Expression and characterization of pro alpha 2-plasmin inhibitor.
alpha s-Plasmin inhibitor (alpha 2PI), one of the serine protease inhibitors in plasma, was expressed in baby hamster kidney (BHK) cell line. The expression vector was constructed with its genomic DNA and cDNA, and was transfected into BHK cells by the calcium phosphate method. The recombinant alpha 2PI which was secreted from the cells was estimated by SDS-PAGE to have a molecular mass of 67 kDa, which is indistinguishable from that of normal plasma alpha 2PI. The leader peptide of 12 amino acids was retained at the amino terminus of the recombinant alpha 2PI. This finding suggests that alpha 2PI has pre-pro type processing and the propeptide of 12 amino acids is not removed in BHK cells. This pro-alpha 2PI shows essentially the same inhibitory activity on plasmin and the same affinity for plasmin(ogen) as those of normal alpha 2PI. However, the cross-linking ability to fibrin is reduced to less than one-third of that of normal alpha 2PI. The cross-linking site is the glutamine residue located at the second position from the amino terminus of normal alpha 2PI. The conformational change of this region caused by the addition of the propeptide may have affected the cross-linking capacity of the inhibitor.
Sumi
Y
Y
Biotechnology Research Laboratory, Teijin Limited, Hino, Tokyo.
Ichikawa
Y
Y
Nakamura
Y
Y
Miura
O
O
Aoki
N
N
D000596:Amino Acids / Q000032:analysis; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006801:Humans; D010449:Peptide Mapping; D011498:Protein Precursors / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins; D014170:Transformation, Genetic; D000979:alpha-2-Antiplasmin / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification
D000596:Amino Acids; D011498:Protein Precursors; D011994:Recombinant Proteins; D000979:alpha-2-Antiplasmin; C062388:pro-alpha-2-plasmin inhibitor