Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma.
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.
Department of Cardiovascular Research, Genentech, Inc., South San Francisco, California 94080.
D L
DL
Eaton
B E
BE
Malloy
S P
SP
Tsai
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W
Henzel
D
D
Drayna
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification; D002417:Cattle; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007527:Isoenzymes / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010958:Plasminogen / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D014158:Transcription, Genetic
D007527:Isoenzymes; D012333:RNA, Messenger; D010958:Plasminogen; D004247:DNA; D002268:Carboxypeptidases; D043424:Carboxypeptidase B