Regulatory Properties of Magnesium-dependent Guanylate Cyclase in Dictyostelium discoideum Membranes

We have characterized a magnesium-dependent guanylate cyclase in homogenates of Dictyostelium discoi- deum 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 FM guanosine 5’-0-(3-thio)triphosphate (GTPyS) or 100 PM guanosine 5’-(,!?,y-imid0)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’-@,y-imido)triphosphate. 2) Both basal and GTPyS-stimulated guanylate cyclase activity were rapidly lost from homogenates as was the ability of GTPyS to stimulate the enzyme after cell lysis. 3) Inclusion of 25 FM GTPyS during cell lysis reduced the KM for GTP from 340 to 85 PM and in- creased the V,,, from 120 to 255 pmol/min*mg protein, as assayed in homogenates 90 s after cell lysis. 4) Besides acting as an activator, GTPyS was also a substrate for the enzyme with a KM = 120 FM and a V- = 115 pmol/min*mg protein. 5) GTPyS-stimulated, Mg2+-dependent guanylate cyclase was inhibited by

= 115 pmol/min*mg protein. 5) GTPyS-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 GTPyS. 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.
In the cellular slime mold Dictyostelium discoideum, intracellular guanylate cyclase is activated up to 10-fold by chemoattractants that bind to cell surface receptors. Other well studied responses regulated via receptors are the activation of adenylate cyclase, the phosphorylation of chemotactic cAMP receptors, and alterations in the polymerization state of actin (1). Signal transduction in D. discoideum shows remarkable similarities with that in higher eukaryotes with respect to the functioning of cell surface receptors, the regulation of ade-* These investigations were supported by the Foundation for Biological Research (BION) and by the C. & C . Huygens Fund, which are subsidized by the Netherlands Organization for Scientific Research (NWO). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Present address: Dept. of Biochemistry, Nyenborg 16, Groningen, The Netherlands. nylate cyclase by GTP-binding proteins, the mechanism of desensitization, and the likely presence of a phosphatidylinositol cycle (2). On the other hand, mechanisms have been proposed to operate in Dictyostelium which have no precedent in other organisms. Examples are an involvement of the phosphatidylinositol cycle in regulation of guanylate cyclase (3,4), and the role that ras proteins might have in the desensitization of this enzyme (5). The latter findings have attracted attention, also because limited insight exists into the functioning of receptor-regulated guanylate cyclases in eukaryotes in general (cf Ref. 6).
Despite extensive studies on the activation and desensitization of guanylate cyclase in intact D. discoideum cells (see Ref. l), little is known about the direct regulation of the enzyme. There exists only one report on the modulation of guanylate cyclase activity in cell-free preparations via cell surface receptors: when cells were stimulated with cAMP and rapidly lysed, the enzyme was found to be stimulated 3-6-fold (7). In vitro, chemicals like adenosine trisphosphates and glycerol modulate the activity of guanylate cyclase (8)(9)(10). However, in all these investigations (7-10) millimolar concentrations of Mn2+ ions have been used in the enzyme assays, because with M$+-GTP little guanylate cyclase activity was found (9, 10). As the intracellular Mn2+ concentrations in Dictyostelium are in the micromolar range (9) the relevance of these studies for insight into the regulation of guanylate cyclase may be called into question. Permeabilized cells have been an alternative tool to the study of Dictyostelium guanylate cyclase. It was described that the elevation of the cGMP concentration evoked by chemoattractants was mimicked by addition of Ca2+ ions or inositol 1,4,5-trisphosphate to permeabilized cells (3, 4 ) . Permeabilized cells, however, offer limited possibilities for manipulation of the enzyme and control of its environment. We have recently managed to identify a guanylate cyclase in cell-free preparations that is active with M p -G T P (11). This enzyme was observed in homogenates, immediately after cell lysis. In this report we describe some salient features of this enzyme, which is present in membranes, has increased activity in the presence of guanosine triphosphates, and is inhibited by Ca2+ ions.

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M p -d e p e n d e n t Guanylate Cyclase in D. discoideum density of 10' cells/ml in lysis buffer (40 mM Hepes'/NaOH, 3 mM MgSO,, 1 mM EGTA, M App(NH)p, pH 7.0, unless otherwise indicated). Homogenization was performed at 0 "C by rapid elution of cell suspensions through polycarbonate filters (pore size 3 pm), by a method developed before for lysis of small cell volumes (100-300 p l ) (13, 14). This method resulted in >99% cell lysis, as estimated by phase contrast microscopy.
Guanylate Cyclase Assay-Guanylate cyclase activity was measured at 25 "C using the method briefly described before (11). Standard reaction mixture contained 20 mM Hepes/NaOH, 1.5 mM MgSO,, 0.5 mM EGTA, 5 X lo-' M App(NH)p, 5 mM dithiothreitol, and 0.3 mM GTP (final concentrations), pH 7.0. For investigation of the enzyme kinetics the GTP or GTPyS concentrations were varied, while keeping the Mg2' concentration at 1.2 mM in excess to the guanine nucleotides by adding extra MgS04. MnCI2, if added, was present at a concentration of 5 mM. Reactions were started between 40 and 120 s after cell lysis, unless otherwise indicated; samples were routinely taken at 0, 40, and 60 s after initiation of the reaction and quenched in 4 N HCIO4. Samples were processed, and cGMP produced was measured in a radioimmunoassay (15, 16). Cyclic GMP synthesis was linear up to about 1.5 mg of protein/ml in the guanylate cyclase assay. Cyclic AMP was measured according to the method of Tovey et al.

(17).
High Performance Liquid Chromatography Analysis-Reaction products formed during the guanylate cyclase reaction were analyzed by high performance liquid chromatography on Lichrosorb RP-18. Samples from the cyclase reaction in HC10, were neutralized with KOH/KHCO3 (15) and either treated with phosphodiesterase or directly applied to the high performance liquid chromatography column. Phosphodiesterase treatment was done for 1 h at 22 "C with 0.1 mg/ml beef heart phosphodiesterase in the presence of 4 mM MgC12; the reaction was arrested by 3 min heating at 99 "C. The high performance liquid chromatography column was eluted with 20% methanol, 1 mM K+ phosphate, pH 6.5, at 1 ml/min. Fractions were collected, lyophilized, and their cGMP content was measured by radioimmunoassay (15, 16).

Increased M$+-dependent Guanylate Cyclase Activity in the Presence of GTPyS
For obtaining Me-dependent guanylate cyclase, cells in a buffer containing M e ions and various additions were lysed by elution through polycarbonate filters, and shortly after lysis homogenates were transferred to a reaction mixture, as described before (11).
When the GTP analogue GTPrS (25 @M) was present during lysis, a marked, about 3-fold higher cGMP production was observed (Table I). It was indeed the activity of a guanylate cyclase that was detected here under cell-free conditions: no cGMP production was obtained with intact cells, and omission of GTP from the reaction mixture resulted in a strong reduction of the cGMP synthesis (Table I), (11). When GTP was replaced by ATP in the reaction mixture a similar reduction of the cGMP production was obtained, while concomitantly the synthesis of cAMP was stimulated strongly (Table I). Although the elimination of GTP from reaction mixtures resulted in reduced cGMP production, this produc-   tion, especially in the presence of GTPrS, was not zero (Table  I). By analysis of high performance chromatography the reaction product synthesized under these conditions was definitely identified as cGMP: reaction products coeluted with authentic cGMP and were degraded by incubation with a cyclic nucleotide phosphodiesterase (data not shown; see also The effect of GTPyS on cGMP production resulted from stimulation of a guanylate cyclase and not from inhibition of a phosphodiesterase. This was inferred from measurements of the phosphodiesterase activity under the reaction conditions, which were done by replacing GTP by cGMP. The degradation of cGMP was about 15%/min and was not reduced in the presence of GTPrS (Table I). Furthermore, stimulation of cGMP production by GTPyS was also observed in homogenates from a Streamer F mutant of D. discoideum (not shown), which is a strain that lacks cGMP-specific phosphodiesterase (18,19).

Factors Affecting the Activity of M$+-dependent Guanylate
Cyclose in the Presence of GTPrS Time after Cell Lysis-Cell homogenates were prepared in the presence or absence of GTPyS (25 g~) and the activity of Me-dependent guanylate cyclase was measured at different times after cell lysis (Fig. 1). Under both conditions the enzyme activity was rapidly lost. In homogenates prepared in the absence of GTP@ no guanylate cyclase activity was detected at 50 min after cell lysis, whereas in homogenates prepared in the presence of GTPrS the activity was decreased to 10% of the activity directly after cell lysis. The loss of M$+-dependent guanylate cyclase in homogenates prepared in the presence of GTPyS showed characteristics of a first order exponential decay, described by a k-l = 0.115 min-' (t0.6 = 6 min), (Fig. 1, inset). The decay of guanylate cyclase activity in homogenates not containing GTPyS appeared to be multiphasic (Fig. 1, inset). Shortly after cell lysis it oc- curred with a to.5 < 3 min and it approached a to.5 GZ 6 min around 8 min after lysis. About the same decay kinetics were observed when the guanylate cyclase activity in homogenates was measured in the presence of Mn2+ ions and 2 mM GTP (Fig. 1). These assay conditions were used as a measure of the total Mg+and Mn2+-dependent guanylate cyclase activities, in case that there might exist distinct M P -and Mn2+-dependent enzymes. However, no evidence for distinct M$+-and Mn2+dependent enzymes was found. GTPyS also stimulated guanylate cyclase assayed with Mn2+-GTP (2 mM), but the relative stimulation by GTPyS was lower than as measured with Mg2"GTP ( Fig. 1). This was due to the higher basal guanylate cyclase activity in the former condition as compared to the latter.
GTPyS also stimulated guanylate cyclase when added to homogenates after cell lysis. However, the longer the time elapsed between cell lysis and the addition of 25 p~ GTPyS, the lower the stimulation (Fig. 2). Thus, in addition to the instability of the catalytic activity of the enzyme as demonstrated in Fig. 1, the potential of the enzyme to be stimulated by GTPyS fades after cell lysis ( to.s = 30-60 s) (Fig. 2). Ca2+ Ions and Inositol Trisphosphate-Ca2+ ions and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) deserve special attention as potential regulators of Dictyostelium guanylate cyclase, as it was reported that these compounds induce a cGMP response in saponin-permeabilized Dictyostelium cells (3, 4). The effect of Ca2+ ions was investigated by varying the free Ca2+ concentration in homogenates using Ca2+-EGTA buffers (20). Both basal and GTPyS-stimulated, Mg+-dependent guanylate cyclase were strongly inhibited by Ca2+ ions (Fig.  3). Half-maximal inhibition of both forms of guanylate cyclase occurred at about 50 nM free Ca2+ ions. At free Ca2+ concentrations higher than about 200 nM no Mg+-dependent guanylate cyclase activity could be detected (Fig. 3).
When lysates were prepared in the presence of excess CaClz (100 gM), and, subsequently, 15 s before the guanylate cyclase assay excess EGTA (5.6 mM) was added to reduce the free Ca2+ concentration, the inhibition of the enzyme by Ca2+ ions was partially (about 30%) reversed. This observation dem-    (Fig. 3b). Rather, in the absence of EGTA, inhibition of guanylate cyclase was observed (significance p < 0.1). This may be explained by an Ins( 1,4,5)P3-mediated release of Ca2+ ions from intracellular stores present in the homogenates. Such a release has been demonstrated in permeabilized D. discoideum cells (21). Considering the sensitivity of Mg'+-dependent guanylate cyclase to Ca2+ ions (Fig. 3a), the Ca'+ release induced by Ins(1,4,5)P3 would be predicted to be about 40 nM. The above explanation agrees with the observation that Ins(1,4,5)P3 did not inhibit guanylate cyclase in the presence of EGTA (Fig. 3b).
Ca'+ ions (2 or 100 PM) also strongly inhibited M$+dependent guanylate cyclase in homogenates, prepared from cells that had been treated with saponin (1 mg/ml, 30 min). The Mc-dependent enzyme was also inhibited by Ca'+ ions when saponin (1 mg/ml) was added to homogenates immediately after cell lysis and prior to the guanylate cyclase assay (data not shown). Thus, the difference in the effects of Ca2+ on cGMP production in permeabilized cells (3,4) and in cell homogenates does not result from the use of saponin in the former system. When we tried to reproduce the previous findings with saponin-permeabilized cells (3,4), we were able to induce a slight cGMP accumulation using Ins(1,4,5)P3 (maximally 10-25% of the accumulation induced by chemoattractants); however, we were unable to induce a cGMP response by use of Ca'+ ions. ' Effect of pH and Adenine Nucleotides-Guanylate cyclase in homogenates prepared both in the absence and presence of GTPyS had optimal activity between pH 7.5 and 8.0 (not shown). This is the same pH optimum as reported for Mn'+dependent guanylate cyclase in Dictyostelium (10). Below pH 7.0 almost no cyclase activity was detectable. Above pH 7.0 the cyclase activity in homogenates not containing GTPyS was also less than about 50% of the activity in the presence of GTPyS. Hence, no evidence was found for pH-dependent uncoupling of guanylate cyclase from its GTPyS sensitivity.
The effect of adenine nucleotides on the GTPyS-stimulated guanylate cyclase was the same as the effect on the enzyme in the absence of GTPyS, described before (11). Thus, App(NH)p stimulated guanylate cyclase in the presence of GTPyS the same amount (2-3-fold) as in its absence, whereas ATP and ATPyS had no effect on the basal or GTPySstimulated enzyme activity (11, and data not shown). As noted before (ll), the latter observation suggests that the stimulation of the enzyme by guanine nucleotides (see below) is not mechanistically related to that by adenine nucleotide(s). Inclusion of GTP at concentrations of about M during cell lysis also resulted in a small increase in the cGMP production by homogenates (Fig. 4). This effect, however, may be explained by an increase in the substrate concentration for guanylate cyclase, taking into account the KM of the enzyme for GTP (see below) and the subsaturating GTP concentration normally used in the enzyme reactions. Therefore, in some experiments, the GTP concentration in the reaction mixtures was adjusted so that homogenates prepared in the presence of GTP were assayed at the same GTP concentration as controls from homogenates that had not received GTP during lysis. In these experiments the presence of 4 mM GTP during cell lysis resulted in only 40% more cGMP synthesis as compared to the synthesis by lysates prepared in the absence of GTP.
Guanosine diphosphates, like GDP and GDPPS, and guanosine monophosphate (GMP) did not activate guanylate cyclase (Fig. 4). Rather, they tended to inhibit the enzyme. GDPPS also antagonized the activation by GTPyS, when present in excess of this compound (Fig. 4, inset). This might be explained by competition of GDPPS with GTPyS for a regulatory site or by competition of GDPPS with GTP or GTPyS at the catalytic site of the enzyme. The latter explanation is favored by the observation that GDPPS inhibited the cGMP synthesis in the presence and absence of GTPyS to relatively the same amount (about 50% at 100 p M GDPPS, Fig. 4, inset).
The activation of Dictyostelium guanylate cyclase by guanosine triphosphates has some characteristics similar to the activation of Dictyosteliurn adenylate cyclase, with regard to its nucleotide specificity and the property to give highest activity shortly after cell lysis (13). A difference, however, is Mg+-dependent Guanylate Cyclase in D. discoideum 4333 that guanylate cyclase activity decreased rather than increased during incubation at 0 "C in the presence of GTPyS (Figs. 1 and 2), while for adenylate cyclase such an incubation is required to obtain maximal activity (13). Another difference between both enzymes was observed when studying the aggregation-less mutant synag 7 (also called N7). Activation of membrane-bound adenylate cyclase requires the presence of a soluble cytosolic factor and does not occur in this mutant, which has a defect in this factor (13, 22). As shown in Table  11, homogenates of mutant synag 7 contained about 5-fold less guanylate cyclase activity than wild type homogenates, as measured with Mn2'-GTP as a substrate. Basal M$+-dependent guanylate cyclase activity could not be detected. However, when GTPyS was included in homogenates a Me-dependent guanylate cyclase activity, comparable to the activity in the presence of Mn2+ ions, was apparent. Thus, in contrast to adenylate cyclase in mutant synag 7 (13,22), guanylate cyclase showed no defect in its activation by GTPyS (Table 11). These observations suggest that, despite some superficial resemblances, Dictyostelium guanylate cyclase and adenylate cyclase are activated by guanine nucleotides by different mechanisms.
Kinetics of GTPyS-stimulated, M$+-dependent Guanylate Cyclase The kinetics of M$+-dependent guanylate cyclase were studied under three different conditions. Either homogenates were prepared in the absence of exogenously added guanine nucleotides and the activity of the enzyme was measured with various concentrations of GTP or GTPyS, or homogenates were prepared in the presence of a low concentration of GTPyS and the enzyme activity was determined with various concentrations of GTP during the reaction. These experiments might shed light on the mechanism of activation of the enzyme by GTPyS and answer the question whether GTPyS was (also) a substrate for the enzyme.
The results of these kinetic measurements are compiled in Table 111. Under each condition tested, the behavior of guanylate cyclase conformed to Michaelis-Menten kinetics. Relatively, the lowest activity was detected with the enzyme prepared in the absence of GTPyS, using GTP as substrate (Table 111, part I). Guanylate cyclase also effectively used GTPyS as a substrate, showing the same Vmax and about a 3fold lower KM with GTPyS than with GTP (Table 111, part 11). When GTPyS was present during cell lysis and the enzyme activity was subsequently measured with GTP the highest activity was detected, resulting from both a high V,,,

TABLE I1 Activation of Mg2+-dependent guanylate cyclase by GTP-yS in cellfree preparations of Dictyostelium mutant synag 7 and wild type NC4
Lysis and reactions were as described in Table I. GTP-yS, if indicated, was present at 25 pM; when MnC12 (5 mM) was present in the reaction mixture, the GTP concentration was 2 mM, instead of 0.3 mM, as under standard conditions. Synug 7 cells were grown and developed until aggregation competence as described before (22

discoideum homogenates under various conditions
The kinetic parameters are the mean f S.E. from 3-6 experiments.
Homogenates were prepared in the absence or presence of GTP-yS, kept at 0 "C, and 90 s after cell lysis the guanylate cyclase activity was measured in reaction mixtures containing 0.05-2 mM GTP or 0.0125-0.9 mM GTP-yS. In all cases analyzed the data showed good fits to linear Michaelis-Menten kinetics. 1 mg of protein is equivalent to 3.8 X lo7 cells. These results suggest that relatively low GTPyS concentrations (1 PM ) affect guanylate cyclase in two ways, both leading to increased cGMP synthesis in homogenates with GTP. Firstly, GTPyS lowers the KM of the enzyme for GTP. Secondly, GTPyS increases the V,,, of guanylate cyclase for GTP. At relatively high concentrations (~100 FM) GTPyS also becomes a substrate for the enzyme.
GTPyS Stimulates Particulate Guanylate Cyclase Mnz+-dependent guanylate cyclase in Dictyostelium has been found in both soluble and particulate cell fractions (7,9, 10, 16). To investigate which of these enzyme forms corresponded to the Me-dependent enzyme and which form was activated by GTPyS, cell homogenates were made in the presence or absence of GTPyS, rapidly centrifuged, and the cyclase activity in sediment and supernatant was measured with M$+-GTP at pH 7.0. Measurements were also done with M$+ ions at pH 8.0 and with Mn2+/2 mM GTP, to investigate whether the enzyme activity observed under these conditions behaved differently from the activity detected with M e -GTP at pH 7.0.
Me-dependent guanylate cyclase was found both in pellet and supernatant fractions after 1 min centrifugation at 10,000 x g (Fig. 5). When homogenates prepared in the presence of GTPyS were fractionated, increased guanylate cyclase activity was measured only in 10,000 X g pellets, not in supernatants (Fig. 5). The same results were obtained when the enzyme was measured with M e ions at pH 8.0 and with Mn2+/2 m M GTP. No evidence was found, using these assay conditions, for a guanylate cyclase other than the M edependent enzyme and behaving differently from the latter enzyme. Only, measuring at pH 8.0 or with Mn2+/2 mM GTP, the activity in preparations not containing GTPyS was higher than when measured with M$+-GTP at pH 7.0 (Fig. 5). This apparently results from a higher intrinsic activity of M edependent guanylate cyclase.

DISCUSSION
In this paper we report on some of the characteristics of a highly active Me-dependent guanylate cyclase in D. discoideum membranes. This enzyme may be identical to the enzyme regulated in vivo via cell surface receptors, as we suggested before (11). The enzyme is stimulated in uitro by the GTP analogues GTPyS and Gpp(NH)p, the ATP analogue App(NH)p, and inhibited by nanomolar concentrations of Ca2+ ions. These effects may reflect regulatory mechanisms that operate in uiuo, especially because they were established under in vitro conditions that are more physiologic than were used previously, i.e. using M$+-GTP instead of Mn2+-GTP as a substrate for the enzyme. Optimal enzyme activity was obtained in homogenates, immediately after lysis of cells in the presence of GTPyS, App(NH)p, and the Ca2+ chelator EGTA. Under these conditions a VmBX = 66 pmol/min. lo7 cells was estimated with Mg2f-GTP as substrate. This activity is close to the activity estimated for guanylate cyclase in uiuo that is stimulated via cell surface receptors (60-90 pmol/min . lo7 cells; 12,19,23).
Several mechanisms can be envisaged to explain the higher activity of Me-dependent guanylate cyclase in the presence of GTPyS: 1) GTPyS may be a substrate for the enzyme; 2) GTPyS may be stabilizing the enzyme; and 3) GTPyS may be an allosteric activator. There is evidence for each of these mechanisms.
( a ) That GTPyS might be a substrate is strongly suggested by the observation of GTPyS-dependent cGMP synthesis, characterized by a V,,, similar to the VmaX with GTP (Table   111). This GTPyS-dependent synthesis was obtained when GTPyS was the sole (exogenous) guanosine triphosphate in the reaction.
( b ) In favor of the idea that GTPyS stabilizes the enzyme is the observation that the preserze of GTPyS during cell lysis resulted in higher cGMP synthesis than presentation of GTPyS only at the time of the reaction (Fig. 2). Furthermore, the enzyme activity decayed more rapidly in the absence than in the presence of GTPyS (Fig. 1).
(c) Suggesting allosteric activation of guanylate cyclase by GTPyS, finally, is the observation that in the presence of GTPyS the enzyme has a significantly lower KM for substrate than in its absence (Table 111). This lower K M was observed when GTPyS was used as the sole substrate for the enzyme, as well as with GTP as a substrate in the presence of a subsaturating concentration of GTPyS (Table 111, part 111).
The kinetic data rule out the possibility that the higher activity of the enzyme under the latter conditions was only because GTPyS was a substrate in addition to GTP.
We propose the following explanation for the effect of GTPyS on the activity of M$+-dependent guanylate cyclase in homogenates. In addition to the catalytic site there is an allosteric site on the enzyme with high affinity for guanine nucleotides. This allosteric site may be located on the enzyme itself or on a separate component, e.g. a G-protein (24). Occupation of the allosteric site by GTP+ lowers the KM of the enzyme and in addition leads to its stabilization. The latter effect is observed as an increase in the Vmx after some time of incubation with GTPyS, for instance of the labile enzyme that is present in homogenates immediately after cell lysis. GTPyS is also a substrate of the enzyme and therefore does not competitively inhibit the cGMP synthesis when GTP is present as substrate. GTP apparently has a much lower affinity for the allosteric site than GTPyS. The affinity of the enzyme for the natural substrate GTP is increased by the presence of GTPyS because this nucleotide occupies the allosteric site more permanently than GTP. Whether this occurs by a mechanism analogous to G-proteins (24) remains to be investigated. As yet, we have no direct evidence for the involvement of G-proteins in the regulation of Mg?"-dependent guanylate cyclase; the properties of the enzyme were not found to be altered by treatment of cells with cholera or pertussis toxin (data not shown).
The role of Ca2+ in the regulation of guanylate cyclase appears to be contrary to the role of guanine nucleotides, i.e.
inhibitory. It appears that the M$+-dependent guanylate cyclase is much more sensitive to Ca2+ than the Mn2+-dependent enzyme, as Mn2+-dependent guanylate cyclase is only weakly inhibited by Ca2+ ions