The association of phosphorylase kinase with rabbit muscle T-tubules

V. Dombrádi, S. R. Silberman, E. Y C Lee, A. H. Caswell, N. R. Brandt

Research output: Contribution to journalArticle

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Abstract

Evidence is presented for the association of a phosphorylase kinase activity with transverse tubules as well as terminal cisternae in triads isolated from rabbit skeletal muscle. This activity remained associated with T-tubules throughout the purification of triad junctions by one cycle of dissociation and reassociation. The possibility that the presence of phosphorylase kinase in these highly purified membrane vesicle preparations was due to its association with glycogen was eliminated by digestion of the latter with α-amylase. The phosphorylase kinase activity associated with the T-tubule membranes was similar to that reported for other membrane-bound phosphorylase kinases. The enzyme had a high pH 6.8 pH 8.2 activity ratio (0.4 - 0.7) and a high level of Ca2+ independent activity (EGTA Ca2+ = 0.3-0.5). The kinase activated and phosphorylated exogenous phosphorylase b with identical time courses. When mechanically disrupted triads were centrifuged on continuous sucrose gradients, the distribution of phosphorylase kinase activity was correlated with the distribution of a Mr 128,000 polypeptide in the gradients. This polypeptide and a Mr 143,000 polypeptide were labeled with 32P by endogenous and exogenous protein kinases. These findings suggest that the membrane-associated phosphorylase kinase may be similar to the cytosolic enzyme. Markers employed for the isolated organelles included a Mr 102,000 membrane polypeptide which followed the distribution of Ca2+-stimulated 3-O-methylfluorescein phosphatase activity, which is specific for the sarcoplasmic reticulum. A Mr 72,000 polypeptide was confirmed to be a T-tubule-specific protein. Several proteins of the triad component organelle were phosphorylated by the endogenous kinase in a Ca2+/calmodulin-stimulated manner, including a Mr ca. 72,000 polypeptide found only in the transverse tubule.

Original languageEnglish
Pages (from-to)615-630
Number of pages16
JournalArchives of Biochemistry and Biophysics
Volume230
Issue number2
DOIs
Publication statusPublished - May 1 1984

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Phosphorylase Kinase
Muscle
Association reactions
Rabbits
Muscles
Peptides
Membranes
Organelles
Phosphotransferases
Phosphorylase b
Egtazic Acid
Sarcoplasmic Reticulum
Enzymes
Calmodulin
Amylases
Glycogen
Protein Kinases
Purification
Sucrose
Digestion

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

The association of phosphorylase kinase with rabbit muscle T-tubules. / Dombrádi, V.; Silberman, S. R.; Lee, E. Y C; Caswell, A. H.; Brandt, N. R.

In: Archives of Biochemistry and Biophysics, Vol. 230, No. 2, 01.05.1984, p. 615-630.

Research output: Contribution to journalArticle

Dombrádi, V. ; Silberman, S. R. ; Lee, E. Y C ; Caswell, A. H. ; Brandt, N. R. / The association of phosphorylase kinase with rabbit muscle T-tubules. In: Archives of Biochemistry and Biophysics. 1984 ; Vol. 230, No. 2. pp. 615-630.
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