Particulate guanylate cyclase of skeletal muscle: effects of Ca2+ and other divalent cations on enzyme activity.

The properties of particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from purified rabbit skeletal muscle membrane fragments were studied. Four membrane fractions were prepared by sucrose gradient centrifugation and the fractions characterized by analysis of marker enzymes. Guanylate cyclase activity was highest in the fraction possessing enzymatic properties typical of sarcolemma, while fractions enriched with sarcoplasmic reticulum had lower activities. In the presence of suboptimal Mn2+ concentrations, Mg2+ stimulated particulate guanylate cyclase activity both before and after solubilization in 1% Triton X-100. Guanylate cyclase activity was biphasic in the presence of Ca2+. Increasing the Ca2+ concentration from 10(-8) to 10(-5) M decreased the specific activity. As the Ca2+ concentration was further increased to 5 . 10(-4) M enzyme activity again increased. After solubilization of the membranes in 1% Triton X-100, Ca2+ suppressed enzyme activity. Studies utilizing ionophore X537A indicated that the altered effect of Ca2+ upon the solubilized membranes was independent of asymmetric distribution of Ca2+ and Mg2+.     

A proper transmembrane Ca2+ gradient is essential for the stimulation of adenylate cyclase activity by stimulatory GTP-binding protein

Stimulatory GTP-binding Protein (Gs) and adenylate cyclase prepared from bovine brain cortices were co-reconstituted into asolectin vesicles with or without 1000-fold transmembrane Ca²⁺ gradient. The results showed that both basal activity and Gs-stimulated activity of adenylate cyclase were highest in proteoliposomes with a transmembrane Ca²⁺ gradient similar to physiological condition (1 M Ca²⁺ outside and 1 mM Ca²⁺ inside) and lowest when the transmembrane Ca²⁺ gradient was in the inverse direction. Such a difference could be diminished following dissipation of the transmembrane Ca²⁺ gradient by A23187. Comparable conformational changes of Gs in proteoliposomes were also observed when Gs was labeled with the fluorescence probe, acrylodan. These results may indicate that a proper transmembrane Ca²⁺ gradient is essential not only for higher adenylate cyclase activity but also for its stimulation by Gs.     

Passive Ca2+ permeability of vesicular sarcolemmal preparations from myocardium

Vesicular preparations of sarcolemma isolated from rat myocardium possessed high ATPase (4.32 +/0 0.57 micromole/min per mg), adenylate cyclase (121 +/- 11 pmole/min per mg) and creatine kinase (1.74 +/- 0.35 micromole/min per mg) activities and a Na-Ca exchange activity specific for sodium. The ATPase activity was inhibited by digitoxigenin by 50-70% and was not changed by ouabain, EGTA, ionophore A23187 and oligomycin, thus showing the absence of mitochondrial and sarcoplasmic reticulum contaminations in the sarcolemmal preparations. The preparations consisted mostly of closed inside-out vesicles. The preparation was used to study the mechanism of Ca2+ penetration across the sarcolemmal membrane. For this purpose the vesicles were load with 45Ca2+, which relatively slowly diffused from the medium into the vesicles, and which was bound to the binding sites inside the vesicles (n = 20.5 +/- 4.6 nmoles per mg of protein, Kd approximately equal to 1.8 +/- 0.21 mM). The transmembrane movement of Ca2+ was demonstrated by the following findings: 1) the ionophore A23187 only insignificantly increased the total vesicular Ca2+ content, but strongly accelerated Ca2+ efflux from the vesicles along its concentration gradient; 2) gramicidin and osmotic shock caused a similar acceleration of Ca2+ efflux. Ca2+ efflux from these vesicles along Ca2+ concentration gradient was studied under conditions, when the extravesicular Ca2+ content was lowered due to its binding to EGTA and by dilution. The gradient of Ca2+ concentration was from 2.0 mM inside to approximately 0.1 micro M outside. The rate of 45Ca2+ efflux depended hyperbolically on the intravesicular Ca2+ efflux from the vesicles was inhibited by Mn2+, Co2+ and verapamil when they acted from the inside of the vesicles. An increase in ionophore A23187 concentration increased the efflux of Ca2+ hyperbolically and enhanced only the maximal rate of the efflux. It is concluded that the passive permeability of Ca2+ across the sarcolemmal membrane along its concentration gradient is controlled by Ca2+ binding to the membrane.     

Calcium-dependent adenylate cyclase of pituitary tumor cells

Effects of Ca2+ and calmodulin on the adenylate cyclase activity of a prolactin and growth hormone-producing pituitary tumor cell strain (GH3) were examined. The adenylate cyclase activity of homogenates was stimulated approx. 60% by submicromolar free Ca2+ concentrations and inhibited by higher (microM range) concentrations of the cation. A 2-3-fold stimulation of the activity in response to Ca2+ was observed at physiologic concentrations of KCl, with both the stimulatory and inhibitory responses occurring at respectively higher free Ca2+ concentrations. Calmodulin in incubations at low KCl concentrations increased the enzyme activity at all Ca2+ concentrations tested. In incubations conducted at physiologic KCl concentrations, both the inhibitory and stimulatory responses to Ca2+ were shifted by calmodulin to lower respective concentrations of the cation, without significant change occurring in the maximal rate of enzymic activity at optimal free Ca2+ X Mg2+ concentrations in the incubation also influenced the Ca2+ concentration dependence of adenylate cyclase; at high Mg2+ more Ca2+ was required to obtain maximal activity. Trifluoperazine inhibited adenylate cyclase of GH3 cells only in the presence of Ca2+; as Ca2+ concentrations in the assay were increased, higher drug concentrations were required to inhibit the enzyme. Ca2+ was also observed to reduce the extent of enzyme destabilization which occurred during pretreatments at warm temperatures. Vasoactive intestinal polypeptide and phorbol myristate acetate, which stimulate prolactin secretion in intact GH3 cells, enhanced enzyme activity 4- and 2.5-fold, respectively, without added Ca2+. Increasing free Ca2+ concentrations reduced the enhancement by VIP and eliminated the stimulation by PMA.     

Solubilization of guanylyl cyclase from bovine rod outer segments and effects of lowering Ca2+ and nitro compounds.

Guanylyl cyclase from bovine rod outer segments was solubilized using Triton X-100 and a high concentration of KCl, and its regulation was studied. The efficiency of solubilization was about 50-90% of total activity. When the Ca2+ content was lowered (less than 80 nM), guanylyl cyclase was activated about 2-fold. In the presence of higher concentrations of Ca2+ (greater than 140 nM), the activity was decreased. The regulation by Ca2+ was also demonstrated with solubilized preparations. In the presence of 186 nM Ca2+ which inhibited guanylyl cyclase, La3+ activated the enzyme about 2-fold, suggesting that the Ca2(+)-binding protein similar to other Ca2(+)-binding proteins associates with guanylyl cyclase regulation. Sodium nitroprusside and nitric oxide which are activators of soluble guanylyl cyclase in other tissues also activated the retinal guanylyl cyclase. Maximum activation by sodium nitroprusside was 20-fold using Mg2+ as a cofactor. Activation by nitric oxide and related compounds suggests that retinal guanylyl cyclase contains a heme prosthetic group that may participate in a novel regulatory mechanism for this enzyme.     

Properties of particulate, membrane-associated and soluble guanylate cyclase from cardiac muscle, skeletal muscle, cerebral cortex and liver.

1. Guanylate cyclase of washed particles and plasma membranes showed S-shaped progress curves when titrated with either GTP or Mn2+ ions; similar results were obtained with Triton X-100-solubilized enzyme preparation from washed particles. Hill plots of these data revealed multiple metal-nucleotide and free-metal binding sites. 2. Guanylate cyclase of supernatant fractions displayed typical Michaelis-Menten properties when enzyme required excess of (free) Mn2+ (over GTP) for maximal activities; Ka (free Mn2+) was about 0.15-0.25 mM at subsaturating concentrations of GTP. 4 MnATP, MnADP, and MnGDP were found to increase the activities of both particulate and superantant enzyme, when MnGTP concentration was below saturation and free Mn2+ ion concentration was low (less than 100 muM); MnATP (50muM-1 mM) inhibited both these activities at high free Mn2+ concentration (1.5 mM) and inhibition of the particulate enzyme was greater than that of supernatant enzyme. 5. Ca2+ ions stimulated supernatant-enzyme activity; the stimulatory concentration of Ca2+ ions depended on the concentration of Mn2+ and GTP. 6. A modest stimulation of particulate guanylate cyclase by pyrophosphate (0.02-1 mM) was observed; the pyrophosphate effect appeared to be competitive with respect to GTP. At a higher concentration (2 mM), pyrophosphate produced a marked inhibition of particulate enzyme; the nature of inhibitory effect appeared complex. 7. Inorganic salts (e.g. NaCl, KCl, LiBr, NaF) produced inhibition of particulate enzyme; the degree of inhibition of Triton X-100-stimulated activity was less than that of unstimulated activity. 9. Treatment of sarcolemmal or microsomal membranes with either phospholipase C or trypsin decreased, whereas phospholipase A increased, the activity of guanylate cyclase.     

Characterization of the cyclic AMP-independent actions of somatostatin in GH cells. II. An increase in potassium conductance initiates somatostatin-induced inhibition of prolactin secretion

The neuropeptide somatostatin inhibits prolactin release from GH4C1 pituitary cells via two mechanisms, inhibition of stimulated adenylate cyclase activity and an undefined cAMP-independent process. Somatostatin also hyperpolarizes GH4C1 cells and reduces their intracellular free Ca2+ concentration ([Ca2+]i) in a cAMP-independent manner. To determine whether these ionic changes were involved in the cAMP-independent mechanism by which somatostatin inhibited secretion, changes in cAMP levels were prevented from having any biological consequences by performing experiments in the presence of a maximal concentration of a cAMP analog. Under these conditions, inhibition of prolactin release by somatostatin required a transmembrane concentration gradient for K+ but not one for either Na+ or Cl-. However, elimination of the outward K+ gradient did not prevent somatostatin inhibition of vasoactive intestinal peptide-stimulated hormone release. Therefore, somatostatin's cAMP-mediated mechanism does not require a K+ gradient, whereas its cAMP-independent inhibition of secretion appears to result from a change in K+ conductance. Consistent with this conclusion, membrane hyperpolarization with gramicidin (1 microgram/ml) mimicked somatostatin inhibition of prolactin release. In addition, the K+ channel blocker tetrabutylammonium prevented the effects of somatostatin on the membrane potential, the [Ca2+]i and hormone secretion. Nonetheless, a K+ gradient was not sufficient for somatostatin action. Even in the presence of a normal K+ gradient, somatostatin was only able to inhibit prolactin release when the extracellular Ca2+ concentration was at least twice the [Ca2+]i. Furthermore, the calcium channel blocker, nifedipine (10 microM), which prevents the action of somatostatin to reduce the [Ca2+]i, specifically blocked inhibition of prolactin release via somatostatin's cAMP-independent mechanisms. Therefore, a decrease in Ca2+ influx through voltage-dependent Ca2+ channels produces both the fall in [Ca2+]i and inhibition of hormone secretion in response to somatostatin.     

Calmodulin-dependent adenylate cyclase activity in rat cerebral cortex: effects of divalent cations, forskolin and isoprenaline

The role of calcium-calmodulin (Ca2+-CaM) in the modulation of beta-adrenergic adenylate cyclase activity in rat cerebral cortex has been studied. In addition, the effects of manganese (Mn2+) and forskolin on CaM-dependent enzyme activity were investigated. At 2 mM magnesium (Mg2+) low concentrations of Ca2+ stimulated the enzyme activity (Ka 0.25 +/- 0.08 microM), whereas higher Ca2+ levels (greater than 2 microM) inhibited the activity. No activating effect of Ca2+ was observed in CaM-depleted membranes, but the inhibitory effect persisted and the stimulatory action of Ca2+ could be restored by addition of exogenous CaM. The ability of Ca2+ to activate the enzyme was reduced by increasing concentrations of Mg2+. At 10 mM Mg2+ the apparent Ka of Ca2+ was 0.55 +/- 0.16 microM and half-maximal inhibition was observed at 80-120 microM Ca2+. A synergistic effect was observed between Ca2+ and isoprenaline on the adenylate cyclase activity. Calcium did not alter the apparent Ka of isoprenaline (0.9 +/- 0.27 microM) and isoprenaline did not change the apparent Ka of Ca2+. However, isoprenaline decreased the apparent Ka of CaM; 0.11 +/- 0.07 micrograms vs. 0.32 +/- 0.1 micrograms (0.5 ml assay mixture)-1, with and without isoprenaline, respectively. A synergistic effect was also observed between Ca2+ and forskolin, but no change in their apparent Ka values was found. Furthermore, Mn2+ was found to activate the enzyme through CaM. These data demonstrate that Ca2+ -CaM potentiates beta-adrenergic adenylate cyclase activity and thus is able to modulate neurotransmitter stimulation in cortex. Furthermore, both forskolin and Mn2+ affect CaM-dependent enzyme activity. Forskolin potentiates Ca2+-CaM stimulation, while Mn2+ increases the activity by activating the enzyme through CaM.     

Regulation by calcium and calmodulin of adenylate cyclase from rabbit intestinal epithelium

The effect of calcium on adenylate cyclase from rabbit small intestine has been studied using a particulate preparation obtained from isolated epithelial cells. Both basal and vasoactive intestinal peptide-stimulated activities were inhibited by calcium concentrations in the micromolar range. In the presence of calmodulin, a biphasic response was obtained. At low calcium concentration (4 X 10(-9)-6 X 10(-8) M) the enzyme was activated up to 50%. As the Ca2+ concentration was increased, the enzyme was concomitantly inhibited. Half-maximal inhibition of calmodulin-dependent activity was obtained at 1 microM free Ca2+. The activation of the enzyme was also dependent on the concentration of Mg2+. At less than 1 microM Ca2+, the enzyme exhibited a biphasic response, being activated at below 3 mM Mg2+ and inhibited at higher concentrations. At Ca2+ concentrations that were inhibitory, the enzyme did not show the biphasic response to Mg2+. At concentrations above 3 mM, the maximal rate (Vmax) remained constant. Vmax was inversely proportional to the concentration of Ca2+ present. Calmodulin altered Vmax when acting on vasoactive intestinal peptide-stimulated enzyme. Calmodulin had no effect on the Km for hormone activation. The calmodulin-dependent activity was inhibited by incubation with trifluoperazine.     

Ca~(2+)通过膜脂影响肌质网Ca~(2+)-ATP酶的活性与Ca~(2+)转运功能

重建在大豆磷脂脂质体上的兔骨骼肌肌质网Ca~(2+)—ATP酶在ATP驱动下可将溶液中的Ca~(2+)转运到脂酶体内部;外加EGTA则可除去脂酶体外部的Ca~(2+),由此可得到四种含Ca~(2+)状态不同的脂酶体:(1)内、外都无Ca~(2+);(2)仅外部有Ca~(2+);(3)内、外都有Ca~(2+);(4),仅内部有Ca~(2+).用DPH和AS系列萤光探针对这四种含Ca~+状态不同的脂酶体的膜脂流动性进行了测定,结果表明:脂酶体外部加入Ca~(2+),脂双层外表面的流动性降低.当Ca~(2+)进入脂酶体内部后,内表面膜脂的流动性也降低,而且外层膜脂流动性进一步降低.脂酶体内、外的Ca~(2+)含量不同时,Ca~(2+)—ATP酶功能状态也不同.转运到脂酶体内部的ca~(2+)积累到一定浓度后,通过Ca~(2+)泵向内转运的Ca~(2+)及Ca~(2+)—ATP酶活力都受到了抑制.转运进行到第四分钟时的酶活只有第一分钟的9%.但在相同的实验条件下,失去了完整的膜结构的纯化的Ca~(2+)—ATP酶蛋白没有被抑制.这提示完整的膜结构是这种抑制作用所必需的,而且膜两侧Ca~(2+)浓度的梯差可通过影响膜脂来调节Ca~(2+)—ATP酶的功能.     

A Quantitative Study of the Ca2+/Calmodulin Sensitivity of Adenylyl Cyclase in Aplysia, Drosophila, and Rat

Studies in Aplysia and Drosophila have suggested that Ca2+/calmodulin-sensitive adenylyl cyclase may act as a site of convergence for the cellular representations of the conditioned stimulus (Ca2+ influx) and unconditioned stimulus (facilitatory transmitter) during elementary associative learning. This hypothesis predicts that the rise in intracellular free Ca2+ concentration produced by spike activity during the conditioned stimulus will cause an increase in the activity of adenylyl cyclase. However, published values for the Ca2+ sensitivity of Ca2+/calmodulin-sensitive adenylyl cyclase in mammals and in Drosophila vary widely. The difficulty in evaluating whether adenylyl cyclase would be activated by physiological elevations in intracellular Ca2+ levels is in part a consequence of the use of Ca2+/EGTA buffers, which are prone to several types of errors. Using a procedure that minimizes these errors, we have quantified the Ca2+ sensitivity of adenylyl cyclase in membranes from Aplysia, Drosophila, and rat brain with purified species-specific calmodulins. In all three species, adenylyl cyclase was activated by an increase in free Ca2+ concentration in the range caused by spike activity. Ca2+ sensitivity was dependent on both calmodulin concentration and Mg2+ concentration. Mg2+ raised the threshold for adenylyl cyclase activation by Ca2+ but also acted synergistically with Ca2+ to activate maximally adenylyl cyclase.     

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.     

Mechanism of passive Ca2+ permeability of vesicular sarcolemmal preparations from rat hearts

Vesicular sarcolemmal preparations isolated from rat hearts were characterized by high total ATPase (4.32 +/- 0.57 mumol/min per mg), adenylate cyclase (121 +/- 11 pmol/min per mg) and creatine kinase (1.73 +/- 0.35 mumol/min per mg) activities as well as Na-Ca exchange specific to sodium. ATPase activity was inhibited with digitoxigenin by 50-70% and was not changed by ouabain, ionophore A23187 or oligomycin. Sarcolemmal vesicles bound [3H]digitoxigenin and [3H]ouabain in isotonic medium in the presence of Pi and Mg2+. The number of binding sites for hydrophobic digitoxigenin (N = 237 pmol/mg) was several-times higher than that for hydrophilic ouabain (N = 32.7 pmol/mg). These data show that sarcolemmal preparations were not significantly contaminated by mitochondria and sarcoplasmic reticulum and consisted mostly of inside-out vesicles. Incubation of these vesicles with 45Ca2+ (0.5-10 mM) led to penetration of the latter into the vesicles with the following binding characteristics: number of binding sites (N = 20.5 +/- 4.6 nmol/mg, Kd approximately equal to 2.0 mM). Ca2+ binding to the inner surface of vesicles was proved by the following facts: (1) Ca2+ ionophore A23187 increased slightly total intravesicular Ca2+ content but markedly accelerated Ca2+ efflux along its concentration gradient; (2) gramicidin and osmotic shock showed a similar accelerating effect. Ca2+ efflux from the vesicles along its concentration gradient ([Ca2+]i/[Ca2+]e = 2.0 mM/0.1 microM) was inhibited by Mn2+, Co2+, and verapamil when they acted inside the vesicles. The rate of Ca2+ efflux was hyperbolically dependent on intravesicular Ca2+ concentration (Km approximately equal to 2.9 mM). These data reveal that Ca2+ efflux from sarcolemmal vesicles is controlled by Ca2+ binding to the sarcolemmal membrane. Ca2+ efflux from the vesicles was stimulated 1.7--times after incubation of vesicles with 0.2 mM MgATP or MgADP and 15-times after treatment with 0.2 mM adenylyl beta, gamma-imidodiphosphate. Enhancement in the rate of Ca2+ efflux correlated with the increase in the intravesicular Ca2+ content. ATP-stimulated Ca2+ efflux was suppressed by verapamil and was nonmonotonically dependent upon the transmembrane potential created by the K+ concentration gradient in the presence of valinomycin, Ca2+ efflux being slower at extreme values of membrane potential (+/- 80 mV).     

Divalent cation and lipid-protein interactions of biomembranes

Divalent cations play an important role in the functions of biomembranes. This review deals with three topics: (1) Mg²⁺-mediated change in physical state of phospholipid induces conformation and activity change of reconstituted mitochondrial H⁺-ATPase, (2) a proper transmembrane Ca²⁺ gradient is essential for the higher enzymatic activity of adenylate cyclase, and (3) role of transmembrane Ca²⁺ gradient in the modulation of reconstituted sarcoplasmic reticulm Ca²⁺-ATPase activity.     

Guanylate cyclase: assay and properties of the particulate and supernatant enzymes in mouse parotid.

A new, very sensitive, rapid and reliable assay for guanylate cyclase has been established based on conversion of [32P]GTP to [32P]guanosine 3':5'-monophosphate and its separation on Dowex 50 and aluminium oxide columns. The optimum conditions for the assay of mouse parotid guanylate cyclase have been established and using this procedure the properties of the enzyme have been investigated. The enzyme was found in both the particulate and supernatant fractions. The particulate enzyme was activated 12-fold by Triton X-100 and the supernatant enzyme activity increased 2-fold. In the presence of detergent guanylate cyclase activity was distributed 85% in the particulate and 15% in the supernatant fractions, respectively. The particulate activity was localised in a plasma membrane fraction. Guanylate cyclase activity was also assayed in a wide variety of other tissues. In all cases enzymatic activity was found in both the particulate and supernatant fractions. The distribution varied with the tissue but only the intestinal mucosa had a greater proportion of total guanylate cyclase activity in the particulate fraction than the parotid. The two enzymes showed some similar properties. Their pH optima were pH 7.4, both enzymes were inhibited by ATP, dATP, dGTP and ITP, required Mn2+ for activity and plots of activity versus Mn2+ concentration were sigmoidal. However, in many properties the enzymes were dissimilar. The ratios of Mn2+ to GTP for optimum activity were 4 and 1.5 for the supernatant and plasma-bound enzymes, respectively. The slope of Hill plots for the supernatant enzyme with varying Mn2+ was 2. The particulate enzyme plots also had a slope of 2 at low Mn2+ concentration but at higher concentrations (above 0.7 mM) the Hill coefficient shifted abruptly to 4. Calcium ions reduced sigmoidicity of the kinetics lowering the Hill coefficient, activated the enzyme at all Mn2+ concentrations but had no effect on the Mn2+:GTP ratio with the supernatant enzyme while with the plasma membrane enzyme Ca2+ had no effect on the sigmoid form of the kinetics at low Mn2+ but prevented the shift to a greater Hill coefficient at higher Mn2+, inhibited the activity at low Mn2+ and shifted the Mn2+:GTP optimum ratio to 4. For the particulate enzyme plots of activity versus GTP concentration were sigmoid (n = 1.3), while the supernatant enzyme exhibited hyperbolic kinetics.     

Investigation of the role of Ca2+ and calmodulin in the regulation of platelet guanylate cyclase activity.

Both soluble and particulate forms of human platelet guanylate cyclase were found to be sensitive to sub-micromolar concentrations of free Ca2+; soluble enzyme activity increased as Ca2+ was increased from 10 nM to 1 microM; particulate enzyme activity showed a biphasic response to Ca2+, with maximal enzyme activity between 1 and 10 nM free Ca2+ and inhibition occurring at higher Ca2+ concentrations. Neither Ca2+-sensitivity appeared to be calmodulin-dependent.     

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.     

The metabolism of cyclic nucleotides in the guinea-pig pancreas. Adenylate cyclase and guanylate cyclase.

Adenylate cyclase from the guinea-pig pancreas was activated in a dose-dependent manner by both secretin and cholecystokinin-pancreozymin, but in contrast with results in other species the hormones were approximately equipotent. All other hormones and transmitter substances tested were without any effect on adenylate cyclase activity. Guanylate cyclase activity was shown to have both particulate and supernatant components in the guinea-pig pancreas. The particulate enzyme, but not the supernatant enzyme, was markedly activated by Triton X-100, and most of the induced activity was released into the supernatant. The supernatant enzyme was specifically Mn2+-dependent, but, even though Mn2+ was maximally effective at a concentration of 3 mM, activity could be raised further by increasing Ca2+ concentration. The particulate enzyme, by contrast, was relatively Mn2+-independent. Activity of the particulate guanylate cyclase was enhanced by phosphatidylserine. The supernatant enzyme displayed classical Michaelis-Menten kinetics, but the particulate enzyme deviated markedly from such kinetics. Under none of the conditions used was any significant activation of guanylate cyclase observed with any of the secretogen hormones or transmitter substances.     

Subcellular distribution and properties of guanylate cyclase in rat cerebellum.

In rat cerebellum the major portion of guanylate cyclase was found to be particulate-bound. The properties of particulate and supernatant guanylate cyclases from the cerebellum were comparatively examined. Both enzymes required the same optimal concentration of Mn2+ and were stimulated by Ca2+ in the presence of a low concentration of Mn2+. But dispersion of the particulate enzyme with Triton X-100 altered the Mn2+ concentration producing maximum activity and the inhibitory effect of Ca2+. The subcellular distributions of guanylate and adenylate cyclases were also studied in rat cerebellum. The major portions of the two cyclases were found in the mitochondrial fraction. The submitochondrial fractions separated by sucrose gradient showed that the major activities of both cyclases were concentrated in the fraction containing mainly nerve ending particles.     

Potent and cooperative feedback inhibition of adenylate cyclase activity by calcium in pituitary-derived GH3 cells

Calcium (Ca2+) ion concentrations that are achieved intracellularly upon membrane depolarization or activation of phospholipase C stimulate adenylate cyclase via calmodulin (CaM) in brain tissue. In the present study, this range of Ca2+ concentrations produced unanticipated inhibitory effects on the plasma membrane adenylate cyclase activity of GH3 cells. Ca2+ concentrations ranging from 0.1 to 0.8 microM exerted an increasing inhibition on enzyme activity, which reached a plateau (35-45% inhibition) at around 1 microM. This inhibitory effect was highly cooperative for Ca2+ ions, but was neither enhanced nor dependent upon the addition of CaM (1 microM) to EGTA-washed membranes. The inhibition was greatly enhanced upon stimulation of the enzyme by vasoactive intestinal peptide (VIP) and/or GTP. Prior exposure of cultured cells to pertussis toxin did not affect the inhibition of plasma membrane adenylate cyclase activity by Ca2+, although in these membranes, hormonal (somatostatin) inhibition was significantly attenuated. Maximally effective concentrations of Ca2+ and somatostatin produced additive inhibitory effects on adenylate cyclase. The addition of phosphodiesterase inhibitors demonstrated that inhibitory effects of Ca2+ were not mediated by Ca2(+)-dependent stimulation of a phosphodiesterase activity. These observations provide a mechanism for the feedback inhibition by elevated intracellular Ca2+ levels on cAMP-facilitated Ca2+ entry into GH3 cells, as well as inhibitory crosstalk between Ca2(+)-mobilizing signals and adenylate cyclase activity.