Ozols J
Department of Biochemistry, University of Connecticut Health Center, Farmington 06032.
J Biol Chem. 1987 Nov 5;262(31):15316-21.
A glycoprotein having a subunit weight of approximately 60,000 was isolated from rabbit liver microsomes. It is a predominant component of the hepatic microsomal membrane and reacts rapidly with diisopropylphosphorofluoridate (DFP), resulting in the loss of enzymatic activity toward artificial substrates such as acyl esters of o-nitrophenols. Automated Edman degradation of this protein together with sequence analysis of peptides provided the NH2-terminal sequence of some 70 residues as follows: His-Pro-Ser- Ala-Pro-Pro-Val-Val-Asp-Thr-Val-Lys-Gly-Lys-Val- Leu-Gly-Lys-Phe-Val-Ser-Leu-Glu-Gly-Phe-Ala-Gln- Pro-Val-Ala-Val-Phe-Leu-Gly-Val-Pro-Phe-Ala-Lys- Pro-Pro-Leu-Gly-Ser-Leu-Arg-Phe-Ala-Pro-Pro-Gln- Pro-Ala-Glu-Ser-Trp-Ser-His-Val-Lys-Asn (CHO)- Thr-Thr-Ser-Tyr-Pro-Pro-Met-Cys-Ser-Ser. A carbohydrate attachment was identified at asparaginyl residue 61. The COOH-terminal peptide of the protein was isolated from two independent enzymatic digests, and its sequence was established as Arg-Glu-Thr-Glu-His-Ile-Glu-Leu. In order to isolate the DFP binding peptide, liver microsomes were labeled with [3H]DFP and the 60-kDa protein containing covalently bound DFP isolated in pure form. Following reduction and carboxymethylation, the DFP-labeled protein was fragmented with trypsin and the digest subjected to gel filtration. Digestion of the labeled peptide preparations with chymotrypsin followed by chromatography of the digest yielded two diisopropylphosphoryl (DIP) peptides. Automated Edman degradation of these peptides provided the following amino acid sequences: Gly-Glu-DIPSer- Ala-Gly-Gly-Gln-Ser-Val-Ser-Ile-Leu-Leu-Leu-Ser- Pro and Thr-Val-Ile-Gly-Asp-DIPHis-Gly-Asp-Glu-Ile-Phe. The active site serine peptide of the 60-kDa protein shows some 70% similarity to the active center region of choline esterases. While the postulated active histidyl residue in choline esterases has not been identified, it is proposed that the DFP binding histidine of the 60-kDa protein corresponds to His-438/440 of choline esterases.
从兔肝微粒体中分离出一种亚基分子量约为60,000的糖蛋白。它是肝微粒体膜的主要成分，能与二异丙基氟磷酸酯（DFP）迅速反应，导致对诸如邻硝基苯酚酰酯等人工底物的酶活性丧失。对该蛋白进行自动Edman降解并结合肽段序列分析，得到了约70个残基的NH2末端序列如下：His-Pro-Ser-Ala-Pro-Pro-Val-Val-Asp-Thr-Val-Lys-Gly-Lys-Val-Leu-Gly-Lys-Phe-Val-Ser-Leu-Glu-Gly-Phe-Ala-Gln-Pro-Val-Ala-Val-Phe-Leu-Gly-Val-Pro-Phe-Ala-Lys-Pro-Pro-Leu-Gly-Ser-Leu-Arg-Phe-Ala-Pro-Pro-Gln-Pro-Ala-Glu-Ser-Trp-Ser-His-Val-Lys-Asn（CHO）-Thr-Thr-Ser-Tyr-Pro-Pro-Met-Cys-Ser-Ser。在天冬酰胺残基61处鉴定出一个碳水化合物连接位点。该蛋白的COOH末端肽段从两次独立的酶切消化产物中分离出来，其序列确定为Arg-Glu-Thr-Glu-His-Ile-Glu-Leu。为了分离DFP结合肽段，用[3H]DFP标记肝微粒体，然后以纯形式分离出含有共价结合DFP的60 kDa蛋白。经过还原和羧甲基化后，用胰蛋白酶切割DFP标记的蛋白，并对消化产物进行凝胶过滤。用胰凝乳蛋白酶消化标记的肽段制剂后，对消化产物进行色谱分析，得到两个二异丙基磷酰基（DIP）肽段。对这些肽段进行自动Edman降解，得到以下氨基酸序列：Gly-Glu-DIPSer-Ala-Gly-Gly-Gln-Ser-Val-Ser-Ile-Leu-Leu-Leu-Ser-Pro和Thr-Val-Ile-Gly-Asp-DIPHis-Gly-Asp-Glu-Ile-Phe。60 kDa蛋白的活性位点丝氨酸肽段与胆碱酯酶的活性中心区域有大约70% 的相似性。虽然胆碱酯酶中假定的活性组氨酸残基尚未确定，但有人提出60 kDa蛋白的DFP结合组氨酸对应于胆碱酯酶的His-438/440。
