Sarcoplasmic reticulum, ATPase from

FIGURE 10.15 A mechanism for Ca -ATPase from sarcoplasmic reticulum. Note the similarity to the mechanism of Na, K -ATPase (see also Figure 10.11). ( Out here represents the cytosol In represents the lumen of the SR.)... 

In the Ca-ATPase from sarcoplasmic reticulum, oligonucleotide-directed, site-specific mutagenesis has been applied to identify amino acids involved in Ca binding. Mutation of 30 glutamate and aspartate residues, singly or in groups, in a stalk sector near the transmembrane domain has little effect on Ca " -transport. In contrast mutations to Glu ° , Glu, Asn , Thr , Asp ° or Glu ° resulted in loss... 

Mutagenesis in yeast H-ATPase and Ca-ATPase from sarcoplasmic reticulum... 

O. T. Jones, R. J. Froud, and A. G. Lee, Interactions of hexachlorocyclohexanes with the (Ca2t+Mg2)-ATPase from sarcoplasmic reticulum, Biochim. Biophys. Acta 812, 740-751 (1985). 

In order to characterize the active site structure of Ca ATPase from sarcoplasmic reticulum, we have employed Gd + as a paramagnetic probe of this system in a series of NMR and EPR investigations. Gadolinium and several other lanthanide ions have been used in recent years to characterize Ca + (and in some cases Mg2+) binding sites on proteins and enzymes using a variety of techniques, including water proton nuclear relaxation rate measurements (35,36,37), fluorescence (38) and electron spin resonance (39). In particular Dwek and Richards (35) as well as Cottam and his coworkers (36,37) have employed a series of nuclear relaxation measurements of both metal-bound water protons and substrate nuclei to characterize the interaction of Gd + with several enzyme systems. 

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... 

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... 

For systems that are naturally and reversibly photosensitive such as the carbon monoxide complexes of heme proteins [18,. 30, 31], initiation by a light pulse is possible. By preparation of inert but photoactivable reactant or cofactor precursors such as caged ATP [32-34], photosensitivity may be conferred on otherwise photoinert systems [21], thus extending the generality of this approach. This approach was first combined with x-ray monitoring by Blasie [35, 36], in studies of oriented multilayers containing the Ca2+-ATPase from sarcoplasmic reticulum. 

The system is, of course, not totally natural in that one surface of the membrane is blocked by the electrode. Interactions may exist between the receptor and the electrode surface that inhibit functionality. We have, however, found that aspects of the function of several receptors, including rhodopsin (8), the nicotinic acetylcholine receptor, and the Ca-ATPase from sarcoplasmic reticulum can be retained in these systems (unpublished results). 

There are two ways in which membranes of diacetylenic lipids containing intrinsic membrane proteins can be obtained either proteins extracted from natural membranes with detergent can be reconstituted into synthetic diacetylenic phosphatidylcholines or the growth medium of micro-organisms incapable of synthesizing their own fatty acids can be enriched with diacetylenic fatty acid. In this laboratory, Ca2+-ATPase from sarcoplasmic reticulum and bacteriorhodopsin from the purple membrane of Halobacterium halobium have been reconstituted into diacetylenic phosphatidylcholines. Provided the more reactive mixed-chain lipids are used polymerisation can be achieved before the protein is denatured by the UV irradiation. Both proteins remain active within polymeric bilayers. 

Pick, U. Interaction of fluorescein isothiocyanate with nucleotide-binding sites of the calcium ATPase from sarcoplasmic reticulum. Eur. J. Biochem. 1981,121, 187-195. 

Luedi, H. Hasselbach, W. Excimer formation of ATPase from sarcoplasmic reticulum labeled with N-(3-pyrene)maleinimide. Fur. J. Biochem. 1983, 130,5-8. 

Correlations between critical temperatures for membrane lipid phases determined with EPR techniques and discontinuities in Arrhenius plots have also been shown for the ATPase from sarcoplasmic reticulum (Eletr and Inesi, 1972), for UDP-glucuronyltransferase, and for G-6-Pase (Eletr et al., 1973) from liver microsomes. In the case of the microsomal membranes, perturbation of the membrane lipids by treatment with detergents or phospholipase A leads to linear Arrhenius plots for both enzyme activities and Tq between 5° and 30 C. For UDP-glucuronyltransferase, the phase change in the lipids also results in a loss of substrate specificity and a loss of sensitivity to an allosteric effector (Vessey and Zakim, 1974). 

Tamburini, R., E. X. Albuquerque, J. W. Daly, and F. C. Kauffman Inhibition of calcium-dependent ATPase from sarcoplasmic reticulum by a new class of indolizidine alkaloids pumiliotoxins A, B and 251D. J. Neurochem. 37, 775—780 (1981). 

Enzyme Assay. Na , K -ATPase, and sarcoplasmic reticulum Ca - ATPase were prepared from rat hearts (22) and dog hearts (23), respectively. Bovine heart cyclic AMP phosphodiesterase was purchased from Sigma. The enzyme reaction was carried out after 5-min pretreatment with the drug, and the amount of inorganic phosphate liberated during the reaction period was determined. 

Fig. 2b. The appearance of two crystal forms shows that the protein in the membrane exists in equilibrium between the protomeric aj8 unit and oligomeric (aj8>2 forms. The high rate of crystal formation of the protein in vanadate solution shows that transition to the E2 form reduces the difference in free energy required for self association of the protein. This vanadate-method for crystallization has been very reproducible [34-36] and it also leads to crystalline arrays of Ca-ATPase in sarcoplasmic reticulum [37] and H,K-ATPase from stomach mucosa [38]. 

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],... 

Professor Eisenman, there is a large body of results indicating the existence of channel systems. One could mention the Ca2+ ATPase of sarcoplasmic reticulum, the FF transporting ATPase of the inner mitochondrial membrane, the purple protein system of halobacteria, the Na and K+ channels of the axonal membranes. Apart from the classical type of evidence provided, for example, by the noise fluctuation technique, we now even begin to see direct electron microscopic evidence for the existence of transport-related openings in biological membranes. On the other hand, solid evidence for the existence of mobile carriers in eucaryotic cell membranes is very scarce, if not outright absent. 

Relaxation of the muscle is brought about by removal of the ionic calcium from the sarcoplasm. This calcium is transported across the membrane of the sarcoplasmic reticulum, in an energy requiring process. In addition to the calcium pumping ATPase, the sarcoplasmic reticulum also contains a calcium binding protein called calsequestrin (Section 4.3.3). Some of the calcium segregated by the sarcoplasmic reticulum is apparently bound to this protein within the lumen of the sarcoplasmic reticulum. As sequestration of calcium ions into sarcoplasmic reticulum proceeds, more calcium ions dissociate from their binding sites on troponin C, re-... 

Pumiliotoxin B has both cardiotonic and myotonic activity in isolated atrial or rat phrenic nerve diaphragm preparations (97). The cardiotonic activity is markedly dependent on the structure of the pumiliotoxin (95). Subsequent studies on the activity of pumiliotoxin B in neuromuscular preparations were interpreted as due to an apparent facilitation of calcium translocation from internal storage sites (99 see review in Ref. 5). Inhibitory effects on the calcium-dependent ATPase of sarcoplasmic reticulum were shown to be due not to pumiliotoxin B, but to phenolic impurities, namely, fcis(2-hydroxy-3-terf-butyl-5-methylphenyl)methane, 3,5-di-/ert-butyl-4-hydroxytoluene (BHT), and nonylphenols (100). 

In muscle contraction, Ca is released from sarcoplasmic reticulum (SR) into muscle cells via a Ca -release channel. Ca -ATPase then pumps back the released Ca into the SR to cause relaxation. SERCAla is both... 

Synthetic membranes as used for filtration and microfiltration were analyzed by STM " 80-nm pores were observed. The three-dimensional crystals — Ca-adenosine triphosphatase (Ca-ATPase) — of the calcium pump from sarcoplasmic reticulum were imaged by AFM. ... 

The identity of two other reported binding proteins from sarcoplasmic reticulum is in doubt. Tropocalcin, isolated by Benson and Han may be identical to calsequestrin (78) and cardioglobulin-C (79) may be identical to the calcium transport ATPase (80), although the cardioglobulin has also been demonstrated in blood plasma (79). 

If one role of PKG is to decrease smooth muscle intracellular Ca levels, then the mechanism by which this occurs is still not completely defined. Several protein substrates that are known to be phosphorylated by activation of PKG have been identified in SMCs, platelets, and other tissues. Interestingly, some of these proteins have been shown by others to regulate intracellular Ca levels in various cell types. For example, phospholamban, when phosphorylated by PKA in cardiac myocytes, dissociates from sarcoplasmic reticulum (SR) Ca -ATPase resulting in the enhanced sequestration of Ca into the SR (Lindemann et al., 1983). A similar effect of PKG activation has been shown in SMCs (Cornwell et al., 1991 Karczewski et al., 1992 Raeymaekers et al., 1988 Sarcevic et al., 1989). Using another example, Supattopone et al. (1988) reported that PKA-dependent phosphorylation of the inositol 1,4,5-trisphosphate (IP3) receptor in broken-cell fractions decreased Ca release from brain microsomes. Our laboratory has recently shown that the IP3 receptor is also phosphorylated by PKG in vitro on the major PKA phosphorylation site (i.e., Ser-1755) (Komalavilas and Lincoln,... 

The cardiac glycosides (e.g., digoxin) improve myocardial contractility. Although the mechanism is not yet clear, it has been demonstrated that these drugs inhibit sodium-potassium ATPase and enhance release of intracellular calcium from sarcoplasmic reticulum. 

The presence of excessive THs in the thyrotoxic state induces significant changes in the neuromuscular system. THs cause a predominantly catabolic state, resulting in increased muscle breakdown, wasting and weakness due to an accelerated metabolic rate. There is often an increase in efflux of branched-chain amino acids, phenylalanine and tyrosine from muscle bed, with a net muscle loss. THs also cause an increase in the activity of uncoupled ATPase and sarcoplasmic reticulum vesicles. This increase is much more in the red muscle types than in white ones, which is... 

Otherwise, the observed plateau could suggest an association of these aromatic amines with membrane components. In fact, it was shown that azido acridines do interact with proteins of submitochondrial particles (9) and that an acridine derivative covalently bounded to impermeable molecules did retain the uncouling property (10). Moreover, quinacrine is an inhibitor of the FI ATPase solubilized from bovine heart mitochondria (11) and this property can be extended to other kind of ATPase, like the calcium dependent enzyme purified from sarcoplasmic reticulum (12). 

Fig. 5. Freeze-fracture profile of the membranes formed from the ATPase of sarcoplasmic reticulum. [Courtesy of Dr. MacLennan and the Journal of Biological Chemistry (MacLennan et al, 1971).]...

Sarcoplasmic calcium ATPase this enzyme utilizes the energy gained from hydrolysis of ATP to pump calcium from the cytosol into the stores of the sarcoplasmic reticulum. Its activity is negatively regulated by the closely associated protein phospholamban, and this inhibition is relieved upon phosphorylation of phospholamban by protein kinase A (PKA). 

Figure 49-8. Diagram of the relationships among the sarcolemma (plasma membrane), a T tubule, and two cisternae of the sarcoplasmic reticulum of skeletal muscle (not to scale). The T tubule extends inward from the sarcolemma. A wave of depolarization, initiated by a nerve impulse, is transmitted from the sarcolemma down the T tubule. It is then conveyed to the Ca release channel (ryanodine receptor), perhaps by interaction between it and the dihydropyridine receptor (slow Ca voltage channel), which are shown in close proximity. Release of Ca from the Ca release channel into the cytosol initiates contraction. Subsequently, Ca is pumped back into the cisternae of the sarcoplasmic reticulum by the Ca ATPase (Ca pump) and stored there, in part bound to calsequestrin.

Relaxation occurs when sarcoplasmic Ca falls below 10 mol/L owing to its resequestration into the sarcoplasmic reticulum by Ca ATPase. TpC.dCa thus loses its Ca. Consequently, troponin, via interaction with tropomyosin, inhibits further myosin head and F-actin interaction, and in the presence of ATP the myosin head detaches from the F-actin. 

A decrease in the concentration of ATP in the sarcoplasm (eg, by excessive usage during the cycle of con-traction-relaxation or by diminished formation, such as might occur in ischemia) has two major effects (1) The Ca ATPase (Ca + pump) in the sarcoplasmic reticulum ceases to maintain the low concentration of Ca + in the sarcoplasm. Thus, the Interaction of the myosin heads with F-actin is promoted. (2) The ATP-depen-dent detachment of myosin heads from F-actin cannot occur, and rigidity (contracmre) sets in. The condition of rigor mortis, following death, is an extension of these events. 

Information about the putative folding of the H,K-ATPase catalytic subunit through the membrane has been obtained by the combined use of hydropathy analysis according to the criteria of Kyte and Doolittle [51], identification of sites sensitive to chemical modification [46,48,50,52-55], and localization of epitopes of monoclonal antibodies [56]. The model of the H,K-ATPase catalytic subunit (Fig. 1) which has emerged from these studies shows ten transmembrane segments and contains cytosolic N- and C-termini [53]. This secondary structure of the catalytic subunit is probably a common feature of the catalytic subunits of P-type ATPases, since evidence supporting a ten a-helical model with cytosolic N- and C-termini has also been published recently for both Ca-ATPase of the sarcoplasmic reticulum and Na,K-ATPase [57-59]. 

The Ca transport ATPase of the surface membrane is a Ca -calmodulin-dependent enzyme of approximately 138-kDa mass that is structurally distinct from the sarcoplasmic reticulum Ca -ATPase, but shares with it some similarities in the mechanism of Ca translocation [2,3,34]. In both enzymes the Ca -dependent phosphorylation of an aspartyl-carboxyl-group by ATP leads to the formation of an acyl phosphate intermediate that provides the coupling between ATP hydrolysis and Ca translocation. 

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