Jr. Arbona et M. Koohmaraie, TECHNICAL NOTE - QUANTIFICATION OF MULTICATALYTIC PROTEINASE COMPLEX (PROTEASOME) ACTIVITY BY ION-EXCHANGE CHROMATOGRAPHY, Journal of animal science, 71(12), 1993, pp. 3301-3306
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.