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  • Title: A covalent molecular weight approximately 92,000 hybrid plasminogen activator derived from human plasmin fibrin-binding and tissue plasminogen activator catalytic domains.
    Author: Robbins KC, Boreisha IG.
    Journal: Biochemistry; 1987 Jul 28; 26(15):4661-7. PubMed ID: 3117104.
    Abstract:
    A covalent hybrid plasminogen activator was prepared from the sulfhydryl forms of the NH2-terminal heavy (A) chain of human plasmin (PlnA) containing the fibrin-binding domain and the COOH-terminal B chain of tissue plasminogen activator (t-PAB) containing the catalytic domain. The sulfhydryl form of PlnA [PlnA(SH)2] was isolated from reduced Lys-2-plasmin on an L-lysine-substituted Sepharose column, and the sulfhydryl form of t-PAB [t-PAB(SH)] was prepared from reduced two-chain tissue plasminogen activator (t-PA) by removing the tissue plasminogen activator NH2-terminal A chain (t-PAA) on an L-lysine-substituted Sepharose column from the chain mixture. The specific plasminogen activator activity, with soluble fibrin, of the isolated t-PAB(SH) chain was determined to be 62,700 international units (IU)/mg of protein, about 13% of the specific plasminogen activator activity of the parent t-PA. The PlnA(SH)2 and the t-PAB(SH) chains were mixed in a 1:1 molar ratio, and hybridization (reoxidation) was allowed to proceed by first dialyzing out the reducing agent at 4 degrees C and then concentrating the mixture. The time for maximum hybridization, or formation of the covalent hybrid activator, was 6 days, as determined by both specific plasminogen activator activity, with soluble fibrin, and specific amidolytic activity; sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the continual formation of an Mr approximately 92,000 hybrid. The covalent PlnA-t-PAB hybrid activator was isolated from the 6-day hybridization mixture by a two-step affinity chromatography method.(ABSTRACT TRUNCATED AT 250 WORDS)
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