Summary
Purified 2-chain recombinant tissue-type plasminogen activator (t-PA) was reduced
under mild conditions - 10 mM dithiothreitol/ 5° C/1.5 h - and the two chains were
separated by chromatography on lysine Sepharose. The t-PA B chain was fully active
as determined by its activity towards the chromogenic substrate S-2288 (H-D-ile-pro-arg
p-nitroanilide). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis
under reducing or non-reducing conditions revealed a single polypeptide at Mr = 35,000
or 29,000 respectively. In addition, under non-reducing conditions a fibrinolytic
band at apparent Mr = 29,000 was present after fibrin zymography. The N-terminal sequence
was confirmed as ile-lys-gly. The t-PA B chain had a specific amidolytic activity,
using S-2288, of 170,000 to 210,000 SU/mg protein. (This compares to a specific activity
of the native 2-chain t-PA of 170,000 SU/mg). It resembles urokinase-type plasminogen
activator in its inability to be stimulated by fibrin and its dose response on human
fibrin plates. However, t-PA B-chain was stimulated to almost the same extent as t-PA
by poly-D-lysine. The isoelectric points, at pH 5.6 and 5.7, fall outside the range
generally quoted for t-PA preparations (pH 7.8-8.8).
Key words
Tissue-type plasminogen activator - Fibrinolysis - Urokinase-type plasminogen activator
