Guanylate cyclase of isolated bovine retinal rod axonemes - American

The guanylate cyclase activityof axoneme-basal apparatus complexes isolated from bovine retinal rods has been investigated. The Mg2+ and Mn2+ complexe...
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BIOCHEMISTRY

& Fridovich, I., Eds.) pp 215-216, Academic Press, London. Cohn, E. J., & Edsall, J. T. (1943) Proteins, Amino Acids and Peptides, pp 370-381, Reinhold, New York. Edwards, Y. H., Hopkinson, D. A., & Harris, H. (1978) Nature (London) 271, 84-87. Evans, H. J., Steinman, H. M., & Hill, R. L. (1974) J . Bioi. Chem. 249, 7315-7325. Fielden, E. M., Roberts, P. B., Bray, R. C., Lowe, D. J., Mautner, G. N., Rotilio, G., & Calabrese, L. (1974) Biochem. J . 139, 49-60. Forman, H. J., & Fridovich, I. (1973) J . Biol. Chem. 248, 2645-2649. Gurd, F. R. N. (1972) Methods Enzymol. 25, 424-438. Habeeb, A. F. S. A. (1972) Methods Enzymol. 25, 457-464. Harris, C. M., & Hill, R. L. (1969) J . Bioi. Chem. 244, 21 95-2203. Hartz, J. W., & Deutsch, H. F. (1972) J. Bioi. Chem. 247, 7043-7050. Hubbard, R. W. (1965) Biochem. Biophys. Res. Commun. 19, 679-685. Katz, S., & Ferris, T. G. (1966) Biochemistry 5, 3246-3253. Keele, B. B., Jr., McCord, J. M., & Fridovich, I. (1971) J. Bioi. Chem. 246, 2875-2880. Klotz, I. M., Darnall, D. W., & Langerman, N. R. (1975) Proteins, 3rd Ed. 1 , 293-41 1. Klug, D., Rabani, J., & Fridovich, I. (1972) J. Bioi. Chem. 247, 4839-4842. Klug-Roth, D., Fridovich, I., & Rabani, J. (1973) J . Am. Chem. SOC.95, 2786-2790. Lippard, S. J., Burger, A. R.,Ugurbil, K., Pantoliano, M. W., & Valentine, J. S. (1977) Biochemistry 16, 1136-1141. Lowry, 0. H., Rosebrough, N . J., Farr, A. L., & Randall, R.

FLEISCHMAN AND DENISEVICH

J. (1951) J. Bioi. Chem. 193, 265-275. Malinowski, D. P., & Fridovich, I. (1979) Biochemistry 18, 237-244. Marmocchi, F., Venardi, G., Bossa, F., Rigo, A., & Rotilio, G. (1978) FEBS Lett. 94, 109-111. McCord, J. M., & Fridovich, I. (1969) J. Biol. Chem. 244, 6049-6055. Moore, S., & Stein, W. H. (1963) Methods Enzymol. 6 , 819-831. Murphy, J. B., & Kies,M. W. (1960) Biochim. Biophys. Acta 45, 382-384. Richardson, J. S., Thomas, K. A,, Rubin, B. H., & Richardson, D. C. (1975a) Proc. Natl. Acad.Sci. U.S.A. 72, 1349-1353. Richardson, J. S., Thomas, K. A., & Richardson, D. C. (1975b) Biochem. Biophys. Res. Commun. 63, 986-992. Rotilio, G., Bray, R. C., & Fielden, E. M. (1972) Biochim. Biophys. Acta 268, 605-609. Steinhardt, J. (1938) J . Bioi. Chem. 123, 543-575. Steinman, H. M., Naik, V. R., Abernethy, J. L., & Hill, R. L. (1974) J. Bioi. Chem. 249, 7326-7338. Tanford, C., Nozaki, Y., Reynolds, J. A,, & Makino, S. (1974) Biochemistry 13, 2369-2375. Tegelstrom, H. (1975) Hereditas 81, 185-198. Waud, W. R., Brady, F. O., Wiley, R. O., & Rajagopalan, K. V. (1975) Arch. Biochem. Biophys. 169, 695-701. Weber, K., & Osborn, M. (1969) J . Biol. Chem. 244, 4406-44 12. Weser, U., Bunnenberg, E., Cammack, R., Djerassi, C., FlohE, L., Thomas, G., & Voelter, W. (1971) Biochim. Biophys. Acta 243, 203-213. Yphantis, D. A. (1964) Biochemistry 3, 297-317.

Guanylate Cyclase of Isolated Bovine Retinal Rod Axonemest Darrell Fleischman* and Michael Denisevich

The guanylate cyclase activity of axoneme-basal apparatus complexes isolated from bovine retinal rods has been investigated. The Mg2+and Mn2+complexes of GTP" serve as substrates. Binding of an additional mole of Mg2+or Mn2+ per mole of enzyme is required. Among cations which are ineffective are Ca2+,Ni2+,Fez+, Fe3+, ZnZ+,and Co2+. The kinetics are consistent with a mechanism in which binding of Mg2+or Mn2+to the enzyme must precede binding of MgGTP or MnGTP. The apparent dissociation constants of the Mgenzyme complex and the Mn-enzyme complex are 9.5 X lo4 and 1.1 X lo4 M, respectively. The apparent dissociation constants for binding of MgGTP and MnGTP to the complex ABSTRACT:

It

has been apparent that cGMP must play an important role in the function or development of vertebrate retinal rods. Rod outer segments (ROS)' contain high levels of a cGMP-specific phosphodiesterase (Pannbacker et al., 1972; Chader et al., 1974a,b; Robb, 1974; Pannbacker & Lovett, 1977) which is activated by bleached rhodopsin in the presence of GTP or ATP (Miki et al., 1973; Chader et al., 1974a; Bitensky et al.,

of the enzyme with the same metal are 7.9 X lo4 and 1.4 X M, respectively. The cyclase activity is maximal and independent of pH between pH 7 and 9. KCl and NaCl are stimulatory, especially at suboptimal concentrations of Mg2+ or Mn2+. Ca2+and high concentrations of Mg2+and Mn2+ are inhibitory. Ca2+inhibition appears to require the binding of 2 mol of Ca2+per mol of enzyme. The dissociation constant of the Ca2-enzyme complex is estimated to be 1.4 X 10" M2. The axoneme-basal apparatus preparations contain adenylate cyclase activity whose magnitude is 1-10% that of the guanylate cyclase activity.

1975; Manthorpe & McConnell, 1975; Sitaramayya et al., 1977). Recent experiments demonstrate that the cGMP hydrolysis is quite rapid; bleaching of a single rhodopsin molecule may result in the hydrolysis of as many as 4 X lo5 cGMP molecules/s (Woodruff et al., 1977; Yee & Liebman, 1978). I

Abbreviations used: CAMP,cyclic adenosine 3',S'-monophosphate;

ROS, rod outer segments; Pipes, 1,4-piperazinediethanesulfonicacid; Contribution No. 672 from the Charles F. Kettering Research Laboratory, Yellow Springs, Ohio 45387. Received May 2, 1979. This work was supported in part by National Institutes of Health Grant EY00847.

0006-2960/79/0418-5060$01 .OO/O

DTT, dithiothreitol; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; PEI, polyethylenimine; Mes, 2-(N-morpholino)ethanesulfonic acid.

0 1979 American Chemical Society

VOL. 1 8 , N O . 2 3 , 1 9 7 9

GUANYLATE CYCLASE KINETICS

Furthermore, there appears to be a correlation between the cGMP content of isolated ROS and their sodium permeability [Woodruff et al., 1977;see also Lipton et al. (1977)l. For those reasons, several groups have proposed that a change in cGMP concentration may be the link between rhodopsin bleaching and rod hyperpolarization. The detailed mechanism may involve cGMP-stimulated phosphorylation of a small protein (Pannbacker & Schoch, 1973;Lolly et al., 1977;Farber et al., 1978, 1979), phosphorylation and dephosphorylation of bleached rhodopsin (Kuhn et al., 1973;Frank & Bensinger, 1974;Chader et al., 1976;Weller et al., 1976;Kiihn, 1978) and a light-activated GTPase (Robinson & Hagins, 1977; Wheeler & Bitensky, 1977;Bignetti et al., 1978). Some sort of interaction with CaZCappears to be involved (Hagins, 1972; Lipton et al., 1977). In addition, inherited defects in cGMP metabolism are implicated in a number of animal retinal degenerative diseases [see Farber et al. (1978)and references therein], ROS originate as modified cilia, as do several other vertebrate sensory receptors (Wiederhold, 1976), and may have evolved from phototactic microorganisms which respond to light by changing the mode of their ciliary beat (Eakin, 1965). We have been examining axoneme-basal apparatus complexes isolated from bovine retinal rods (Raveed & Fleischman, 1975) with the hope of determining whether this conserved structure plays a biochemical as well as a structural role in vision. The complex includes the outer segment microtubule doublets, the basal body, and occasionally the centriole and part of the ciliary rootlet. In a preliminary report (Pannbacker & Fleischman, 1972), we noted that bovine ROS contain high levels of guanylate cyclase and that the activity seemed to be enriched in partially purified axonemes. More highly purified axoneme preparations, obtained by density gradient centrifugation of detergent-solubilized ROS, retain most of the guanylate cyclase activity of the ROS. The cyclase activity cannot be separated from the axonemes by extraction with nonionic detergents, alkali metal salts, or EDTA (D. Fleischman, M. Denisevich, D. Raveed, and R. G. Pannbacker, unpublished experiments). In this communication, some of the kinetic characteristics of the axoneme-associated guanylate cyclase will be described. There have been a number of studies of guanylate cyclase activity in unfractionated ROS (Pannbacker, 1973;Goridis et al., 1973;Krishna et al., 1976;Krishnan et al., 1978). In most respects, we have found the behavior of cyclase in the isolated axoneme to resemble that reported for intact ROS. However, the results of the present experiments suggest alternative interpretations of certain of the earlier experiments. Materials and Methods Reagents. Inorganic chemicals were reagent grade. For the experiments in which metal and GTP concentrations were varied independently, the metal solutions were standardized against EDTA, using Eriochrome Black T as indicator (Vogel, 1961). For these experiments, Sigma Type I11 GTP (shipped and stored at dry ice temperature) was used. GTP solutions were prepared immediately before use. Radioactively labeled nucleotides were purchased from the New England Nuclear Corp. Other nucleotides and biochemicals were purchased from Sigma Chemical Co. Preparation of Rod Outer Segments. Retinas were removed from freshly enucleated bovine eyes in dim light and collected in a solution of 10 mM Pipes and 5 mM MgC12,pH 7.0(PM), containing 50% (w/w) sucrose at 4 OC. The retinal suspension was swirled vigorously and filtered through cheesecloth. The filtrate was centrifuged for at least 3 h at 13OOOg. ROS were

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Table I: Nucleotide Transformation in the Presence of Rod Axonemesa nucleotide

initial (cpm)

final (CPm)

change (cpm)

expt l : b GTP 24828 + 145 24485 f 567 3943 f 585 cGMP (144 + 17) 2304 f 26 2160 f 31 guanylate cyclase activity: 28 nmol min-' (mg of protein)" expt 2:= GTP 3709k 30 3455 k 6 7 254f73 cGMP ( 2 0 + 1) 185 + 6 165 f 6 2 9 7 f 31 72f42 GDP 225 + 2 8 5'-GMP 143 + 8 163+12 2 0 f 14 guanylate cyclase activity: 10 nmol min-' (mg of protein)-' GDP formation: 4 nmol min-' (mg of protein)-' 5'-GMP formation: 1 nmol min-' (mg of protein)-' a Values reported for expt 1 and 2 are the averages of three samples, plus or minus standard deviations. Nucleotides were sepaAxonemes rated and counted as described under Methods. (50.1 pg) were incubated with 20 mM Hepes, 2 mM GTP (containing [a-'*P]GTP), and 4.0 mM MnCI,, pH 7.5, in a final volume of 125 p L for 11 min at 37 "C. The reactions were stopped by adding 50 p L of a solution containing 35 mM EDTA, 10 mM cGMP, and 9 mM 5'-GMP and boiling for 5 min. For blanks, the stopping solution was added and the mixtures were boiled immediately after adding the final reagent. Axonemes (89 pg) were incubated with 20 mM Hepes, 2 mM GTP (containing [a-"P]GTP), 1 mM cGMP, and 4 mM acetate, pH 7.5, for 10 min at 37 "C. The stopping solution contained 35 mM EDTA and 10 mM cGMP.

removed from the surface and purified by centrifugation in a linear 2538% (w/w) sucrose gradient in PM buffer for at least 3 h at 13000g. ROS were removed from the gradient, diluted with PM, underlaid with a layer of 50% sucrose in PM, and collected at the interface after centrifugation for 1 h at 13000g. Isolation of Axoneme Basal Apparatus Complexes. Purified ROS were dissolved in PM buffer containing 2% Triton X-100and 1 mM dithiothreitol (DTT) at a concentration of 5 mg of protein per mL or less. Ten milliliters of the solution of dissolved ROS was layered onto a 10-mL linear 45-65% (w/w) sucrose gradient made up in the PM-DTT-Triton X-100 solution. After centrifugation at 13000g for 6-1 2 h, the axonemebasal apparatus complexes were found in a turbid band at -55% sucrose. The turbid band was collected, resuspended in at least 5 volumes of the PM-DTT-Triton x-100 solution, and pelleted by centrifugation at 13000g for 1 h. Axonemes were stored at 4 OC (frozen axonemes cannot be resuspended) and were used within 3 days of isolation. Guanylate Cyclase Assay. Guanylate cyclase activity of axonemes was determined by a modification of the method of Pannbacker (1973). Axonemes (usually -2 pg) were incubated for 1 1 min at 37 OC with GTP (containing 0.1-10 Ci/mol [cx-~~PIGTP) and the appropriate reagents in a volume of 120-200 pL. Unless otherwise specified, the mixtures were buffered at pH 7.5with 50 mM Hepes. In some of the earliest experiments GTP-regenerating systems and phosphodiesterase inhibitors were included, but these were omitted after it became apparent that GTPase and phosphodiesterase activities of the axonemes were negligible (Table I). Reactions were stopped by adding 50 pL of 35 mM EDTA (containing 8 mM cold cGMP to permit localization of the cGMP spots after thin-layer chromatography) and boiling for 5 min. Higher EDTA concentrations were used for experiments involving high Mg2+or Mn2+concentrations. For zero time blanks, EDTA and cGMP were injected and the mixtures were boiled as soon as the last reagent was added. Nucleotides were separated by thin-layer chromatography on PEI-cellulose as described by Pannbacker (1973). cGMP spots were located under ul-

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B IO C H E M 1 S T R Y

traviolet light, removed, and counted in a Nuclear Chicago 1043 low-background Geiger-Mueller counter. In experiments in which [3H]cGMP was included, the cGMP spots were eluted with 1 mL of 0.02 M Tris, pH 7.5, containing 0.7 M MgC12, and 3H and 32Pwere counted in a Packard Tri-Carb 3255 liquid scintillation spectrometer after the addition of 10 mL of Aquasol (New England Nuclear). Cyclase activity was constant for at least 20 min and diminished only as the GTP was consumed. Identification of cGMP. In order to establish that the major reaction product was indeed cGMP, an incubated reaction mix was chromatographed on a PEI-cellulose sheet in two dimensions. The sheet was placed in contact with X-ray film as described by Pannbacker (1973) and Randerath & Randerath (1964). The blackened area of the X-ray film was found to coincide with the cGMP spot located on the thin-layer sheet with ultraviolet light. Competing Reactions. Autoradiography of developed thin layer sheets followed by counting of the GTP, GDP, 5'-GMP, and cGMP spots in incubated samples and blanks furnished assurance that the cyclase kinetics were not distorted by phosphodiesterase or GTPase activity. Table I, expt 2, presents an accounting of the fate of GTP during the course of a typical guanylate cyclase assay. (The protein concentration in this experiment is 10 times that employed in most of the other experiments reported here.) Protein Assay. Protein was assayed by a modified Lowry technique employing sodium dodecyl sulfate (Peterson, 1977). General. Preliminary experiments were performed under a number of conditions (e.g., range of the varied parameter, divalent cation concentration, and protein concentration). Once the reproducibility of the results was assured (or the reason for apparent inconsistencies was identified), the conditions for the reported experiments were chosen best to illustrate the behavior of the enzyme.

-

Results Effect of Triton X-100. Since axoneme isolation involved the use of Triton X-100, it was important to determine whether cyclase activity was substantially affected by this detergent. There have been conflicting reports about the effect of Triton X-100 on guanylate cyclase activity in ROS (Bensinger et al., 1974; Krishnan et al., 1978). Our results indicate only a small influence of Triton X-100 on guanylate cyclase activity when corrections are made for phosphodiesterase activity (Table 11). It seems probable that Bensinger et al. (1974) underestimated the phosphodiesterase activity in their control ROS. Since it was not yet recognized that GTP stimulates phosphodiesterase, GTP was not included in the separate phosphodiesterase assay mixture. We are as yet unable to explain why our results differ from those of Krishnan et al. (1978), who found inhibition at Triton X- 100 concentrations comparable to those employed here. Perhaps differences in the method of ROS isolation are responsible. Stability of Guanylate Cyclase. The guanylate cyclase activity of axonemes suspended in water in contact with air diminished steadily, reaching half the initial activity in 9 days. The loss of activity was unchanged when 1 mM DTT was included in the suspension. Divalent Cation Dependence. Axonemes and GTP were incubated with Mn2+,Mg2+,Zn2+,Co2+,Ni2+,Fez+,and Fe3+ at concentrations ranging from 17 to 0.17 mM. There was no measurable activity with metals other than MnZ+and Mgz+. Axonemes and GTP were also incubated with Ca2+at concentrations ranging from 6.7 to 0.053 mM. Again there was no detectable activity.

FLEISCHMAN AND DENISEVICH

Table 11: Effect of Triton X-100 on the Guanylate Cyclase Activity of ROSa

Triton X-100 cGMP accumu(%) lated (32Pcounts) 0 0.1 0.2 0.5 1.0 2.0

1875 f 9 2933 i 104 2768 i. 38 2541 i. 124 2391 i: 8 2387 * 63

cor cyclase fraction of act. [nmol [ 3H] cGMP inin-' (mg hydrolyzed protein)-' ] 0.530 0.119 0.080 0.093 0.099 0.131

0.71 0.86 0.80 0.74 0.70 0.7 1

a Bleached ROS (0.075 pg) were incubated with 20 mM Hepes, 1.3 mM GTP (containidg [ W ~ ~ P I G T P0.6 ) , mM cGMP (containing -6 Ci/mol [3H] cGMP), 1.8 mM MnCl,, 2.5 mM isobutylmethylxanthine, 1.3 mg of phosphocreatine, 0.1 mg of creatine phosphokinase, and the indicated concentrations of Triton X-100 in a final volume of 100 p L at 37 "C for 11 min. The reactions were stopped by adding 25 p L of a solution containing 35 mM EDTA, 11 mM 5'-GMP, and 15 mM cGMP and boiling for 5 min. For blanks, the stopping solution was added and the mixtures were boiled immediately after adding the final reagent. cGMP was separated, and 32Pand 3H were counted as described under Materials and Methods Reactions were run in duplicate, with one blank for each Triton X-100 concentration. Reported activities are the average number of counts per minute minus the corresponding blanks, plus or minus standard deviations. Corrections are based on the assumption that the fraction of newly formed cGMP destroyed by phosphodiesterase is half the fraction of the initally present [3H] cGMP lost during the incubation.

Dependence of Guanylate Cyclase Activity upon the Concentration of GTP, M$+, and Mnz+. G T P forms very stable 1:l complexes with Mg2+and Mn2+. Under conditions of ionic strength comparable to those in the cyclase assays, their association constants have been estimated to be 65 000 M-' and 353 000 M-I, respectively (Garbers et al., 1975). It seemed reasonable to suspect that Mgz+ or MnZ+complexes of GTP (MgGTP and MnGTP) are the physiological substrates of the guanylate cyclase. In the earliest experiments, it was apparent that the cyclase was stimulated by total magnesium concentrations in excess of the GTP concentration, suggesting a requirement for uncomplexed Mgz+. Increasing the total manganese concentration above the GTP concentration had little stimulatory effect, however. We suspected that the low concentration of Mn2+in equilibrium with MnGTP, even when GTP was in excess, was sufficient to activate the enzyme. To perform the kinetic experiments properly, it was therefore necessary to vary Mn2+while maintaining MnGTP constant and vice versa. A computer program was written, employing the MnGTP and MgGTP association constants, which allowed us to calculate the amounts of GTP and metal salt required to achieve the desired concentrations of MnGTP and Mn2+ or MgGTP and Mgz+. Representative double-reciprocal plots in which Mn2+and MnGTP were varied independently are displayed in parts a and b of Figure 1, and corresponding plots in which Mgz+ was varied while MgGTP was held constant are displayed in Figure IC. In each case the plots are linear (except at high metal concentrations), in agreement with the concept that MnGTP and MgGTP serve as substrates and that binding of 1 mol of MnZ+or Mg2+ per mol of enzyme is required. When MnGTP or MgGTP is varied, the doublereciprocal plots intersect on the 1/ Vaxis; when Mg2+or Mn2+ is varied, they do not. The kinetics of enzyme-catalyzed reactions involving activators such as Mg2+and Mn2+have been considered in detail by Segal et al. (1952). Linear double-reciprocal plots which

VOL. 18, NO. 23, 1979

GUANYLATE CYCLASE KINETICS CMn2' 1.mM I

I

1

1

,

I

CGTP MnPl,mM

, I

I

I

I

I

I

1

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Table 111: Kinetic Constants of Axoneme Guanylate Cyclase Vm"

rsp

[nmol min-' (mg of [El [ M e l / W e I (M) protein)-']

Me

(MI 1.9 x 10-4 1.4 x 10-4

9.5 x 10-4 1.1 x 10-4

30

Mg Mn

[EM1 [EMS1

30

Values ranging from 10 to 50 nmol min-' (mg of protein)-' have been found. [SI = MeGTP. a

t 2

4 6 8 10 CGTP M n l - l , m M - '

12

*

1 02

06

04

0.8

1.0

C Mn2'l-l , mM'I CGTP M g 2 - l ,mM

z

I

I

I

I

I

I

I

I

I

-I "re I

0.5mM GTP.Mn

I59 I .o

0.5

0

C Mg2+1-' ,rnM-I FIGURE 1: (a) Dependence of axoneme guanylate cyclase activity upon the concentration of MnGTP at various concentrations of free Mn2+. (b) Dependence of axoneme guanylate cyclase activity upon the concentration of free Mn2+ at various concentrations of MnGTP. (c) Dependence of axoneme guanylate cyclase activity upon the concentration of free Mg2+ at various concentrations of MgGTP.

intersect on the l/Vaxis when the substrate concentration is varied but do not do so when the activator concentration is varied are predicted if the activator must bind to the enzyme before the substrate can bind. An additional condition is that the rate constant for product formation must be small compared to the rate constant for dissociation of the substrate from the enzyme. Guanylate cyclase kinetics are therefore consistent with the following mechanism (E = enzyme, Mez+ = Mgz+ or Mn2+,S = MgGTP or MnGTP, and V, is the velocity when all of the enzyme is in the form EMeS).

E EMe

k + Mez+$ EMe k-1

k +S & EMeS k-2

k,

-+ k*

E

(1) products



I

0

02

,

I

04

,

I

0.6

I

l

0.0

I

I

1.0

CMn2'1-', mM-I

2: Inhibition of guanylate cyclase activity by high concentrations of Mgz+ or MnZ+. In (a), the MgGTP concentration was 2 mM. FIGURE

displayed in Table 111. (Numbers were obtained by linear regression analysis of the linear portions of the curves. For the Mn slope replot the curve is linear, with a correlation coefficient of 0.990, between MnZ+concentrations of 0.1 and 0.5 mM, but the slope deviates upward at [Mn2+] = 2 mM as inhibition by excess Mn2+ becomes significant.) V, is independent of which divalent cation (MgZ+ or MnZ+) is present (data not shown). It must be stressed that the mechanism we propose and the kinetic constants we have derived are based upon the assumption that an equilibrium, rather than a steady state, analysis is justifiable, i.e., that eq 3 is valid. At very high concentrations of Mgz+or MnZ+,the enzymatic activity is diminished (Figures 1b and 2). High Mn2+concentrations appear to decrease V, (note the increased 1/V intercept at 0.5 mM MnZ+in Figure la) and increase the apparent K,. The complexity of the system prevents our drawing firm conclusions about the mechanism of the inhibition. For example, GTP could form complexes with two Mg2+ or two MnZ+ions. Such complex formation could lower the substrate concentration, or the MezGTP could act as a competitive inhibitor (Garbers et al., 1975). The fact that the MeZ+concentration at which inhibition becomes apparent is approximately independent of the MeGTP concentrations (Figures l b and 4b) argues against an involvement of MezGTP. Krishnan et al. (1978) have suggested that Ca2+, MnZ+,and Mgz+ inhibit guanylate cyclase in ROS by a common mechanism. This idea seems reasonable; perhaps Caz+ is a physiological regulator and other divalent cations at high concentrations can inhibit by binding to the Ca2+site on the enzyme.

5064

FLEISCHMAN AND DENISEVICH

B IOCH E M ISTR Y

b\

61

4

I

I

I

I

I

I

I

1

/

l

'

"

'

~

'

"

'

~

"

'

'

~

b

\

i i

I 0

I

0

I

I

I

2

3

4

5

C C a C l 2 1 ,mM

FIGURE 3: Calcium inhibition of guanylate cyclase. Closed circles: cyclase activity when the indicated concentration of CaC12was added to a solution containing 1 rnM GTP and 5 mM MgCI2. Open circles: cyclase activity as a function of [CaCI,] when [MgGTP] and [Mg2+] were held constant by the addition of enough extra GTP to recomplex the Mg2+ which had been displaced from the MgGTP complex by the added Ca2+; [MgGTP] was 1.0 mM and [Mg2+] was 4.0 mM. Dashed line: predicted cyclase activity if binding of one CaZ+inactivates the enzyme and [CaZ+][E]/[CaE] = 1.2 X lo-' M. Solid line: predicted cyclase activity if binding of two Ca2+inactivates the enzyme and [CaI2[E]/[Ca2E] = 1.44 X 10" M2.

Ca2+Inhibition. It has been reported that Ca2+stimulates or inhibits guanylate cyclase in ROS (Pannbacker, 1973; Krishnan et al., 1978). In addition, Ca2+ and cGMP seem to exert opposite influences upon some electrophysiological processes in retinal rods (Lipton et al., 1977). GTP4- forms a complex with Ca2+. Garbers et al. (1975) estimated its association constant to be 25 000 M-I, It seemed possible that Ca2+could inhibit cyclase by displacing Mg2+or Mn2+from its complex with GTP, thereby lowering the substrate concentration. The resulting increase in Mg2+or Mn2+concentration could either be inhibitory or stimulatory (Figure 2 ) . Guanylate cyclase activity of axonemes was examined over the range of Ca2+concentrations reported to be stimulatory or inhibitory in ROS. The closed circles in Figure 3 represent cyclase activity in an experiment in which the indicated concentration of CaC12was simply added to a mixture containing axonemes, 4 mM MgC12, and 1 mM GTP. The open circles indicate cyclase activity in an experiment in which just enough additional GTP was added to recomplex the Mg2+which had been displaced from the MgGTP complex by Ca2+. The association constants of MgGTP and CaGTP cited above were used to calculate the appropriate amounts of additional GTP. In this experiment then, the concentrations of MgGTP and Mg2+ were held constant as the total concentration of Ca2+ was varied. Ca2+ inhibits (and does not stimulate) the axoneme guanylate cyclase at all concentrations examined. Clearly Ca2+does not exert its influence by displacing Mg2+ from the MgGTP complex. In the simplest alternative mechanism, CaZ+might completely inhibit cyclase by binding to a regulatory site on the enzyme. The dashed line represents the cyclase activity predicted if binding of a single Ca2+ inactivates the enzyme and [Ca2+][E]/[CaE] = 1.2 mM (this dissociation constant was chosen to fit the data at 50% inhibition). The rather sigmoidal inhibition pattern observed does not conform to the calculated curve. A second mechanism would require the binding of two Ca2+for inhibition. The solid line represents the cyclase activity predicted in this case if [Ca2+]Z[E]/[Ca2E]= 1.44 X M2, again normalizing to

1

I

M

100 CKCI1,mM

IM

200

j

, , , , , , , / , , , , , , , ,

5

0

10

j

15

CM$'l,mM

FIGURE 4: Effect of monovalent cations upon guanylate cyclase activity. (a) Dependence of cyclase activity upon KCI concentration. Reaction mixes contained 1.7 mM MgGTP and 2 mM free Mg2+. (b) Dependence of KCI stimulation of guanylate cyclase upon Mg2+ concentration. Reaction mixes contained 2.0 mM MgGTP. Closed circles, 100 rnM KCI; open circles, no KCI.

lo

-

1

i. i

i

/-

70

8.0

9.0

PH

FIGURE 5 : pH dependence of guanylate cyclase. Reaction mixes contained 20 mM each Tris, Hepes, and Mes, 2 mM GTP, and 1 mM MnCI, or MgCI2. Curves are arbitrarily displaced along the log V axis.

fit the data at 50% inhibition. This time the fit is rather good. This mechanism presupposes that CaE is completely active or that the association constants for binding of the first and second Ca2+ are such that [CaE] is negligible. Effect of Monovalent Cations. Since the binding of acetylcholine to muscarinic cholinergic receptors may activate guanylate cyclase in some cells (Goldberg & Haddox, 1977), we examined the effect of acetylcholine on the axoneme guanylate cyclase. Acetylcholine caused some stimulation. However, since the concentration required to achieve half-maximal stimulation (-0.5 mM) seemed too high to be of physiological significance, we suspected a nonspecific stimulation by salt. Alkali metal salts were in fact found to stimulate the cyclase (Figure 4a). The salt was most effective at suboptimal concentrations of Mg2+ or Mn2+ (Figure 4b). There was no measurable effect of alkali metal salts at Mg2+concentrations higher than those displayed in Figure 4b. p H Dependence. A plot of log I/ vs. pH is presented in Figure 5. The data conform well to Dixon's rules for the behavior of an enzyme whose activity requires that a single acidic group be ionized (Fromm, 1975) (strict application of Dixon's rules would require the use of V, measured at each pH). According to these rules, a plot of log V , against pH should consist of straight line segments of integral slope connected by short curved sections. Slope changes correspond to the ionization of acidic groups; the tangents to the curves above and below the slope change intersect at the pK of the

GUANYLATE CYCLASE KINETICS

acidic group. Integral slope changes imply that a single acidic group is involved. Negative slope changes imply ionization of a group in the ES complex rather than in the free enzyme or substrate. The data in Figure 5 then suggest that the guanylate cyclase can function only when a basic group with a pK near 7.0 (e.g., histidine) in the EMeS complex is not protonated. Again, this is the least conservative interpretation. Complicated equilibria exist among Me2+,MeGTP complexes, and GTP in several states of protonation. A large number of such species whose concentrations are pH dependent are potential substrates or inhibitors of the enzyme. Adenylate Cyclase. The axoneme preparations contained an adenylate cyclase whose sctivity was 1-10% of the activity of the guanylate cyclase, depending upon the concentrations of ATP, GTP, Mn2+,and Mg2+. Adenylate cyclase activity in the presence of 8 mM Mg2+ was 1.1% that of guanylate cyclase in the presence of 4 mM Mn2+;nucleotide triphosphate concentrations were 1 mM in each case. Since a similar adenylate cyclase/guanylate cyclase ratio has been found in ROS (Manthorpe & McConnell, 1975), the two activities seem to have been purified together. This could suggest that cAMP and cGMP are formed by the same enzyme. We had hoped to compare the K , of adenylate cyclase with the KI for inhibition of guanylate cyclase by ATP. However, it was found that ATP at certain concentrations stimulated the guanylate cyclase rather than inhibiting it. This phenomenon has been observed with ROS as well (Krishnan et al., 1978) and deserves further study. Interestingly, Mg2+seems to active the adenylate cyclase more effectively than does Mn2+. Miscellaneous Inhibitors and Activators. Fluoride (2 mM) increased guanylate cyclase activity by less than 10%. Since the axoneme preparations contained microtubules and probably actin (D. Fleischman, M. Denisevich, D. Raveed, and R. G. Pannbacker, unpublished experiments), the effects of colchicine (2 mM) and cytochalaisin B (20 pg/mL) upon the guanylate cyclase were examined. Both were without measurable effect. A number of detergents and chaotropic agents have been employed in attempts to dissociate the guanylate cyclase from the axoneme preparations. During these experiments we have found (Fleischman et al., unpublished experiments) that sodium dodecyl sulfate (0.2%), sodium lauroyl sarcosinate (0.03%), lauryldimethylamine N-oxide (1%), sodium azide (2%), and potassium thiocyanate (2%) all inhibit the axoneme guanylate cyclase by more than 50%. Metrizamide (10%) causes 50% inhibition; sucrose solutions of comparable density are not inhibitory. Discussion In almost every respect the guanylate cyclase of the isolated axoneme-basal apparatus preparation behaves like the cyclase of unfractionated ROS. The single exception is inhibition, rather than stimulation, of cyclase by Ca2+at concentrations below 1 mM. Since there have been conflicting reports about the effect of Ca" on guanylate cyclase in unfractionated ROS, this phenomenon deserves further study. It has recently been reported that ROS isolated with osmotically intact plasma membranes contain a number of soluble proteins (Godchaux & Zimmerman, 1979), and evidence has been presented that a calcium-dependent modulator protein exists in ROS (Liu et al., 1979). It is entirely possible that such proteins could modulate guanylate cyclase in vivo and could be lost during axoneme isolation. The loss of such proteins in the course of ROS isolation could explain the variability in the reported effects of Triton X-100 and exposure to light as well. While our data largely agree with the published descriptions of the behavior of ROS guanylate cyclase, our interpretations

VOL. 18, NO. 23, 1979

5065

differ somewhat. By separately controlling the concentrations of Me2+and MeGTP, we were able to show that free Me2+ (in addition to the Me2+complexed with GTP4-) is required for activity. The reported values of the K, of guanylate cyclase are thus somewhat misleading, since the apparent K , depends upon the concentration of free Me2+. The association constants of the enzyme-metal and enzyme-metalsubstrate complexes are perhaps more useful. Their physical interpretation of course depends upon the validity of our proposed mechanism. This in turn is based upon the assumption that we are justified in using the equilibrium rather than the steady-state method of analysis. At best we can only state that the mechanism is consistent with the data obtained thus far. The value of such experiments may be phenomenological rather than mechanistic; they at least allow us to predict how rapidly cGMP will be made under a variety of conditions. V, is independent of the Me2+concentration and independent of whether Me2+is Mg2+or Mn2+. Earlier experiments had led us to wonder whether Mn2+might be the physiologically active divalent cation. The current experiments demonstrate that Mg2+-activatedcyclase can be almost as active as Mn2+-activatedcyclase at Mg2+and GTP concentrations near plausible in vivo values. We therefore suggest that Mg2+is the physiologically active divalent cation. The apparent cooperativity displayed by ROS guanylate cyclase (Krishnan et al., 1978) can be explained readily since increasing the concentration of MnGTP (when total Mn equals total GTP) also increases the concentration of free Mn2+. As Figure 1 demonstrates, double-reciprocal plots are linear when the concentrations of MnGTP and free Mn2+are controlled independently. The fact that Ca2+ inhibition seems to require the binding of two Ca2+allows cyclase activity to be modulated by small changes in Ca2+concentration. The effective range (1-2 mM) seems quite high, however. It remains to be shown whether such Ca2+concentrations exist at the in vivo location of the guanylate cyclase. Several additional questions remain unresolved. The adenylate cyclase activity of the axonemes and the effects of ATP on cyclase are small and probably are not physiologically significant. Still, they have received only cursory study and might be important under some conditions. Lipton et al. (1977) found that prostaglandin Fk exerts an effect similar to that of cGMP or phosphodiesterase inhibitors upon the electrophysiological properties of retinal rods. Its effect upon guanylate cyclase should be examined, probably in unfractionated ROS or in intact retinas. These authors also reported that cAMP and prostaglandin El exerted effects antagonistic to those of cGMP and prostaglandin F2a. Therefore, the influence of prostaglandin E, on adenylate cyclase perhaps should be examined as well. Acknowledgments We thank Dr. R. G. Pannbacker for valuable discussions and Rob Shapiro, Katherine Leung, and Cindi Jay for assisting with the experiments. References Bensinger, R. E., Fletcher, R. T., & Chader, G. J. (1974) Science 183, 86-87. Bignetti, E., Cavaggioni, A., & Sorbi, R. T. (1978) J . Physiol. (London) 279, 55-69. Bitensky, M. W., Miki, N., Keirns, J. J., Keirns, M., Baraban, J. M., Freeman, J., Wheeler, M. A., Lacey, J., & Marcus, F. R. (1975) Adv. Cyclic Nucleotide Res. 5 , 213-240. Chader, G. J., Herz, L. R., & Fletcher, R. T. (1974a) Biochim. Biophys. Acta 347, 491-493.

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