Deletion of residues K296-G302 from the slowly-cleared tissue-type plasminogen activator t-PA del (G) leads to partial loss of plasminogen activating activity.
Plasmids encoding tissue-type plasminogen activators lacking residues K296-G302 were constructed and were expressed in a Hela cell transient expression system. Conditioned media from the cultures were tested in a number of systems designed to detect function or antigen. Functional assays comprised the chromogenic substrate S2288 and three plasminogen activation assays. All t-PA variants were active in all assays but to varying degrees. The results are consistent with the conclusion that deletion of residues K296-G302 from the slowly-cleared t-PA mutant t-PA del(G) adversely affects the plasminogen activating ability of the molecule without altering the integrity of the active site. This is in contrast to the situation in native t-PA where the same mutation has little overall effect on activity.
Dodd
I
I
Department of Biotechnology, SmithKline Beecham Pharmaceuticals, Surrey, U.K.
Browne
M J
MJ
Chapman
C G
CG
Cronk
D
D
Hastwell
C
C
Wilson
S
S
Robinson
J H
JH
D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D015153:Blotting, Western; D017077:Culture Media, Conditioned; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D010958:Plasminogen / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D017384:Sequence Deletion; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000502:physiology*
D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; D010958:Plasminogen; D010959:Tissue Plasminogen Activator