Technical note: quantification of multicatalytic proteinase complex (proteasome) activity by ion-exchange chromatography.
Within 1 h after slaughter, two 10-g samples of longissimus muscle were obtained from four crossbred beef cattle. Samples were homogenized in three or six volumes of extraction solution that consisted of 50 mM Tris base, 10 mM EDTA, and 10 mM 2-mercaptoethanol, pH adjusted to 8.3 with 6 N HCl. After centrifugation the supernatant from the three-volume extract was fractionated by addition of solid (NH4) 2SO4. Proteins that precipitate between 40 and 65% (NH4) 2SO4 were dialyzed and then loaded onto a DEAE-Sephacel column and eluted with a continuous gradient of NaCl from 100 to 400 mM (125 mL of each; Method A). The six-volume extract was loaded onto a DEAE-Sephacel column and eluted with a continuous gradient of NaCl from 0 to 350 mM (250 mL of each; Method B). Total peptidase activity eluted from the column was determined using the synthetic peptide N-CBZ-Gly-Gly-Leu-p-nitroanilide. Method B yielded greater multicatalytic proteinase complex (MCP) activities (picomoles of p-nitroaniline released/hour-1) per gram of muscle (1,538.25 +/- 105.15) than did Method A (1,195.05 +/- 86.55; P < .05). In addition, Method B permitted the quantification of calpain activity from the same fractions eluted. The relationship between enzyme activity and assay time (up to 45 min) and protein concentration (up to 10 micrograms) in the assay was linear. Studies indicated that the optimum temperature is in the range of 50 to 60 degrees C and the optimum pH in the range of 7.5 to 8.5.(ABSTRACT TRUNCATED AT 250 WORDS)
Arbona
J R
JR
Roman L. Hruska U.S. Meat Animal Research Center, ARS, USDA, Clay Center, NE 68933-0166.
Koohmaraie
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle / Q000378:metabolism*; D002852:Chromatography, Ion Exchange / Q000662:veterinary*; D003546:Cysteine Endopeptidases / Q000032:analysis* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000032:analysis* / Q000378:metabolism; D009132:Muscles / Q000201:enzymology*; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D013696:Temperature
D009097:Multienzyme Complexes; D009842:Oligopeptides; C037286:benzyloxycarbonylglycyl-glycyl-leucine-4-nitroanilide; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex