Chicken skeletal muscle has three Ca2+-dependent proteinases.
Chicken breast muscle has three Ca2+-dependent proteinases, two requiring millimolar Ca2+ (m-calpain and high m-calpain) and one requiring micromolar Ca2+ (mu-calpain). High m-calpain co-purifies with mu-calpain through successive DEAE-cellulose (steep gradient), phenyl-Sepharose, octylamine agarose, and Sephacryl S-300 columns, but elutes after mu-calpain when using a shallow KCl gradient to elute a DEAE-cellulose column. The mu- and m-calpains have 80 and 28 kDa polypeptides and are analogous to the mu- and m-calpains that have been purified from bovine, porcine and rabbit skeletal muscle. High m-calpain, which seems to be a new Ca2+-dependent proteinase, is still heterogeneous after the DEAE-cellulose column eluted with a shallow KCl gradient. Additional purification through two successive HPLC-DEAE columns and one HPLC-SW-4000 gel permeation column produces a fraction having six major polypeptides and 6-8 minor polypeptides on SDS-PAGE. A 74-76 kDa polypeptide in this fraction reacts in Western blots with monospecific, polyclonal anti-calpain antibodies that react with both the 80 kDa and the 28 kDa polypeptides of mu- or m-calpain. High m-calpain also is related to mu- and m-calpain in that it causes the same limited digestion of skeletal muscle myofibrils, has a similar pH optimum near pH 7.9-8.4, requires Ca2+ for activity, and reacts with the calpain inhibitor, calpastatin, and a variety of serine and cysteine proteinase inhibitors in a manner identical to mu- and m-calpain. High m-calpain differs from mu- and m-calpain in its elution off DEAE-cellulose columns and its requirement of 3800 microM Ca2+ for one-half maximal activity compared with 5.35 microM Ca2+ for mu-calpain and 420 microM Ca2+ for m-calpain. The physiological significance of high m-calpain in unclear. The presence of mu-calpain in chicken breast muscle suggests that all skeletal muscles contain both mu- and m-calpain, although the relative proportions of these two proteinases may vary in different species.
Wolfe
F H
FH
Department of Animal Sciences, University of Arizona, Tucson 85721.
Sathe
S K
SK
Goll
D E
DE
Kleese
W C
WC
Edmunds
T
T
Duperret
S M
SM
D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000032:analysis; D002154:Calpain / Q000302:isolation & purification* / Q000378:metabolism; D002645:Chickens; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D009210:Myofibrils / Q000201:enzymology; D009218:Myosins / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D014336:Troponin / Q000378:metabolism; D019210:Troponin I; D020107:Troponin T
D000199:Actins; D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; D014336:Troponin; D019210:Troponin I; D020107:Troponin T; C033668:calpastatin; D002154:Calpain; D009218:Myosins; D002118:Calcium