The association of phosphorylase kinase with rabbit muscle T-tubules.
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 alpha-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.
D000818:Animals; D002499:Centrifugation, Density Gradient; D006003:Glycogen / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D009124:Muscle Proteins / Q000378:metabolism; D009132:Muscles / Q000201:enzymology* / Q000648:ultrastructure; D010764:Phosphorylase Kinase / Q000302:isolation & purification*; D011817:Rabbits
D008565:Membrane Proteins; D009124:Muscle Proteins; D006003:Glycogen; D010764:Phosphorylase Kinase