Purification and characterization of biologically active scatter factor from ras-transformed NIH 3T3 conditioned medium

Coffer, A., Fellows, J., Young, S., Pappin, D., Rahman, D. (August 1991) Purification and characterization of biologically active scatter factor from ras-transformed NIH 3T3 conditioned medium. Biochem J, 278 ( . pp. 35-41. ISSN 0264-6021 (Print)0264-6021 (Linking)

URL: http://www.ncbi.nlm.nih.gov/pubmed/1831975

Abstract

Scatter factor (SF), a glycoprotein produced by cultured fibroblasts, acts in vitro on epithelial cells causing separation and increased local motility. In this study, the polypeptide was purified to apparent homogeneity in high yields with conserved biological activity from medium conditioned by ras-transformed NIH 3T3 cells, by a three-step procedure involving ammonium sulphate fractionation, cation-exchange and hydroxyapatite chromatography. After purification, SF specific activity increased from approximately 0.3 units/microgram in unprocessed conditioned medium to approximately 5 units/ng, and cumulative recovery of biological activity was approximately 38%. Treatment of pure SF with N-glycanase resulted in a decreased Mr, but no concomitant effect was observed on biological activity. Proteolytic activity was absent from samples of both partially purified and pure SF. Our biochemical studies showed that SF, which is highly aggregated in low-ionic-strength media, is not aggregated in 0.4 M-salt. Under non-reducing conditions, pure SF migrated as a single stained band at Mr 67,000 on SDS/PAGE, and biological activity was eluted from unstained gels with an identical Mr. SF was electrofocused sharply at pI 8.5 with no degradation of activity. From ultracentrifugation studies (under non-aggregating conditions), the sedimentation coefficient of active SF was 3.7 S and f.p.l.c. molecular sieve chromatography indicated a Stokes' radius of 2.95 nm. The calculated Mr from these data was 61,400. The appearance of three stained polypeptides of Mr 82,000, 57,000 and 32,000 derived from the Mr-67,000 constituent after reduction with mercaptoethanol suggests that SF may be a heterodimer of Mr-57,000 and -32,000 subunits. Data from protein sequence analysis of the hydroxyapatite-purified protein confirms that SF has sequence identity with both rat hepatocyte growth factor and human fibroblast tumour cytotoxic factor.

Item Type: Paper
Uncontrolled Keywords: Amidohydrolases/metabolism Amino Acid Sequence Animals Cell Line, Transformed Chemistry, Physical Chromatography Culture Media Cytokines/chemistry/ isolation & purification/metabolism Electrophoresis, Polyacrylamide Gel Fibroblasts/ metabolism Genes, ras Hepatocyte Growth Factor Macromolecular Substances Mice Molecular Sequence Data Molecular Weight Osmolar Concentration Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Physicochemical Phenomena
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein characterization
biotechnology > chromatography > protein purification
CSHL Authors:
Communities: CSHL labs > Pappin lab
Depositing User: Matt Covey
Date: 15 August 1991
Date Deposited: 10 Sep 2014 15:07
Last Modified: 10 Sep 2014 15:07
PMCID: PMC1151445
Related URLs:
URI: http://repository.cshl.edu/id/eprint/30776

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