Purification of soluble guanylate cyclase enzyme from human platelets

Soluble guanylate cyclase enzyme was purified from human platelets. The soluble fraction of the lysed platelets was sequentially chromatographed over DEAE-sepharose, GTP-agarose and HPLC size-exclusion columns. About 0.1 mg of purified enzyme could be obtained from 2000 ml of platelet rich plasma. The purified enzyme had the specific activity of 205 nmoles cGMP/mg/min with Mn2+ as cofactor. The enzyme eluted at the 160,000 daltons position from the size-exclusion column. Electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions revealed two subunits of 83,000 and 71,000 daltons respectively.     

An endogenous regulator of the guanylate cyclase activity of human platelets

Chromatography of soluble human platelet guanylate cyclase (105,000 g supernatant) on DEAE-cellulose in a linear gradient of NaCl (0-0.5 M) in 50 mM Tris-HCl buffer pH 7.6 gave two protein peaks, I and II, of which only peak II possessed the guanylate cyclase activity (0.18-0.22 M NaCl). The protein fraction I was found to possess an inhibiting activity; its addition to the partially purified enzyme decreased the guanylate cyclase activity by 60-70% in the presence of Mg2+ with no effect on the enzyme activity in the presence of Mn2+. The isolated enzyme lost (by approximately 80%) its ability to be activated by sodium nitroprusside; the latter was reconstituted after addition of the inhibiting fraction. The data obtained testify to the heme origin of the endogenous inhibitor of human platelet guanylate cyclase.     

Purification of guanylate cyclase from human platelets and effect of arachidonic acid peroxide.

Guanylate cyclase of human platelets was separated from cyclic nucleotide and GTP hydrolytic activities with a 104-fold purification over the homogenate. The purified guanylate cyclase preparation requires neither the GTP regenerating system nor cyclic GMP but is stimulated by about 2-fold by 2.5 mM cyclic GMP. The molecular weight of the enzyme was estimated as 180,000 and the Km value for GTP was 95 μM. Arachidonic acid peroxide stimulated the purified enzyme by increasing maximum velocity without changing Km value.     

Receptor-mediated activation of spermatozoan guanylate cyclase

The sea urchin egg peptides speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Arg-Leu-NH2) bind to spermatozoa of the homologous species (Lytechinus pictus or Arbacia punctulata, respectively) and cause transient elevations of cyclic GMP concentrations (Hansbrough, J. R., and Garbers, D. L. (1981) J. Biol. Chem. 256, 1447-1452). The addition of these peptides to spermatozoan membrane preparations caused a rapid and dramatic (up to 25-fold) activation of guanylate cyclase. The peptide-induced activation of guanylate cyclase was transient, and the subsequent decline in enzyme activity coincided with conversion of a high Mr (phosphorylated) form of guanylate cyclase to a low Mr (dephosphorylated) form. When membranes were incubated at pH 8.0, the high Mr form was converted to the low Mr form without substantial changes in basal enzyme activity. However, the peptide-stimulated activity of the low Mr form of guanylate cyclase was much less than the peptide-stimulated activity of the high Mr form. Activation of the low Mr form by peptide was not transient and persisted for at least 10 min. In addition, the pH 8.0 treatment that caused the Mr conversion of guanylate cyclase also caused an increase in the peptide-binding capacity of the membranes. We propose a model in which activation of the membrane form of guanylate cyclase is receptor-mediated; the extent of enzyme activation is modulated by its phosphorylation state.     

Cation-dependent prolactin activation of guanylate cyclase

Prolactin enhanced guanylate cyclase [E.C.4.6.1.2] two- to threefold in ovary, testis, mammary gland, liver and kidney. Dose response relationships revealed that maximal activation of this enzyme was at a concentration of one nanomolar and that increasing prolactin's concentration to the millimolar range caused no further increase in activity. There was an absolute cation requirement for prolactin's enhancement of guanylate cyclase. Calcium or manganese allowed prolactin to increase guanylate cyclase activity. Greater enhancement of this enzyme's activity by prolactin was observed when manganese was the co-factor. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of prolactin.     

Receptor-mediated activation of detergent-solubilized guanylate cyclase

Here for the first time we report the successful detergent-solubilization of the speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) receptor and the subsequent activation of guanylate cyclase in response to receptor occupation. Sea urchin sperm membranes treated with a solution containing 0.5% LubrolR PX and 0.5% EmulphogeneR in the presence of MgCl2 and NaF released both the speract receptor and guanylate cyclase activity into solution. The solubilized apparent receptor was not sedimented at 400,000 x g x 15 min and was not retained by glass microfiber filters. In the presence of 125I-GGG(Y2)speract and dissuccinimidyl suberate, a major radioactive band at about Mr = 77,000 and minor bands at Mr = 35,000 and 150,000 were cross-linked. Speract but not resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-LeuNH2) competed in the cross-linking reaction. The amount of 125I-GGG(Y2)speract bound to solubilized receptor did not increase in a linear manner as a function of added protein but instead was concave upward. The addition of speract but not resact to the solubilized preparation resulted in the activation of the enzyme guanylate cyclase; the extent of stimulation was dependent on the amount of enzyme protein added and also was concave upward. Approximately 900 nM speract half-maximally activated guanylate cyclase. These data suggest that the speract receptor and guanylate cyclase are closely apposed, even in detergent, or that they are the same molecule.     

Butadiene diepoxide activation of guanylate cyclase.

A variety of nitroso chemical carcinogens increase the activity of guanylate cyclase (EC 4.6.1.2), the enzyme catalyzing the production of guanosine 3',5'-monophosphate. In the present report, the first non-nitroso chemical carcinogen, butadiene diepoxide, was shown to activate guanylate in a variety of tissues over the concentration range 1-100 mmol/l. At 20 mmol/l concentration, increases were 2- to 17-fold above control. These observations have potential importance since guanosine 3',5'-monophosphate may be involved in cell growth and malignant transformation.     

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.     

Factors affecting the activity of guanylate cyclase in lysates of human blood platelets.

1. Under optimal ionic conditions (4 mM-MnCl2) the specific activity of guanylate cyclase in fresh platelet lysates was about 10nmol of cyclic GMP formed/20 min per mg of protein at 30 degrees C. Activity was 15% of optimum with 10mM-MgCl2 and negligible with 4mM-CaCl2. Synergism between MnCl2 and MgCl2 or CaCl2 was observed when [MnCl2] less than or equal to [GPT]. 2. Lower than optimal specific activities were obtained in assays containing large volumes of platelet lysate, owing to the presence of inhibitory factors that could be removed by ultrafiltration. Adenine nucleotides accounted for less than 50% of the inhibitory activity. 3. Preincubation of lysate for 1 h at 30 degrees C increased the specific activity of platelet guanylate cyclase by about 2-fold. 4. Lubrol PX (1%, w/v) stimulated guanylate cyclase activity by 3--5-fold before preincubation and by about 2-fold after preincubation. Triton X-100 was much less effective. 5. Dithiothreitol inhibited the guanylate cyclase activity of untreated, preincubated and Lubrol PX-treated lysates and prevented activation by preincubation provided that it was added beforehand. 6. Oleate stimulated guanylate cyclase activity 3--4-fold and arachidonate 2--3-fold, whereas palmitate was almost inactive. Pretreatment of lysate with indomethacin did not inhibit this effect of arachidonate. Oleate and arachidonate caused marked stimulation of guanylate cyclase in preincubated lysate, but inhibited the enzyme in Lubrol PX-treated lysate. 7. NaN3 (10mM) increased guanylate cyclase activity by up to 7-fold; this effect was both time- and temperature-dependent. NaN3 did not further activate the enzyme in Lubrol PX-treated lysate. 8. The results indicated that preincubation, Lubrol PX, fatty acids and NaN3 activated platelet guanylate cyclase by different mechanisms. 9. Platelet particulate fractions contained no guanylate cyclase activity detectable in the presence or absence of Lubrol PX that could not be accounted for by contaminating soluble enzyme, suggesting that physiological aggregating agents may increase cyclic GMP in intact platelets through the effects of intermediary factors. The activated and inhibited states of the enzyme described in the present paper may be relevant to the actions of these factors.     

Particulate guanylate cyclase and adenylate cyclase activities after activation with various agents in rabbit platelets. An ultracytochemical study

Summary The cytochemical localization of particulate guanylate cyclase and adenylate cyclase activities in rabbit platelets were studied after stimulation with various agents, at the electron microscope level. In the presence of platelet aggregating agents such as thrombin and ADP, the particulate reaction product of guanylate cyclase activity was detectable on plasma membrane and on membranes of the open canalicular system. In contrast, samples incubated with platelet-activating factor showed no activation of the cyclase activity. Atrial natriuretic factor stimulated the particulate guanylate cyclase. The ultracytochemical localization of this activated cyclase was the same as that of thrombin-or ADP-stimulated guanylate cyclase. Adenylate cyclase activity was studied in platelets incubated with prostaglandin E₁ plus or minus insulin. The enzyme reaction product was found at the same sites where guanylate cyclase was detected. Therefore guanylate and adenylate cyclase activities do not seem to be preferentially localised in platelet membranes.     

Heme deficiency of soluble guanylate cyclase in rat platelets]

Analysis of soluble guanylate cyclase of rat platelets (105,000 g supernatant) revealed no activating effect of sodium nitroprusside on the enzyme activity. Dithiothreitol (2 x 10(-4) H) added to the sample stimulated the basal activity of guanylate cyclase in the presence of Mg2+ but did not induce the enzyme activation by sodium nitroprusside. Hemoglobin added to the enzyme did not influence its basal activity or the activating effect of sodium nitroprusside. DEAE-Cellulose chromatography of the 105,000 g supernatant revealed two protein peaks, I and II, of which only peak II possessed a guanylate cyclase activity. Fraction I added to a partly purified enzyme did not change the enzyme activity, nor did it enhance the sodium nitroprusside-induced activation of guanylate cyclase. Spectral analysis of the 105,000 g supernatant revealed that the presence of a maximum at 415-425 nm (Soret band) depended on the degree of plasma hemolysis. In the absence of hemolysis the Soret band was unobserved either in the 105,000 g supernatant or in fractions I and II. It is suggested that rat platelet guanylate cyclase is present in these cells in a heme-deficient state.     

Fatty acid activation of guanylate cyclase from fibroblasts and liver.

Sodium arachidonate and sodium oleate increased particulate guanylate cyclase activity from homogenates of Balb 3T3 cells or rat liver. The fatty acids were about equipotent and were maximally effective at about 100 μm concentrations. Higher concentrations were less effective or inhibitory. Activation was similar in an air or nitrogen atmosphere and was unaltered by KCN, aspirin, or indomethacin. The dose-response curve was shifted to the right when arachidonate was preincubated prior to its addition to guanylate cyclase assays. Agents that facilitate fatty acid oxidation and the formation of malonyldialdehyde during preincubation such as glutathione, hemoglobin, Mn²⁺, Fe³⁺, or lipoxygenase shifted the dose-response curve further to the right. In contrast, agents that decreased or prevented arachidonate oxidation and malonyldialdehyde formation during preincubation such as butylated hydroxyanisole, propyl gallate, hydroquinone, and diphenylfuran prevented the shift in the dose-response curve or in some instances shifted the dose-response curve to the left. Activation of guanylate cyclase by arachidonate was reversed by the addition of lipoxygenase to incubations. These studies indicate that unsaturated fatty acids and not their oxidation products activate particulate enzyme from Balb 3T3 cells. The mechanism of fatty acid activation appears to be different from activation by nitro compounds. Fatty acids but not nitro compounds activated fibroblast preparations, and the effect of fatty acids in contrast to the activation by nitroprusside in liver preparations was not prevented with Lubrol PX.     

Studies on cytosolic guanylate cyclase from human placenta.

We have purified the soluble form of guanylate cyclase from human placenta greater than 2400-fold. The enzyme shared several characteristics with the enzyme purified from other sources including molecular mass and subunit composition, activation by divalent cations, inhibition by ATP and Michaelis constants. The enzyme, however, had a lower absorption maximum in the Soret region (417 +/- 1 nm) than the enzyme from other sources and was activated only one-fifth as much by nitric oxide as the bovine lung enzyme. It appears that the heme prosthetic group in the human placental enzyme may be hexa-coordinate and in the bovine lung enzyme the heme group may be penta-coordinate.     

Cation-dependent gonadotropin releasing hormone activation of guanylate cyclase

Summary Gonadotropin releasing hormone enhanced guanylate cyclase [E.C.4.6.1.2] two- to threefold in pituitary, testis, liver and kidney. Dose response relationships revealed that at a concentration of 1 nanomolar, gonadotropin releasing hormone caused a maximal augmentation of guanylate cyclase activity and that increasing its concentration to the millimolar range caused no further enhancement of this enzyme. There was an absolute cation requirement for gonadotropin releasing hormone's enhancement of guanylate cyclase activity as there was no increase without any cation present. Gonadotropin releasing hormone could increase guanylate cyclase activity with either calcium or manganese in the incubation medium but more augmentation was observed with manganese. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of gonadotropin releasing hormone.     

Superoxide dismutase catalyzes activation of synaptosomal soluble guanylate cyclase from rat brain

The hydrogen ion changes resulting from the photolysis of the rod visual pigment, rhodopsin, were investigated at acidic pH (5.2–6.5). After light-induced proton uptake, slow proton release occurred both in the dark and in the light. It was found that the amount of proton release in the dark was not equal to that in the light; about 0.9 proton remained bound to rhodopsin bleached in the dark, while all the bound protons were released in the light. Furthermore, the time course of proton release in the dark is not related to the decay of metarhodopsin II₃₈₀, but is closely related to the formation of metarhodopsin III₄₆₅.     

Cation-dependent substance P activation of the enzyme guanylate cyclase

Substance P enhanced guanylate cyclase (E.C.4.6.1.2) two- to fourfold in pancreas, small intestine, cerebellum, liver, kidney, and lung. Dose response relationship revealed that substance P caused a maximal augmentation of guanylate cyclase activity at concentration of 1 micromolar. Increasing substance P's concentration to the millimolar range caused no further increase in activity. There was an absolute cation requirement for substance P's enhancement of guanylate cyclase activity. Substance P could increase guanylate cyclase activity with either calcium or manganese in the incubation medium but more augmentation was observed with manganese. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of substance P.     

Potentiation of NO-dependent activation of soluble guanylate cyclase by polyamines

The influence of polyamines (putrescine, spermidine, and spermine) on the activity of human platelet soluble guanylate cyclase and the stimulation of the enzyme by sodium nitroprusside (SNP), YC-1 and their combination was investigated. All these polyamines stimulated the guanylate cyclase activity and potentiated its activation by sodium nitroprusside. The stimulatory effects of sodium nitroprusside and putrescine (or spermine) were addidive; spermidine produced a synergistic activation and increased the additive effect. All the polyamines inhibited the enzyme activation by YC-1 and decreased the synergistic activation of SNP-stimulated guanylate cyclase activity by YC-1 with nearly the same potency. The ability of the investigated polyamines to potentiate and to increase synergistically (similar to to YC-1, but less effective) NO-dependent activation of soluble guanylate cyclase represents a new biochemical effect of these compounds; this effect should be taken into consideration, especially due to the endogenous nature of polyamines. The data obtained suggest, that specific biological functions of polyamines in the processes of growth and differentiation of cells may be also related to the ability of compounds to activate soluble guanylate cyclase and to increase intracellular cGMP level.     

Reversible activation of soluble guanylate cyclase by oxidizing agents.

Soluble guanylate cyclase of human platelets was stimulated by thiol oxidizing compounds like diamide and the reactive disulfide 4, 4'-dithiodipyridine. Activation followed a bell-shaped curve, revealing somewhat different optimum concentrations for each compound, although in both cases, higher concentrations were inhibitory. Diamide at a concentration of 100 microM transiently activated the enzyme. In the presence of moderate concentrations of diamide and 4,4'-dithiodipyridine, causing a two- to fourfold activation by themselves, the stimulatory activity of NO-releasing compounds like sodium nitroprusside was potentiated. In contrast, higher concentrations of thiol oxidizing compounds inhibited the NO-stimulated activation of soluble guanylate cyclase. Activation of guanylate cyclase was accompanied by a reduction in reduced glutathione and a concomitant formation of protein-bound glutathione (protein-SSG). Both compounds showed an activating potency as long as reduced glutathione remained, leading to inhibition of the enzyme just when all reduced glutathione was oxidized. Activation was reversible while reduced glutathione recovered and protein-SSG disappeared. We propose that diamide or reactive disulfides and other thiol oxidizing compounds inducing thiol-disulfide exchange activate soluble guanylate cyclase. In this respect partial oxidation is associated with enzyme activation, whereas massive oxidation results in loss of enzymatic activity. Physiologically, partial disulfide formation may amplify the signal toward NO as the endogenous activator of soluble guanylate cyclase.     

Identification of residues crucially involved in soluble guanylate cyclase activation

The ubiquitous heterodimeric nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in various signal transduction pathways. Binding of NO takes place at the prosthetic heme moiety at the N-terminus of the beta(1)-subunit of sGC. The induced structural changes lead to an activation of the catalytic C-terminal domain of the enzyme and to an increased conversion of GTP into the second messenger cyclic GMP (cGMP). In the present work we selected and substituted different residues of the sGC heme-binding pocket based on a sGC homology model. The generated sGC variants were tested in a cGMP reporter cell for their effect on the enzyme activation by heme-dependent (NO, BAY 41-2272) stimulators and heme-independent (BAY 58-2667) activators. The use of these experimental tools allows the enzyme's heme content to be explored in a non-invasive manner. Asp(44), Asp(45) and Phe(74) of the beta(1)-subunit were identified as being crucially important for functional enzyme activation. beta(1)Asp(45) may serve as a switch between different conformational states of sGC and point to a possible mechanism of action of the heme dependent sGC stimulator BAY 41-2272. Furthermore, our data shows that the activation profile of beta(1)IIe(145) Tyr is unchanged compared to the native enzyme, suggesting that Tyr(145) does not confer the ability to distinguish between NO and O(2). In summary, the present work further elucidated intramolecular mechanisms underlying the NO- and BAY 41-2272-mediated sGC activation and raises questions regarding the postulated role of Tyr(145) for ligand discrimination.