Effects of Metals and Nucleotides on the Inactivation of Sea Urchin Sperm Guanylate Cyclase by Heat and N-Ethylmaleimide”

SUMMARY Preincubation of sea urchin sperm guanylate cyclase at 35, 37, 40, or 43” resulted in inactivation. Various metals were able to protect guanylate cyclase against heat inactivation. Estimated binary enzyme-metal dissociation constants for Mn2+, Fe*+, La3+, Ca2+, Ba2+, Mg2+, Co2+, and Ni2+ were 123,361, 5.5, 692,984,335,79, and 47 pM, respectively. Extrapolated rates of enzyme denaturation in the presence of saturating concentrations of metal divided by the rates of enzyme denaturation in the absence of metal gave values of 0.13, 0.08, -0.1, 0.30, 0.59, 0.66, 0.28, and 0.42 for Mn2+, Fez+, La3+, Cat+, BaZf, Mg2+, Co2+, and Ni2+, respectively. GTP, MgGTP, and SrGTP protected the enzyme only slightly against heat inactivation, but CaGTP and MnGTP protected substantially. Neither CaGTP nor MnGTP protected maximally, however, unless the metal concentration exceeded that of GTP. At fixed free Mn*+ or free Ca*+ concentrations, protection

Various metals were able to protect guanylate cyclase against heat inactivation. Estimated binary enzyme-metal dissociation constants for Mn2+, Fe*+, La3+,Ca2+,Ba2+,Mg2+,Co2+,and Ni2+ were 123,361,5.5,692,984,335,79, and 47 pM, respectively. Extrapolated rates of enzyme denaturation in the presence of saturating concentrations of metal divided by the rates of enzyme denaturation in the absence of metal gave values of 0.13, 0.08, -0. 1,0.30,0.59,0.66,0.28,and 0.42 for Mn2+,Fez+,La3+,Cat+,BaZf,Mg2+,Co2+,and Ni2+,respectively respect to Mn2+ at fixed GTP concentrations, it has been suggested that the enzyme binds free metal in addition to MnGTP (1, 2) ; enzyme inhibition by free GTP, however, also could explain these results. The enzyme displays positive cooperative kinetic patterns with respect to MnGTP at fixed free Mn2+ concentrations, which suggests that MnGTP serves as both an activator and a substrate for the enzyme (1). Guanylate cyclase activity from sea urchin sperm and other sources also can be increased by the addition of Ca2+ in the presence of hln2+ (1,(3)(4)(5). The apparent complex interactions of metals and nucleotides with sea urchin sperm guanylate cyclase have been examined further in this study by subjection of the enzyme to denaturation conditions.

Enzyme
Preparation-Particulate enzyme was prepared as described in another paper (6).
Triton X-100 previously has been shown to disperse and increase the apparent activity of guanylate cyclase from particulate fractions of sea urchin sperm and other sources (l-5). Triton-dispersed enzyme preparations were made by the following procedures: Triton X-100 (final concentration 1%) was added to suspensions of washed sperm particles prepared as described in another paper (6). The suspension then was sonicated (approximately 15 s) and centrifuged at 100,000 X g for 1 hour. Greater than 90% of the enzyme activity was recovered in the supernatant fraction. The specific activity of these preparations ranged from 50 to 100 nmol of cyclic GMP' formed min-1 mg of protein-l at 30" and pH 7.8.
Assay-The usual assay mixture contained 32 mM triethanolamine (TEA) buffer at pH 7.8, 0.8 mM dithiothreitol, 8 mM theophylline, 8 mM sodium azide (NaNa), 6.5 mM MnC12, and 0.32 mM GTP containing 1 to 3 X 106 dpm of ["H]GTP in a volume of 0.5 to 0.6 ml. Assays were done at 30", and the determination of the cyclic [3H]GMP formed was carried out as described previously (6).

Preincubalion
Conditions-The conditions of each preincubation are described in the corresponding figure legend. Controls were run for all preincubation conditions. The control samples in heat denaturation experiments were preincubated at O-2"; those in Nethylmaleimide experiments were preincubated at O-2" in the absence of N-ethylmaleimide or in the presence of N-ethylmaleimide with excess dithiothreitol.
The volume of preincubation mixture (40 ~1) transferred to the assay mixture represented 5 to 10% of the assay volume. where E = free enzyme, A = effector molecule, EA = enzyme-A complex, K,+ = binary enzyme-A dissociation constant, kl = rate of denaturation of E, kt = rate of denaturation of EA, and D = denatured enzyme. If the concentration of enzyme is assumed to be much lower than the concentration of A, and the equilibration between A and enzyme is assumed to be more rapid than the enzyme denaturation rate, the following equation derived by Scrutton and Utter (7) applies, where V, and Vo are the inactivation rates in the presence and absence of A, respectively. When V,,/V, is plotted against (1 - the slope of the line is Kd and the intercept is kdkl.

Materials
Metals and nucleotides were purchased from the sources described in another paper (3). N-Ethylmaleimide was purchased from Eastman Kodak.

Inactivation of Enzyme by Heat-I'reincubation
of sea urchin sperm guanylate cyclase at various temperatures resulted in initial first order rates of inactivation ( Fig. 1). The values of t I,2 for inactivation were approximately 35, 14, 5, and 1.5 min at 35, 37, 40, and 43", respectively. Arrhenius plots of the initial rates of inactivation between 35 and 43" were straight, suggesting that thermodynamic properties of the enzyme inactivation do not change between these temperatures. The inclusion of 1% Triton X-106 labilized the enzyme to heat, decreasing the h/z for inactivation at 37" from 11.5 to 4 min. The rate of heat inactivation was also influenced by pH. The first order rates of inactivation at 37" at various pH values were as follows: pH 6.9, 0.066 min-l; pH 7.2, 0.081 min-I; pH 7.4, 0.115 min-I; and pH 7.6, 0.141 min-i. Thus, sea urchin sperm guanylate cyclase is more stable at slightly acid pH despite optimal catalytic activity at distinctly alkaline pH values (8) and 500 pg of protein in a volume of 0.5 ml. 2483 rates of enzyme inactivation also were observed in the presence of various metals. Guanylate cyclase from sea urchin sperm is dispersed by Triton X-100 and remains in the supernatant fraction after centrifugation at 100,000 x g for 1 hour (2, 8). Mn2+ protected Triton-dispersed guanylate cyclase against heat inactivation, but the degree of protection declined with repeated freezing and thawing (Fig. 2). When the data shown in Fig. 2 were replotted according to Equation 1, Kd was found to increase from 0.22 mM after one freeze-thaw to 0.4 mM after six freezethaws. When Triton-dispersed preparations were stored at -70" for 1 to 2 months, the estimated & for Mnz+ increased to 1.2 mM. These results suggest that conformational changes occur upon freezing, thawing, or storage of the Triton-dispersed enzyme preparations, such that afhnity of the enzyme for hIn2+ decreases. Because of these effects of freezing and thawing, both the particulate and Triton-dispersed enzyme preparations were routinely frozen and thawed only one time.
Protective effects of Mn2+ and several other metals were compared in preliminary experiments in the presence or absence of Triton. There was no appreciable effect of Triton on metal protection except with Las+, which was the most protective metal tested in the absence of Triton but was completely ineffective in the presence of detergent (not shown).
K,j and k2/kl values for several metals were determined in experiments carried out in the absence of Triton. An example of how metal protection data were treated is shown in Fig. 3 at a protein concentration of 2.5 mg ml-i and containing 0.5yo Triton X-100 and 50 mM TEA buffer at pH 7.0 were frozen rapidly in a mixture of Dry Ice and alcohol and thawed to a temperature of 0 to 3" for the number of times shown in the figure. The preincubation volume of 0.58 ml contained 43 mM TEA buffer at pH 7.8,1'% Triton X-100,8.5 mM dithiothreitol, 100 pg of protein and various concentrations of Mn2+. After preincubation at 37" for 10 min, 40 ~1 of the preincubation mixture was assayed as described under "Methods." vertical lines represent standard deviations.
The inset represents data replotted according to Equation 1. The estimated Kd was 123 PM, and kz/kl was 0.13. To determine if metals acted in an additive or synergistic manner to protect the enzyme against heat, Ca*, Mg*+, and Ba2+ (0.05 to 1.5 mM) were tested in combination with 0.39 mu Mn2+, which protected the enzyme to a greater extent than did t,he highest concentrations of the other metals. There were no additive or synergistic effects of the metal combinations; in fact, the combined effects of the ions were either no greater or  Table II. Initial first order rates of inactivation were again observed. MnGTP and MnATP protected the enzyme to a greater extent than did Mn*+ or nucleotides alone. FeGTP and FeATP protected to about the same degree as did Fe*+ alone, and CaATP and MgATP provided only slightly more protection than did metal or ATP alone. Although neither GTP nor Ca*+ provided appreciable protection, CaGTP provided substantial protection, and with 0.43 mM CaGTP, some activation of the enzyme occurred. This CaGTP activation was not due to transfer of Ca*+ to the assay mixture since all preincubations were compared to control preincubations run at O-2". The difference between the protective abilities of CaGTP and CaATP apparent from Table II was not a fortuitous result of the concentrations selected. The protection by CaGTP and CaATP was studied further as a function of the calcium-nucleotide concentration at a fixed free Ca2+ concentration of 0.7 mM (Fig. 4). These experiments clearly demonstrated a marked difference between the abilities of CaATP and CaGTP to protect the enzyme against heat. The apparent sigmoidicity of the CaGTP protection curve could indicate multiple binding sites for CaGTP.
Similar experiments were done with MnATP and MnGTP at a fixed concentration of free J:n*+ (Fig. 5). The results with MnGTP were similar to t,hose seen with CaGTP, in that a sigmoidal curve suggesting multiple binding sites was observed. MnATP provided slight protection at concentrations less than 20 /.m.
Experiments were next designed to determine if free Mn*+ was required for protection by MnGTP. GTP was fixed at 0.43 mM (a concentration of MnGTP giving maximal protection in the presence of 0.28 mM free M&), and the Mn*+ concentration was varied (Fig. 6). Complete protection by MnGTP occurred only After preincubation at 0" for 90 min, 50 ~1 of the preincubation mixture was transferred to an assay mixture containing 7 mM dithiothreitol and assayed for guanylate cyclase activity as described under the "Methods." when the Mn*+ concentration exceeded the GTP concentration. Similarly, maximum protection by CaGTP was observed only when Ca2+ concentrations exceeded those of GTP (data not shown).
When AD1 or ATP concentrations were fixed, various metals were added, and the enzyme protection then measured, the behavior was quite different from that seen with GTP (data not shown). Although the combination of ADP or ATP with Mn2+, Ca2+, or Mg*+ protected to a greater degree than did the free metals alone, there was no further increase in protection when concentrations of the metals exceeded those of ADP or ATP. Inactivation of Enzyme with N-EthylmaleimioS-Sea urchin sperm guanylate cyclase was very susceptible to inactivation by N-ethylmaleimide or p-hydroxymercuribenzoate. Dithiothreitol in excess of either agent completely protected the enzyme against inactivation.
Inactivation rates in the presence of N-ethylmaleimide concentrations greater than 20 mM followed pseudofirst order kinetics at 0".
In contrast to their abilities to protect guanylate cyclase against inactivation by heat, neither Ca"+ nor Mn*+ provided any detectable protection against inactivation by N-ethylmaleimide when the metals were present in concentrations from 0.1 to 3.0 rnM. Protection against N-Ethylmaleimide-induced Inactivation by Metal-Nucleotides-CaGTP provided substantial protection against N-ethylmaleimide whereas MnGTP, in contrast to its affects with heat, protected only slightly, if at all (Fig. 7). CaGDP and CaGMP did not substantially protect against N-ethylmaleimide .
MnATP provided more protection against N-ethylmaleimide than did MnGTP (Fig. 8), and as observed with heat inactivation, MnATP did not further protect when MnZ+ concentrations exceeded the ATP concentrations.
Both CaGTP and CaATP protected against N-ethylmaleimide in the absence of substantial free Ca2+, but CaGTP was more potent. CaGTP was also much more potent than MnGTP in protecting against N-ethylmaleimide (note scale difference on figure). Concentrations of MnGTP at least 1 to 2 orders of magnitude greater than those of CaGTP Mn*+(AM) Co*+ (AMI FIG. 8. Protection by metal-nucleotide chelates against Nethylmaleimide inactivation of sea urchin sperm guanylate cyclase. Preincubation mixtures (0.52 ml) contained 38 mM TEA (pH 7.8), 16 mM or no N-ethylmaleimide, 1% Triton X-100, 100 pg of protein, and the concentration of ATP, GTP, and metals shown in the figure. After preincubation for 90 min at O-2", 40.~1 aliquots were transferred to an assay mixture (0.54 ml) containing 7 mM dithiothreitol and assayed as described under the "Methods." were required to give equivalent protection. The estimated Kd for CaGTP was 40 PM, and that for MnATP was 170 C(M; no attempt was made to determine dissociation constants for CaATP and MnGTP. DISCUSSION

REFERENCES
Protection of an enzyme against heat implies enzyme-effector molecule interaction. While protection of membrane-bound enzymes by effector molecules could involve nonspecific membrane stabilization phenomena, the protection data presented here are consistent with kinetic and other data (1),2 and suggest that the enzyme stabilization patterns observed reflect direct molecular interactions with the enzyme. The estimated Kd of 123 to 210 PM for enzyme-Mn2+ agrees closely with estimates of dissociation constants of about 320 PM from kinetic data (9). This suggests that protection of the enzyme by Mn2+ and activation of the enzyme by Mn* involve the same binding site. The failure of Ca2+, Ba2+, and Mgz+ to act additively with Mn2+ in protecting the enzyme against heat inactivation suggests that these metals bind at one site.