Dannenberg A J, Kavecansky J, Scarlata S, Zakim D
Department of Medicine, Cornell University Medical College, New York, New York 10021.
Biochemistry. 1990 Jun 26;29(25):5961-7. doi: 10.1021/bi00477a012.
Treatment of microsomes at pressures as high as 2.25 kbar led to an apparent irreversible activation of UDP-glucuronylsyltransferase 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 degrees 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 degrees 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-glucuronosyltransferase 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.(ABSTRACT TRUNCATED AT 250 WORDS)
在高达2.25千巴的压力下处理微粒体，当压力释放时会导致UDP-葡萄糖醛酸基转移酶出现明显的不可逆激活。压力释放后测得的活性反映出，该酶对压力的反应似乎是不连续的，因为在压力低于1.2千巴时，任何制剂都未观察到激活现象。此外，激活作用还依赖于温度。在2.25千巴时，最大激活发生在约12摄氏度；在10分钟内，较高或较低温度下的激活程度都较小。激活作用也依赖于时间。在2.25千巴和9摄氏度时，最大激活需要90分钟的压力处理。在较低压力下，激活似乎发生得更慢。压力诱导的激活与酶对UDP-N-乙酰葡糖的变构激活敏感性丧失以及动力学模式从非米氏-门坦型转变为米氏-门坦型有关。压力不会激活之前通过向微粒体中添加去污剂而最大程度激活的酶。压力也不会激活重构到二油酰磷脂酰胆碱单层囊泡中的纯UDP-葡萄糖醛酸基转移酶。压力处理不会使微粒体中的UDP-葡萄糖醛酸基转移酶释放到水中。压力对掺入微粒体中的荧光探针的极化和准分子/单体形成有持续影响，当压力释放时，这些特性基本恢复到1个大气压下的值。在2.2千巴下的活性测量表明，微粒体中UDP-葡萄糖醛酸基转移酶的压力诱导激活通过两个无活性的中间体发生，并且酶的激活状态是在压力释放期间/之后产生的。（摘要截断于250字）
