Separation of polar lipids by two‐dimensional thin layer chromatography providing resolution of all the lipid classes commonly encountered in animal cells and a sensitive, rapid, reproducible procedure for determination of phospholipids by phosphorus analysis of spots are described. Values obtained for brain and mitochondrial inner membrane phospholipids are presented.

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We have developed a procedure to isolate, from skeletal muscle, enriched terminal cisternae of sarcoplasmic reticulum (SR), which retain morphologically intact junctional "feet" structures similar to those observed in situ. The fraction is largely devoid of transverse tubule, plasma membrane, mitochondria, triads (transverse tubules junctionally associated with terminal cisternae), and longitudinal cisternae, as shown by thin-section electron microscopy of representative samples. The terminal cisternae vesicles have distinctive morphological characteristics that differ from the isolated longitudinal cisternae (light SR) obtained from the same gradient. The terminal cisternae consist of two distinct types of membranes, i.e., the junctional face membrane and the Ca 2+ pump protein-containing membrane, whereas the longitudinal cisternae contain only the Ca 2÷ pump protein-containing membrane. The junctional face membrane of the terminal cisternae contains feet structures that extend ~12 nm from the membrane surface and can be clearly visualized in thin section through using tannic acid enhancement, by negative staining and by freeze-fracture electron microscopy. Sections of the terminal cisternae, cut tangential to and intersecting the plane of the junctional face, reveal a checkerboardlike lattice of alternating, square-shaped feet structures and spaces each 20 nm square. Structures characteristic of the Ca 2+ pump protein are not observed between the feet at the junctional face membrane, either in thin section or by negative staining, even though the Ca 2÷ pump protein is observed in the nonjunctional membrane on the remainder of the same vesicle. Likewise, freeze-fracture replicas reveal regions of the P face containing ropelike strands instead of the high density of the 7-8-nm particles referable to the Ca ~+ pump protein. The intravesicular content of the terminal cisternae, mostly Ca2+-binding protein (calsequestrin), is organized in the form of strands, sometimes appearing paracrystalline, and attached to the inner face of the membrane in the vicinity of the junctional feet. The terminal cisternae preparation is distinct from previously described heavy SR fractions in that it contains the highest percentage of junctional face membrane with morphologically well-preserved junctional feet structures.The muscle fiber contains an intricate membraneous network that controls muscle contraction and relaxation by regulating the intracellular calcium concentration. The plasma membrane or plasmalemma invaginates transversely into the muscle sarcoplasm to form transverse tubules, which are connected to an internal reticular membrane system, the sarcoplasmic reticulum (SR). 1 The SR surrounds the sarcomere in Abbreviation used in this paper. SR, sarcoplasmic reticulum. a sleevelike manner, and is composed of two distinct portions: (a) the terminal cisternae which are junctionally associated with the transverse tubule, and (b) the longitudinal cisternae or longitudinal SR, which connect medially with the two term...

The calcium release channels (CRC)/ryanodine receptors of skeletal (Sk) and cardiac (C) muscle sarcoplasmic reticulum (SR) are hetero-oligomeric complexes with the structural formulas (ryanodine recepter (RyR)1 protomer) 4 (FKBP12) 4 and (RyR2 protomer) 4 (FKBP12.6) 4 , respectively, where FKBP12 and FKBP12.6 are isoforms of the 12-kDa receptor for the immunosuppressant drug FK506. The sequence similarity between the RyR protomers and FKBP12 isoforms is 63 and 85%, respectively. Using 35 S-labeled FKBP12 and 35 S-labeled FKBP12.6 as probes to study the interaction with CRC, we find that: 1) analogous to its action in skeletal muscle sarcoplasmic reticulum (SkMSR), FK506 (or analog FK590) dissociates FKBP12.6 from CSR; 2) both FKBP isoforms bind to FKBP-stripped SkMSR and exchange with endogenously bound FKBP12 of SkMSR; and 3) by contrast, only FKBP12.6 exchanges with endogenously bound FKBP12.6 or rebinds to FKBP-stripped CSR. This selective binding appears to explain why the cardiac CRC is isolated as a complex with FKBP12.6, whereas the skeletal muscle CRC is isolated as a complex with FKBP12, although only FKBP12 is detectable in the myoplasm of both muscle types. Also, in contrast to the activation of the channel by removal of FKBP from skeletal muscle, no activation is detected in CRC activity in FKBPstripped CSR. This differential action of FKBP may reflect a fundamental difference in the modulation of excitation-contraction coupling in heart versus skeletal muscle.FK506 is a potent immunosuppressive drug that binds to a family of related intracellular receptors termed FK506-binding proteins, varying in size from 12 to 54 kDa. Among these FKBPs, 1 FKBP12 is the most abundant and is involved in mediating the immunosuppressive action of FK506 in T lymphocytes. All of the known FKBP family members display cis-trans peptidyl-prolyl isomerase (PPIase) activity that is inhibited by FK506 and a structurally related compound, rapamycin (1). However, neither the immunosuppressive nor toxic side effects (including severe neuro-and nephrotoxicity) associated with FK506 therapy result from the inhibition of PPIase activity. Rather, the action of FK506 results from the specific inhibition of calcineurin by the FKBP12⅐drug complex. Calcineurin is a calcium-dependent protein phosphatase involved in the activation of T-lymphocytes (2). FKBP12 is a mere bystander protein during T-cell activation that becomes involved in blocking activation after it binds FK506. To date, the only physiological function directly associated with FKBP12 is its tight binding to (3, 4) and modulation of the ryanodine receptor (RyR-1) or calcium release channel of skeletal muscle SR (5-10). The purified SkM CRC 2 isolated from the CHAPS-solubilized TC is a hetero-oligomeric complex, with the structural formula of (RyR-1 protomer) 4 (FKBP12) 4 (5). Both FK506 and rapamycin bind to and dissociate FKBP12 from the SkM CRC (5). Both the native and FKBP-stripped CRCs have similar unitary conductance and sensitivity to ruthenium red (6). However, in co...

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