title: Fibre type-specific expression of p94, a skeletal muscle-specific calpain.
abstract: Members of the calpain proteinase family are present in all mammalian cells, although a novel calpain 94 kDa isoform is found almost exclusively in skeletal muscle. p94 is difficult to purify from muscle and recombinant p94 autolyses rapidly when expressed in COS cells. However, in vivo the enzyme may be stabilised by interaction with titin, which has two well-characterised binding sites for p94 at the N2- and M-lines. Both these titin subdomains are subject to muscle-specific alternative splicing, which could be related to p94 expression level or stability in muscles of different fibre type. In this study, porcine longissimus dorsi (LD), trapezius (TZ) and adductor longus (AL) were characterised as fast, intermediate and slow using commercially available specific anti-human fast- and slow-myosin heavy chain mAbs and also by conventional histochemistry. p94 was quantified both in whole muscle preparations and single fibres by western blotting using an anti-p94 antiserum generated by expressing a recombinant p94 sequence as a GST fusion protein antigen. SDS PAGE and immunoblotting revealed a single band of approximately 94 kDa with identical mobility in all muscle and fibre preparations. The intensity of the 94 kDa band was greater in LD (22 +/- 1.7 densitometric units mean +/- SEM, n = 3) than TZ and AL (10 +/- 2.3 and 6 +/- 0.9 units, respectively). Expressed as a ratio relative to actin immunoreactivity, p94 is present in all types of single fibres isolated from TZ, but at a significantly lower level (P < 0.01) in slow type I (0.08 +/- 0.01, n = 9), compared to fast IIA/IIB fibres (0.22 +/- 0.02, n = 26). No evidence was seen for rapid or variable rate of p94 degradation in either type of fibre. These data suggest a positive correlation between p94 expression level and fast glycolytic characteristics in porcine muscle.
authors.affiliation: Division of Nutritional Biochemistry, School of Biological Sciences, University of Nottingham, Loughborough, Leicestershire, UK.
authors.forename: S W
authors.initials: SW
authors.lastname: Jones
authors.forename: T
authors.initials: T
authors.lastname: Parr
authors.forename: P L
authors.initials: PL
authors.lastname: Sensky
authors.forename: G P
authors.initials: GP
authors.lastname: Scothern
authors.forename: R G
authors.initials: RG
authors.lastname: Bardsley
authors.forename: P J
authors.initials: PJ
authors.lastname: Buttery
mesh_terms: D000818:Animals; D002154:Calpain / Q000096:biosynthesis*; D018656:Muscle Fibers, Fast-Twitch / Q000378:metabolism* / Q000473:pathology; D018657:Muscle Fibers, Slow-Twitch / Q000378:metabolism* / Q000473:pathology; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000473:pathology; D020033:Protein Isoforms / Q000378:metabolism; D013552:Swine; D013997:Time Factors
chemical_list: D020033:Protein Isoforms; D002154:Calpain; C078134:calpain p94