Stoichiometry of the interaction of human plasma alpha 1-proteinase inhibitor with subtilisin BPN'.
Interaction of human plasma alpha 1-proteinase inhibitor (alpha 1PI) with subtilisin BPN' was assessed by spectrophotometric determination of the inhibitory capacity and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). During the course of incubation of the enzyme and the inhibitor (E : I = 1 : 7.5) at pH 8.0 about 17% of the enzyme activity which had been inhibited initially was regenerated, indicating a temporary type of inhibition. The results of the titration experiments indicate that 9.8 mol of the inhibitor is required to inhibit 1 mol of the enzyme completely. However, patterns of 5% disc SDS-PAGE under non-reducing conditions revealed only an equimolar complex (Mr80K) of alpha 1PI with the enzyme and no other higher Mr component than the native inhibitor (Mr 56K). On the other hand, complete dissociation of the complex occurred under reducing conditions, producing an enzymatically modified inhibitor. When 5 21% gradient slab SDS-PAGE was employed, no complex formation was observed under either reducing or non-reducing conditions. With the gradient gel system, dissociation of the equimolar complex produced different forms of the inhibitor, that is, regeneration of an intact alpha 1PI under non-reducing conditions and an enzymatically modified form under reducing conditions. All these results indicate that the complex formed between subtilisin BPN' and human alpha 1PI is not so stable as that of the inhibitor with bovine chymotrypsin and that no covalent bond may be involved in the complex formation. The results also indicate that human alpha 1PI is not an effective inhibitor of subtilisin BPN' and behaves like a substrate for the enzyme.
Abe
O
O
Laboratory of Chemistry, Fukuoka Dental College.
Kuromizu
K
K
D001665:Binding Sites; D001798:Blood Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D013053:Spectrophotometry; D013381:Subtilisins / Q000378:metabolism*; D000515:alpha 1-Antitrypsin
D001798:Blood Proteins; D000515:alpha 1-Antitrypsin; D013381:Subtilisins