Hind-limb protein metabolism and calpain system activity influence post-mortem change in meat quality in lamb
Introduction
It is well established that the proteolysis of myofibrillar proteins by endogenous proteases during post-mortem aging is primarily responsible for the tenderisation of meat (Penny, 1980). The major proteins associated with myofibrillar contraction, actin, myosin and α-actinin, are not degraded during the aging process. Tenderisation is therefore thought to result from the cleavage of key structural cytoskeletal proteins such as the intermediate filament and costamere proteins (Taylor et al., 1995). Of the known endogenous proteolytic systems, the calcium activated proteolytic (calpain) system (E.C. 3.4.22.17) is thought to be responsible for the specific cleavage of these proteins during aging (Koohmaraie, 1994). It is becoming increasingly clear that calpains constitute a large family of enzymes (Suzuki et al., 1995). At present, skeletal muscle is known to contain the ubiquitous μ- and m-calpains, active at micro- and milli-molar Ca2+ concentrations, respectively, the muscle-specific p94 calpain, and the calpain-specific inhibitor calpastatin.
Apart from their effects on muscle during post-mortem storage, calpains play an important physiological role in intracellular protein degradation. The calpain system was first implicated in initiating metabolic turnover of myofibrillar proteins by Dayton et al., 1975. Since then, considerable evidence has been accumulated to support the involvement of calpains in skeletal muscle growth and development (Goll et al., 1991, Goll et al., 1992). Lysosomal proteolytic mechanisms account for approximately 25–30% of intracellular protein degradation (Reeves et al., 1981, Lowell et al., 1986), which corresponds to the levels of sarcoplasmic protein in muscle cells (Goll et al.). Also, although the proteasome is thought to be responsible for much of the degradation of myofibrillar proteins to their constituent amino acids through the ubiquitin system (Ciechanover, 1995), it is unable to initiate the disassembly of myofibrillar proteins from the myofibre (Koohmaraie, 1992). Accordingly, it is not unreasonable to believe that if calpains are involved in initiation of protein degradation in muscle in vivo, they may be altered in concert by factors known also to alter protein degradation.
The purpose of this study was to determine associations between variation in protein degradation in muscle of live animals, differences in calpain system activity and the rate of meat tenderisation during aging. Experimental treatments used to alter muscle protein degradation were level of nutrition and two endocrine regulators (clenbuterol, a β-agonist, and Long R3IGF-1, an analog of insulin-like growth factor 1) previously shown to alter muscle protein degradation (Bohorov et al., 1987, Oddy and Owens, 1996).
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Animals and experimental design
Twenty six Dorset–Merino–Border Leister-cross castrate lambs with an average weight of 27.0 ± 3.3 kg (s.e.) were used in this study. The experiment was based on a two by three factorial design. Lambs were divided into two groups and fed either at a high or low plane of nutrition. At each plane, animals were subdivided randomly into three groups of at least four lambs for endocrine (control, β-agonist, or IGF-1 analog) treatment.
All lambs were fed a high quality pelleted diet (Thompson et al., 1985
Protein kinetics in hind-limb muscle
No interactions between level of FI and endocrine treatment were observed for hind-limb protein turnover, oxygen consumption and blood flow determinations. Level of FI had no effect on hind-limb arterial–venous oxygen difference, oxygen uptake or blood flow through the infused hind-limb (Table 1). However, nutritional restriction reduced the difference in phenylalanine concentration between the arterial and venous bloods (p < 0.1), and had significant effects on hind-limb protein turnover.
Discussion
It is clear that nutritional and endocrine influences play an important role in regulating protein turnover (Reeds, 1989). This work describes the influences of short-term nutritional restriction, IGF-1 analog and clenbuterol treatments on the rate of protein turnover in the hind limb, levels of calpain system activity, and tenderisation (rate of proteolysis during aging). These observations are more striking given that rate of protein turnover was measured in the entire hind-limb muscle bed,
Acknowledgements
The authors are grateful for the expert technical assistance provided by C. Quinn, R. Woodgate and K. Zirkler. The Long-R3-IGF-1 was kindly donated by Dr. P.C. Owens, Department of Obstetrics and Gynecology, School of Medicine, University of Adelaide, Australia. The authors wish to acknowledge the financial support from the Cattle and Beef Industry Co-operative Research Centre (Meat Quality), and from NSW Agriculture. M.B. McDonagh was in receipt of a Meat Quality CRC postgraduate scholarship
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