Sarcoplasmic membrane proteins

The number of different proteins in a membrane varies from less than a dozen in the sarcoplasmic reticulum to over 100 in the plasma membrane. Most membrane proteins can be separated from one another using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), a technique that has revolutionized their study. In the absence of SDS, few membrane proteins would remain soluble during electrophoresis. Proteins are the major functional molecules of membranes and consist of enzymes, pumps and channels, structural components, antigens (eg, for histocompatibility), and receptors for various molecules. Because every membrane possesses a different complement of proteins, there is no such thing as a typical membrane structure. The enzymatic properties of several different membranes are shown in Table 41-2. 

Purified membrane proteins or enzymes can be incorporated into these vesicles in order to assess what factors (eg, specific lipids or ancillary proteins) the proteins require to reconstitute their function. Investigations of purified proteins, eg, the Ca " ATPase of the sarcoplasmic reticulum, have in certain cases suggested that only a single protein and a single lipid are required to reconstitute an ion pump. 

The Ca transport ATPase of sarcoplasmic reticulum is an intrinsic membrane protein of 110 kDa [8-11] that controls the distribution of intracellular Ca by ATP-dependent translocation of Ca " ions from the cytoplasm into the lumen of the sarcoplasmic reticulum [12-16],... 

Phospholamban (PLB or PLN) is a single-pass, 52-residue integral membrane protein that regulates myocardial contractility by direct physical interaction with sarco(endo)plasmic reticulum Ca-ATPase (SERCA), a 110-kDa enzyme that maintains calcium homeostasis in the sarcoplasmic... 

The low lipid-protein ratio of 0.5 together with the size of the sarcoplasmic vesicles implies that only approximately 30% of their membranes can be occupied by a regular lipid bilayer structure. Consequently, a large fraction of the membrane protein must interrupt the lipid bilayer and reach throughlt. The fact that only one polypeptide chain constitutes the structural unit of the calcium transport protein strong-... 

The calcium-independent ATPase of the lipid modified preparations is not only different from the calcium-dependent ATPase but also from the calcium-independent ATPase of native preparations — the basic ATPase — which has a lower nucleotide specificity126. The experiments in which the lipid matrix of the sarcoplasmic membranes has been replaced by lipid compounds not present in native membranes reveal a high degree of functional flexibility of the enzyme. On the other hand, a few residual lipids in the protein are sufficient to prevent these changes in the structure of the enzyme and to preserve its calcium sensitivity. 

The transporting protein in the sarcoplasmic membrane can be phosphorylated by ATP as well as by inorganic phosphate (cf.2,174 ). In the forward running mode of the pump, i. e. when the calcium pump accumulates calcium and concomitantly hydrolyzes ATP, the terminal phosphate residue of ATP is transferred to the transport protein. The reaction depends on the presence of calcium ions in the external medium. In the reverse mode of the pump inorganic phosphate is incorporated into the transport protein. This reaction is inhibited when calcium ions are present in the external medium,... 

High affinity Ca2+ binding sites amounting to about 10 nmoles per mg of protein are exposed on the exterior of sarcoplasmic reticulum membranes. They can solely be accounted for by the ATPase which binds two moles of Ca2+ per mole and constitutes between 5 and 7 nmoles per mg of intact sarcoplasmic reticulum protein. Inside the vesicles the binding capacities for ATP-involved calcium can be as high as 100 nmoles per mg protein. 

A biologic reason for the abundance of nonlamellar lipids in membranes is that they possess the ability to modulate the activities of membrane proteins (15, 16). It has been recognized that membranes exist in a state of curvature frustration, which may be sufficiently large to have significant effect on certain protein conformations (17). Many examples show that the lipid bilayer elastic curvature stress indeed couples to conformational changes of membrane proteins (15, 18, 19). Protein kinase C is one such example of an enzyme activated by lipids that exhibit a propensity for nonlamellar phase formation (20). The activity of Ca " -ATPase from sarcoplasmic reticulum membranes also strongly correlates with the occurrence of nonbilayer lipids in the membrane and increases with the increase of their amount. It is noteworthy that the protein activity does not depend on the chemical structure of the lipids but only on their phase propensity thus specific binding interactions are ruled out. The list of proteins with activities that depend on the phase properties... 

Figure 12.16. Sds-Acrylamide Gel Patterns of Membrane Proteins. (A) The plasma membrane of erythrocytes. (B) The photoreceptor membranes of retinal rod cells. (C) The sarcoplasmic reticulum membrane of muscle cells. [Courtesy of Dr. Theodore Steck (Part A) and Dr. David MacLennan (Part C).]...

The Sarcoplasmic Reticulum Ca + ATPase Is an Integral Membrane Protein... 

We will consider the structural and mechanistic features of these enzymes by examining the Ca2+ ATPase found in the sarcoplasmic reticulum (SR Ca2+ ATPase) of muscle cells. This enzyme, which constitutes 80% of the sarcoplasmic reticulum membrane protein, plays an important role in muscle contraction, which is triggered by an abrupt rise in the cytosolic calcium level. Muscle relaxation depends on the rapid removal of Ca + from the cytosol into the sarcoplasmic reticulum, a specialized compartment for calcium storage, by the SR Ca + ATPase. This pump maintains a Ca2+ concentration of approximately 0.1 iM in the cytosol compared with 1.5 mM in the sarcoplasmic reticulum. 

Active transport is often carried out at the expense of ATP hydrolysis. P-type ATPases pump ions against a concentration gradient and become transiently phosphorylated on an aspartic acid residue in the process of transport. P-type ATPases, which include the sarcoplasmic reticulum Ca2+ ATPase and the Na+-K+ ATPase, are integral membrane proteins with conserved structures and catalytic mechanisms. 

Relatively large changes in membrane thickness have been demonstrated to alter the function of integral membrane proteins. An example of the magnitude of the change in membrane thickness needed to alter protein function is provided by studies of the sarcoplasmic reticulum calcium ATPase. Activity of this integral membrane protein in bilayers with symmetrically substituted, monounsaturated acyl chains with 16, 18, or 20 carbons is nearly constant. However, when the acyl chains are shortened to 14 carbons or lengthened to 22 carbons, activity is reduced by more than a factor of 3 (Lee, 1998). 

The effect of hydrogenation on membrane protein function has been examined using the Ca2+ pump of sarcoplasmic reticulum of rabbit hind-leg muscle [36, 37]. Up to 35% of the unsaturated bonds of the membrane lipids could be saturated in the presence of Wilkinson s catalyst. ATPase activity was completely inhibited on adding catalyst but this could be prevented by preserving the catalyst in its hydride form. When the effect of hydrogenation of sarcoplasmic reticulum on calcium pump activity was assayed in buffers saturated with H2, it was found that removal of 25% of cis double bonds did not affect the activity of Ca2+-ATPase. 

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... 

Two examples of membrane proteins physically associated to lipids will be discussed bovine erythrocyte acetylcholinesterase and skeletal muscle sarcoplasmic reticulum Ca -Mg " ATPase. 

The Ca -Mg ATPase is the major protein constituent of the skeletal muscle sarcoplasmic membranes. This enzyme can be solubilized... 

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