Affinity labeling of calmodulin-binding proteins in skeletal muscle sarcoplasmic reticulum.
125I-Calmodulin (125I-CaM) binding to sarcoplasmic reticulum (SR) membranes isolated from skeletal muscle cells was investigated, and the CaM receptors associated with the membrane were identified by using the photoaffinity cross-linker methyl-4-azidobenzimidate or the chemical cross-linker dithiobis-N-hydroxysuccinimidyl propionate. Exogenous CaM binds to CaM-depleted membranes in a Ca2+- or Mg2+-dependent way. When both cations are added together to the reaction medium, the stimulatory effects appear to be additive, suggesting that Ca2+ and Mg2+ act by two distinct mechanisms. The Ca2+/Mg2+-dependent binding of CaM is specific since it is inhibited by unlabeled CaM or by trifluoperazine. Furthermore, it is saturable and shows one class of high affinity binding sites with a KD of about 52 nM and a beta max of about 5 pmol/mg of protein. The sensitivity of Ca2+ is expressed in two steps reaching half-saturation at free Ca2+ concentrations of about 1.6 x 10(-7) and 3 x 10(-5) M, respectively. On the other hand, the sensitivity to Mg2+ is expressed in one step with a half-saturation Mg2+ concentration of about 2 x 10(-3) M. Electrophoretic analysis in a polyacrylamide gradient and subsequent autoradiography demonstrated a major CaM-binding protein of about 60 kDa and five minor CaM receptors of about 148, 125, 41, 33, and 23 kDa, respectively. The major labeled protein (60 kDa) probably represents the CaM-dependent component involved in Ca2+ release from SR, whereas the others represent a previously unrecognized class of CaM receptors in skeletal SR.
Department of Zoology, University of Coimbra, Portugal.
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MG
Vale
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D008970:Molecular Weight; D009132:Muscles / Q000187:drug effects / Q000648:ultrastructure*; D011817:Rabbits; D014268:Trifluoperazine / Q000494:pharmacology
D000345:Affinity Labels; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D014268:Trifluoperazine; D008274:Magnesium; D002118:Calcium