Characterization of recombinant human. cap alpha. /sub 2/-antiplasmin and of mutants obtained by site-directed mutagenesis of the reactive site

Journal Article · Tue Aug 11 04:00:00 EDT 1987 · Biochemistry; (United States)

OSTI ID:5879346

Holmes, W E; Lijnen, H R; Collen, D

Human ..cap alpha../sub 2/-antiplasmin (..cap alpha../sub 2/AP) has been expressed in Chinese hamster ovary cells and purified from conditioned media. The recombinant protein (r..cap alpha../sub 2/AP) is immunologically identical with natural ..cap alpha../sub 2/AP and indistinguishable with respect to plasmin(ogen) binding properties. Second-order rate constants (k/sub 1/) for the interaction of ..cap alpha../sub 2/AP and r..cap alpha../sub 2/AP with plasmin are both (1-2) x 10/sup 7/ M/sup -1/s/sup -1/. In order to examine the effects of alterations within the reactive site of ..cap alpha../sub 2/AP, deletions of the P/sub 1/ residue Arg-364 (r..cap alpha../sub 2/AP-..delta..Arg364) of the P'/sub 1/ residue Met-365 (r..cap alpha../sub 2/AP-..delta..Met365) were introduced by in vitro site-directed mutagenesis. r..cap alpha../sub 2/AP-..delta..Met365 completely retains its ability to inhibit both plasmin and trypsin, indicating that ..cap alpha../sub 2/AP has no absolute requirement for Met in the P'/sub 1/ position. Unexpectedly, no increase in antithrombin activity was observed. r..cap alpha../sub 2/AP-..delta..Arg364 has lost the ability to inhibit plasmin, trypsin, and thrombin, but unlike the wild-type protein, this variant is an effective elastase inhibitor.

• Research Organization: Univ. of Leuven, Belgium
• OSTI ID: 5879346
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:16; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACID SEQUENCE
ANIMAL CELLS
AUTORADIOGRAPHY
BIOCHEMICAL REACTION KINETICS
CHO CELLS
DNA
DNA SEQUENCING
DRUGS
ELECTROPHORESIS
ENZYME IMMUNOASSAY
ENZYME INHIBITORS
ENZYMES
FIBRINOLYSIN
FIBRINOLYTIC AGENTS
HEMATOLOGIC AGENTS
HYDROLASES
IMMUNOASSAY
KINETICS
MOLECULAR STRUCTURE
MUTAGENESIS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PEPTIDE HYDROLASES
PURIFICATION
REACTION KINETICS
RECOMBINANT DNA
SERINE PROTEINASES
STRUCTURAL CHEMICAL ANALYSIS