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 wer e obtained from four crossbred beef cattle. Samples were homogenized i n 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 HCI. After centrifugation the supernatant from the three-vol ume 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 gradi ent of NaCl from 100 to 400 mM (125 mL of each; Method A). The six-vol ume extract was loaded onto a DEAE-Sephacel column and eluted with a c ontinuous 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 yiel ded greater multicatalytic proteinase complex (MCP) activities (picomo les 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 sa me fractions eluted. The relationship between enzyme activity and assa y time (up to 45 min) and protein concentration (up to 10 mug) 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. Also, MCP activity was unaffected by postmortem aging up to 14 d. This procedure should be beneficial to elucidate further the rol e of MCP in muscle growth and protein turnover.