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1.
Guanylate cyclase activity was determined in a 1000g particulate fraction derived from rabbit heart homogenates using Mg2+ or Mn2+ as sole cation in the presence and absence of Triton X-100. With Mg2+, very little guanylate cyclase activity could be detected in the original particulate fraction assayed with or without Triton, or in the particulate fraction treated with varying concentrations of Triton (detergent-treated mixture) prior to enzyme assay. However, the detergent-solubilized supernatants as well as the detergent-insoluble residues (pellets) derived from detergent-treated mixtures possessed appreciable Mg2+-supported enzyme activity. With Mn2+, significant enzyme activity was detectable in the original particulate fraction assayed without Triton. Much higher activity was seen in particulate fraction assayed with Triton and in detergent-treated mixtures; the supernatants but not the pellets derived from detergent-treated mixtures possessed even greater activity. The sum of enzyme activity in pellet and supernatant fractions greatly exceeded that of the mixture. When the pellets and supernatants derived from detergenttreated mixtures were recombined, measured enzyme activities were similar to those of the original mixture. With Mg2+ or Mn2+, the specific activity of guanylate cyclase in pellet and supernatant fractions varied considerably depending on the concentration of Triton used for treatment of the particulate fraction; treatment with low concentrations of Triton (0.2–0.7 μmol/mg protein) gave supernatants showing high activity whereas treatment with relatively greater concentrations of the detergent (>0.7 μmol/mg protein) gave pellets showing high activity. The relative distribution of guanylate cyclase in pellet and supernatant fractions expressed as a function of Triton concentration during treatment (of the particulate fraction) showed that 50 to 80% of the recovered enzyme activity remained in supernatants at low detergent concentrations whereas 50 to 80% of the recovered activity resided in the pellets at higher detergent concentrations. Inclusion of excess Triton in the enzyme assay medium did not alter the specific activity profiles and the relative distribution patterns of the cyclase in pellet versus supernatant fractions. The results demonstrate the inherent potential of cardiac particulate guanylate cyclase to utilize Mg2+ in catalyzing the synthesis of cyclic GMP. However, it appears that some factor(s) endogenous to the cardiac particulate fraction severely impairs the expression of Mg2+-dependent activity; Mn2+-dependent activity is also affected by such factor(s) but apparently less severely. Further, the results suggest that previously reported activities of cardiac particulate guanylate cyclase, despite being assayed with Mn2+ and in the presence of Triton X-100, represent underestimation of what otherwise appears to be a highly active enzyme system capable of utilizing physiologically relevant divalent cation such as Mg2+.  相似文献   

2.
Abstract— Mn2+ caused an 8-to 16-fold stimulation of adenylate cyclase activity in homogenates as well as synaptosomcs. isolated synaptic membranes, and slices prepared from rat brain. The stimulation occurred at low concentrations of Mn2+. with a doubling of activity at 50-60μM. and was unaffected by a 60-fold excess of Mg2+. Whether or not Mg2+ was added, inclusion of a low concentration of Mn2+ reduced, but did not prevent the stimulation of adenylate cyclase caused by dopaminc in homogenates of corpus striatum. In contrast, Ca2+. at a concentration that had little effect on basal cyclase activity, completely prevented the stimulation by dopamine. The increase of cyclase activity produced by Mn2+ in brain homogenates was potentiated by F?. Other ions, notably Hg2+. Pb2+. Cu2+ and Zn2+. in order of decreasing potency, inhibited both basal and Mn2--stimulated cyclase activity. It is proposed that the effect of Mn2+ on adenylate cyclase activity may involve only the catalytic subunit of the enzyme, and that the mechanism is different from that by which either dopamine or F? stimulates the enzyme. These results suggest that the effects of low concentrations of Mn2+ and certain other divalent metal ions on adenylate cyclase activity may be involved in their neuropsychiatrie or other toxic effects, and that such ions may also participate in normal physiological mechanisms involving cyclic nucleotides.  相似文献   

3.
Certain biochemical characteristics of an adenylate cyclase that is activated by low concentrations of histamine (Ka, 8 μm) and that is present in cell-free preparations from the dorsal hippocampus of guinea pig brain have been studied. Histamine increased the maximal reaction velocity of adenylate cyclase without altering the Km (0.18 mm) for its substrate, MgATP. Increasing concentrations of free Mg2+ stimulated enzymatic activity; the kinetic properties of this activation by Mg2+ suggest the existence of a Mg2+ allosteric site on the enzyme. Histamine increased the affinity of this apparent site for free Mg2+. Free ATP was a competitive inhibitor with respect to the MgATP substrate. The apparent potency of free ATP as an inhibitor increased in the presence of histamine. In the presence of Mg2+, low concentrations of Ca2+ markedly inhibited adenylate cyclase activity; half-maximal inhibition of both basal and histamine-stimulated enzyme activity occurred at 40 μm Ca2+. Other divalent cations, including Zn2+, Cu2+, and Cd2+, were also inhibitory. Of the divalent cations tested, only Co2+ and Mn2+ could replace Mg2+ in supporting histamine-stimulated adenylate cyclase activity. The nucleoside triphosphates GTP and ITP increased basal adenylate cyclase activity and markedly potentiated the stimulation by histamine. Preincubation of adenylate cyclase with 5′-guanylylimidodiphosphate dramatically increased enzyme activity; in this activated state, the adenylate cyclase was relatively refractory to further stimulation by histamine or F?. The subcellular distribution of histamine-sensitive adenylate cyclase activity was studied in subfractions from guinea pig cerebral cortex. The highest total and specific activities were observed in those fractions enriched in nerve endings, while adenylate cyclase activity was not detectable in the brain cytosol fraction. A possible physiological role for this histamine-sensitive adenylate cyclase in neuronal function is discussed.  相似文献   

4.
Low concentrations of Mn2+ supported the basal adenylate cyclase activity in crude and purified sarcolemmal membranes from cardiac muscle more effectively than did relatively high concentrations of Mg2+; at saturating concentrations the cyclase activities obtained with Mg2+ or Mn2+ were similar. In contrast, Mg2+ supported the basal cyclase activities of crude membrane fractions and purified sarcolemmal membranes from skeletal muscle far more effectively than did Mn2+; at saturating concentrations of either metal ion the Mg2+-supported cyclase activities were 5- to 10-fold greater than Mn2+-supported activities. Further, compared to Mg2+, Mn2+ supported the cyclase activities very poorly in all the primary subcellular fractions of skeletal muscle, whereas this cation was at least as effective as Mg2+ in all fractions of cardiac muscle. The apparent affinities of the cyclase for Mn2+ in heart as well as skeletal muscle appeared to be greater compared to those for Mg2+. The skeletal muscle cyclase displayed greater apparent affinity for MnATP2? (app. Km 0.10 mm) compared to MgATP2? (app. Km 0.32 mm) whereas the heart enzyme displayed greater apparent affinity for MgATP2? (app. Km 0.07 mm) compared to MnATP2? (app. Km 0.19 mm). Following preactivation with guanyl-5′-yl imidodiphosphate and isoproterenol, Mn2+ (0.15 to 2 mm) supported the cyclase activity of skeletal muscle even more effectively than did optimally effective concentrations of Mg2+. With the heart enzyme the relatively greater potency of Mn2+ persisted following preactivation. Significant enhancement in the Mn2+-sensitivity of skeletal muscle cyclase was also observed when assayed in the presence of GTP and isoproterenol or in the presence of NaF. Preactivation of both heart and skeletal muscle cyclases caused selective enhancement in the enzyme's apparent affinity for free Me2+ (Mg2+ or Mn2+) without influencing the apparent Km for MeATP2? (MgATP2? or MnATP2?). Evidences were obtained to show that the poor effectiveness of Mn2+ in supporting the basal activity of skeletal muscle cyclase is not related to (a) potentiation by Mn2+ of adenosine-mediated inhibition of the cyclase, (b) Mn2+-induced lability of the cyclase, (c) indirect effects of Mn2+ on ATP-regenerating system, or (d) the presence of different cation-specific molecular forms of the cyclase. It is also shown that the onset of enhanced Mn2+ sensitivity of the skeletal muscle enzyme following preactivation is not accompanied by a general loss of cation specificity of the cyclase. These results suggest that cations support the catalytic activity of adenylate cyclase by interacting with an enzymeregulatory free metal binding site and that the differential cation sensitivity of nonactivated (basal) cyclases from heart and skeletal muscle is likely due to differences in the properties of such an allosteric metal site. Furthermore, the metal site appears to undergo a conformational change following interaction of the cyclase system with the guanyl nucleotide and isoproterenol since the cation sensitivity of the cyclase and the relative potency of cations depend on the conformational status of the enzyme.  相似文献   

5.
In hamster adipocyte ghosts, ACTH stimulates adenylate cyclase by a GTP-dependent process, whereas prostaglandin E E1, α-adrenergic agonists and nicotinic acid inhibit the enzyme by a mechanism which is both GTP- and sodium-dependent. The influence of the divalent cations Mn2+ and Mg2+, was studied on these two different, apparently receptor-mediated effects on the adipocyte adenylate cyclase. At low Mn2+ concentrations, GTP (1 μM) decreased enzyme activity by about 80%. Under this condition, ACTH (0.1 μM) stimulated the cyclase by 6- to 8-fold, and NaCl (100 mM) caused a similar activation. In the presence of both GTP and NaCl, prostaglandin E1 (1 or 10 μM) and nicotinic acid (30 μM) inhibited the enzyme by about 70–80% and epinephrine (300 μM, added in combination with a β-adrenergic blocking agent) by 40–50%. With increasing concentrations of Mn2+, the GTP-induced decrease and the NaCl-induced increase in activity diminished, with a concomitant decrease in prostaglandin E1?, nicotinic acid- and epinephrine-induced inhibitions as well as in ACTH-induced stimulation. At 1 mM Mn2+, inhibition of the enzyme was almost abolished and stimulation by ACTH was largely reduced, whereas activation of the enzyme by KF (10 mM) was only partially impaired. The uncoupling action of Mn2+ on hormone-induced inhibition was half-maximal at 100–200 μM and appeared not to be due to increased formation of the enzyme substrate, Mn · ATP. It occurred without apparent lag phase and could not be overcome by increasing the concentration of GTP. Similar but not identical findings with regard to adenylate cyclase stimulation and inhibition by hormonal factors were obtained with Mg2+, although about 100-fold higher concentrations of Mg2+ than of Mn2+ were required. The data indicate that Mn2+at low concentrations functionally uncouples inhibitory and stimulatory hormone receptors from adenylate adenylate cyclase in membrane preparations of hamster adipocytes, and they suggest that the mechanism leading to uncoupling involves an action of Mn2+ on the functions of the guanine nucleotide site(s) in the system.  相似文献   

6.
Sperm from several invertebrates contained guanylate cyclase activity several-hundred-fold greater than that in the most active mammalian tissues; the enzyme was totally particulate. Activity in the presence of Mn2+ was up to several hundred-fold greater than with Mg2+ and was increased 3–10-fold by Triton X-100. Sperm from several vertebrates did not contain detectable guanylate cyclase. Sperm of both invertebrates and vertebrates contained roughly equal amounts of Mn2+-dependent adenylate cyclase activity; in invertebrate sperm, this enzyme was generally several hundred-fold less active than guanylate cyclase. Adenylate cyclase was particulate, was unaffected by fluoride, and was generally greater than 10-fold more active with Mn2+ than with Mg2+. Invertebrate sperm contained phosphodiesterase activities against 1.0 μm cyclic GMP or cyclic AMP in amounts greater than mammalian tissues. Fish sperm, which did not contain guanylate cyclase, had high phosphodiesterase activity with cyclic AMP as substrate but hydrolyzed cyclic GMP at a barely detectable rate. In sea urchin sperm, phosphodiesterase activity against cyclic GMP was largely particulate and was strongly inhibited by 1.0% Triton X-100. In contrast, activity against cyclic AMP was largely soluble and was weakly inhibited by Triton. The cyclic GMP and cyclic AMP contents of sea urchin sperm were in the range of 0.1–1 nmol/g. Sea urchin sperm homogenates possessed protein kinase activity when histone was used as substrate; activities were more sensitive to stimulation by cyclic AMP than by cyclic GMP.5  相似文献   

7.
Restriction endonucleases of the PD…D/EXK family need Mg2+ for DNA cleavage. Whereas Mg2+ (or Mn2+) promotes catalysis, Ca2+ (without Mg2+) only supports DNA binding. The role of Mg2+ in DNA cleavage by restriction endonucleases has elicited many hypotheses, differing mainly in the number of Mg2+ involved in catalysis. To address this problem, we measured the Mg2+ and Mn2+ concentration dependence of DNA cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me2+ per active site. DNA cleavage experiments were carried out at various Mg2+ and Mn2+ concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg2+ and Mn2+ concentration dependence. In general, the Mg2+ concentration optimum (between ∼ 1 and 10 mM) is higher than the Mn2+ concentration optimum (between ∼ 0.1 and 1 mM). At still higher Mg2+ or Mn2+ concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca2+. Based on these results, we propose that one Mg2+ or Mn2+ is critical for restriction enzyme activation, and binding of a second Me2+ plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg2+ or Mn2+ mainly leads to an increase in Km, such that the inhibitory effect of excess Mg2+ or Mn2+ can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me2+ binding to these enzymes.  相似文献   

8.
  • 1.1. The enzyme fructose-1,6-bisphosphatase was purified from the mantle of the sea mussel Mytilus galloprovincialis Lmk. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis. The mol. wt and subunit mol. wt of the enzyme were 105,000 and 27,000, respectively.
  • 2.2. Divalent cations are essential for the enzyme activity. In the absence of chelating agents, FBPase 1 exhibits hyperbolic kinetics with respect to Mn2+, Zn2+ and Mg2+. The Km for Mg2+ is lower than the physiological concentration of cation in the tissue, whereas its Km for Mn2+ and Zn2+ is greater than the respective in vivo concentrations.
  • 3.3. The joint action of Mg2+ and Zn2+ increases the affinity of the enzyme for the substrate Fru-1,6-P2, though Vmax is reduced.
  • 4.4. Na+ strongly inhibits the enzyme even at very low concentrations. K+ has no effect whatsoever.
  相似文献   

9.
Summary Smooth Muscle Phosphatases II (SMP-I1) which has been purified from turkey gizzards and previously classified as protein phosphatase 2C, is inactive in the absence of divalent cations. Study of the activation of SMP-II by Mg2+ and Mn2+ revealed differences in the modes of activation by these cations. The maximal activation elicited by Mg2+ is 1.5–2.5-fold higher than the maximal Mn2+ activation. However, the latter is achieved at a lower concentration than the maximal Mg2+-activation. Furthermore, at low cation concentrations ( 2 mM), the Mn2+-activated activity is higher than the Mg2+-activated activity. In the presence of both cations, the effect of Mn2+ predominates suggesting that the affinity of the enzyme for Mn2+ is greater than for Mg2+. In contrast to Mg2+ and Mn2+, Ca2+ does not activate SMP-II but it was observed to antagonize the effects of Mg2+ and Mn2+. Ca2+ acts as a competitive inhibitor of Mg2+. However, the inhibitory effect at high Ca2+ concentrations is not completely reversed by increasing the Mg2+ concentration. Mn2+ activation is also inhibited by Ca2+ but to a lesser extent. Ca2+ cannot completely inhibit Mn2+-activation suggesting that SMP-I1 has greater affinity for Mn2+ than for Ca2+. The finding that Ca2+ inhibits the activation of SMP-II raises the possibility that Ca2+ may be a regulator of SMP-II in vivo.Abbreviations SMP-II Smooth Muscle Phosphatase-II - MOPS 3-[N-Morpholine]propane Sulfonic Acid - PLC Phosphorylated Myosin Light Chains  相似文献   

10.
dCMP deaminase was partially purified from BHK-21/C13 cells grown in culture. The molecular weight of the enzyme was estimated by gel filtration and gradient centrifugation to be 130000 and 115000 respectively. The enzyme had a pH optimum of 8.4. Its activity versus substrate concentration curve was sigmoid, the substrate concentration at half-maximal velocity being 4.4mm. dCTP activated the deaminase maximally at 40μm, gave a hyperbolic curve for activity versus dCMP concentration and a Km value for dCMP of 0.91mm. dCTP activation required the presence of Mg2+ or Mn2+ ions. dTTP inhibited the deaminase maximally at 15μm; the inhibition required the presence of Mg2+ or Mn2+ ions. The enzyme was very heat-labile but could be markedly stabilized by dCTP at 0.125mm and ethylene glycol at 20% (v/v).  相似文献   

11.
The catalytic activity of guanylate cyclase (GCase) coupled to atrial natriuretic peptide (ANP) receptor depends on the metal co-factor, Mn2+ or Mg2+. ATP synergistically stimulates the ANP-stimulated GCase in the presence of Mg2+. We have now shown the ATP regulation of the ANP-stimulated GCase in the presence of Mn2+ in rat lung membranes. ANP stimulated the GCase 2.1-fold compared to the control. ATP enhanced both the basal (basal-GCase) and the ANP-stimulated GCase maximally 1.7- and 2.3- fold compared to the control, respectively, at a concentration of 0.1 mM. The stimulation by ATP was smaller in the presence of Mn2+ than in the presence of Mg2+. The addition of inorganic phosphate to the reaction mixture altered the GCase activities in the presence of Mn2+ with or without ANP and/or ATP. In the presence of 10 mM phosphate, ATP dose-dependently stimulated the basal GCase 5-fold compared to the control at a concentration of 1 mM and augmented the ANP-stimulated GCase, which was 4.2-fold compared to the basal-GCase, 5.5-fold compared to the control at a concentration of 0.5 mM. Protein phosphatase inhibitors, okadaic acid (100 nM), H8 (1 M) and staurosporin (1 M), did not alter the activity. Orthovanadate (1 mM), an inorganic phosphate analogue, significantly stimulated both the basal-GCase and the ANP-stimulated GCase, which were inhibited by ATP. It was assumed that phosphate and orthovanadate might interact with the GCase to regulate the activity in the opposite manner. This was the first report that inorganic phosphate and orthovanadate affected the ATP-regulation of the ANP-stimulated GCase in the presence of Mn2+.  相似文献   

12.
O'neal D  Joy KW 《Plant physiology》1974,54(5):773-779
Purified glutamine synthetase from pea seedlings was most active with Mg2+ as the metal activator, but Mn2+ and Co2+ were 45 to 60% and 30 to 45% as effective, respectively, when assayed at the optimal pH for each cation. The Mg2+ saturation curve was quite sigmoid, and evidence indicates that MgATP is the active ATP substance. Co2+ also gave a sigmoidal saturation curve, but when Mn2+ was varied only slightly sigmoidal kinetics were seen. Addition of Mn2+, Ca2+, or Zn2+ at low concentrations sharply inhibited the Mg2+ -dependent activity, partially by shifting the pH optimum. Addition of Co2+ did not inhibit Mg2+-dependent activity. The nucleotide triphosphate specificity changed markedly when Co2+ or Mn2+ replaced Mg2+. Using the Mg2+-dependent assay, the Michaelis constant (Km) for NH4+ was about 1.9 × 10−3 M. The Km for l-glutamate was directly proportional to ATP concentration and ranged from 3.5 to 12.4 mm with the ATP levels tested. The Km for MgATP also varied with the l-glutamate concentration, ranging from 0.14 mm to 0.65 mm. Ethylenediaminetetracetic acid activated the enzyme by up to 54%, while sulfhydryl reagents gave slight activation, occasionally up to 34%.  相似文献   

13.
Summary The effects of divalent metals, metal chelators (EDTA, EGTA) and sodium dodecyl sulfate were investigated on the phosphatase activity of isolated bovine brain calcineurin assayed in the absence (called intrinsic) and presence of calmodulin. Intrinsic phosphatase was increased by Mn2+, was unaffected by Mg2+, Ca2–, and Ba+, and was markedly inhibited by Ni2–, Fe2+, Zn2+ and Cu2–. When assayed in the presence of calmodulin, many divalent metals (Ni2–, Zn2+, Pb2+, Cd2+), besides Mn2+, increased modestly the phosphatase activity at low concentrations (10–100 M) and inhibited it markedly at high concentrations. Ca2–-calmodulin stimulated phosphatase activity was antagonized by Ni2+, Zn2+, Fe2+, Cu2+, Pb2+, at low concentrations (50 M), and by Ba2+, Cd2+ at slightly higher concentrations (> 100 M); Mn2+ and Co2– (50 M to 1 mM) in fact augmented it. EDTA and EGTA in a concentration and time dependent fashion inhibited the intrinsic phosphatase activity, particularly that of trypsinized calcineurin. SDS in low concentrations (0.005%) augmented the phosphatase activity and inhibited it at high concentrations. Mn2+ (± calmodulin) and Ca2+ only with calmodulin present increased the phosphatase activity assayed with low concentrations of SDS. The EDTA dependent inhibition of intrinsic phosphatase was almost abolished in assays containing SDS. Prior exposure of calcineurin to Mn2+ led to a high activity conformation state of calcineurin that was long-lived or pseudo-irreversible. Such Mn2+-activated state of calcineurin exhibited no discerbible change in the affinity towards myelin basic protein or its inhibition by trifluoperazine. At alkaline pH, Mg2+ supported the intrinsic phosphatase activity, although to a lesser degree than Mn2+. The latter cation, compared to Mg2+ and Ni2+, was also a more powerful stimulator of the calcineurin phosphatase assayed with histone (III-S) and myosin light chain as substrates.  相似文献   

14.
  • 1.1. The native rat-kidney cortex Fructose-1,6-BPase is differentially regulated by Mg2+ and Mn2+.
  • 2.2. Mg2+ binding to the enzyme is hyperbolic and large concentrations of the cation are non-inhibitory.
  • 3.3. Mn2+ produces a 10-fold rise in Vmax higher than Mg2+. [Mn2+]0.5 is much larger than [Mg2+]0.5. At elevated [Mn2+] inhibition is observed.
  • 4.4. Mg2+ and Mn2+ produce antagonistic effects on the inhibition of the enzyme by high substrate.
  • 5.5. Fru-2,6-P2 inhibits the enzyme by rising the S0.5 and favouring a sigmoidal kinetics.
  • 6.6. The inhibition by Fru-2,6-P2 is released by Mg2+ and more powerfully by Mn2+ increasing the I0.5.
  相似文献   

15.
—Some basic kinetic properties of adenylate cyclase in cell free preparations of mouse neuroblastoma were investigated. Production of cAMP from ATP by the enzyme requires the presence of either Mg2+ or Mn2+ in addition to ATP. In the presence of Mg2+, the Km for ATP is 120 ± 15 μM and the interaction of ATP and adenylate cyclase appears to be non-cooperative (Hill coefficient of 1). Magnesium ion concentrations in excess of the ATP concentration cause stimulation although similar excess concentrations of Mn2+ cause inhibition. Prostaglandin E1 and 2-chloroadenosine activate the enzyme. The Km of the cyclase for 2-chloroadenosine is 6 μm . Activation by 2-chloroadenosine leads to an increase in Vmax but does not effect the Km for ATP. At a fixed ATP concentration, the extent of activation caused by prostaglandin E1 and 2-chloroadenosine is inversely related to the Mg2+ concentration. Calcium ion causes inhibition of adenylate cyclase from 0.1 to 4mM with a Ki of 5 ± 10?4m . Ca2+ interaction with the enzyme in the absence or presence of either 2-chloroadenosine or prostaglandin E1 appears cooperative (i.e. Hill coefficients of ?2). Ca2+ inhibition is non-competitive with respect to either ATP or 2-chloroadenosine but is progressively diminished by increasing Mn2+ concentrations. Divalent cation effects and activation by 2-chloroadenosine and prostaglandin E1 of the neuroblastoma adenylate cyclase are compared with ion effects and hormone activation of the enzyme obtained from non-neuronal tissue.  相似文献   

16.
GTP has been shown to inhibit AlF4-stimulated, and to activate forskolin-stimulated adenylyl cyclase activity in the presence of Mg2+ in cell membranes from human embryonic kidney 293 cells. The maximal inhibitory response of AlF4-stimulated adenylyl cyclase activity by GTP was not dependent on the concentration of Mg2+, but was so in the case of forskolin-activated activity at all forskolin concentrations assayed. Mn2+ ions stimulated AlF4- or forskolin-activated adenylyl cyclase activity to a greater extent than Mg2+. The inhibition of AlF4-stimulated cyclase by GTP was still observed with Mn2+, but the activation of forskolin-stimulated cyclase by GTP was not. When assayed together, Mn2+ and Mg2+ showed non-additive behaviours with respect to the amount of cyclic AMP formed after AlF4-stimulation of adenylyl cyclase. The temperature dependence of the activation of adenylyl cyclase by forskolin, AlF4 or under basal conditions was observed to be somehow different in the presence of Mn2+ than in the presence of Mg2+ ions. Cholera toxin treatment produced a markedly increased cyclase activity, specially when assayed with AlF4. In the case of forskolin-activated adenylyl cyclase, UTP and CTP were unable to reproduce the cyclase activation detected with GTP. However, in the case of AlF4-stimulated adenylyl cyclase, UTP was as good as GTP at inhibiting cyclase activity, and CTP virtually eliminated the activation of the cyclase with AlF4.  相似文献   

17.
Mg2+-ATPase activity was identified in the cytosol of human erythrocytes. A partial purification of this activity was achieved by an initial DEAE-Sephadex column chromatography, followed by gel filtration on Sephadex G-100 and then a second DEAE-Sephadex chromatography procedure. The enzyme appeared in the void volume of the Sephadex G-100 column and was retained on an Amicon XM100A ultrafiltration membrane. The molecular weight of the enzyme was estimated to be 113 000 from SDS gels. The above purification protocol yielded an enzyme with an optimal pH between 7.6 and 8.2. The enzyme activity increased linearly between 30 and 44°C. It was stable for several months at ?20°C. Magnesium was essential for activity, but the rate attainable with Mn2+ was at least as great as that due to Mg2+. No other divalent cation was able to substitute for Mg2+ or Mn2+. Neither low nor high Ca2+ concentrations significantly affected the enzymatic activity. Substrate specificity studies showed that ATP was the preferred substrate followed by CTP (46% of the rate produced by ATP). Hydrolysis of GTP, UTP, ITP and ADP was less than 10% of the rate seen with ATP. No phosphatase, pyrophosphatase, phosphodiesterase, hexokinase, phosphofructokinase or adenylate cyclase activity could be detected in this enzyme preparation. Calmodulin, which stimulates the (Ca2+ + Mg2+)-ATPase of the human erythrocyte membrane, failed to enhance the Mg2+-ATPase activity. Of considerable interest, the activity of this Mg2+-ATPase was enhanced approximately 5-fold by low concentrations of mercuric ion, p-hydroxymercuribenzoate and DTNB, but was much less sensitive to iodoacetamide.  相似文献   

18.
Two major peaks of RNA polymerase activity have been routinely separated by diethylaminoethyl cellulose chromatography following solubilization from soybean (Glycine max L. var. Wayne) chromatin. The relative amounts of these two peaks depend upon the manner in which the chromatin is purified. Pelleting the chromatin through dense sucrose solutions results in not only a loss of total solubilized RNA polymerase activity but also a selective loss of the α-amanitin-sensitive form of the enzyme. Peak I elutes from a diethylaminoethyl cellulose column at a KCl concentration of approximately 0.27 m, is insensitive to α-amanitin and rifamycin, and has Mg2+ + Mn2+ optima of 5 mm and 1.25 mm, respectively. The enzyme is inhibited by KCl concentrations of about 0.03 m or greater. Peak II elutes from the column at a KCl concentration of approximately 0.35 m, is sensitive to α-amanitin, insensitive to rifamycin, and has Mg2+ + Mn2+ optima of 2 mm and 1.0 mm, respectively. Activity is inhibited by KCl concentrations of about 0.06 m or greater. Both enzymes prefer denatured calf thymus DNA, but peak II exhibits a stronger preference.  相似文献   

19.
Streptozotocin, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and N-methyl nitrosourea, compounds with both oncogenic and cytotoxic properties, increased guanylate cyclase activity in the 100 000 × g soluble fractions of rat renal cortex and liver 35- to 65-fold over basal values. Particulate enzyme activities of these tissues were increased 2- to 4-fold by a maximally effective concentration of the nitrosoureas. In the presence of the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, maximally effective concentrations of these nitrosoureas increased cyclic GMP accumulation of hepatic and renal cortical slices to peak levels 7- to 10-fold over control in 30 min. By contrast, with the structurally related carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) peak increases occurred in 5–10 min and were 40- to 70-fold over control levels in renal cortex and liver, respectively. Unlike the Ca2+-dependent actions of cholinergic stimuli on cyclic GMP, the nitrosoureas and MNNG increased cyclic GMP in either the presence or absence of extracellular Ca2+. Moreover, while basal soluble guanylate cyclase of renal cortex was highly Mn2+-dependent and decreased 85% when either Mg2+ or Ca2+ was employed as sole divalent cation in reaction mixtures, the actions of nitrosoureas on enzyme activity were well expressed with either Mn2+ or Mg2+, but not with Ca2+, as sole divalent cation. Improved utilization of Mg2+ by guanylate cyclase in the presence of nitrosoureas would favor enhanced enzyme activity under cellular conditions where Mg2+ is abundant. In the presence of maximally stimulatory concentrations of streptozotocin or BCNU, high concentrations of Mg2+ or Mn2+ further increased soluble guanylate cyclase, suggesting important differences in metal and nitrosourea stimulation of enzyme activity.Preincubation of supernatant fractions with nitrosoureas plus dithiothreitol inhibited the action of the N-nitroso compounds to increase renal cortical guanylate cyclase. Glutathione and cysteine were also inhibitory, but less effective than dithiothreitol. Initial incubation of nitrosoureas with dithiothreitol in buffer alone similarly suppressed the subsequent action of the N-nitroso compounds on guanylate cyclase, and implicated direct chemical interactions. Prior incubation of renal cortical supernatant fractions with the SH blockers N-ethylmaleimide or maleimide significantly suppressed guanylate cyclase activation mediated by streptozotocin or BCNU. Direct drug interactions seemed unlikely, since effects of the inhibitors were optimally expressed by initial exposure of the supernatant fraction of tissue to the SH blockers and were not potentiated by a 30 min preincubation of the SH blockers and nitrosoureas in buffer alone.Thus, nitrosoureas activate and alter the metal requirements of soluble guanylate cyclase and increase cellular cyclic GMP in the presence or absence of extracellular Ca2+. Activation of soluble guanylate cyclase by nitrosoureas may involve an interaction of these agents with tissue SH groups, and possibly SH to SS transformation. Stimulation of the guanylate cyclase system by nitrosoureas could be related to the oncogenic actions of these agents.  相似文献   

20.
YHS-domains are small protein modules which have been proposed to bind transition-metal ions like the related TRASH-domains. They are found in a variety of enzymes including copper-transporting ATPases and adenylyl cyclases. Here we investigate a class IIIc adenylyl cyclase from Mycobacterium phlei which contains a C-terminal YHS-domain linked to the catalytic domain by a peptide of 8 amino acids. We expressed the isolated catalytic domain and the full-length enzyme in E. coli. The catalytic domain requires millimolar Mn2+ as a cofactor for efficient production of cAMP, is unaffected by low micromolar concentrations of Cu2+ and inhibited by concentrations higher than 10 μM. The full-length enzyme also requires Mn2+ in the absence of an activator. However, 1–10 μM Cu2+ stimulate the M. phlei adenylyl cyclase sixfold when assayed with Mn2+. With Mg2+ as the probable physiological cofactor of the adenylyl cyclase Cu2+ specifically switches the enzyme from an inactive to an active state. Other transition-metal ions do not elicit activity with Mg2+. We favor the view that the YHS-domain of M. phlei adenylyl cyclase acts as a sensor for copper ions and signals elevated levels of the transition-metal via cAMP. By analogy to TRASH-domains binding of Cu2+ probably occurs via one conserved aspartate and three conserved cysteine-residues in the YHS-domain.  相似文献   

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