Organization of microsomal UDP-glucuronosyltransferase. Activation by treatment at high pressure

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Abstract
Treatment of microsomes at pressures as high as 2.25 kbar led to an apparent irreversible activation of UDP-glucuronosyltransferase when pressure was released. The response of the enzyme to pressure, as reflected by activity measured after release of pressure, appeared to be discontinuous in that no activation was seen for any preparation at pressures less than 1.2 kbar. In addition, activation was temperature dependent. Maximum activation at 2.25 kbar occurred at about 12.degree. C; the extent of activation in 10 min was less for either higher or lower temperatures. Activation was also time dependent. Maximum activation at 2.25 kbar and 9.degree. C required 90 min of pressure treatment. Activation appeared to occur more slowly at lower pressure. Pressure-induced activation was associated with a loss of sensitivity of the enzyme to allosteric activation by UDP-N-Ac-Glc and a conversion of the kinetic pattern from non-Michaelis-Menten to Michaelis-Menten. Pressure did not activate enzyme that had previously been activated maximally by adding detergent to microsomes. Pressure also did not activate pure UDP-glucuronosyltransferse reconstituted into unilamellar vesicles of dioleoylphosphatidylcholine. Pressure treatment did not release UDP-glucuronosyltransferase from microsomes into water. Pressure had a continuous effect on the polarization and excimer/monomer formation of fluorescent probes incorporated into microsomes, and the properties returned essentially to their values at 1 atm when pressure was released. Measurements of activity at 2.2 kbar showed that pressure-induced activation of UDP-glucuronosyltransferase in microsomes occurred via two intermediates that were inactive and that the activated state of the enzyme was generated during/after release of pressure. These experiments also showed that the temperature dependence of pressure-induced activation was due primarily to stabilization of the native state of UDP-glucuronosyltransferase at T > 20.degree. C.