New methods for the isolation of skeletal muscle sarcolemma and sarcoplasmic reticulum allowing a comparison between the mammalian and amphibian beta(2)-adrenergic receptors and calcium pumps

New methods were established for the rapid and simultaneous isolation of mu ltiple sarcolemmal and sarcoplasmic reticular fractions from very small amo unts (0.25-2.0 g) of skeletal muscle. The beta (2)-adrenergic receptor and calcium transport systems were used as indices of purity and functional int egrity as well as being the focal points of the study. These methods were f ound to be suitable for the special needs of small tissue samples, allowed rapid preparation and were appropriate for skeletal muscle from various spe cies, frogs to mammals. The sarcolemmal beta (2)-adrenergic receptor was ex pressed in frogs and mammals at similar levels of expression (336-454 fmol. mg(-1)). The calcium pump was also present in sarcolemmal and sarcoplasmic reticular fractions in all species but notable species differences were fou nd. In sarcolemmal fractions, while calcium binding was uniformly low (<1 n mol.mg(-1)), oxalate stimulation was variable: low in frogs (<similar to>1. 05-fold) high in mammals (120-450-fold). In sarcoplasmic reticular fraction s, calcium binding was low in frogs (4-9 nmol.mg(-1)) and much higher in ma mmals (322-383 nmol.mg(-1)); oxalate stimulated calcium transport to a much greater extent in frogs (>70-fold) than in mammals (1.6-2-fold). It is con cluded that the beta (2)-adrenergic receptor appears to be strongly conserv ed in skeletal muscle while the use of calcium pumps evolves from reliance in Amphibia on the sarcoplasmic reticular calcium pump to the use in Mammal ia of calcium pumps from both the sarcoplasmic reticulum and the plasma mem brane. Copyright (C) 2001 John Wiley & Sons, Ltd.