The effects of high pressure on the kinetic properties of microsomal UDP-glucuronosyltransferase (assayed with 1-naphthol as aglycon) were studied in the range of 0.001-2.2 kbar to clarify further the basis for regulating this enzyme in untreated microsomes. Activity changed in a discontinuous manner as a function of pressure. Activation occurred at pressure as low as 0.1 kbar, reaching one of two maxima at 0.2 kbar. As pressure was increased above 0.2 kbar, activity decreased, reaching a minimum at about 1.4 kbar followed by a second activation. The pathway for activation at pressure > 1.4 kbar was complex. The immediate effect of 2.2 kbar was nearly complete inhibition of activity. The inhibited state relaxed, however, over about 10 min (at 10°C), to a state that was activated as compared with enzyme at 0.001 kbar or enzyme at pressures between 1.4 and 2.2 kbar, which was the highest pressure we could test. Examination of the detailed kinetic properties of UDP-glucuronosyltransferase indicated that the effects of pressure were due to selective stabilization of unique functional states of the enzyme at 0.2 and 2.2 kbar. Activation at 0.2 kbar was reversible when pressure was released. This was true as well for activation at pressure > 1.4 kbar, but after prolonged treatment at 2.2 kbar, UDP-glucuronosyltransferase became activated irreversibly on release of pressure. The process by which prolonged treatment at 2.2 kbar led to permanent activation of UDP-glucuronosyltransferase after release of pressure was not reflected, however, by time-dependent changes in the functional state of UDP-glucuronosyltransferase at this pressure. Thus, appearance of the unique functional state of UDP-glucuronosyltransferase at 2.2 kbar occurred within about 10 min after reaching this pressure, and this state of the enzyme persisted for as long as 60 min (longest time studied). By contrast, the distribution of UDP-glucuronosyltransferase between native state and permanently activated state, after release of 2.2 kbar, shifted in favor of the latter with increasing time of treatment at 2.2 kbar. When pressure was released after 60 min at 2.2 kbar, about 80% of UDP-glucuronosyltransferase became permanently activated. We have interpreted this result to mean that treatment at high pressure perturbs interactions between UDP-glucuronosyltransferase and an undefined regulatory factor in microsomes that is important for maintaining the enzyme in its native conformational state.
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