Eaton D L, Malloy B E, Tsai S P, Henzel W, Drayna D
Department of Cardiovascular Research, Genentech, Inc., South San Francisco, California 94080.
J Biol Chem. 1991 Nov 15;266(32):21833-8.
A novel plasminogen-binding protein has been isolated from human plasma utilizing plasminogen-Sepharose affinity chromatography. This protein copurified with alpha 2 antiplasmin when the plasminogen affinity column was eluted with high concentrations of epsilon-aminocaproic acid (greater than 20 mM). Analysis by sodium dodecyl sulfate suggests this protein has an apparent Mr of 60,000. The amino-terminal amino acid sequence showed no similarity to other protein sequences. Based on the amino-terminal amino acid sequence, oligonucleotide probes were designed for polymerase chain reaction primers, and an approximately 1,800 base pair cDNA was isolated that encodes this Mr 60,000 protein. The deduced amino acid sequence reveals a primary translation product of 423 amino acids that is very similar to carboxypeptidase A and B and consists of a 22-amino acid signal peptide, a 92-amino acid activation peptide, and a 309-amino acid catalytic domain. This protein shows 44 and 40% similarity to rat procarboxypeptidase B and human mast cell procarboxypeptidase A, respectively. The residues critical for catalysis and zinc and substrate binding of carboxypeptidase A and B are conserved in the Mr 60,000 plasminogen-binding protein. The presence of aspartic acid at position 257 of the catalytic domain suggests that this protein is a basic carboxypeptidase. When activated by trypsin, it hydrolyzes carboxypeptidase B substrates, hippuryl-Arg and hippuryl-Lys, but not carboxypeptidase A substrates, and it is inhibited by the specific carboxypeptidase B inhibitor (DL-5-guanidinoethyl)mercaptosuccinic acid. We propose that the Mr 60,000 plasminogen-binding protein isolated here is a novel human plasma carboxypeptidase B and that it be designated pCPB.
利用纤溶酶原-琼脂糖亲和层析从人血浆中分离出一种新型纤溶酶原结合蛋白。当用高浓度的ε-氨基己酸（大于20 mM）洗脱纤溶酶原亲和柱时，该蛋白与α2抗纤溶酶共纯化。十二烷基硫酸钠分析表明该蛋白的表观分子量为60,000。其氨基末端氨基酸序列与其他蛋白序列无相似性。基于氨基末端氨基酸序列，设计了用于聚合酶链反应引物的寡核苷酸探针，并分离出一个约1800碱基对的cDNA，其编码这种分子量为60,000的蛋白。推导的氨基酸序列显示，其初级翻译产物为423个氨基酸，与羧肽酶A和B非常相似，由一个22个氨基酸的信号肽、一个92个氨基酸的激活肽和一个309个氨基酸的催化结构域组成。该蛋白与大鼠羧肽酶原B和人肥大细胞羧肽酶原A的相似性分别为44%和40%。羧肽酶A和B催化、锌结合及底物结合的关键残基在分子量为60,000纤溶酶原结合蛋白中保守。催化结构域第257位存在天冬氨酸表明该蛋白是一种碱性羧肽酶。经胰蛋白酶激活后，它能水解羧肽酶B的底物马尿酰-精氨酸和马尿酰-赖氨酸，但不能水解羧肽酶A的底物，且被特异性羧肽酶B抑制剂（DL-5-胍基乙基）巯基琥珀酸抑制。我们认为这里分离出的分子量为60,000的纤溶酶原结合蛋白是一种新型人血浆羧肽酶B，命名为pCPB。
