Purification of a form of protease nexin 1 that binds heparin with a low affinity.
A form of protease nexin 1 (PN-1) that binds heparin with a low affinity (L-PN-1) was purified and studies since altered interactions with glycosaminoglycans could affect its inhibition of certain serine proteases. Purification of L-PN-1 and PN-1 was achieved by fractionating serum-free conditioned culture medium from human fibroblasts over dextran sulfate-Sepharose followed by immunoaffinity fractionation over a PN-1 monoclonal antibody-Sepharose column. The first step separated L-PN-1 from PN-1, and the second step resulted in apparently homogeneous L-PN-1 and PN-1. Comparisons of the two proteins showed that they could not be distinguished by the following properties: (a) molecular weight; (b) proteases complexed; (c) molecular weights of protease-L-PN-1 and protease-PN-1 complexes; (d) CNBr peptide maps; and (e) immunological cross-reactivity. Studies on activities that depend on the heparin binding domain revealed that heparin equally accelerated the rate of formation of 125I-thrombin-L-PN-1 and 125I-thrombin-PN-1 complexes even when the ratio of heparin to L-PN-1 or PN-1 was varied from 0.01 to 100. A functional difference, however, between L-PN-1 and PN-1 was observed in studies on the ability of the fibroblast surface to accelerate their reactions. Fixed fibroblasts accelerated the formation of 125I-thrombin-L-PN-1 complexes 2-fold, whereas they accelerated the formation of 125I-thrombin-PN-1 complexes 5-fold. The availability of purified L-PN-1 will permit studies on its functional relationship to PN-1.
Van Nostrand
W E
WE
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717.
Wagner
S L
SL
Cunningham
D D
DD
D016564:Amyloid beta-Protein Precursor; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003488:Cyanogen Bromide; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007231:Infant, Newborn; D007700:Kinetics; D008297:Male; D010449:Peptide Mapping; D011480:Protease Inhibitors / Q000302:isolation & purification*; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D058258:Serpin E2; D012867:Skin / Q000378:metabolism
D016564:Amyloid beta-Protein Precursor; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D011480:Protease Inhibitors; D058255:Protease Nexins; D011956:Receptors, Cell Surface; C545486:SERPINE2 protein, human; D058258:Serpin E2; D006493:Heparin; D003488:Cyanogen Bromide