Exchangers 7 and 8 pm and m voltage sensi tive ca 2

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exchangers; 7 and 8, PM and M voltage-sensi- tive Ca 2+ channels. In addition, some not-wellCdefined "passive" transport pathways are indi- cated by dashed arrows. Figure 3.10. 47 The sarcroplasmic reticulum in muscle cells is abundant in Ca 2 +- ATPase. It is estimated that this protein constitutes more than 80 percent of the integral membrane proteins, and covers a third of the surface area. The sarcoplasmic reticulum Ca 2 + -ATPase (SR Ca 2 + -ATPase) was first pu- rified by MacLennan in 1970. 48 Presently it is the best characterized Ca 2 + - ATPase. A schematic model and a summary of some properties are given in Figure 3.11. 49 Ten hydrophobic segments of about 20 amino-acid residues each are revealed by hydropathy plots, and these segments are assumed to span the membrane as a-helices. (For the one-letter codes for amino acids, see Appendix B in Section IX.) The phosphorylation site has been identified as Asp-35I, and the nucleotide binding domain is following the phosphorylation domain. The Ca 2 + -binding sites are located within the predicted trans-membrane domains
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126 N 1 phospholipid sensitive domain 2 calmodulin binding domain 3 cAMP phosphorylation domain 4 hinge Figure 3.10 Schematic structure of the calmodulin (CaM)-activated plasma membrane Ca 2 +-ATPase of erythrocytes. Some molecular characteristics are: My = 138,000: transport rate (30°C), 20-70 Ca 2 + ions per protein molecule per second; K M (Ca 2 +) = 0.5 J.LM (cytoplasmic side in high- affinity form); Ca 2 +/ATP ratio, I(?); activated not only by CaM but also by acidic phospho- lipids and unsaturated fatty acids. Figure kindly provided by R. Moser and E. Carafoli. (see Figure 3.11). This was shown through a series of site-directed mutations in which likely Ca 2+ -liganding residues like Asp, GIu, and Thr were mutated into residues lacking possible side-chain ligands (e.g., Asn, GIn, and Ala).50 The presently accepted reaction cycle involves two main alternative confor- mations, E] and E 2 , the former with two high-affinity sites (K m ;5 1 J.LM)4 on the cytoplasmic side, which in E 2 are open to the luminal side with K m ~ 1 mM. 49 ,5] The mechanism suggested for Ca 2 + transport (Figure 3.12) has many features similar to that suggested by Williams for H + translocation in the mitochondrial ATPase. 52 It is instructive to consider briefly the thermodynamic limits of the transport. (The discussions about the thermodynamics behind Ca2+ IN a + transport pertain to Na + IK + gradients in excitable tissues as well). Let us define an "inside" and an "outside" separated by a membrane, as shown in Figure 3.13, where [Ca 2 +] and l/J denote activities and membrane potentials, respectively. The dif- ference in electrochemical potential, /).p" , across the membrane for a Ca 2+ ion is given by (3.4)
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127 ATP \ I ~0351 /. C E982 0981 K972 E90 R63 stalk domain N trans- membrane domain Figure 3.11 Schematic structure of the Ca 2 +- ATPase of sarcoplasmic reticulum. Some molecular characteris- tics are: My = 110,000; K m < li-LM (two Ca 2+ sites on cytoplasmic side in high-affinity form); Ca 2 +/ATP ratio, 2; Mg 2 + required for activity. The amino-acid residues labeled were mutated to a residue lacking side chains capable of
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