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New methods for the isolation of skeletal muscle sarcolemma and sarcoplasmic reticulum allowing a comparison between the mammalian and amphibian β 2 ‐adrenergic receptors and calcium pumps
Author(s) -
Hemmings Susan J.
Publication year - 2001
Publication title -
cell biochemistry and function
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.933
H-Index - 61
eISSN - 1099-0844
pISSN - 0263-6484
DOI - 10.1002/cbf.909
Subject(s) - sarcolemma , calcium , sarcoplasm , endoplasmic reticulum , skeletal muscle , medicine , reticular connective tissue , chemistry , endocrinology , calsequestrin , biology , biochemistry , biophysics , anatomy , ryanodine receptor
New methods were established for the rapid and simultaneous isolation of multiple sarcolemmal and sarcoplasmic reticular fractions from very small amounts (0.25–2.0 g) of skeletal muscle. Theβ 2 ‐adrenergic receptor and calcium transport systems were used as indices of purity and functional integrity as well as being the focal points of the study. These methods were found to be suitable for the special needs of small tissue samples, allowed rapid preparation and were appropriate for skeletal muscle from various species, frogs to mammals. The sarcolemmalβ 2 ‐adrenergic receptor was expressed 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 found. In sarcolemmal fractions, while calcium binding was uniformly low (<1 nmol·mg −1 ), oxalate stimulation was variable: low in frogs (∼1.05‐fold) high in mammals (120–450‐fold). In sarcoplasmic reticular fractions, calcium binding was low in frogs (4–9 nmol·mg −1 ) and much higher in mammals (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 concluded that theβ 2 ‐adrenergic receptor appears to be strongly conserved in skeletal muscle while the use of calcium pumps evolves from reliance in Amphibia on the sarcoplasmic reticular calcium pump to the use in Mammalia of calcium pumps from both the sarcoplasmic reticulum and the plasma membrane. Copyright © 2001 John Wiley & Sons, Ltd.