Junctional transverse tubules (TT) isolated from triads of rabbit skeletal muscle by centrifugation in an ion-free sucrose gradient were compared with membrane subfractions, predominantly derived from the free portion of TT, that had been purified from sarcoplasmic reticulum membrane contaminants by three different methods. The markers used were diagnostic membrane markers and the dihydropyridine (DHP) receptor, which is a specific marker of the junctional membrane of TT. Junctional TT have a high membrane density (Bmax. 60 pmol/mg of protein) of high-affinity (Kd 0.25 nM) DHP-binding sites using [3H]PN200-110 as the specific ligand. When analysed by SDS/PAGE under reducing conditions and by Western blot techniques, the TT were found to contain a concanavalin A-binding 150 kDa glycoprotein which probably corresponds to the alpha 2-subunit of the DHP receptor. This conclusion was supported by correlative immunoblot experiments with a specific antibody. Junctional TT are further distinguished from free TT by the presence of a high number (Bmax. 20 pmol/mg of protein) of [3H]cyclic AMP receptor sites, as determined by the Millipore filtration technique of Gill & Walton [(1974) Methods Enzymol. 38, 376-381]. Use of this method means that the number of receptors may have been underestimated. The TT-bound cyclic AMP receptor was identified as a 55 kDa protein by specific photoaffinity labelling with 8-N3-[3H]cyclic AMP, and had similar phosphorylation properties and apparent molecular mass to the RII form of the regulatory subunit of cyclic AMP-dependent protein kinase. Co-localization of the intrinsic cyclic AMP-dependent protein kinase and of the DHP receptor complex to the junctional membrane of TT supports the hypothesis that the 170 kDa alpha 1-subunit of the receptor is a substrate for the kinase.
COLOCALIZATION OF THE DIHYDROPYRIDINE RECEPTOR AND THE CYCLIC AMP-BINDING SUBUNIT OF AN INTRINSIC PROTEIN-KINASE TO THE JUNCTIONAL MEMBRANE OF THE TRANSVERSE TUBULES OF SKELETAL- MUSCLE
SALVATORI, SERGIO;DAMIANI, ERNESTO;MARGRETH, ALFREDO
1990
Abstract
Junctional transverse tubules (TT) isolated from triads of rabbit skeletal muscle by centrifugation in an ion-free sucrose gradient were compared with membrane subfractions, predominantly derived from the free portion of TT, that had been purified from sarcoplasmic reticulum membrane contaminants by three different methods. The markers used were diagnostic membrane markers and the dihydropyridine (DHP) receptor, which is a specific marker of the junctional membrane of TT. Junctional TT have a high membrane density (Bmax. 60 pmol/mg of protein) of high-affinity (Kd 0.25 nM) DHP-binding sites using [3H]PN200-110 as the specific ligand. When analysed by SDS/PAGE under reducing conditions and by Western blot techniques, the TT were found to contain a concanavalin A-binding 150 kDa glycoprotein which probably corresponds to the alpha 2-subunit of the DHP receptor. This conclusion was supported by correlative immunoblot experiments with a specific antibody. Junctional TT are further distinguished from free TT by the presence of a high number (Bmax. 20 pmol/mg of protein) of [3H]cyclic AMP receptor sites, as determined by the Millipore filtration technique of Gill & Walton [(1974) Methods Enzymol. 38, 376-381]. Use of this method means that the number of receptors may have been underestimated. The TT-bound cyclic AMP receptor was identified as a 55 kDa protein by specific photoaffinity labelling with 8-N3-[3H]cyclic AMP, and had similar phosphorylation properties and apparent molecular mass to the RII form of the regulatory subunit of cyclic AMP-dependent protein kinase. Co-localization of the intrinsic cyclic AMP-dependent protein kinase and of the DHP receptor complex to the junctional membrane of TT supports the hypothesis that the 170 kDa alpha 1-subunit of the receptor is a substrate for the kinase.
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