Purification of protease nexin II from human fibroblasts.
Normal human fibroblasts secrete a protein named protease nexin II (PN II) which previously was shown to form sodium dodecyl sulfate (SDS)-stable complexes with epidermal growth factor-binding protein (EGF-BP). These complexes then bind to the same cells and are rapidly internalized and degraded (Knauer, D.J., and Cunningham, D.D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2310-2314). Here we describe a procedure for purifying PN II to apparent homogeneity from serum-free culture medium conditioned by human fibroblasts. The first step employed dextran sulfate-Sepharose affinity chromatography. Further purification was achieved by ion-exchange chromatography on DEAE-Sepharose followed by gel filtration on Sephacryl S-400. Sequence analysis of purified PN II identified 33 amino-terminal amino acids; a computer search of several protein sequence data banks failed to reveal homologies with other reported amino acid sequences. Purified PN II had an apparent Mr of 106,000 and an isoelectric point of approximately 7.2. It retained full activity after incubation in the presence of 0.05% SDS or at a pH of 1.5. PN II formed SDS-stable complexes with EGF-BP, the gamma subunit of 7 S nerve growth factor, and trypsin with estimated Mr of 120,000, 120,000, and 110,000, respectively. PN II was metabolically labeled with [35S]methionine and purified; the metabolically labeled protein formed complexes with EGF-BP. Complexes between purified PN II and EGF-BP bound to human fibroblasts. These results show that the purified protein possesses the properties previously attributed to PN II in cell culture medium.
D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D008970:Molecular Weight; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D012867:Skin / Q000201:enzymology
D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; D058255:Protease Nexins; D011956:Receptors, Cell Surface