Structural determinants of the noncatalytic chain of tissue-type plasminogen activator that modulate its association rate with plasminogen activator inhibitor-1.
In order to identify the regions of recombinant (r) tissue plasminogen activator (tPA) that mediate its kinetically relevant interaction with r-plasminogen activator inhibitor-1 (rPAI-1), we have determined the second-order association rate (k1) constants of domain-altered variants of tPA with rPAI-1, at 10 degrees C. With two-chain, wild-type recombinant tPA (tcwt-rtPA), obtained by expression of the human cDNA for tPA in five different cell systems (viz. insect cells, human kidney 293 cells, Chinese hamster ovary cells, human melanoma cells, and mouse C127 cells), the average k1 was 1.45 x 10(7) M-1 s-1 (range, 1.34 10(7) M-1 s-1-1.68 x 10(7) M-1 s-1). Since this value was not significantly different for the different tcwt-rtPA preparations, it appears as though the nature of the glycosylation of tPA plays little role in its initial interaction with PAI-1. The k1 determined for tcwt-rtPA was slightly higher than that of 0.87 x 10(7) M-1 s-1, obtained for a similar inhibition of human urokinase by rPAI-1. The k1 value obtained for single-chain (sc) wt-rtPA was approximately 6-fold lower than that of the two-chain molecules, results consistent with previous conclusions on this matter. The k1 value for tcwt-rtPA was not influenced by the presence of epsilon-aminocaproic acid, suggesting that the lysine-binding site associated with the kringle 2 (K2) region of tPA does not modulate the rate of its initial interaction with rPAI-1. Removal of the K2 domain from tPA, by recombinant DNA technology, results in a protein, F-E-K1-P (tc-r delta K2-tPA), containing only the finger (F), growth factor (E), kringle 1 (K1), and serine protease (P) domains. This variant protein was more rapidly inhibited by rPAI-1 (k1 = 3.00 x 10(7) M-1 s-1) than its wild-type counterparts. Deletion of both the K1 and K2 domains resulted in a variant molecule, F-E-P (tc-r delta K1 delta K2-tPA), that was slightly more rapidly inhibited by rPAI-1 (k1 = 2.01 x 10(7) M-1 s-1).(ABSTRACT TRUNCATED AT 400 WORDS)
de Serrano
V S
VS
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Castellino
F J
FJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D014644:Genetic Variation; D006801:Humans; D007313:Insecta; D007700:Kinetics; D046911:Macromolecular Substances; D008545:Melanoma; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D015849:Plasminogen Inactivators / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism
D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D015849:Plasminogen Inactivators; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator