Comparisons of four methods for quantification of lysosomal cysteine proteinase activities
Journal of Animal Science
Four methods were compared to optimize the measurement of the activities of cathepsin B and cathepsin L in porcine skeletal muscle. These methods were: Method A (lysosomal enriched fraction obtained by differential centrifugation), Method B (muscle extract in the absence of detergent), Method C (muscle extract in the presence of detergent) and Method D (the same as method C, but passed through a Scarboxymethylated-papain-Sephamse affinity column). Results indicated that, of the methods tested,
... he methods tested, Method D yielded greater cathepsin B and consistently greater cathepsin B + L activities per gram of muscle. Hence, Method D is the method of choice for quantification of these enzyme activities. Studies indicated that for cathepsin B with Z-Arg-Arg-NMec as substrate, Km and V" values were .416 mM and 4,405 pmol-min-'.mg proteid, r e s F l y . The Km and Vmax for cathepsins B + L were .132 mM and 9,346 pmol.min--mg protein-', respectively. The relationship between enzyme activity and incubation time was linear for the incubation times studied (up to 60 min). Also, the relationship between enzyme activities and amount of protein in the assay was linear at the concentrations studied (up to 20 pg protein). The same preparations were assayed by conditions commonly used by many investigators (.005 mM substrate, approximately 75 pg protein, 30 min at 37'C) and by conditions established in this study (1.0 mM substrate, 10 pg protein and 15 min at 37°C). Results indicated that the activities (nmole NMec re1eased.g of tissue-l.min-l) of cathepsin B and B + L were 48-and 10-fold greater, respectively, when assayed under optimal conditions determined in this study compared with the other assay conditions. (NE 68933. 2We would like to thank M. G. %e, Dept. of Food Sci., University of Nebraska, Lincoln, Sue Hauver for her a&tance. in conducting these experiments and Caw1 Grummert for p i n g this manuscript. Mention of trade names, proprietary products or spec-Sic equipment does not constitute a guarantee or w-ty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable.