Membrane-Bound Trypsin-Like Enzyme Functioning in Degradation of Dynorphin in Neuroblastoma Cells. Purification and Characterization1

TL;DRAbstract

A trypsin-like enzyme has been purified to apparent homogeneity from neuroblastoma cell membranes by a procedure including extraction with Triton X-100, soybean trypsin inhibitor-immobilized Sepharose 4B affinity chromatography, and gel filtration. SDS-polyacrylamide gel electrophoresis under reducing conditions of the purified enzyme gave a single band corresponding to a molecular weight of 28,000. The molecular weight of the enzyme was also estimated to be 32,000 by gel filtration. The pH optimum of the activity was 8.5-9.0. The purified enzyme was inhibited by diisopropylphosphorofluoridate, p-aminobenzamidine, and leupeptin, and moderately by chymostatin, but not, or only scarcely, by bestatin, phosphoramidon, p-chloromercuribenzoate, and N-ethylmaleimide. The substrate subsite specificity of the purified enzyme was broad toward various peptidyl-arginine (or lysine) 4-methylcoumaryl-7-amides, but it cleaved dynorphin(1-17) only at two sites, i.e., between the Arg6-Arg7 and Lys11-Le

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