P. Gaussem et al., SODIUM DODECYL SULFATE-INDUCED DISSOCIATION OF COMPLEXES BETWEEN HUMAN TISSUE-PLASMINOGEN ACTIVATOR AND ITS SPECIFIC INHIBITOR, The Journal of biological chemistry, 268(16), 1993, pp. 12150-12155
The stability of complexes between serine proteinases and their inhibi
tors after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophor
esis has been claimed to indicate covalent bond formation. In this wor
k we have investigated the effects of SDS on the stability of complexe
s between single-chain or two-chain tissue plasminogen activator (t-PA
) and its inhibitor (PAI-1). Complexes formed by incubation of t-PA wi
th PAI-1 for 15 min at 22-degrees-C were further incubated with variou
s amounts of SDS before being subjected to SDS-polyacrylamide gel elec
trophoresis. The molecular species in the gels were identified both by
zymography or by autoradiography after immunoblotting with antibodies
directed against either t-PA or PAI-1. It was demonstrated that the i
nteraction of SDS with t-PA.PAI-1 complexes before electrophoresis res
ulted in a transition from the complexed state to the free forms of t-
PA and PAI-1 in a time- and dose-dependent manner. The first-order dis
sociation rate constant in the presence of 35 mm SDS at 22-degrees-C h
ad a k(off) value of 1.4 x 10(-2) min-1, which corresponds to a half-l
ife of 49.5 min. The t-PA released from the complexes was fibrinolytic
ally active, whereas the released PAI-1 inhibited activator-dependent
fibrinolysis. In a similar fashion, the well characterized non-acylate
d pair alpha1-proteinase inhibitor-elastase was dissociated by SDS tre
atment, confirming the validity of our experimental approach to demons
trate the reversibility of t-PA.PAI-1 complexes. These results demonst
rate that SDS-polyacrylamide gel electrophoresis traps the molecular s
pecies in the state in which the proteins existed prior to the analysi
s, and they suggest that under the conditions used, the interaction of
t-PA with PAI-1 results in the formation of nonacylated reversible co
mplexes. This phenomenon may be relevant to the pathophysiology of fib
rinolysis and to the general mechanism of serine proteinase-inhibitor
complex formation.