Preparation of a novel streptokinase mutant with improved stability.
The novel mutant streptokinase, SK-K59E, can activate human plasminogen as efficiently as the purified commercially available streptokinase. Several peptide bonds including Lys59-Ser60 in native streptokinase were hydrolyzed in reaction with plasmin and peptides of small molecular masses were generated. The plasminogen activator activity of native streptokinase in reaction with human plasmin declined to 25% of the original activity in a 120-min incubation. On the other hand, the NH2-terminal peptide of SK-K59E remained intact in reaction with plasmin and the activator activity of streptokinase decreased to 75% of the original activity in 120 min. The major degraded peptide fragments of native streptokinase in reaction with plasmin had molecular masses of 36 and 30 kDa. However, two major peptide fragments of 42 and 34 kDa were observed in the reaction of SK-K59E with human plasmin. The 42 kDa peptide fragment, which contained NH2-terminal of streptokinase, could activate human plasminogen as efficiently as the native streptokinase. SK-K59E can induce greater degree of caseinolysis and fibrinolysis than the native streptokinase. In conclusion, the results demonstrate that the prevention of cleavage at Lys59 of streptokinase prolongs the half-life of streptokinase in complex with plasmin and that the NH2-terminal of streptokinase (Ile1-Lys59) plays an important role in maintaining its stability.
Shi
G Y
GY
Department of Biochemistry, Medical College, National Cheng Kung University, Tainan, Taiwan, Republic of China.
Chang
B I
BI
Su
S W
SW
Young
K C
KC
Wu
D H
DH
Chang
L C
LC
Tsai
Y S
YS
Wu
H L
HL
D004795:Enzyme Stability; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D010958:Plasminogen / Q000378:metabolism; D010960:Plasminogen Activators / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology*; D013300:Streptokinase / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology*
D010446:Peptide Fragments; D010958:Plasminogen; D013300:Streptokinase; D010960:Plasminogen Activators; D005341:Fibrinolysin