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Criteria for Identification of α1 and α2 Subunits in a Purified Skeletal Muscle Calcium Channel Preparation

  • Pal L. Vaghy
  • Kunihisa Miwa
  • Kiyoshi Itagaki
  • Ferenc GubaJr.
  • Edward McKenna
  • Arnold Schwartz
Part of the GWUMC Department of Biochemistry Annual Spring Symposia book series (GWUN)

Abstract

Skeletal and cardiac muscle Ca channels have been isolated in several different laboratories (Curtis and Catterall, 1984; Borsottoet al., 1985; Striessniget al., 1986a; Cooperet al., 1987a; Nakayamaet al., 1987). The procedures are modified versions of one of the two originally described methods, i.e., the Curtis and Catterall (1984) and the Lazdunski pro cedure (Borsottoet al., 1985). Curtis and Catterall (1984) isolated T-tubular membranes from fresh (not frozen) rabbit skeletal muscle, solubilized the membranes with 1% digitonin, and employed a four-step purification procedure, which included the combined use of wheat germ agglutinin affinity chromatography and ion exchange chromatography followed by sucrose density gradient centrifugation. This procedure resulted in the enrichment of the preparation in three different polypeptides termedα(135-kDa),ß(55-kDa), andγ(33-kDa) subunits. Theαsubunit had a characteristic behavior under sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); its apparent molecular weight was higher (165 kDa) under nonreducing conditions than under disulfide-reducing conditions (135 kDa) that were in the presence of dithiothreitol orß-mercaptoethanol. The Lazdunski group isolated Ttubular membranes from frozen rabbit skeletal muscle thawed at room temperature, solubilized the T-tubular membranes with 1% CHAPS, and employed a combination of Chromatographie steps utilizing anion exchange, lectin affinity, and gel filtration (Borsottoet al.,

Keywords

Apparent Molecular Weight Sucrose Density Gradient Nonreducing Condition Rabbit Skeletal Muscle Sucrose Density Gradient Centrifugation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Plenum Press, New York 1989

Authors and Affiliations

  • Pal L. Vaghy
    • 1
  • Kunihisa Miwa
    • 1
  • Kiyoshi Itagaki
    • 1
  • Ferenc GubaJr.
    • 1
  • Edward McKenna
    • 1
  • Arnold Schwartz
    • 1
  1. 1.Department of Pharmacology and Cell BiophysicsUniversity of Cincinnati College of MedicineCincinnatiUSA

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