Comparisons of four methods for quantification of lysosomal cysteine proteinase activities.
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 S-carboxymethylated-papain-Sepharose affinity column). Results indicated that, of the 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 Vmax values were .416 mM and 4,405 pmol.min-1.mg protein-1, respectively. The Km and Vmax for cathepsins B + L were .132 mM and 9,346 pmol.min-1.mg protein-1, 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 micrograms protein). The same preparations were assayed by conditions commonly used by many investigators (.005 mM substrate, approximately 75 micrograms protein, 30 min at 37 degrees C) and by conditions established in this study (1.0 mM substrate, 10 micrograms protein and 15 min at 37 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)
Koohmaraie
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M
U.S. Department of Agriculture, Clay Center, NE 68933-0166.
Kretchmar
D H
DH
D000818:Animals; D002401:Cathepsin B / Q000032:analysis / Q000302:isolation & purification; D056668:Cathepsin L; D002403:Cathepsins / Q000032:analysis / Q000302:isolation & purification; D002498:Centrifugation; D002846:Chromatography, Affinity; D003546:Cysteine Endopeptidases / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D009132:Muscles / Q000201:enzymology*; D013552:Swine / Q000378:metabolism*
D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; D056668:Cathepsin L