Isolation of sarcoplasmic reticulum by zonal centrifugation and purification of Ca2+-pump and Ca2+-binding proteins

Since this is the first review on lipids to appear in the Annual Review of Physiology, it should cover the spectrum of the functional roles of lipids: (a) as fuel; (b) as membrane components; (c) in blood coagulation; (d) as components of immune systems; (e) as vitamins and hormones; and (j) in interfacial phenomena (e.g. in digestive processes and at the gas-liquid interface of lung alveoli). Space limitations make it impossible to consider all of these areas. Fortunately, the latter four have been the subject of many reviews, not as lipids per se but as components of physiologi�al systems. Therefore this discussion is devoted to two of the functions: as fuel ,lU ld as membrane components. The following review articles are offered as a guide to the other four areas: blood coagulation (110, 204); immune systems (157); hormones and vitamins (21, 39, 55, 60, 84, 103, 154, 203); interfacial phenomena in digestive processes (15, 16); lung surfactant (91, 127, 195).

Lipids and Lipid Metabolism

A fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene, was incorporated in four different biological membranes, the purple membrane of Halobacterium halobium, human erythrocyte membrane, rabbit sarcoplasmic reticulum membrane, and rat liver mitochondrial membrane. Time-resolved fluorescence depolarization of the probe suggested that the rotational Brownian motion of the probe in the membranes was restricted in the angular range. The motion of the rod-shaped, lipophilic probe molecule, expected to reflect closely the motion of neighboring lipid hydrocarbon chains, was analyzed in terms of the wobbling-in-cone model in which the major axis of the probe was assumed to wobble freely in a cone of semiangle theta c with a wobbling diffusion constant Dw. At 35 degrees C, Dw in the four membranes, in the above order, ranged between 0.048 and 0.15 ns-1 and theta c between 31 and 53 degrees. From the rotational rate Dw, the viscosity against the wobbling motion was calculated to be 0.9-0.3 P. When the temperature was raised from 10 to 35 degrees C, Dw in all membranes increased approximately 3-fold, corresponding to activation energies of 7-8 kcal/mol, and theta c increased by about 10 degrees, except for the purple membrane in which the angular range remained narrow. The same characteristic temperature dependence has been found in many model membrane systems that contain unsaturated lecithins, suggesting an important role of unsaturated phospholipids in the dynamic structure of the lipid hydrocarbon chain region of biological membranes at physiological temperatures. Comparison with model systems suggests that proteins and cholesterol act mainly as barriers that narrow the angular range.

Dynamic structure of biological membranes as probed by 1,6-diphenyl-1,3,5-hexatriene: a nanosecond fluorescence depolarization study.

Publisher Summary This chapter discusses a critical appraisal of information about the possible involvement of sarcoplasmic reticulum in various muscle diseases such as cardiac failure, muscular dystrophy, myotonia, and denervation atrophy. Because of the singular concentration of attention on the role of sarcoplasmic reticulum in the regulation of sarcoplasmic Ca 2+ concentration, conspicuous gaps developed in the knowledge regarding other possible functions of sarcoplasmic reticulum such as its involvement in the energy metabolism of muscle cells. Recent evidence indicates that the sarcoplasmic reticulum may represent a compartment for hexokinase, glyceraldehydephosphate dehydrogenase, and phosphofructokinase activities. Other metabolic functions of sarcoplasmic reticulum include the synthesis of phospholipids and proteins, as suggested by their turnover in Sarcoplasmic reticulum membrane. Finally, a detailed biochemical analysis of the function of transverse tubular system and the triad may soon become possible. The distribution of sarcoplasmic reticulum in the muscle cell and its relationship to the myofibrils, surface membranes, and the T-system tubules bear considerable importance in relation to its physiological function.

Biochemical and Clinical Aspects of Sarcoplasmic Reticulum Function

A multiple equilibrium binding model is used to examine phospholipid and cholesterol binding with the transmembranous protein Ca2+-ATPase (calcium pump). The protein was reconstituted in egg phosphatidylcholine bilayers by lipid substitution of rabbit muscle sarcoplasmic reticulum. Electron spin resonance spectra of a phosphatidylcholine spin-label and a recently developed cholesterol spin-label show two major spectral contributions, a motionally restricted component consistent with interactions between the label and the protein surface and another component characteristic of motion of the label in a fluid lipid bilayer. The number of lipid binding (or contact) sites at the hydrophobic surface of the protein is calculated to be N = 22 +/- 2. Experiments with intact sarcoplasmic reticulum membranes give approximately the same value for N. The relative binding constants are Kav approximately 1 for the phosphatidylcholine label and Kav approximately 0.65 for the cholesterol spin-label. Thus, cholesterol does contact the surface of the protein, but with a somewhat lower probability than phosphatidylcholine. This is confirmed by competition experiments where unlabeled cholesterol and the phospholipid spin-label are both present in the bilayer. Evidently the flexible acyl chains of the phospholipid molecules accommodate more readily to the irregular surface of the protein than does the rigid steroid structure of cholesterol.

Competition between cholesterol and phosphatidylcholine for the hydrophobic surface of sarcoplasmic reticulum Ca2+-ATPase.

The lipid composition of a highly purified preparation of rat skeletal muscle sarcoplasmic reticulum was studied. These membranes contain per mg of protein 0.50 ± 0.02 μmoles of phosphatidylcholine, 0.13 ± 0.02 μmoles of phosphatidylethanolamine and 0.07 ± 0.02 μmoles ofphosphatidylinositol. These three components account for 97.3% of total lipid phosphorus. Unlike other mammalian membranes so far studied, this preparation contains neither sphingomyelin nor phosphatidylserine. Neural lipids were also measured and it is concluded that neither cholesterol nor other neutral lipids are components of the membranes studied. The results of this study indicate therefore that the lipid profile of sarcotubular membranes is relatively simple compared with most other membranes.

B. P. Yu

Papers

Lipid components of sarcotubular membranes.