Isolation and characterization of streptokinases of E. coli HB101 transformant and streptococcus equisimilis

Abstract

The bacterial protein streptokinase functions in the species-specific conversion of human plasminogen to plasmin. The activation of plasminogen by streptokinase occurs indirectly via a plasmiogen activator complex. All of the assays of streptokinase activity detected the presence of streptokinase indirectly through its capacity to activate plasminogen to plasmin. The assays conditions were established using CLN as a substrate for plasmin. Recently a streptokinase gene from Streptococcus equisimilis was cloned and expressed in E. coli. For the comparative studies of two kinds of streptokinase produced by the transformant and Streptococcus equisimilis, they were purified by isoelectric precipitation, ethanol precipitation, DEAE-Sephadex A-50 ion exchange chromatography. Streptokinase-like protein isolated from the transformant showed no detectable amount of enzymatic activity. Only when it was incubated with human plasminogen, casein hydrolytic and CLN esterolytic activities developed. In studies on the effect of heat on streptokinase-like protein, the capacity of streptokinase-like protein to hydrolyze CLN was appreciably decreased only upon long exposure to temperatures of 75-95 C. With the concentration of human plasminogen fixed at a constant value, the amount of CLN esterolytic activity inhibited by soybean trypsin inhibitor was decreased with increasing streptokinase-like protein concentration. At high streptokinase-like protein levels, the inhibitable CLN esterolytic activity fell to near zero. The CLN esterolytic activity of some component which was generated by activating human plasminogen with catalytic levels of streptokinase-like protein was competitively inhibited by -amino caproic acid. Also with streptokinase produced by streptococcus equisimilis the same results were obtained.