Acyl-enzyme complexes between tissue-type plasminogen activator and neuroserpin are short-lived in vitro.
The serine protease tissue-type plasminogen activator (t-PA) initiates the fibrinolytic protease cascade and plays a significant role in motor learning, memory, and neuronal cell death induced by excitotoxin and ischemia. In the fibrinolytic system, the serpin PAI-1 negatively regulates the enzymatic activity of both single-chain and two-chain t-PA (sct-PA and tct-PA). In the central nervous system, neuroserpin (NSP) is a serpin thought to regulate t-PA enzymatic activity. We report that although both sct-PA and tct-PA rapidly form acyl-enzyme complexes with NSP in vitro, the interactions are short-lived, rapidly progressing to complete cleavage of NSP and regeneration of fully active enzyme. All NSP molecules appear to transit through the detectable acyl-enzyme intermediate and progress to completion of cleavage; no subpopulation that functions as a pure substrate was detected. Likewise, all molecules were reactive, with no evidence of a latent subpopulation. The interactions between NSP and t-PA were distinct from those between plasmin and NSP, wherein the same peptide bond was cleaved but there was no evidence of a detectable plasmin-NSP acyl-enzyme complex. The interactions between t-PA and NSP contrast with the formation of long-lived, physiologically irreversible acyl-enzyme complexes between t-PA and PAI-1, suggesting that the physiologic effect of t-PA-NSP interactions may be more complex than previously thought.
Barker-Carlson
Karen
K
Department of Biomolecular Chemistry, Medical Scientist Training Program, University of Wisconsin, Madison, Wisconsin 53706, USA.
Lawrence
Daniel A
DA
Schwartz
Bradford S
BS
D001665:Binding Sites; D015153:Blotting, Western; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D009479:Neuropeptides / Q000737:chemistry* / Q000502:physiology*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015843:Serpins / Q000737:chemistry* / Q000502:physiology*; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000502:physiology*; D000097522:Neuroserpin
D018076:DNA, Complementary; D005341:Fibrinolysin; D009479:Neuropeptides; D010455:Peptides; D012313:RNA; D015843:Serpins; D010959:Tissue Plasminogen Activator; D000097522:Neuroserpin