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1.
Denninger JW Schelvis JP Brandish PE Zhao Y Babcock GT Marletta MA 《Biochemistry》2000,39(14):4191-4198
The enzyme-soluble guanylate cyclase (sGC), which converts GTP to cGMP, is a receptor for the signaling agent nitric oxide (NO). YC-1, a synthetic benzylindazole derivative, has been shown to activate sGC in an NO-independent fashion. In the presence of carbon monoxide (CO), which by itself activates sGC approximately 5-fold, YC-1 activates sGC to a level comparable to stimulation by NO alone. We have used kinetic analyses and resonance Raman spectroscopy (RR) to investigate the interaction of YC-1 and CO with guanylate cyclase. In the presence of CO and 200 microM YC-1, the V(max)/K(m GTP) increases 226-fold. While YC-1 does not perturb the RR spectrum of the ferrous form of baculovirus/Sf9 cell expressed sGC, it induces a shift in the Fe-CO stretching frequency for the CO-bound form from 474 to 492 cm(-1). Similarly, YC-1 has no effect on the RR spectrum of ferrous beta1(1-385), the isolated sGC heme-binding domain, but shifts the nu(Fe-CO) of CO-beta1(1-385) from 478 to 491 cm(-1), indicating that YC-1 binds in heme-binding region of sGC. In addition, the CO-bound forms of sGC and beta1(1-385) in the presence of YC-1 lie on the nu(Fe-CO) vs nu(C-O) correlation curve for proximal ligands with imidazole character, which suggests that histidine remains the heme proximal ligand in the presence of YC-1. Interestingly, YC-1 does not shift nu(Fe-CO) for the CO-bound form of H105G(Im), the imidazole-rescued heme ligand mutant of beta1(1-385). The data are consistent with binding of CO and YC-1 to the sGC heme-binding domain leading to conformational changes that give rise to an increase in catalytic turnover and a change in the electrostatic environment of the heme pocket. 相似文献
2.
Soluble guanylate cyclase (sGC, EC 4.6.1.2) acts as a sensor for nitric oxide (NO), but is also activated by carbon monoxide in the presence of an allosteric modulator. Resonance Raman studies on the structure-function relations of sGC are reviewed with a focus on the CO-adduct in the presence and absence of allosteric modulator, YC-1, and substrate analogues. It is demonstrated that the sGC isolated from bovine lung contains one species with a five-coordinate (5c) ferrous high-spin heme with the Fe-His stretching mode at 204 cm(-1), but its CO adduct yields two species with different conformations about the heme pocket with the Fe-CO stretching (nuFe-CO) mode at 473 and 489 cm(-1), both of which are His- and CO-coordinated 6c ferrous adducts. Addition of YC-1 to it changes their population and further addition of GTP yields one kind of 6c (nuFe-CO=489 cm(-1)) in addition to 5c CO-adduct (nuFe-CO=521 cm(-1)). Under this condition the enzymatic activity becomes nearly the same level as that of NO adduct. Addition of gamma-S-GTP yields the same effect as GTP does but cGMP and GDP gives much less effects. Unexpectedly, ATP cancels the effects of GTP. The structural meaning of these spectroscopic observations is discussed in detail. 相似文献
3.
E M Becker F Wunder R Kast C Robyr M Hoenicka R Gerzer H Schr?der J P Stasch 《Nitric oxide》1999,3(1):55-66
A stably transfected soluble guanylate cyclase (sGC, alpha1 and beta1 subunits of the rat lung enzyme)-overexpressing CHO cell line was generated for the characterization of different types of activators of the soluble guanylate cyclase. Polyclonal antibodies directed against both subunits of the rat enzyme were used to detect both subunits in the cytosol of the transfected CHO cells. We studied the effects of different nitric oxide (NO) donors like SNP and DEA/NO and, in particular, the direct, NO-independent stimulator of the soluble guanylate cyclase 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1), on intracellular guanosine 3',5'-cyclic monophosphate (cGMP) production. DEA/NO (0.01-3 microM), SNP (1-10 microM), and YC-1 (1-10 microM) induced a concentration-dependent intracellular cGMP increase with maximal effects of 16-fold (3 microM DEA/NO), 8-fold (10 microM SNP), and 6-fold (10 microM YC-1) stimulation compared to controls, respectively. In addition, a synergistic effect of the combination of the NO donor and YC-1 could be observed with a maximal stimulation of 64-fold by SNP (10 microM) and YC-1 (10 microM). 1H-(1,2,4)-Oxadiazolo-(4,3-a)-6-bromo-quinoxazin-1-one (ODQ, 10 microM), a potent and selective inhibitor of sGC, inhibited both the single effects of NO donors [DEA/NO (3 microM), 77%; SNP (3 microM), 83%] and YC-1 [YC-1 (3 microM), 82%], but moreover the synergistic effects between NO donors and YC-1 [DEA/NO (3 microM) + YC-1 (3 microM), 81%; SNP (3 microM) + YC-1 (3 microM),89%] on intracellular cGMP production. In summary,we have generated a simple, sensitive, and useful bioassay method to characterize all types of sGC activators on the cellular level without the need of primary cell culture, several transfections, or purifying enzyme from biological materials. 相似文献
4.
Miñana MD Corbalán R Montoliu C Teng CM Felipo V 《Biochemical and biophysical research communications》1999,257(2):405-409
Chronic hyperammonemia impairs the glutamate-nitric oxide-cGMP pathway in rat brain in vivo. The aims of this work were to assess whether hyperammonemia impairs modulation of soluble guanylate cyclase, and to look for a peripheral marker for impairment of this pathway in brain. We activated the pathway at different steps using glutamate, SNAP, or YC-1. In control neurons these compounds increased cGMP by 7.4-, 9.7- and 7.2-fold, respectively. In ammonia-treated neurons formation of cGMP induced by glutamate, SNAP, and YC-1 was reduced by 50%, 56%, and 52%, respectively, indicating that hyperammonemia impairs activation of guanylate cyclase. This enzyme is also present in lymphocytes. Activation of guanylate cyclase by SNAP or YC-1 was impaired in lymphocytes from hyperammonemic rats. These results suggest that determination of the activation of soluble guanylate cyclase in lymphocytes could serve as a peripheral marker for impairment of the neuronal glutamate-nitric oxide-cGMP pathway in brain. 相似文献
5.
I. S. Severina N. V. Pyatakova A. Ya. Shchegolev 《Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry》2007,1(3):254-257
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. 相似文献
6.
Particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing] has been cytochemically evidentiated in the cells which make-up the lung air-blood barrier. The cytochemical procedure utilized demonstrates the presence of membrane-bound guanylate cyclase activity through precipitation of lead pyrophosphate in tissues incubated with GTP or with guanylyl imidodiphosphate. Electron microscopic examination reveals that guanylate cyclase (GC) is localized, as micropinocytic vesicles, within endothelial components of small blood vessels, in basal lamina and in the flat alveolar cells. The secretory alveolar cells also exhibit the positive GC reactivity in their peripheric cytoplasm and in their microvilli. The observations support that GC and cGMP are involved in cellular transport phenomena. The enzyme might play a role in the secretion process of surface active material. Positive staining has been found also in other types of cells, namely alveolar macrophages and fibroblasts. A biochemical evaluation of GC activity shows that about 30-40% of this activity is associated with the particulate fraction, which justifies its abundance in the cytochemical reports shown in the paper. 相似文献
7.
Resonance Raman (RR) spectra of soluble guanylate cyclase (sGC) reported by five independent research groups have been classified as two types: sGC(1) and sGC(2). Here we demonstrate that the RR spectra of sGC isolated from bovine lung contain only sGC(2) while both species are observed in the spectra of the CO-bound form (CO-sGC). The relative populations of the two forms were altered from an initial composition in which the CO-sGC(2) form predominated, with the Fe-CO (nu(Fe)(-)(CO)) and C-O stretching modes (nu(CO)) at 472 and 1985 cm(-)(1), respectively, to a composition dominated by the CO-sGC(1) form with nu(Fe)(-)(CO) and nu(CO) at 488 and 1969 cm(-)(1), respectively, following the addition of a xenobiotic, YC-1. Further addition of a substrate, GTP, completed the change. GDP and cGMP had a significantly weaker effect, while a substrate analogue, GTP-gamma-S, had an effect similar to that of GTP. In contrast, ATP had a reverse effect, and suppressed the effects of YC-1 and GTP. In the presence of both YC-1 and GTP, vinyl vibrations of heme were significantly influenced. New CO isotope-sensitive bands were observed at 521, 488, 363, and 227 cm(-)(1). The 521 cm(-)(1) band was assigned to the five-coordinate (5c) species from the model compound studies using ferrous iron protoporphyrin IX in CTAB micelles. Distinct from the 472 cm(-)(1) species, both the 488 and 521 cm(-)(1) species were apparently un-photodissociable when an ordinary Raman spinning cell was used, indicating rapid recombination of photodissociated CO. On the basis of these findings, binding of YC-1 to the heme pocket is proposed. 相似文献
8.
Soluble guanylate cyclase (GC) from bovine lung is activated 4-fold by carbon monoxide (CO) and 400-fold by nitric oxide (NO). Spectroscopic and kinetic data for ligation of CO and NO with GC are summarized and compared with similar data for myoglobin (Mb), hemoglobin (Hb), and heme model compounds. Kinetic, thermodynamic, and structural data form a basis on which to construct a model for the manner in which the two ligands affect protein structure near the heme for heme proteins in general and for GC in particular. The most significant datum is that although association rates of ligands with GC are similar to those with Mb and Hb, their dissociation rates are dramatically faster. This suggests a delicate balance between five- and six-coordinate heme iron in both NO and CO complexes. Based on these and other data, a model for GC activation is proposed: The first step is formation of a six-coordinate species concomitant with tertiary and quaternary structural changes in protein structure and about a 4-fold increase in enzyme activity. In the second step, applicable to NO, the bond from iron to the proximal histidine ruptures, leading to additional relaxation in the quaternary and tertiary structure and a further 100-fold increase in activity. This is the main event in activation, available to NO and possibly other activators or combinations of activators. It is proposed, finally, that the proximal base freed in step 2, or some other protein base suitably positioned as a result of structural changes following ligation, may provide a center for nucleophilic substitution catalyzing the reaction GTP --> cGMP. An example is provided for a similar reaction in a derivatized protoheme model compound. The reaction mechanism attempts to rationalize the relative enzymatic activities of GC, heme-deficient GC, GC-CO, and GC-NO on a common basis and makes predictions for new activators that may be discovered in the future. 相似文献
9.
The review highlights the molecular mechanism underlying the physiological effects of nitric oxide (NO), the role of signaling system: NO-soluble guanylate cyclase-cyclic 3′,5′-guanosine monophosphate (cGMP) in the realization of NO action. This review considers data on basic chemical characteristics of guanylate cyclase, such as the subunits structure, isoforms, modern concepts of the catalytic and regulatory centers of this enzyme. Realization of physiological effects of NO by guanylate cyclase depends on its heme prostetic group. NO-dependent activation of guanylate cyclase may be synergistically increased by a new NO-independent, allosteric activator of soluble guanylate cyclase-YC-1-(benzyl indasol derivative). Special attention is paid to the data on guanylate cyclase sites responcible for binding of the enzyme with YC-1 and the possible molecular mechanism underlying the synergistic increase of NO-dependent activation of soluble guanylate cyclase by YC-1. New compounds of endogenous nature capable to potentiate and synergistically increase the activation of guanylate cyclase by NO-donors have been found and investigated. The important physiological, pharmacotherapeutical and pathophysiological significance of this new fact is discussed. 相似文献
10.
Regulatory properties of magnesium-dependent guanylate cyclase in Dictyostelium discoideum membranes 总被引:6,自引:0,他引:6
P M Janssens C C De Jong A A Vink P J Van Haastert 《The Journal of biological chemistry》1989,264(8):4329-4335
We have characterized a magnesium-dependent guanylate cyclase in homogenates of Dictyostelium discoideum cells. 1) The enzyme shows an up to 4-fold higher cGMP synthesis in the presence of GTP analogues with half-maximal activation at about 1 microM guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or 100 microM guanosine 5'-(beta, gamma-imido)triphosphate; little or no stimulation was observed with GTP, guanosine mono- and diphosphates or with adenine nucleotides, with the exception of the ATP analogue adenosine 5'-(beta, gamma-imido)triphosphate. 2) Both basal and GTP gamma S-stimulated guanylate cyclase activity were rapidly lost from homogenates as was the ability of GTP gamma S to stimulate the enzyme after cell lysis. 3) Inclusion of 25 microM GTP gamma S during cell lysis reduced the KM for GTP from 340 to 85 microM and increased the Vmax from 120 to 255 pmol/min.mg protein, as assayed in homogenates 90 s after cell lysis. 4) Besides acting as an activator, GTP gamma S was also a substrate for the enzyme with a KM = 120 microM and a Vmax = 115 pmol/min.mg protein. 5) GTP gamma S-stimulated, Mg2+-dependent guanylate cyclase was inhibited by submicromolar concentrations of Ca2+ ions, and by inositol 1,4,5-trisphosphate in the absence of Ca2+ chelators. 6) Guanylate cyclase activity was detected in both supernatant and pellet fractions after 1 min centrifugation at 10,000 x g; however, only sedimentable enzyme was stimulated by GTP gamma S. We suggest that the Mg2+-dependent guanylate cyclase identified represents the enzyme that in intact cells is regulated via cell surface receptors, and we propose that guanine nucleotides are allosteric activators of this enzyme and that Ca2+ ions play a role in the maintenance of the enzyme in its basal state. 相似文献
11.
Iu Iu Chirkov I A Tyshchuk N N Belushkina I S Severina 《Biokhimii?a (Moscow, Russia)》1987,52(6):956-963
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. 相似文献
12.
The increase of cGMP by atrial natriuretic factor correlates with the distribution of particulate guanylate cyclase 总被引:20,自引:0,他引:20
We have demonstrated previously that atrial natriuretic factor (ANF) augments urinary, plasma and kidney cGMP levels but has no significant effect upon cAMP. Using cGMP as a marker, we searched for specific target sites involved in the action of ANF in the dog kidney, and observed no change of cGMP in the proximal tubules, a 2-fold increase over basal levels in the thick loop of Henle and a 3-fold elevation in the collecting duct. The most striking action on cGMP occurred in the glomeruli with a rise of up to 50-fold being evident at 1-2 min. after the addition of ANF. The results obtained in the absence or presence of a phosphodiesterase inhibitor support the notion that the effects of ANF were exerted at the level of guanylate cyclase stimulation rather than cGMP phosphodiesterase inhibition. The action of sodium nitroprusside (SNP), a direct stimulator of soluble guanylate cyclase, differed from that of ANF. The ability of the factor to enhance cGMP levels was correlated with the distribution of particulate guanylate cyclase. This study identifies the glomeruli and the distal part of the nephron as specific targets of ANF and implicates particulate guanylate cyclase as the enzyme targetted for the expression of its action. 相似文献
13.
Soluble guanylate cyclase (sGC) uses a ferrous heme cofactor as a receptor for NO and once bound activates the enzyme for the conversion of GTP to cGMP. The heme cofactor in sGC does not bind oxygen, thereby allowing it to selectively bind NO despite a cellular concentration of oxygen (microM) that is much higher than signaling concentrations of nitric oxide (nM). The molecular details of this ligand discrimination against oxygen have emerged and allowed for predictions regarding ligand specificity in the sGC family. The results reported here show that Gyc-88E from Drosophila is a hemoprotein that binds oxygen, as well as NO and CO. All three ligands form 6-coordinate complexes. Gyc-88E is active as a homodimer (5600 +/- 243 nmol min(-1) mg(-1)) and is inhibited by O2, CO, and NO (3.2-, 2.9-, and 2-fold, respectively). The Km for GTP was 0.66 +/- 0.15 mM in air (273 microM oxygen) and 0.82 +/- 0.15 mM under anaerobic conditions. The Ki for oxygen was calculated to be 51 +/- 28 microM. The biochemical properties of Gyc-88E are unique for guanylate cyclases and suggest a possible function as an oxygen sensor. 相似文献
14.
Hu X Murata LB Weichsel A Brailey JL Roberts SA Nighorn A Montfort WR 《The Journal of biological chemistry》2008,283(30):20968-20977
Soluble guanylyl/guanylate cyclase (sGC), the primary biological receptor for nitric oxide, is required for proper development and health in all animals. We have expressed heterodimeric full-length and N-terminal fragments of Manduca sexta sGC in Escherichia coli, the first time this has been accomplished for any sGC, and have performed the first functional analyses of an insect sGC. Manduca sGC behaves much like its mammalian counterparts, displaying a 170-fold stimulation by NO and sensitivity to compound YC-1. YC-1 reduces the NO and CO off-rates for the approximately 100-kDa N-terminal heterodimeric fragment and increases the CO affinity by approximately 50-fold to 1.7 microm. Binding of NO leads to a transient six-coordinate intermediate, followed by release of the proximal histidine to yield a five-coordinate nitrosyl complex (k(6-5) = 12.8 s(-1)). The conversion rate is insensitive to nucleotides, YC-1, and changes in NO concentration up to approximately 30 microm. NO release is biphasic in the absence of YC-1 (k(off1) = 0.10 s(-1) and k(off2) = 0.0015 s(-1)); binding of YC-1 eliminates the fast phase but has little effect on the slower phase. Our data are consistent with a model for allosteric activation in which sGC undergoes a simple switch between two conformations, with an open or a closed heme pocket, integrating the influence of numerous effectors to give the final catalytic rate. Importantly, YC-1 binding occurs in the N-terminal two-thirds of the protein. Homology modeling and mutagenesis experiments suggest the presence of an H-NOX domain in the alpha subunit with importance for heme binding. 相似文献
15.
The effects of sodium azide on guanylate cyclase activity of homogenates of rat renal cortex and on the guanosine 3':5'-monophosphate (cGMP) content of cortical slices were examined and compared to those of carbamylcholine and NaF. In complete Krebs-Ringer bicarbonate buffer containing 10 mM theophylline, tissue cGMP content was increased 5- to 6-fold by 0.05 mM carbamylcholine or 10 mM NaN3, and 3-fold by 10 mM NaF. Increases in cGMP were maximal in response to these concentrations of the agonists and occurred within 2 min. Exclusion of Ca2+ from the incubation media reduced basal cGMP by 50% in 20 min and abolished responses to carbamylcholine and NaF, while exclusion of Mg2+ was without effect. Analogous reductions in cGMP were observed in complete buffer containing 1 mM tetracaine, an agent which blocks movement of Ca2+ across and binding to biologic membranes. By contrast, exclusion of Ca2+ or addition of tetracaine did not alter relative cGMP responses to NaN3 (6-fold increase over basal), although levels were reduced in slices exposed to these buffers for 20 min. When slices were incubated without Ca2+ or with tetracaine for only 2 min prior to addition of agonists, basal cGMP did not decline. Under these conditions, both absolute and relative increases in cGMP in response to NaN3 were comparable to those of slices incubated throughout in complete buffer, while carbamylcholine and NaF effects on cGMP were abolished. NaN3 increased guanylate cyclase activity of whole homogenates (10- to 20-fold), and of the 100,000 X g soluble (20-fold) and particulate (4-fold) fractions of cortex. Prior incubation of slices with NaN3 in the presence or absence of Ca2+ or with Ca2+ plus tetracaine also markedly enhanced enzyme activity in homogenates and subcellular fractions subsequently prepared from these slices. In the presence of 3 mM excess MnCl2, NaN3 raised the apparent Km for MnGTP of soluble guanylate cyclase from 0.11 mM to 0.20 mM, and reduced enzyme dependence on Mn2+. Thus, when Mg2+ was employed as the sole divalent cation in the enzyme reaction mixture basal and NaN3-responsive activities were 7% and 30% of those seen with optimal concentrations of Mn2+, respectively. Under a variety of assay conditions where responses to NaN3 were readily detectable, alterations in guanylate cyclase activities could not be demonstrated in response to carbamylcholine or NaF. By contrast Ca2+ increased the guanylate cyclase activity 6- to 7-fold over basal under conditions of reduced Mn2+ (0.75 mM Mn2+/1 mM GTP). This latter effect of Ca2+ was shared by Mg2+ and not blocked by tetracaine. Carbamylcholine, NaF, Ca2+, and NaN3 all failed to alter cGMP phosphodiesterase activity in cortex. Thus, while carbamylcholine and NaF enhance renal cortical cGMP accumulation through actions which are dependent upon the presence of extracellular Ca2+, NaN3 stimulates cGMP generation in this tissue through an apparently distinct Ca2+-independent mechanism. 相似文献
16.
The guanosine 3',5'-cyclic monophosphate (cGMP) level in the mouse splenic lymphocytes was increased about 2- to 3-fold by concanavalin A. This increase was completely dependent on the presence of Ca2+ in the medium. Homogenates of mouse splenic lymphocytes contained significant guanylate cyclase [EC 4.6.1.2] activity in both the 105,000 X g (60 min) particulate and supernatant fractions and both fractions required Mn2+ for full activity. Calcium ion (3mM) activated soluble guanylate cyclase 3-fold at a relatively low concentration of Mn2+ (less than 1mM) but inhibited the particulate enzyme slightly at all Mn2+ concentrations tested. Concanavalin A itself did not stimulate either fraction of guanylate cyclase. Thus these results suggest that elevation of the cGMP level in lymphocytes by concanavalin A might be brought about by stimulation of Ca2+ uptake and activation of soluble guanylate cyclase by the latter. 相似文献
17.
18.
Regulation of intracellular cyclic GMP concentration by light and calcium in electropermeabilized rod photoreceptors 总被引:2,自引:0,他引:2
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《The Journal of general physiology》1994,103(1):67-86
This study examines the regulation of cGMP by illumination and by calcium during signal transduction in vertebrate retinal photoreceptor cells. We employed an electropermeabilized rod outer segment (EP-ROS) preparation which permits perfusion of low molecular weight compounds into the cytosol while retaining many of the features of physiologically competent, intact rod outer segments (ROS). When nucleotide-depleted EP-ROS were incubated with MgGTP, time- and dose- dependent increases in intracellular cGMP levels were observed. The steady state cGMP concentration in EP-ROS (0.007 mol cGMP per mol rhodopsin) approached the cGMP concentration in intact ROS. Flash illumination of EP-ROS in a 250-nM free calcium medium resulted in a transient decrease in cGMP levels; this occurred in the absence of changes in calcium concentration. The kinetics of the cGMP response to flash illumination of EP-ROS were similar to that of intact ROS. To further examine the effects of calcium on cGMP metabolism, dark-adapted EP-ROS were incubated with MgGTP containing various concentrations of calcium. We observed a twofold increase in cGMP steady state levels as the free calcium was lowered from 1 microM to 20 nM; this increase was comparable to the behavior of intact ROS. Measurements of guanylate cyclase activity in EP-ROS showed a 3.5-fold increase in activity over this range of calcium concentrations, indicating a retention of calcium regulation of guanylate cyclase in EP-ROS preparations. Flash illumination of EP-ROS in either a 50- or 250-nM free calcium medium revealed a slowing of the recovery time course at the lower calcium concentration. This observation conflicts with any hypothesis whereby a reduction in free calcium concentration hastens the recovery of cytoplasmic cGMP levels, either by stimulating guanylate cyclase activity or by inhibiting phosphodiesterase activity. We conclude that changes in the intracellular calcium concentration during visual transduction may have more complex effects on the recovery of the photoresponse than can be accounted for solely by guanylate cyclase activation. 相似文献
19.
The partially purified soluble guanylate cyclase (GTP pyrophosphatelyase(cyclizing), EC 4.6.1.2) from human caudate nucleus is stimulated from 2 to 4-fold by metal chelating agents. EDTA (K 1/2 - 4.8 microM) is more potent than CDTA (K 1/2 = 13.2 microM) or EGTA (K 1/2 = 21.8 microM) at stimulating activity. Stimulation by chelating agents is apparently not due to removal of inhibitory divalent cations which contaminate the enzyme or reaction mixture. EDTA increases guanylate cyclase activity in part by increasing the affinity of the enzyme for the substrate (MgGTP) 10-fold. Dopamine inhibits partially purified guanylate cyclase in the presence or absence of EDTA. Dopamine increases the Ka of guanylate cyclase for the activator, free Mn2+, more than 50-fold, from 3 to 150 microM. 相似文献
20.
Guanylate cyclase activity (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2.), measured in purified rat liver plasma membranes, was markedly increased by treatment with various purified proteases. The effect was maximal with trypsin, alpha-chymotrypsin, papain, and thermolysin (6- to 8-fold increase with 5 to 20 microgram of protease/ml) and lower with subtilisin and elastase (3- to 4-fold increase). The activation was due to an increase in the maximal velocity of the cyclizing reaction. No modification was observed either in the apparent affinity for the substrate MnGTP or in the cooperative behavior of the enzyme kinetics which displayed Hill coefficients of 1.6 for both basal and activated states. The Triton X-100-dispersed guanylate cyclase remained sensitive to papain, which suggests that the action of proteases was not restricted to an indirect action upon the membranous environment of the guanylate cyclase. In contrast, the cytosolic soluble guanylate cyclase, assayed in the presence or absence of sodium azide, was absolutely insensitive to papain. Thus, proteolysis represents a previously undescribed mechanism for activating membranous guanylate cyclase systems, which might be of importance in the physiological regulation of this enzyme. 相似文献