Structural determinants of the noncatalytic chain of tissue-type plasminogen activator that modulate its association rate with plasminogen activator inhibitor-1

• Published in: The Journal of biological chemistry Vol. 265; no. 18; pp. 10473 - 10478
• Main Authors: DE SERRANO, V. S, CASTELLINO, F. J
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25-06-1990
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Binding Sites; Biological and medical sciences; Cell Line; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Genetic Variation; Humans; Hydrolases; Insecta; Kinetics; Macromolecular Substances; Melanoma; Molecular Sequence Data; Oligonucleotide Probes; plasminogen activator inhibitor I; Plasminogen Inactivators - metabolism; Recombinant Proteins - metabolism; Tissue Plasminogen Activator - genetics; Tissue Plasminogen Activator - metabolism; Tumor Cells, Cultured - metabolism; Urokinase-Type Plasminogen Activator - genetics; Urokinase-Type Plasminogen Activator - metabolism; Regulation(control); Enzyme; Enzyme inhibitor; Immunoblotting assay; Plasminogen activator; Binding site; Kinetics; Inhibition; Recombinant protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)86971-X

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).