Baker R R, Chang H Y
Department of Medicine, University of Toronto, ON, Canada.
Biochim Biophys Acta. 1997 Apr 1;1345(2):197-206. doi: 10.1016/s0005-2760(96)00178-6.
Neuronal nuclear fraction N1 was isolated from cerebral cortices of 15-day-old rabbits, and nuclear subfractions prepared, in order to study the location of nuclear lyso platelet-activating factor (lyso-PAF) acetyltransferase and alkylglycerophosphate (AGP) acetyltransferase, and factors that affect the loss of these two nuclear activities. Subfractionation of prelabelled N1 indicated that the nuclear envelope had the highest percentage of the radioactive acetylated products alkylacetylglycerophosphate (AAGP) and PAF, and the distribution of these phospholipids reflected phospholipid distributions in the nuclear subfractions. The majority (95%) of radioactive AAGP and PAF was also recovered in Triton X-100 extracts of prelabelled nuclei, suggesting that these acetylated lipids are located in nuclear membranes rather than in the nuclear matrix/chromatin. Of the nuclear subfractions, the envelope had the highest AGP and lyso-PAF acetyltransferase specific activities which were close to corresponding values seen in the parent N1 fraction. Thus the nuclear AGP and lyso-PAF acetyltransferases were principally localized to the nuclear membranes. Differentials in activity loss were seen for the two acetyltransferase activities. In the nuclear envelope fractions, the lyso-PAF acetyltransferase was the more susceptible to oxidation reactions which could be reversed or blocked by the use of reducing agents. In preincubations, N1 showed greater losses in lyso-PAF acetyltransferase activity than in AGP acetyltransferase activity, losses which were not attributable to oxidation. Addition of cytosolic fraction S3 to preincubations promoted losses for each acetyltransferase in N1, and gave evidence for cytosolic and endogenous nuclear contributions to the activity loss. Addition of okadaic acid to the preincubations did not prevent the decline of either acetyltransferase in intact nuclei, but did diminish the loss of nuclear lyso-PAF acetyltransferase activity promoted by S3 addition, and also blocked the loss of this acetyltransferase seen in preincubations of isolated nuclear envelopes. This suggests that nuclear lyso-PAF acetyltransferase is susceptible to okadaic acid-sensitive nuclear and cytosolic protein phosphatase activities, while AGP acetyltransferase may lose activity by the action of other phosphatases or by other mechanisms within the nucleus.
从15日龄兔的大脑皮层中分离出神经元核组分N1，并制备核亚组分，以研究核溶血血小板活化因子（lyso-PAF）乙酰转移酶和烷基甘油磷酸（AGP）乙酰转移酶的定位，以及影响这两种核活性丧失的因素。对预先标记的N1进行亚组分分离表明，核膜中放射性乙酰化产物烷基乙酰甘油磷酸（AAGP）和PAF的百分比最高，这些磷脂的分布反映了核亚组分中的磷脂分布。大部分（95%）放射性AAGP和PAF也在预先标记的细胞核的Triton X-100提取物中回收，这表明这些乙酰化脂质位于核膜中而非核基质/染色质中。在核亚组分中，核膜具有最高的AGP和lyso-PAF乙酰转移酶比活性，接近在亲本N1组分中观察到的相应值。因此，核AGP和lyso-PAF乙酰转移酶主要定位于核膜。两种乙酰转移酶活性在活性丧失方面存在差异。在核膜组分中，lyso-PAF乙酰转移酶更容易受到氧化反应的影响，使用还原剂可使其逆转或阻断。在预孵育中，N1中lyso-PAF乙酰转移酶活性的丧失比AGP乙酰转移酶活性的丧失更大，这种丧失不归因于氧化。向预孵育中添加胞质组分S3会促进N1中每种乙酰转移酶的活性丧失，并证明了胞质和内源性核成分对活性丧失的作用。向预孵育中添加冈田酸并不能阻止完整细胞核中任何一种乙酰转移酶的活性下降，但确实减少了添加S3所促进的核lyso-PAF乙酰转移酶活性的丧失，并且也阻断了在分离的核膜预孵育中观察到的这种乙酰转移酶的活性丧失。这表明核lyso-PAF乙酰转移酶易受冈田酸敏感的核和胞质蛋白磷酸酶活性的影响，而AGP乙酰转移酶可能通过其他磷酸酶的作用或通过细胞核内的其他机制丧失活性。
