Factors Affecting the Activity of Guanylate Cyclase in Lysates of Human Blood Platelets

Abstract

<p>The mechanism by which physiological stimuli increase cyclic GMP formation in platelets or in other cells is unknown. Agents that promote the formation of cyclic GMP in intact cells have in general not been found to stimulate the activity of guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2.] in broken cell preparations. Therefore, possible mechanisms for the activation and control of guanylate cyclase activity in platelets were investigated in this thesis.</p> <p>It was found that over 90% of the total guanylate cyclase activity is present in supernatant fractions of hypotonically lysed platelets. Platetet particulate fractions contained no guanylate cyclase activity that could not be accounted for by contaminating soluble enzyme, suggesting that physiological aggregating agents may increase cyclic GMP levels in intact platelets through the effects of intermediary factors. Because of the possibility that soluble as well as particulate factors may be involved in the control of enzyme activity, whole platelet lysate was used in studies of the properties and activation of guanylate cyclase.</p> <p>Under optimal ionic conditions (4.0 mM-MnCl₂), the specific activity of guanylate cyclase in fresh platelet lysates was about 10 nmol of cyclic GMP formed/20 min per mg of protein at 30°C, which is higher than that of any other mammalian cells or tissues studied. Activity was 15% of optimum with 10.0 mM-MgCl₂ and negligible with 4.0 mM-CaCl₂. Synergism between MnCl₂ and MgCl₂ or CaCl₂ was observed when [MnCI₂] ≤ [GTP]; under more physiological ionic conditions (Mg²⁺ present), micromolar concentrations of Ca²⁺ stimulated enzyme activity by about 50%.</p> <p>Lower than optimal specific activities were obtained in assays containing large volumes of platelet lysate, owing to the presence of inhibitory factors that could be removed by ultrafiltration. Adenine nucleotides and glutathione accounted for less than 50% of the inhibitory activity. The combined effects of inhibitory factors and of suboptimal ionic conditions are likely to lower the guanylate cyclase activity in intact platelets to almost negligible values in the absence of activating factors.</p> <p>Dithiothreitol (5.0 mM) and N-ethylmaleimide (0.1 mM) inhibited the activity of platelet lysate by about 70 and 50%, respectively. Preincubation of lysate for 60 min at 37°C increased guanylate cyclase activity on average by 225%. This effect could be blocked with dithiothreitol or N-ethylmaleimide, but dithiothreitol could not fully reverse activation once it had occurred. Oxidants such as 4,4'-dithiodipyridine (0.04 mM), diamide (0.4 mM) and tert-butylhydroperoxide (1.0 mM) increased enzyme activity on average, by 40, 87 and 165% respectively. Neither diamide nor tert-butylhydroperoxide had an effect on enzyme that had been preincubated or treated with N-ethylmaleimide.</p> <p>Sodium azide (10.0 mM) increased guanylate cyclase activity by an average of 335%; this effect was both time- and temperature-dependent. Activation by sodium azide was not prevented by dithiothreitol. Sodium nitroprusside (1.0 mM) increased enzyme activity by about 1000%; this effect could be blocked by preincubation or by tert-butylhydroperoxide, but not by either dithiothreitol or N-ethylmaleimide.</p>