Land S J, Zukowski K, Lee M S, Wang C Y, King C M
Department of Chemical Carcinogenesis, Michigan Cancer Foundation, Detroit 48201.
Carcinogenesis. 1993 Jul;14(7):1441-9. doi: 10.1093/carcin/14.7.1441.
Rat liver cytosol is capable of N-acetylation of arylamines, O-acetylation of arylhydroxylamines and N,O-acyltransfer of arylhydroxamic acids. The objective of this study was to characterize the enzyme(s) responsible for these reactions. A partially purified acetyltransferase preparation from rat liver cytosol was used to produce five mouse monoclonal IgG1S that bound to acetyltransferase on Western blots and affected one or more of the acetylation reactions. Two immunoaffinity columns were prepared by covalently cross-linking monoclonal antibodies to protein A-Sepharose. The first column permitted recovery of a single, immunoreactive 32 kDa protein that was capable of catalyzing all three reactions, while the second removed all three acetylation activities from a partially purified enzyme preparation and yielded a single, immunoreactive 32 kDa protein on elution. The harsh conditions necessary for elution from the latter column precluded recovery of an active enzyme. Although Western blots from SDS-PAGE at all stages of purification showed a single 32 kDa protein, purification was associated with the production of multiple, immunochemically reactive peptides with higher pIs. Direct enzymatic assays of these immunochemically reactive components after isoelectric focusing on polyacrylamide gels demonstrated that a single 32 kDa, pI 4.5 protein is capable of all three cytosolic acetylation activities. A second 32 kDa protein, pI 4.8, was able to carry out N-acetylation but not N,O-acetyltransfer. Immunoreactive components with pIs > 4.8 that were formed during purification were catalytically inactive. However, isoelectric focusing in solution of cytosolic preparations that had been subjected only to gel filtration gave a single 32 kDa immunoreactive peptide that was capable of all three acetylation reactions. Buffer concentration differentially affected the enzymatic activities of the enzyme, i.e. as a pH 7.4 buffer was decreased from 50 mM sodium pyrophosphate to 2 mM, the ability to N-acetylate arylamines was lowered while the abilities for O-acetylation and N,O-acetyltransfer were unaffected. It has been shown that a single 32 kDa protein carries out all of the acetylation reactions in rat liver cytosol. Although it cannot be ruled out that other similarly sized and closely related enzymes that share antigenic sites are also capable of these acetylation reactions, these studies suggest that instabilities of the major peptide responsible for these activities, as reflected in changes in isoelectric point, may be responsible for changes in the enzymatic potentials of this peptide.
大鼠肝细胞溶胶能够对芳胺进行N - 乙酰化、对芳基羟胺进行O - 乙酰化以及对芳基异羟肟酸进行N,O - 酰基转移。本研究的目的是鉴定负责这些反应的酶。使用从大鼠肝细胞溶胶中部分纯化的乙酰转移酶制剂来制备五种小鼠单克隆IgG1，这些单克隆抗体在蛋白质印迹上与乙酰转移酶结合，并影响一种或多种乙酰化反应。通过将单克隆抗体与蛋白A - 琼脂糖共价交联制备了两种免疫亲和柱。第一根柱子可回收一种单一的、具有免疫反应性的32 kDa蛋白质，该蛋白质能够催化所有三种反应，而第二根柱子则从部分纯化的酶制剂中去除了所有三种乙酰化活性，并在洗脱时产生了一种单一的、具有免疫反应性的32 kDa蛋白质。从后一根柱子洗脱所需的苛刻条件妨碍了活性酶的回收。尽管在纯化的各个阶段SDS - PAGE的蛋白质印迹显示有单一的32 kDa蛋白质，但纯化过程伴随着产生多个具有更高pI值的免疫化学反应性肽段。在聚丙烯酰胺凝胶上进行等电聚焦后，对这些免疫化学反应性成分进行直接酶活性测定表明，一种单一的32 kDa、pI 4.5的蛋白质能够进行所有三种胞质乙酰化活性。另一种32 kDa、pI 4.8的蛋白质能够进行N - 乙酰化，但不能进行N,O - 乙酰转移。纯化过程中形成的pI > 4.8的免疫反应性成分没有催化活性。然而，仅经过凝胶过滤的胞质制剂在溶液中进行等电聚焦时，产生了一种单一的32 kDa免疫反应性肽段，该肽段能够进行所有三种乙酰化反应。缓冲液浓度对该酶的酶活性有不同影响，即当pH 7.4的缓冲液从50 mM焦磷酸钠降至2 mM时，芳胺N - 乙酰化的能力降低，而O - 乙酰化和N,O - 乙酰转移的能力不受影响。已经表明，一种单一的32 kDa蛋白质在大鼠肝细胞溶胶中进行所有的乙酰化反应。尽管不能排除其他大小相似且抗原位点相关的酶也能进行这些乙酰化反应，但这些研究表明，负责这些活性的主要肽段的不稳定性，如等电点的变化所反映的，可能是该肽段酶活性变化的原因。
