Identification and purification of a novel serine proteinase inhibitor.
We report the identification, purification, and partial amino acid sequence of a novel serine proteinase inhibitor which is present in extracts from human placentas and in the cytosolic fraction of the leukemic cell line K562. Extracts from these tissues exhibited time-dependent inhibition of the serine proteinase thrombin. This activity was not accelerated by heparin and corresponded to a protein which formed a 67-kDa complex with 125I-thrombin. The complex was stable on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A cleaved and functionally inactive form of the protein was purified from placental tissue by chromatography on DEAE-Sepharose, followed by affinity chromatography on thrombin-Sepharose. Antibodies raised against the placental protein recognized the inhibitor from K562 cells and placental extract. Western blotting experiments using the antibody showed that the uncleaved inhibitor has a molecular mass of 38 kDa. Amino acid sequencing was performed on the purified protein. Sequences of peptides resulting from digestion with cyanogen bromide followed by Endoproteinase Lys-c confirmed that this is a novel inhibitor with significant homology to the serpin family.
Coughlin
P B
PB
Department of Medicine, Monash Medical School, Box Hill Hospital, Melbourne, Australia.
Tetaz
T
T
Salem
H H
HH
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006736:Horses; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010920:Placenta / Q000502:physiology; D011247:Pregnancy; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D015843:Serpins / Q000737:chemistry*; D013917:Thrombin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D015842:Serine Proteinase Inhibitors; D015843:Serpins; D013917:Thrombin