Sarcoplasmic reticulum membranes, isolation

Other fatty acids varied to a lesser extent. The accumulation of unsaturated fatty acids occurs with an increase in chain length which would mantain the fluidity of the membrane constant, suggesting that these variations in fatty acid composition do not contribute significantly to the observed changes in Ca transport during muscle development in vivo. This is supported by the lack of changes in passive Ca permeability observed in sarcoplasmic reticulum membranes isolated from developing embryos and chicks (Martonosi, 1975). 

Fig. lc. Electronmicrograph of isolated sarcoplasmic reticulum membranes loaded with calcium oxalate46. The preparation was negatively stained with potassium phosphotungstate. Size and shape of the calcium oxalate precipitates differ considerably. Fine particles which are present on the surface of most vesicles are clearly visible. In all pictures the length of the bars measures 0.1 pm... 

Isolation and Purification of Fragmented Sarcoplasmic Reticulum Membranes... 

Ghosh et al. [48] have isolated two isoforms of nSMase from rabbit skeletal muscle (92 and 53 kDa). Peptide mapping revealed important structural similarities, and the catalytic activities were also similar, except that the 53 kDa protein was Mg" -independent. These nSMases are located in the transverse tubules of the muscle cells, which may be related to the observation that sphingosine modulates calcium release from sarcoplasmic reticulum membranes [49]. Two Mn -and Mg" -dependent nSMases located in the microsomal membranes of seminiferous tubes of immature Wistar rats have been characterized [50] whose properties do not appear to differ significantly from other mammalian nSMases. Finally, we mention two other nSMases purified from eukaryotic natural sources, namely the Mg -dcpcridcril nSMase isolated from Saccharomyces cerevisiae [51], and that obtained from membrane fractions of intraeryfhrocytic Plasmodium falciparum, the malaria parasite [52]. The latter enzyme was activated by phosphatidylserine and other anionic phosphohpids, and was sensitive to scyphostatin, an inhibitor of mammalian nSMase (see below). 

The transport protein is the main constituent of the sarcoplasmic reticulum membranes and thus can be isolated in large quantities. 

The sarcoplasmic reticulum membranes, a structural component of the muscle cell—organization, isolation and identification... 

For the isolation of sarcoplasmic reticulum membranes the homogenate was subjected to differential centrifugation inmediately after preparation (Boland et al., 1974). 

The techniques employed to measure the ATP dependent, azide insensitive calcium uptake at 25 by whole cell muscle homogenates and isolated SR are described elsewhere (Boland et al., 1974 Martonosi et al., 1976). The concentration of Ca-ATPase in homogenates and sarcoplasmic reticulum membranes was determined by specific labeling of the enz)mie with 32p-ATP (Martonosi et al., 1976). 

The intracellular hgand-gated Ca " channels include the channels in endoplasmic and sarcoplasmic reticulum (SR) membranes that are opened upon binding of the second messenger, inositol triphosphate (IP3). These are intracellular Ca release channels that allow Ca to exit from intracellular stores, and consequently to increase the concentration of cytoplasmic Ca [5]. A second type of intracellular Ca release channel is the Ca - and ryanodine-sensitive channel that was originally characterized and isolated from cardiac and skeletal muscle [5-7] but appears to exist in many types of cells. It has become evident that IP3-gated channels and ryanodine-sensitive channels are structurally related but distinct proteins [8] that are present in many cell types [9]. While very interesting, time and space will not allow for further discussion of these channels. 

Fig. 2 a. Elcctronmicrograph of a freeze fractured preparation of isolated sarcoplasmic reticulum vesicles. 9 nm membrane particles are clearly visible on the concave cytoplasmic fracture faces of the vesicular membranes... 

Fig. 2 b. Electronmicrograph of a sectioned preparation of isolated sarcoplasmic reticulum vesicles decorated with the electron dense thiol reagent mercuri phenylazoferritin. Note the absence of ferritin particles on the internal surface of the membrane fragment in the center of the picture50 ... 

Biochemical alterations have been found in fragmented sarcoplasmic reticulum isolated from dystrophic human, mouse and chicken muscle. Alterations in calcium transport, ATP hydrolysis and phosphoenzyme formation have been reported. Some of these biochemical alterations in the dystrophic sarcoplasmic reticulum are suggested to be due to alterations of the lipid environment of these membranes it has been suggested that the cholesterol content of dystrophic sarcoplasmic reticulum is elevated [182-187]. 

More difficult, but also proniising to be honoured with success proves the isolation of pure, integral membrane proteins and their structure analysis by diffraction techniques. One avenue is given by defined solubilization with detergents and the evaluation of the small-angle (particle) scattering pattern from dilute solution (for reviews on this method, see Refs. and ). This has so far been attempted with bovine rhodopsin, the major protein component of retinal rod outer sement membranes with the Ca -dependent ATPase from sarcoplasmic reticulum... 

Lipids play an essential role in the structure and function of biological membranes. The concept that the lipid enviroment influences the activity of membrane bound enzymes is now generally accepted. The sarcoplasmic reticulum(SR) of skeletal muscle is an intracellular membranous system with an important physiological role in contraction and relaxation (Hasselbach, 1964 Martonosi, 1971). The Ca2+ transport and (Ca2++ Mg2+)-ATPase activities which exhibit isolated SR preparations have been shown to be dependent on membrane phospholipids (Martonosi, 1968). An involvement of the fatty acid component of phospholipids in the functioning of the Ca2+ pump has also been suggested (Seiler et al., 1970 Seiler Hasselbach,... 

The cardiac toxicity of anthracyclines can be reproduced in numerous animal species (Working et al. 1999). Histopathologically, the cardiac effects consist of damage to the sarcoplasmic reticulum (due to drug interaction with ryanodine receptor-mediated calcium-induced calcium release), and, as a consequence, calcium overload results in mitochondrial swelling and altered outer membrane permeability that can occur within hours of administration and progress with repeated exposures. The myocardial damage can also be induced in vitro in isolated heart tissues (Meima et al. 2007 Robert 2007). 

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