Sarcoplasmic membrane isolated

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]. 

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). 

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. 

Mehorta and coworkers (1989) observed that isolated fractions of brain and heart cells from rats orally administered 0.5-10 mg endrin/kg showed significant inhibition of Ca+2 pump activity and decreased levels of calmodulin, indicating disruption of membrane Ca+2 transport mechanisms exogenous addition of calmodulin restored Ca+2-ATPase activity. In vitro exposure of rat brain synaptosomes and heart sarcoplasmic reticuli decreased total and calmodulin-stimulated calcium ATPase activity with greater inhibition in brain preparations (Mehorta et al. 1989). However, endrin showed no inhibitory effects on the calmodulin-sensitive calcium ATPase activity when incubated with human erythrocyte membranes (Janik and Wolf 1992). In vitro exposure of rat brain synaptosomes to endrin had no effect on the activities of adenylate cyclase or 3, 5 -cyclic phosphodiesterase, two enzymes associated with synaptic cyclic AMP metabolism (Kodavanti et al. 1988). 

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... 

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 ... 

Live and deproteinated plasma membranes of Acholeplasma laUawii were investigated by FTIR (Casal et al., 1980 Cameron et al., 1985). The temperature profiles of the gel to liquid crystalline phase transition of intact and deproteinated membranes, monitored by z/as(CD2), differ considerably. In intact membranes, the transition is broad and at temperatures within the range of the phase transition the live mycoplasma is able to keep the fluidity of its plasma membranes at a much higher level than that of the isolated plasma membrane. Native and reconstituted sarcoplasmic recticulum were investigated by Mendelsohn et al. (Mendelsohn et al., 1984). It appears that the protein Ca -ATPase interacts preferentially with the DOPC component of the membrane. A survey of these studies is available (Mantsch and McElhaney, 1991). 

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). 

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... 

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... 

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