MODIFICATION OF GLUTAMIC AND ASPARTIC ACID RESIDUES OF PLASMINOGEN INHIBITS ITS ABILITY TO FORM AN ACTIVE PLASMINOGEN-STREPTOKINASE COMPLEX

Abstract

Plasminogen binds to streptokinase in a 1:1 molar complex that has activity as a plasminogen activator. This function of plasminogen, as a cofactor for streptokinase conversion of plasminogen to plasmin, was studied after treatment of Glu-, Lys-, and Mini-plasminogens with 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide (EDC). Amino acid analysis showed that both aspartic and glutamic acid residues were modified by EDC. Activity of the complex formed between streptokinase and the modified plasminogen was measured using the cfhromogenic substrate H-D-Val-Leu-Lys-pNA. Plasminogen, 2.8 uM, was incubated with 40 mM EDC in 50 mM MES buffer, pH 6.0, at 25°C. At various times while reacting plasminogen with the EDC, aliquots were removed for assay. Plasminogen function was assayed by mixing with a slight molar excess of streptokinase for 1 min at 37 C, followed by reaction with 0.1 M substrate, and absorbancy monitored at 405 nm. Modifications of 20% of the glutamic and aspartic acid residues occurred after treatment of plasminogen with EDC. This resulted in 80 to 90% inhibition of activation in all three types of plasminogen. Glu- and Lys-plasminogens reacted more quickly with the EDC than did Mini-plasminogen, with 50% inhibition occurring after 16 ± 5, 16 ± 4, and 67 ± 13 min reaction time with EDC for Glu-, Lys-, and Mini-plasminogens, respectively. Maximum inhibition of activation occurred within 1 hr reaction with EDC for Glu- and Lys-plasminogens but required 2.5 hr for Mini-plasminogen. The time courses for activation inhibition and the modification of the glutamic and aspartic acids of treated Mini-plasminogen were compared. A significant decrease in activation occurred (52%) concomitant with modification of only one or two glutamic acids, followed on further reaction with EDC by more loss of activatability as more glutamic and aspartic acids were modified. The inability of plasminogen to form an active plasminogen-streptokinase complex after modification with EDC indicates that glutamic and aspartic acid residues are involved in the binding site of plasminogen for streptokinase.