Functional role of proteolytic cleavage at arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis

Journal Article · Tue Jan 27 04:00:00 EST 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00376a002· OSTI ID:6790677

Tate, K M; Higgins, D L; Holmes, W E; Winkler, M E; Heyneker, H L; Vehar, G A

Activation of the zymogen form of a serine protease is associated with a conformational change that follows proteolysis at a specific site. Tissue-type plasminogen activator (t-PA) is homologous to mammalian serine proteases and contains an apparent activation cleavage site at arginine-275. To clarify the functional consequences of cleavage at arginine-275 of t-PA, site-specific mutagenesis was performed to convert arginine-275 to a glutamic acid. The mutant enzyme (designated Arg-275 ..-->.. Glu t-PA) could be converted to the two-chain form by Staphylococcus aureus V8 protease but not by plasmin. The one-chain form was 8 times less active against the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine-rho-nitroanilide (S-2288), and the ability of the enzyme to activate plasminogen in the absence of fibrinogen was reduced 20-50 times compared to the two-chain form. In contrast, one-chain Arg-275 ..-->.. Glu t-PA has equal activity to the two-chain form when assayed in the presence of physiological levels of fibrinogen and plasminogen. Fibrin bound significantly more of the one-chain form of t-PA than the two-chain form for both the wild-type and mutated enzymes. One- and two-chain forms of the wild-type and mutated plasminogen activators slowly formed complexes with plasma protease inhibitors, although the one-chain forms showed decreased complex formation with ..-->../sub 2/-macroglobulin. The one-chain form of t-PA therefore is fully functional under physiologic conditions and has a increased fibrin binding compared to the two-chain form.

• Research Organization: Genentech, Inc., South San Francisco, CA
• OSTI ID: 6790677
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:2; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
ALKALI METAL COMPOUNDS
AMINO ACIDS
ARGININE
AUTORADIOGRAPHY
BACTERIA
BETA DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ENZYMATIC HYDROLYSIS
ENZYMES
FIBRINOLYSIN
FIBRINOLYTIC AGENTS
GLUTAMIC ACID
HALIDES
HALOGEN COMPOUNDS
HEMATOLOGIC AGENTS
HYDROLASES
HYDROLYSIS
HYDROXY ACIDS
INORGANIC PHOSPHORS
INTERMEDIATE MASS NUCLEI
IODIDES
IODINE 125
IODINE COMPOUNDS
IODINE ISOTOPES
ION EXCHANGE CHROMATOGRAPHY
ISOTOPES
LYSIS
MICROORGANISMS
MUTAGENESIS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PEPTIDE HYDROLASES
PHOSPHORS
PLASMINOGEN
RADIOISOTOPES
SEPARATION PROCESSES
SERINE
SERINE PROTEINASES
SODIUM COMPOUNDS
SODIUM IODIDES
SOLVOLYSIS
STAPHYLOCOCCUS