The influence of human fibrinogen (Fg) and its terminal plasminolytic digestion products, fragment D and fragment E, on the kinetics of activation of human plasminogen (Pg) by catalytic levels of streptokinase (SK) has been investigated. Both Fg and fragment D enhanced the rates of activation of human Glu1-Pg, Lys77-Pg, and Val442-Pg. Fragment E was refractive in this regard. In the case of Glu1-Pg, the Km for activation by SK, 0.4 microM, was not affected by the presence of Fg or fragment D. The kcat for this same reaction, 0.12 s-1, was elevated to 0.3 s-1 at saturating levels of these effector molecules. On the other hand, the Km for activation of Lys77-Pg, 0.5 microM, was decreased to 0.09 microM, whereas the kcat, 0.33 s-1, was not altered in the presence of saturating concentrations of Fg or fragment D. In the case of Val442-Pg, the Km for this same activation, 2.0 microM, was lowered to 0.4 microM and 0.25 microM in the presence of Fg and fragment D, respectively. The kcat for this process, 1.0 s-1, was unchanged in the presence of these agents. The concentrations of Fg (KFg) and fragment D (KFD) that led to half-maximal stimulation of the activation rates were determined. For Fg with Glu1-Pg, Lys77-Pg, and Val442-Pg, the KFg values were 0.08 microM, 0.14 microM, and 0.17 microM, respectively. The KFD values for these same plasminogens were 0.25 microM, 2.0 microM, and 1.7 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

We have examined the time-dependent appearance of amidolytic activity in equimolar complexes of streptokinase (SK) and human [Glul]plasminogen (HPg) under various conditions. When stoichiometric levels of the two proteins are incubated and assayed in hypotonic buffers at 4°C, amidolytic activity toward the chromogenic substrate D-ValLeu-Lys-p-nitroanilide (S-2251), within the resulting complex, appears with an observed first-order rate constant of 1.03 ± 0.06/min. On the other hand, when the assay for amidolytic activity is conducted at a Cl-concentration of 0.15 M, this same activity develops with an observed first-order rate constant of 0.13 ± 0.01/min. Under all conditions of assay of importance to the mechanism proposed, the only molecular components present are SK and H]Pg. The rate of appearance of an enzyme species displaying amidolytic activity is dependent on the anion in its assay; a much slower rate constant is obtained with Cl than with AcO-. These observations are consistent with the formation, within the complex, of an early anion-sensitive active site (SK-HPg*) that is converted to a form (SK-HPg') that is much less sensitive to the presence of anions. During the time period of this process, no conversion of plasminogen to plasmin occurs within the complex. Steadystate kinetic properties of SK-HPg* and SK-HPg' have been measured toward the substrate S-2251. Consistent with the mechanism suggested above, the amidolytic activity of SK-HPg* is inhibited by Cl to a much greater extent than is that of SK-HPg'.Fibrinolytic activity in mammalian plasma is primarily expressed through the action of plasmin, a serine protease generated consequent to activation of its plasma protein precursor, plasminogen. This activation occurs as a result of cleavage of a single peptide bond, ArgSWq-Vall6l, in the zymogen (1) and is mediated by a variety of proteins, such as urokinase (2), streptokinase (SK) (3), and tissue activators (4). Of these, the bacterial protein SK is unique in that it does not appear to be a protease, since a synthetic substrate, or a protein substrate, for SK, other than plasminogen, has not as yet been identified.Current understanding of the mechanism whereby SK activates human plasminogen (HPg) is based on studies that correlate appearance of enzymic activity with the molecular species of proteins present in equimolar complexes of HPg and SK (for a comprehensive review of this area, see ref. 5). From such work, it has been concluded that the major activators of HPg are equimolar complexes of SK and HPg (6), in which an active site has developed in the plasminogen moiety of the complex (7), and a similar complex of SK and human plasmin (HPm), which uses the active site present in HPm for its activator activity (8). This latter complex forms by intramolecular (9, 10) peptide bond cleavage of HPg, within the SK-HPg complex, and/or by stoichiometric interaction of SK with previously formed HPm.Recent work from our laboratory has shown that at very early reaction times, at least two types o...

Proteins which produce a thermal hysteresis (difference between the freezing and melting points) in aqueous solution are well-known for their antifreeze activity in polar marine fishes. Much less is known about the biology and biochemistry of similar antifreeze proteins found in certain insects. A thermal hysteresis protein was purified from cold acclimated larvae of the beetle, Tenebrio molitor, by using ethanol fractionation, DEAE ion-exchange chromatography, gel filtration, and high-pressure liquid chromatography. The purified protein had a molecular mass of 17 000 daltons and its N terminus was lysine. The amino acid composition of the antifreeze protein contained more hydrophilic amino acids than the fish antifreezes. This is consistent with the compositions of previously purified insect thermal hysteresis proteins. However, the percentage of hydrophilic amino acids in this Tenebrio antifreeze protein was considerably less than that of other insect thermal hysteresis proteins. The freezing point depressing activity of the Tenebrio antifreeze was less than that of fish proteins and glycoproteins at low protein concentrations but was greater at high protein concentrations.

The effects of human fibrinogen, and several plasmin-derived fragments of fibrinogen, on the streptokinase-induced activation of human plasminogen (Pg) have been investigated. Fibrinogen stimulates the rate of activation of human Glu1-Pg, Lys77-Pg, and Val442-Pg. The cofactor activity of fibrinogen appears to reside mainly in the D-domain region, since purified fragment D is active in this system. Fibrinogen fragment E was not active in this regard. The cofactor activity of fragment D was partially dependent on the presence of Ca2+. This effect of Ca2+ was likely due to its stabilizing influence on fragment D, as revealed by studied employing differential scanning calorimetry. Conversion of fragment D1 to fragments D2--5 did not alter the cofactor activity. Steady-state kinetic analysis of the activation of Val442-Pg by the streptokinase-Val442-plasmin complex demonstrated that the Km decreased approximately 2-fold, in the presence of fragment D1. Very little change in the steady-state kinetic parameters for Glu1-Pg and Lys77-Pg, when activated by the streptokinase-Lys77-plasmin complex, was noted in the presence of fragment D1. It was also found that both fibrinogen and fibrinogen fragment D1 increased the rate of formation of the active site in the streptokinase-plasminogen complex, of all forms of plasminogen, and that this effect was sufficient to explain the overall stimulation of the activation of plasminogen by fibrinogen and fibrinogen fragment D1.

Human plasmin derivatives, possessing heavy chains greatly reduced in size, have been utilized to assess the role of the heavy chain in the steady-and pre-steady-state kinetic parameters of this enzyme, as well as its equimolar complex with streptokinase. Native human plasmin (M, = 85 000) possesses a