Characterization of ATP-stimulated guanylate cyclase activation in rat lung membranes

Many of the effects of ANP are mediated through the elevation of cellular cGMP levels by the activation of particulate guanylate cyclase. While the stimulation of this enzyme is receptor-mediated, the molecular mechanism of activation remains unknown. In this study we present evidence that ATP as well as its analogues adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) and adenylylimidophosphate (AMPPNP) activates guanylate cyclase from rat lung membranes and markedly potentiates the effect of ANP on the enzyme. The order of potency is ATP gamma S greater than ATP greater than AMPPNP. The enzyme activation by adenine nucleotide and ANP together is much more than the sum of the individual activations, suggesting that ATP may be the physiological component essential for the ANP-stimulated guanylate cyclase activation. The ATP gamma S-stimulated guanylate cyclase activity diminishes in the presence of various kinds of detergents, suggesting either that the conformation of an ATP binding site in guanylate cyclase is altered by detergents or that protein-protein interaction may be involved in the activation of guanylate cyclase by ATP. Guanylate cyclase from rat lung membranes is poorly activated by ANP and/or ATP gamma S after removing the cytosolic and weakly membrane-associated proteins or factors by centrifugation. Pre-incubation of the membranes with ATP gamma S retains enzyme activation after membrane washing. These results suggest either that ATP gamma S stabilizes the conformation of nucleotide binding site in guanylate cyclase from denaturation by membrane washing, or that the stimulatory effect of ATP on guanylate cyclase activity may be mediated by accessory proteins or non-protein cofactors which are lost during membrane washing, but remain bound to membranes by ATP gamma S pretreatment.     

Tissue-specific effects of divalent cations and activators on soluble guanylate cyclase

The interactions of divalent cations, Mn²⁺, Mg²⁺, and Ca²⁺, with the cytosolic guanylate cyclase (GTP pyrophosphate-lyase (cyclizing, EC 4.6.1.2) from different tissues were studied. Guanylate cyclase activities of the kidney, liver, and lung were strongly dependent on Mn²⁺. In contrast, the enzyme in smooth muscle of the colon, aorta, and vas diferens was active with Mg²⁺ as well as with Mn²⁺. Ca²⁺ was ineffective in all tissues. Preincubation, at 30°C, of colon extracts, but not those of kidney and liver, increased guanylate cyclase activity. The Mg²⁺-dependent activity was preferentially enhanced by this treatment. These results suggest that when the enzyme was autoactivated by endogenus factors it became more Mg²⁺ dependent. Dithiothreitol strongly inhibited the Mg²⁺-dependent colon enzyme, whereas activities in kidney and liver were not affected and the response of the enzyme in lung was intermediate. This suggests that autoactivation involved an oxidative-reductive alteration of the enzyme. Ca²⁺ markedly inhibited the Mg²⁺-dependent activity in smooth muscles but Mg²⁺-dependent activities in lung, liver, and kidney were not influenced appreciably. Exogenous activators, dehydroascorbate and NaN₃, increased guanylate cyclase, assayed with either Mg²⁺ or Mn²⁺. However, the relative stimulation of the enzyme assayed with Mg²⁺ was greater than with Mn²⁺. When activated by these exogenous agents, guanylate cyclase in all tissues became inhibitable by Ca²⁺. These findings suggest that guanylate cyclase in smooth muscle, as prepared, was in a partially activated form.The endogenous activating factors in colon smooth muscle were heat-stable, largely extractable with chloroform/methanol, and cochromatographed with authentic fatty acids. Arachidonic acid stimulated colon guanylate cyclase and enhancement of the Mg²⁺-dependent activity was blocked by Ca²⁺. This strongly infers that a significant part of the endogenous activating factors in the colon was fatty acids or their derivatives. The colon activators had only minimal affects on the enzyme in the kidney. Similarly prepared activator extracts from the kidney increased colon guanylate cyclase but did not stimulate the renal enzyme. Thus, the ability of the enzyme to be stimulated by endogenous activators was dependent on the tissue from which the enzyme was derived.The possibility that the interactions of Mg²⁺ and Ca²⁺ on guanylate cyclase in smooth muscles are of regulatory significance to the contractile response of the muscle was discussed. It was proposed as a working hypothesis that cyclic GMP and Ca²⁺ might participate in reciprocal negative feedback mechanisms.     

Modulation by substrate and cations of guanylate cyclase activity in detergent-dispersed plasma membranes from rat adipocytes.

Guanylate cyclase activity in Triton X-100-treated plasma membranes exhibits sigmoidal profiles as a function of MgGTP, irrespective of the excess Mg²⁺⁽¹⁾ concentration. In contrast, at low excess Mn²⁺ (0.2 mM) the activity vs substrate (MnGTP) concentration profile corresponds to a michaelian behaviour. In addition the enzyme does not require similar excess Mn²⁺ and Mg²⁺ for optimal activity at various substrate concentrations. Moreover, low concentrations of Ca²⁺ are capable of stimulating guanylate cyclase activity with Mg²⁺ as the major divalent cation.     

Membrane lipids can modulate guanylate cyclase activity of rat intestinal microvillus membranes

Studies of the temperature dependence (10-40 degrees C) of guanylate cyclase in rat intestinal microbillus membranes reveal a change in energy of activation (slope of the Arrhenius plot) at 30 +/- 1 degree C. The break point temperature corresponds to the lipid thermotropic transition in these membranes previously characterized by differential scanning calorimetry (range: 23-39 degrees C; peak temperature, 31 degrees C). The break point temperature for guanylate cyclase also corresponds to that of a number of other microbillus membrane enzymes and of D-glucose transport. These activities are defined as "intrinsic" membrane activities by this operational criterion. Treatment with the nonionic detergent Lubrol WX increased the guanylate cyclase activity 4- to 8-fold and removed the discontinuity in the Arrhenius plot.     

The influence of the divalent cations Mn2+ and Mg2+ on the activation of particulate and digitonin-solubilized adenylate cyclase from rat fat cell membranes

A comparison was made between the activation of membrane-bound adenylate cyclase from rat fat cell membranes and the enzyme solubilized with digitonin. The isoprenaline stimulation of the particulate enzyme was enhanced by GTP, both in the presence of Mg²⁺ and Mn²⁺, but no effect of the metal ion nor of GTP was found on the K_a of isoprenaline. The K_a of sodium fluoride for enzyme stimulation was shifted to 3-fold higher concentrations when Mg²⁺ was replaced by Mn²⁺, whereas V decreased. GTP did not influence the K_a of sodium fluoride but reduced V, irrespective of the metal ion. After digitonin solubilization the enzyme was no longer responsive to isoprenaline or GTP; however, V of the sodium fluoride activation was higher in the presence of Mn²⁺ than in the presence of Mg²⁺, and the K_a was found at 15-fold higher concentrations. Both the solubilized and the particulate adenylate cyclase were inhibited by adenosine; this inhibition was also seen with the fluoride stimulated enzyme. We conclude that solubilization with digitonin did not result in an enzyme preparation which preferentially turns over MnATP²⁺, although the fat cell adenylate cyclase possesses a metal ion regulatory site with a higher affinity for Mn²⁺ than for Mg²⁺. The data suggest that the guanyl nucleotide regulatory site and the sodium fluoride-sensitive site are located on different subunits while there is an interaction between the metal ion regulatory site and the fluoride-sensitive site.     

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.     

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%.     

Protoporphyrin IX activates the Mg dependent guanylate cyclase from rat liver plasma membranes

In the presence of Mg-GTP, the rat liver guanylate cyclase, in either intact membranes or trypsin solubilized form, was stimulated by protoporphyrin IX 6 to 10-fold. However, when Mn-GTP was the substrate, protoporphyrin IX activated the membrane-bound guanylate cyclase only 50%, in contrast to the marked activation reported for the cytosolic enzyme. Meso- and deuteroporphyrin IX, hematoporphyrin and coproporphyrin III also activated membrane guanylate cyclase while uroporphyrin III, and hemin had no effect. Basal, Mg2+-dependent activity exhibited two classes of catalytic sites with apparent Km values of 2 mM and 0.12 mM. Activation by protoporphyrin resulted in the disappearance of the low affinity sites. The activated enzyme exhibited Michaelis-Menten kinetics and no alteration in its requirement for excess Mg2+. These data indicate that, in the presence of Mg2+, a heme-like structure can interact with the membrane-bound guanylate cyclase and regulate 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+.     

Calcium reveals different mechanisms of guanylate cyclase activation by atrial natriuretic factor and ATP in rat lung membranes.

CaCl2 inhibited ATP-stimulated guanylate cyclase activity, but had little effect on basal and atrial natriuretic factor-stimulated guanylate cyclase activity in rat lung membranes. LaCl3 had similar effects as CaCl2 on basal and stimulated guanylate cyclase activity. LiCl and other monovalent salts inhibited ATP-stimulated guanylate cyclase activity more than basal enzyme activity. However, atrial natriuretic factor somehow stabilized the enzyme against the inhibitory effect of LiCl. These results suggest that ATP and atrial natriuretic factor activate the enzyme through different mechanisms. Since the effect of calcium on guanylate cyclase activity is different from that of monovalent salts and can be mimicked by lanthanum, it may be mediated by a specific calcium binding site or binding protein.     

Kinetic studies of adenylyl cyclase of fat cell membranes

Summary The kinetic behavior of the adenylyl cyclase activity associated with fat cell membranes purified by centrifugation on sucrose gradients was studied. Under most of the conditions explored, with either Mn⁺⁺ or Mg⁺⁺ as the divalent cation in the assay mixtures, the time courses of the reaction were not linear. In the absence of modifiers (i.e., basal activity) or in the presence of insulin, the rate tended to decrease with time; on the other hand, with fluoride or GMP-P(NH)P the curves were concave upwards. To simplify analysis of the results, two kinetic components were defined: an initial component corresponding to the transient rate measured between zero time and 1.5 min of assay and a final component corresponding to the transient rate determined between 3 and 5 min.Over the entire range of Mn⁺⁺ concentration explored (0.5 to 6.0mm), the basal initial rates were slightly higher than the final ones. With Mg⁺⁺ in the range between 1.5 and 2.5mm, the final rates were fourfold lower than the initial ones. Higher or lower Mg⁺⁺ concentrations gave velocity ratios equivalent to those observed with Mn⁺⁺.Insulin clearly decreased the final rates at Mn⁺⁺ concentrations up to 2.5mm. With higher concentrations the effects were completely reversed. The effects of insulin on initial rates measured with Mn⁺⁺, or the initial or final rates measured with Mg⁺⁺, were less evident.Stimulation of adenylyl cyclase activity by fluoride was most pronounced on the final rates. In addition, this stimulation was higher with Mg⁺⁺ than with Mn⁺⁺.Isoproterenol stimulation of adenylyl cyclase was negligible in the presence of Mn⁺⁺ (0.5 to 6.0mm). With Mg⁺⁺ (0.5 to 6.0mm), stimulation was more evident on the final rates. *** DIRECT SUPPORT *** A0130063 00002     

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.     

Atrial natriuretic peptide receptors and activation of guanylate cyclase in rat cardiac sarcolemma

Two classes of atrial natriuretic peptide (ANP) receptors are present in purified sarcolemmal membrane fractions isolated from rat ventricle. Scatchard analysis using [125I]-ANP reveals high affinity (Kd approximately 10(-11) M) and low affinity (Kd approximately 10(-9) M) binding sites. Basal guanylate cyclase activities associated with these membrane fractions range from 3.2 +/- 1.3 pmol/min/mg protein in the presence of Mg2+ to 129 +/- 17 pmol/min/mg protein in the presence of Mn2+. Millimolar concentrations of adenosine triphosphate (ATP) potentiates Mg2+- but not Mn2+-supported activity. Binding of ANP to the low affinity site but not the high affinity site results in a maximum 2-fold activation of Mn2+- and up to 6-fold activation of Mg2+/ATP supported guanylate cyclase activities.     

Phlorizin-receptor interactions in fat cell plasma membranes

The rate but not the extent of phlorizin binding to purified fat cell plasma membranes was temperature dependent and this binding was a saturable process. A Scatchard plot revealed a population of sites which exhibited a dissociation constant of about 0.35 mM and a maximum binding capacity of about 8 nmoles/mg membrane protein. Under the conditions of these experiments neither glucose, phloretin, nor cytochalasin B inhibited [³H]phlorizin binding. These data demonstrate the presence in fat cell plasma membrane of specific receptors for phlorizin which may mediate the inhibitory effects of this agent on hexose trasport.