Interactions of divalent cations and nucleotides with solubilized cardiac guanylate cyclase.

Some properties of guanylate cyclase, which was solubilized from the rabbit heart washed particles by the treatment with Triton X-100, were investigated. The solubilized enzyme activity was stimulated by Mg2+ in the presence of low (subsaturating) Mn2+ (GTP is greater than Mn2+); under these conditions, Ga2+ was inhibitory. At subsaturating MnGTP and free Mn2+, the solubilized enzyme was markedly stimulated by MnGDP and MnATP; CaGTP on the other hand, was inhibitory. These results are consistent with the view that the particulate guanylate cyclase may exist in the cell as a metalloenzyme with tightly bound Mn2+ and that Mg2+ supports its catalysis while Ca2+ as well as nucleotides may exert regulatory effects on its activity.     

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+.     

Characterization of guanylate cyclase of rod outer segments of the bovine retina.

Guanylate cyclase (GTP pyrophosphate-lyse (cyclizing), EC 4.6.1.2.) of bovine retinal rod outer segments is almost completely particulate, i.e. associated with rod outer segment membranes. In contrast to particulate guanylate cyclase in other tissues, treatment of rod outer segments with Triton X-100 does not solublize the enzyme but inhibits it. Enzyme activity is dependent on the presence of divalent cation, especially Mn2+ with only poor activation by Mg2+ (10-fold lower) and no activation seen with other cation. Ezpression of maximal activity required Nm2+ and GTP in equimolar concentrations with an apparent Km of 8 . 10(-4) M and V of 10 nmol/min per mg protein. Excess of Mn2+ over that required for the formation of the Mn . GTP complex was inhibitory. Ca2+, Ba2+ and Co2+ inhibited enzyme activity when assayed with the Mn . GTP substrate complex. In the presence of a fixed concentration of 1mM Mn2+, the enzyme exhibited strong negative cooperative interactions with GTP, characterized by an intermediary plateau region in the substrate vs. enzyme activity curve, a curve of downward concavity in the double reciprocal plot and a Hill coefficient of 0.5. Nucleotides such as ITP, ATP and UTP at higher concentrations (1 mM) stimulates activity by 40%. NaN3 has no effect on the guanylate cyclase. It is thus possible that the guanylate cyclase may be regulated in vivo by both the metal : GTP substrate ratio and the free divalent cation concentration as well as by the ATP concentration and thus play an important but yet undefined role in the visual process.     

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.     

Guanylate cyclase. Subcellular distribution in cardiac muscle, skeletal muscle, cerebral cortex and liver.

1. Guanylate cyclase of every fraction studied showed an absolute requirement for Mn2+ ions for optimal activity; with Mg2+ or Ca2+ reaction was barely detectable. Triton X-100 stimulated the particulate enzyme much more than the supernatant enzyme and solubilized the particulate-enzyme activity. 2. Substantial amounts of guanylate cyclase were recovered with the washed particulate fractions of cardiac muscle (63-98%), skeletal muscle (77-93%), cerebral cortex (62-88%) and liver (60-75%) of various species. The supernatants of these tissues contained 7-38% of total activities. In frog heart, the bulk of guanylate cyclase was present in the supernatant fluid. 3. Plasma-membrane fractions contained 26, 21, 22 and 40% respectively of the total homogenate guanylate cyclase activities present in skeletal muscle (rabbit), cardiac muscle (guinea pig), liver (rat) and cerebral cortex (rat). In each case, the specific activity of this enzyme in plasma membranes showed a five- to ten-fold enrichment when compared with homogenate specific activity. 4. These results suggest that guanylate cyclase, like adenylate cyclase, and ouabain-sensitive Na+ + K+-dependent ATPase (adenosine triphosphatase), is associated with the surface membranes of cardiac muscle, skeletal muscle, liver and cerebral cortex; however, considerable activities are also present in the supernatant fractions of these tissues which contain very little adenylate cyclase or ouabain-sensitive Na+ + K+-dependent ATPase activities.     

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.     

Activation of guanylate cyclase from rat liver and other tissues by sodium azide.

Sodium azide, hydroxylamine, and phenylhydrazine at concentrations of 1 mM increased the activity of soluble guanylate cyclase from rat liver 2- to 20-fold. The increased accumulation of guanosine 3':5'-monophosphate in reaction mixtures with sodium azide was not due to altered levels of substrate, GTP, or altered hydrolysis of guanosine 3':5'-monophosphate by cyclic nucleotide phosphodiesterase. The activation of guanylate cyclase was dependent upon NaN3 concentration and temperature; preincubation prevented the time lag of activation observed during incubation. The concentration of NaN3 that resulted in half-maximal activation was 0.04 mM. Sodium azide increased the apparent Km for GTP from 35 to 113 muM. With NaN3 activation the enzyme was less dependent upon the concentration of free Mn2+. Activation of enzyme by NaN3 was irreversible with dilution or dialysis of reaction mixtures. The slopes of Arrhenius plots were altered with sodium azide-activated enzyme, while gel filtration of the enzyme on Sepharose 4B was unaltered by NaN3 treatment. Triton X-100 increased the activity of the enzyme, and in the presence of Triton X-100 the activation by NaN3 was not observed. Trypsin treatment decreased both basal guanylate cyclase activity and the responsiveness to NaN3. Phospholipase A, phospholipase C, and neuraminidase increased basal activity but had little effect on the responsiveness to NaN3. Both soluble and particulate guanylate cyclase from liver and kidney were stimulated with NaN3. The particulate enzyme from cerebral cortex and cerebellum was also activated with NaN3, whereas the soluble enzyme from these tissues was not. Little or no effect of NaN3 was observed with preparations from lung, heart, and several other tissues. The lack of an effect with NaN3 on soluble GUANYLATE Cyclase from heart was probably due to the presence of an inhibitor of NaN3 activation in heart preparations. The effect of NaN3 was decreased or absent when soluble guanylate cyclase from liver was purified or stored at -20degrees. The activation of guanylate cyclase by NaN3 is complex and may be the result of the nucleophilic agent acting on the enzyme directly or what may be more likely on some other factor in liver preparations.     

Purification and properties of guanylate cyclase from the synaptosomal soluble fraction of rat brain.

Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) was purified 2250-fold from the synaptosomal soluble fraction of rat brain. The specific activity of the purified enzyme reached 41 nmol cyclic GMP formed per min per mg protein at 37 degrees C. In the purified preparation, GTPase activity was not detected and cyclic GMP phosphodiesterase activity was less than 4% of guanylate cyclase activity. The molecular weight was approx. 480 000. Lubrol PX, hydroxylamine, or NaN3 activated the guanylate cyclase in crude preparations, but had no effect on the purified enzyme. In contrast, NaN3 plus catalase, N-methyl-N'-nitro-N-nitrosoguanidine or sodium nitroprusside activated the purified enzyme. The purified enzyme required Mn2+ for its activity; the maximum activity was observed at 3-5 mM. Cyclic GMP activated guanylate cyclase activity 1.4-fold at 2 mM, whereas inorganic pyrophosphate inhibited it by about 50% at 0.2 mM. Guanylyl-(beta,gamma-methylene)-diphosphonate and guanylyl-imidodiphosphate, analogues of GTP, served as substrates of guanylate cyclase in the purified enzyme preparation. NaN3 plus catalase or N-methyl-N'-nitro-N-nitrosoguanidine also remarkably activated guanylate cyclase activity when the analogues of GTP were used as substrates.     

Lipids associated with bovine kidney glomerular basement membranes.

1. The activities of the enzymes involved in the metabolism of cyclic nucleotides were studied in sarcolemma prepared front guinea-pig heart ventricle; the enzyme activities reported here were linear under the assay conditions. 2. Adenylate cyclase was maximally activated by 3mM-NaF; NaF increased the Km for ATP (from 0.042 to 0.19 mM) but decreased the Ka for Mg2+ (from 2.33 to 0.9 mM). In the presence of saturating Mg2+ (15 mM), Mn2+ enhanced adenylate cyclase, whereas Co2+ was inhibitory. beta-Adrenergic amines (10-50 muM) stimulated adenylate cyclase (38+/-2%). When added to the assay mixture, guanyl nucleotides (GTP and its analogue, guanylyl imidophosphate) stimulated basal enzyme activity and enhanced the stimulation by isoproterenol. By contrast, preincubation of sarcolemma with guanylyl imidodiphosphate stimulated the formation of an 'activated' form of the enzyme, which did not reveal increased hormonal sensitivity. 3. The guanylate cyclase present in the membranes as well as in the Triton X-100-solubilized extract of membranes exhibited a Ka for Mn 2+ of 0.3 mM; Mn2+ in excess of GTP was required for maximal activity. Solubilized guanylate cyclase was activated by Mg2+ only in the presence of low Mn2+ concentrations; Ca2+ was inhibitory both in the absence and presence of low Mn2+. Acetylcholine as well as carbamolycholine stimulated membrane-bound guanylate cyclase. 4. Cylic nucleotide phosphodiesterase activities of sarcolemma exhibited both high-and low-Km forms with cyclic AMP and with cyclic GMP as substrate. Ca2+ ions increased the Vmax. of the cyclic GMP-dependent enzyme.     

Interaction of Triton X-100 with particulate cardiac guanylate cyclase: comparison between Mg2+- and Mn2+-supported enzyme activities in particulate. detergent-solubilized and detergent-insoluble fractions

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⁺.     

Effects of cations on guanylate cyclase of sea urchin sperm.

Mn2+ and to some degree Fe2+, but not Mg+, Ca2+, ba2+, Sr2+, Co2+, Ni2+, La3+, or Fe3+ were able to serve as effective metal cofactors for sea urchin sperm guanylate cyclase. The apparent Michaelis constant for Mn2+ in the presence of 0.25 mM MnGTP was 0.23 mM. In the presence of a fixed free mn2+ concentration, variation in mngTP resulted in sigmoid velocity-substrate plots and in reciprocal plots that were concave upward. These positive cooperative patterns were observed at both pH 7.0 and 7.8 and in the presence or absence of Triton X-100. When Mn2+ and GTP were equimolar, Ca2+, Ba2+, Sr2+, and Mg2+ increased apparent guanylate cyclase activity. This increase in enzyme activity at least could be accounted for partially by an increase in free Mn2+ concentration caused by the complex formation of GTP with the added metals. However, even at relatively low GTP concentrations and with Mn2+ concentrations in excess of GTP, Ca2+, Sr2+, and Ba2+ significantly increased guanosine 3':5'-monophosphate production. As the total GTP concentration was increased, the degree of stimulation in the presence of Ca2+ decreased, despite maintenance of a fixed total concentration of Ca2+ and a fixed free concentration of Mn2+, suggesting that the concentration of CaGTP and MnGTP were determining factors in the observed response. The concave upward reciprocal plots of velocity against MnGTP concentration were changed to linear plots in the presence of CaGTP or SrGTP. These results suggest that sea urchin sperm guanylate cyclase contains multiple nucleotide binding sites and that stimulation of guanosine 3':5'-monophosphate synthesis by Ca2+, Sr2+, and perhaps other metals may reflect interaction of a metal-GTP complex with enzyme as either an effector or a substrate.     

Myocardial guanylate cyclase: properties of the enzyme and effects of cholinergic agonists in vitro.

The characteristics of myocardial guanylate cyclase (GTP pyrophosphatelyase, EC 4.6.1.2) were studied. Specific activity of the myocardial enzyme in five vertebrate species was guinea pig greater than man greater than cat greater than dog greater than rat. In the guinea pig, guanylate cyclase activity was uniformly distributed throughout the anatomical regions of the heart. The major portion of the enzyme activity was retrieved in the supernatant fraction after centrifugation at 12 000 times g. The Km for GTP was similar in supernatant (0.12 mM) and particulate (0.21 mM) preparations, although the Ka for Mn2+ in particulate preparations (0.3-0.6 mM) was less than that observed for guanylate cyclase in the supernatant fraction (0.8-2.0 mM). ATP competitively inhibited supernatant and particulate activity. Addition of 0.005-10.0 mM Ca2+ to assay incubations did not enhance guanylate cyclase activity. Suspension of 105 000 times g supernatant guanylate cyclase preparations with membrane lipids or phosphatidylserine stimulated activity 1.4-4.3 fold, whereas similar treatment of particulate preparations caused little alteration of enzyme activity. Addition of the cholinergic agonists acetylcholine, carbachol or methacholine (10-4-10-8 M) to homogenate, supernatant, particulate and disrupted tissue slice preparations in the presence of 0.0012-1.2 mM GTP, 0.3-10.0 mM Mn2+ and 0.005-10.0 mM Ca2+ or 0.0012-1.2 mM ATP did not stimulate guanylate cyclase activity. Similarly, further stimulation of guanylate cyclase activity was not elicited when enzyme-lipid suspensions were assayed in the presence of cholinergic agents.     

Guanylate cyclase in Escherichia coli. Purification and properties.

Guanylate cyclase has been purified from extracts of Escherichia coli. After a 1000-fold purification, the enzyme contains only minor contaminants as judged by disc gel electrophoresis. The Km for GTP is approximately 7 times 10(-5) M and the optimal pH is 8.0. More activity is observed with Mn2+ than with Mg2+, and maximal activity is observed at 0.14 mM Mn2+ and 1.4 mM Mg2+. Based on its behavior on Sephadex G-100, the molecular weight of E. coli guanylate cyclase is about 30,000. Disc gel electrophoretic analysis indicates that the enzyme consists of a single polypeptide chain. Guanylate cyclase does not form 3':5'-AMP from ATP, and therefore, is distinct from adenylate cyclase.     

Localization of particulate guanylate cyclase in plasma membranes and microsomes of rat liver.

The subcellular localization of guanylate cyclase was examined in rat liver. About 80% of the enzyme activity of homogenates was found in the soluble fraction. Particulate guanylate cyclase was localized in plasma membranes and microsomes. Crude nuclear and microsomal fractions were applied to discontinuous sucrose gradients, and the resulting fractions were examined for guanylate cyclase, various enzyme markers of cell components, and electron microscopy. Purified plasma membrane fractions obtained from either preparation had the highest specific activity of guanylate cyclase, 30 to 80 pmol/min/mg of protein, and the recovery and relative specific activity of guanylate cyclase paralleled that of 5'-nucleotidase and adenylate cyclase in these fractions. Significant amounts of guanylate cyclase, adenylate cyclase, 5'-nucleotidase, and glucose-6-phosphatase were recovered in purified preparation of microsomes. We cannot exclude the presence of guanylate cyclase in other cell components such as Golgi. The electron microscopic studies of fractions supported the biochemical studies with enzyme markers. Soluble guanylate cyclase had typical Michaelis-Menten kinetics with respect to GTP and had an apparent Km for GTP of 35 muM. Ca-2+ stimulated the soluble activity in the presence of low concentrations of Mn-2+. The properties of guanylate cyclase in plasma membranes and microsomes were similar except that Ca-2+ inhibited the activity associated with plasma membranes and had no effect on that of microsomes. Both particulate enzymes were allosteric in nature; double reciprocal plots of velocity versus GTP were not linear, and Hill coefficients for preparations of plasma membranes and microsomes were calculated to be 1.60 and 1.58, respectively. The soluble and particulate enzymes were inhibited by ATP, and inhibition of the soluble enzyme was slightly greater. While Mg-2+ was less effective than Mn-2+ as a sole cation, all enzyme fractions were markedly stimulated with Mg-2+ in the presence of a low concentration of Mn-2+. Triton X-100 increased the activity of particulate fractions about 3- to 10-fold and increased the soluble activity 50 to 100%.     

Properties of guanylate cyclase from rat kidney cortex and transplantable kidney tumors.

The subcellular distribution and properties of guanylate cyclase was examined in preparations of normal rat renal cortex and Morris renal tumors MK2 and MK3. In normal kidney cortex about two-thirds of guanylate cyclase activity of homogenates was found in soluble fractions. With renal tumors the homogenate activity was less and the enzyme was equally divided between particulate and soluble fractions. The particulate enzyme in kidney cortex and tumors was associated with all particulate fractions. Triton X-100 increased the activity of all preparations. All preparations preferred Mn2+ as the sole cation. The stimulatory effects of Ca2+ on soluble enzyme and inhibitory effects on particulate activity were similar with preparations of renal cortex and tumors. ATP inhibited all preparations. Soluble and particulate guanylate cyclases from renal cortex were activated several-fold with 1 mM NaN3. Preparations of tumor enzymes did not respond to NaN3. Thus, compared to normal renal cortex the subcellular distribution of guanylate cyclase and some of its properties are altered in preparations of renal tumors.     

Guanylate cyclase from human blood platelets

Human blood platelets were disrupted by ultrasonication, and the guanylate cyclase activity was determined in the 105,000 g supernatant. The guanylate cyclase preparation obtained in the absence of dithiothreitol (DTT) was characterized by a nonlinear dynamics of cGMP synthesis during incubation at 37 degrees C. The use of 0.2 mM DTT during platelet ultrasonication stabilized the guanylate cyclase reaction and did not influence the enzyme activity. With a rise in DTT concentration up to 2 mM the guanylate cyclase activity diminished. Sodium nitroprusside stimulated the enzyme; this effect was enhanced in the presence of DTT. The maximum guanylate cyclase activity was revealed at 4 mM Mn2+ or Mg2+ and with 1 mM GTP. In the presence of Mn2+ the enzyme activity was higher than with Mg2+. The apparent Km values for GTP in the presence of 4 mM Mn2+ and Mg2+ was 30 and 200 microM, respectively. At GTP/cation ratio of 1:4 the Km values for Mn2+ and Mg2+ were nearly the same (249 and 208 microM, respectively). It was assumed that besides being involved in the formation of the GTP-substrate complex, Mn2+ exerts a strong influence on guanylate cyclase by oxidizing the SH-groups of the enzyme.     

Effects of metals and nucleotides on the inactivation of sea urchin sperm guanylate cyclase by heat and N-ethylmaleimide.

Preincubation of sea urchin sperm guanylate cyclase at 35, 37, 40, or 43 degrees resultedin inactivation. Various metals were able to protect guanylate cyclase against heat inactivation. Estimated binary enzyme-metal dissociation constants for Mn2+, Fe2+, La3+, Ca2+, Ba2+, Mg2+, Co2+, and Ni2+ were 123, 361, 5.5, 692, 984, 335, 79, and 47 muM, respectively. Extrapolated rates of enzyme denaturation in the presence of saturating concentrations of metal divided by the rates of enzyme denaturation in the absence of metal gave values of 0.13, 0.08, minus 0.1, 0.30, 0.59, 0.66, 0.28, and 0.42 for Mn2+, Fe2+, La3+, Ca2+, Ba2+, Mg2+, Co2+, and Ni2+, respectively. GTP, MgGTP, and SrGTP protected the enzyme only slightly against heat inactivation, but CaGTP and MnGTP protected substantially. Neither CaGTP nor MnGTP protected maximally, however, unless the metal concentration exceeded that of GTP. At fixed free Mn2+ or free Ca2+ concentrations, protection curves as a function of MnGTP or CaGTP appeared to be sigmoidal, suggesting multiple nucleotide binding sites. MnATP also protected against heat, but CaATP was virtually ineffective. Sea urchin sperm guanylate cyclase was inactivated by N-ethylmaleimide; CaGTP and MnATP were effective protectants with estimated binary enzyme-Me2+ nucleoside triphosphate dissociation constants of 40 and 170 muM, respectively. MnGTP protected only slightly or not at all against N-ethylmaleimide. These results suggest that: (a) sea urchin sperm guanylate cyclase binds free metal, (b) the binding of free metal is required for protection by nucleotides, and (c) the enzyme contains multiple nucleotide binding sites.     

Guanylate cyclase in the accessory gland of the cricket, Acheta domesticus

Guanylate cyclase (E.C. 4.6.1.2.) was investigated in the accessory reproductive gland of the male house cricket, Acheta domesticus, which is known to accumulate exceptionally high levels of guanosine 3′,5′-cyclic monophosphate (cyclic GMP). Accessory gland guanylate cyclase activity was linear with time for at least one hour, and with enzyme concentration to about 5 mg soluble protein per ml. Activity was dependent on Mn²⁺ and was maximal at pH 7.3 to 8.0. Sodium fluoride had no effect on activity, but sodium azide was slightly stimulatory. About 80% of the activity was sedimentable at 16,000 g, and both soluble and particulate activities were increased slightly in the presence of Triton X-100. Kinetic analysis indicated half-maximal velocity at 85 μM GTP in the presence of excess Mn²⁺, and reciprocal plots were concave upward. Changes in activity during maturation of the gland were small, and did not provide evidence for a regulatory role of guanylate cyclase in the accumulation of accessory gland cyclic GMP. The regulation and rôle of cyclic GMP in the accessory gland are discussed.     

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.     

Differential effects of manganese ions on Blastocladiella emersonii adenylate cyclase.

Adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity in Blastocladiella emersonii is associated with particulate subcellar fractions. Solubilization after treatment with detergent suggests its localization in a membrane fraction of the zoospore homogenate. The enzyme specifically requires Mn2+ for activity and is not stimulated by NaF. The kinetic characteristics of substrate utilization by B. emersonii adenylate cyclase were investigated with various concentrations of ATP and Mn2+, and in the presence of inhibitors. Plots of enzyme activity versus the actual concentration of the MnATP2- complex give sigmoid curves. An excess of Mn2+ activates the enzyme at low concentrations of substrate and leads to a modification of the enzyme kinetics. The nucleotides 5'-AMP and GTP were shown to be competitive inhibitors of the enzyme. In addition, kinetic data, obtained under conditions in which an inhibitor (ATP) is added in constant proportion to the variable substrate (MnATP2-) concentration, produced reciprocal plots that were linear and intersecting to the right of the ordinate, and secondary replots that were hyperbolic. These kinetic patterns support a model in which: MnATP2- is the substrate; free Mn2+ is an activator at low substrate concentrations, but an inhibitor at high substrate concentrations; and free ATP is not an efficient inhibiyor (Ki greater than 1.10(-4) M).