin-metal chelate for activation. However, agents which interact with the iron nucleus of porphyrins, such as cyanide, had little effect on the ability of hemin to activate guanylate cyclase. The stimulatory effects of hemin were observed in the presence of detergents such as Lubrol-PX, and highly purified particulate enzyme could be activated to the same extent as enzyme in native membranes. These data suggest that the interaction of porphyrins with particulate guanylate cyclase is complex in nature and different from that with the soluble enzyme.     

Purification, properties and enhanced expression under nitrogen starvation of the NADP+-isocitrate dehydrogenase from the cyanobacterium Phormidium laminosum

Nitrogen starvation enhances up to 8-fold the cellular level of the NADP+-dependent isocitrate dehydrogenase activity (isocitrate:NADP+ oxidoreductase (decarboxylating), IDH, EC 1.1.1.42) in the thermophilic filamentous non-N2-fixing cyanobacterium Phormidium laminosum. The enzyme was purified 650-fold to electrophoretic homogeneity from nitrogen-starved cells with an activity yield of 25% and a specific activity of 500 U (mg protein)-1. The native enzyme showed a pI of 5.9 and it was a dimer of 107 kDa consisting of two identical subunits of 53 kDa. The activity required the presence of a divalent metal cation as an essential activator, Mn2+ or Mg2+ being the most effective. The optimum temperature for activity was 55 degrees C and the Ea for catalysis was 39.7 kJ mol-1. An optimum pH for activity of 8.5 was found and the calculated pKE1, pKE2 and pKES1 of enzyme ionisation groups were 6.0, 8.9 and 6.3, respectively. Km values of 22, 50 and 24 microM were calculated for d,l-isocitrate, NADP and Mn2+, respectively, in the Mn2+-dependent reaction and 70, 32 and 159 microM for d,l-isocitrate, NADP and Mg2+, respectively, in the Mg2+-dependent reaction. The decarboxylating activity was inhibited by ATP, ADP and by its reaction products 2-oxoglutarate and NADPH2. Polyclonal antibodies raised against the pure IDH were used to assess the presence of the enzyme in cells subjected to nitrogen starvation.     

Partial purification and characterization of an anion-activated ATPase from radish microsomes

Previous investigation showed two distinct ATP-dependent proton-transporting systems in microsomal vesicle from radish seedlings, one inhibited by vanadate and one inhibited by NO-3. On the bases of the effects of these inhibitors we could discriminate two distinct ATPase activities in the same material. The NO-3 sensitive activity was separated from the vanadate-sensitive activity and partially purified by a single-step chromatographic method, which lead to approx 35-fold purification from the microsomes and to a specific activity of 2.3 mumol Pi X min-1 X mg protein-1, at 30 degrees C. The partially purified activity was specific for ATP, some activity being observed toward GTP, and even less toward CTP, UTP and ITP. No significant Pi hydrolysis was found with ADP, AMP, p-nitrophenylphosphate and glucose 6-phosphate. ADP but not AMP was inhibiting in the presence of ATP. The activity was dependent on divalent cations in the order of preference: Mg2+ greater than Mn2+ greater than Co2+ greater than Ca2+ greater than Zn2+. The activity was unaffected by monovalent cations, strongly activated by Cl-, inhibited by 90% by 50 mM NO-3, virtually unaffected by oligomycin and NaN3. At least 90% of the activity was abolished in the presence of each: 10 microM N,N'-dicyclohexylcarbodiimide, 10 microM erythrosin B, 10 mu mersalyl, 100 microM trimethyltin, 100 microM diethylstilbestrol, 100 microM N-ethylmaleimide. No inhibition has been found in the presence of Ca2+, at a concentration blocking the vanadate-sensitive activity. Nigericin, gramicidin and carbonylcyanide p-trifluoromethoxyphenylhydrazone stimulated the activity of this preparation after it was incubated in the presence of sonicated phospholipids, suggesting the capacity of the ATPase to function as a H+-transporting system. All characteristics mentioned were closely similar to those described in the vacuolar ATPases.     

The role of specific cations in regulation of cyanobacterial glutamine synthetase

Purified glutamine synthetase from the cyanobacterium Anabaena cylindrica required a divalent cation for activity. Maximum biosynthetic activity required Mg2+ (25 mM when supplied alone). Co2+ and Mn2+ each supported up to 20% of this activity; 12 other cations tested were ineffective. At 2.5 - 10 mM Mg2+, 0.1 mM Co2+ or ethylene glycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) stimulated GS activity to maximum rates; other divalent cations (particularly Mn2+) inhibited Mg2+-dependent activity. At 5 mM Mg2+ the Kappm for NH+4 (0.05 mM) was 20-fold lower than at 25 mM Mg2+; added Co2+ did not markedly alter this low Km for NH+4; this could be physiologically important.     

Regulation and properties of glutamine synthetase purified from Bacillus cereus

The glutamine synthetase from Bacillus cereus IFO 3131 was purified to homogeneity. The enzyme is a dodecamer with a molecular weight of approximately 600,000, and its subunit molecular weight is 50,000. Both Mg2+ and Mn2+ activated the enzyme as to the biosynthesis of L-glutamine, but, unlike in the case of the E. coli enzyme, the Mg2+-dependent activity was stimulated by the addition of Mn2+. The highest activity was obtained when 20 mM Mg2+ and 0.5 mM Mn2+ were added to the assay mixture. For each set of optimal assay conditions, the apparent Km values for glutamate, ammonia and a divalent cation X ATP complex were 1.03, 0.34, and 0.40 mM (Mn2+: ATP = 1: 1); 14.0, 0.47, and 0.91 mM (Mg2+: ATP = 4: 1); and 9.09, 0.45, and 0.77 mM (Mg2+: Mn2+: ATP = 4: 0.2: 1), respectively. At each optimum pH, the Vmax values for these reactions were 6.1 (Mn2+-dependent), 7.4 (Mg2+-dependent), and 12.9 (Mg2+ plus Mn2+-dependent) mumoles per min per mg protein, respectively. Mg2+-dependent glutamine synthetase activity was inhibited by the addition of AMP or glutamine; however, this inhibitory effect was suppressed in the case of the Mg2+ plus Mn2+-dependent reaction. These results suggest that the activity of the B. cereus glutamine synthetase is regulated by both the intracellular concentration and the ratio of Mn2+/Mg2+ in vivo. Also in the present investigation, a potent glutamine synthetase inhibitor(s) was detected in crude extracts from B. cereus.     

Inhibition of cytochalasin D-stimulated G-actin ATPase by ADP-ribosylation with Clostridium perfringens iota toxin.

Clostridium perfringens iota toxin belongs to a novel family of actin-ADP-ribosylating toxins. The effects of ADP-ribosylation of skeletal muscle actin by Clostridium perfringens iota toxin on cytochalasin D-stimulated actin ATPase activity was studied. Cytochalasin D stimulated actin-catalysed ATP hydrolysis maximally by about 30-fold. ADP-ribosylation of actin completely inhibited cytochalasin D-stimulated ATP hydrolysis. Inhibition of ATPase activity occurred at actin concentrations below the critical concentration (0.1 microM), at low concentrations of Mg2+ (50 microM) and even in the actin-DNAase I complex, indicating that ADP-ribosylation of actin blocks the ATPase activity of monomeric actin and that the inhibitory effect is not due to inhibition of the polymerization of actin.     

Stimulation and inhibition of rat basophilic leukemia cell adenylate cyclase by forskolin

The influence of the diterpene, forskolin, was studied on adenylate cyclase activity in membranes of rat basophilic leukemia cells. Forskolin increased basal adenylate cyclase activity maximally 2-fold at 100 microM. However, adenylate cyclase activity stimulated via the stimulatory guanine nucleotide-binding protein, Ns, by fluoride and the stable GTP analog, guanosine 5'-O-(3-thiotriphosphate), was inhibited by forskolin. Half-maximal and maximal inhibition occurred at about 1 and 10 microM forskolin, respectively. The inhibition occurred without an apparent lag phase, whereas the enzyme stimulation by forskolin was preceded by a considerable lag period. The inhibition was not affected by treating intact cells or membranes with pertussis toxin and proteolytic enzymes, respectively, which have been shown in other cell types to prevent adenylate cyclase inhibition mediated by the guanine nucleotide-binding regulatory component, Ni. The forskolin inhibition of the stable GTP analog-activated adenylate cyclase was impaired by increasing the Mg2+ concentration and was reversed into a stimulation by Mn2+. Under optimal inhibitory conditions, forskolin even decreased basal adenylate cyclase activity. Finally, forskolin largely reduced the apparent affinity of the rat basophilic leukemia cell adenylate cyclase for its substrate, MgATP, which reduction resulted in an apparent inhibition at low MgATP concentrations and a loss of the inhibition at higher MgATP concentrations. The data indicate that forskolin can cause both stimulation and inhibition of adenylate cyclase and, furthermore, they suggest that the inhibition may not be mediated by the Ni protein, but may be caused by a direct action of forskolin at the adenylate cyclase catalytic moiety.     

Purification of bovine brain inositol 1,4,5-trisphosphate 3-kinase. Identification of the enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.

Treatment of human platelets with concentrations of benzyl alcohol up to 50 mM augmented adenylate cyclase activity when it was assayed in the basal state and when stimulated by prostaglandin E1 (PGE1), isoprenaline or NaF. Benzyl alcohol antagonized the stimulatory effect exerted on the catalytic unit of adenylate cyclase by the diterpene forskolin. Benzyl alcohol did not modify the magnitude of the inhibitory response when the catalytic unit of adenylate cyclase was inhibited by using either low concentrations of guanosine 5'-[beta gamma-imido]triphosphate, which acts selectively on the inhibitory guanine nucleotide-regulatory protein Gi, or during alpha 2-adrenoceptor occupancy, by using adrenaline (+ propranolol). Some 34% of the potent inhibitory action of adrenaline on PGE1-stimulated adenylate cyclase was obliterated in a dose-dependent fashion (concn. giving 50% inhibition = 12.5 mM) by benzyl alcohol, with the residual inhibitory action being apparently resistant to the action of benzyl alcohol at concentrations up to 50 mM. Treatment of membranes with benzyl alcohol did not lead to the release of either the alpha-subunit of Gi or G-protein subunits. The alpha 2-adrenoceptor-mediated inhibition of adenylate cyclase was abolished when assays were performed in the presence of Mn2+ rather than Mg2+ and, under such conditions, dose-effect curves for the action of benzyl alcohol on PGE1-stimulated adenylate cyclase activity were similar whether or not adrenaline (+propranolol) was present. We suggest that (i) alpha 2-adrenoceptor- and Gi-mediated inhibition of PGE1-stimulated adenylate cyclase may have two components, one of which is sensitive to inhibition by benzyl alcohol, and (ii) the Gi-mediated inhibition of forskolin-stimulated adenylate cyclase exhibits predominantly the benzyl alcohol-insensitive component.     

Purification and characterization of cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. Effects of divalent cations on activity

Cyclic GMP-stimulated cyclic nucleotide phosphodiesterase purified greater than 13,000-fold to apparent homogeneity from calf liver exhibited a single protein band (Mr approximately 102,000) on polyacrylamide gel electrophoresis under denaturing conditions. Enzyme activity comigrated with the single protein peak on analytical polyacrylamide gel electrophoresis, sucrose density gradient centrifugation, and gel filtration. From the sedimentation coefficient of 6.9 S and Stokes radius of 67 A, an Mr of 201,000 and frictional ratio (f/fo) of 1.7 were calculated, suggesting that the native enzyme is a nonspherical dimer of similar, if not identical, peptides. The effectiveness of Mg2+, Mn2+, and Co2+ in supporting catalytic activity depended on the concentration of cGMP and cAMP present as substrate or effector. Over a wide range of substrate concentrations, optimal concentrations for Mg2+, Mn2+, and Co2+ were about 10, 1, and 0.2 mM, respectively. At concentrations higher than optimal, Mg2+ inhibited activity somewhat; inhibition by Co2+ (and in some instances by Mn2+) was virtually complete. At low substrate concentrations, activity with optimal Mn2+ was equal to or greater than that with Co2+ and always greater than that with Mg2+. With greater than or equal to 0.5 microM cGMP or 20 to 300 microM cAMP and for cAMP-stimulated cGMP or cGMP-stimulated cAMP hydrolysis, activity with Mg2+ greater than Mn2+ greater than Co2+. In the presence of Mg2+, the purified enzyme hydrolyzed cGMP and cAMP with kinetics suggestive of positive cooperativity. Apparent Km values were 15 and 33 microM, and maximal velocities were 200 and 170 mumol/min/mg of protein, respectively. Substitution of Mn2+ for Mg2+ increased apparent Km and reduced Vmax for cGMP with little effect on Km or Vmax for cAMP. Co2+ increased Km and reduced Vmax for both. cGMP stimulated cAMP hydrolysis approximately 32-fold in the presence of Mg2+, much less with Mn2+ or Co2+. In the presence of Mg2+, Mn2+ and Co2+ at concentrations that increased activity when present singly inhibited cGMP-stimulated cAMP hydrolysis. It appears that divalent cations as well as cyclic nucleotides affect cooperative interactions of this enzyme. Whereas Co2+ effects were observed in the presence of either cyclic nucleotide, Mn2+ effects were especially prominent when cGMP was present (either as substrate or effector).     

Alkaline phosphatase from Paramecium cilia and cell bodies: purification and characterization

A soluble alkaline phosphatase was purified 10 000-fold in an overall yield of 8% from both of the cilia and cell bodies of the protozoan Paramecium tetraurelia. The concentration in cilia (1.7 microM) was 6-fold higher than in cell bodies, although the latter contained most of the activity due to their much greater volume. The purified protein showed a single (36 kDa) protein staining band on SDS-PAGE. This value, in conjunction with the apparent molecular mass of 66 kDa for the native enzyme (gel filtration) suggests a dimeric structure. The specific activity of the purified phosphatase ranged from 10 to 70 mumols.min-1.mg-1 at the pH-optimum of 8.0 and the Km for p-nitrophenyl phosphate was 81 microM. Basal enzyme activity was inhibited by metal chelators and stimulated up to 12-fold by addition of divalent cations. Mg2+ acted as a non-essential mixed-type activator with a half-maximal effect at 7 microM. Ca2+ was inhibitory, the extent of inhibition was dependent on the concentration of Mg2+ in the assay. Furthermore, the kinetics of inhibition by Ca2+ varied with the Mg2+ concentration. Phosphate, pyrophosphate, and SH-group blocking agents also strongly inhibited. The enzyme did not dephosphorylate Tyr- or Ser-/Thr-phosphoproteins. The Paramecium enzyme is not of lysosomal origin and its properties are quite different from all known phosphatases. It is a novel type of phosphatase since it (i) shows F(-)-inhibition like Ser/Thr-phosphatases but (ii) is inhibited by vanadate and molybdate like Tyr-phosphatases, and (iii) inhibition by Ca2+ has not been reported for any other phosphatase.     

Biochemical studies in marine species--I. NADP(+)-dependent isocitrate dehydrogenase from turbot liver (Scophthalmus maximus L.)

1. An NADP+-dependent isocitrate dehydrogenase was extracted from turbot liver. The enzyme required divalent cations (Mg2+ or Mn2+) for its activity but was inhibited by high salt concentrations. 2. The enzyme had an optimum activity in the pH range between 7.5 and 9.0. The enzymic activity increased with temperature (in the range of 5 to 68 degrees C) with an Ea of 23.5 kJ/mol and a Q10 of 1.34. 3. The apparent Km values for the substrates were 6.5 microM for NADP+, 56 microM for Mg2+, 87 microM for Mn2+ and 4.2 and 73.5 microM for the complexes Mg-isocitrate and Mn-isocitrate, respectively. The physiological significance of these results is discussed. 4. The enzyme was inhibited by citrate and adenine nucleotides. The degree of inhibition depended on the relation between the concentrations and that of magnesium. A possible regulating mechanism is proposed.     

Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform.

Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The Km of calcineurin A for 32P-RII pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 microM with or without calmodulin, and calmodulin increased the Vmax about 4-fold. The Km of reconstituted calcineurin A plus B for 32P-RII pep was 20 microM, and calmodulin increased the Vmax 18-fold without affecting the Km. CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 microM and 90 microM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.     

Characterization of a calmodulin-stimulated adenylate cyclase from abalone spermatozoa

Abalone sperm adenylate cyclase activity is particulate in nature and displays a high Mg2+-supported activity (Mg2+/Mn2+ = 0.8) as compared to other sperm adenylate cyclases. Approximately 90% of the enzyme activity in crude homogenates is inhibited by EGTA in a concentration-dependent manner which is overcome by added micromolar free Ca2+. The EGTA-inhibited Ca2+-stimulated enzyme activity is also inhibited by phenothiazines. Added calmodulin, however, has no effect on enzyme activity prepared from crude homogenates. Preparation of a twice EGTA-extracted 48,000 X g pellet fraction yields a particulate enzyme activity that can be stimulated 10-65% by added calmodulin in the presence of micromolar free Ca2+. Detergent extraction (1% Lubrol PX) of the EGTA-washed 48,000 X g pellet solubilizes 2-5% of the total particulate adenylate cyclase activity, and this solubilized enzyme is activated up to 125% by calmodulin. The ability of the different enzyme preparations to be stimulated by calmodulin is inversely proportional to the endogenous calmodulin concentration. Calmodulin stimulation of the Lubrol PX-solubilized enzyme is specific to this Ca2+-binding protein and is mediated as an effect on the velocity of the enzyme. This stimulation is completely Ca2+ dependent and is fully reversible. These data suggest that the control of sperm cAMP synthesis by changes in Ca2+ conductance may be mediated via this Ca2+-binding protein.     

Purification and kinetic properties of polynucleotide kinase from rat testes

Polynucleotide kinase (EC 2.7.1.78) has been purified from rat testes, and an approximately 2000-fold purification was obtained. The purified enzyme had an Mr of 38000 +/- 3800. The enzyme phosphorylated micrococcal nuclease-treated calf thymus DNA and (dT)10 while 5'-HO-tRNA was a very poor substrate. A certain degree of specificity towards purine-containing 5'-HO-nucleotides was observed. The polynucleotide kinase had an absolute requirement for a divalent cation. Both Mg2+ and Mn2+ could be used, but 10 mM MgCl2 gave optimal activity. The monovalent cations Na+, K+ and NH4+ all stimulated enzyme activity, and the optimal concentration was 0.1 M. The enzyme was inhibited by inorganic phosphate, pyrophosphate and sulphate. A 50% inhibition was obtained with 20, 0.3 and 2 mM, respectively. At 2 mM MgCl2, 1 mM spermine enhanced the enzyme activity 3-times. The apparent KATP was estimated to be 36 microM and KHO-DNA was found to be 2 microM.