Calcium dependence of Pi phosphorylation of sarcoplasmic reticulum Ca 2+-ATPase at low water content: water dependence of the E 2→E 1 conversion

• Published in: Biochimica et biophysica acta. Biomembranes Vol. 1419; no. 1; pp. 55 - 63
• Main Authors: Sodré, Cátia L, Scofano, Helena M, Barrabin, Héctor
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 1999
• Subjects: Ca 2+-ATPase; Phosphorylation; Reverse micelle; Sarcoplasmic reticulum; Water; Water; EGTA, ethylene glycol-bis (β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid; Phosphorylation; Tris, Tris(hydroxymethyl) aminomethane; Ca 2+-ATPase; SR, sarcoplasmic reticulum; Triton X-100, p- t-octylphenyl polyoxyethylene ether; FITC, fluorescein-5′-isothiocyanate; Pi, inorganic phosphate; EP, phosphoenzyme; MES, 2-[ N-morpholino] ethanesulfonic acid; Sarcoplasmic reticulum; Wo, water:surfactant molar ratio; pNPP, p-nitrophenylphosphate; AcP, acetylphosphate; TNP-ATP, 2′,3′- O-(2,4,5-trinitrocyclohexadienylidene)adenosine 5′-triphosphate; Reverse micelle
• ISSN: 0005-2736; 1879-2642
• DOI: 10.1016/S0005-2736(99)00051-6

Enzymes entrapped in reverse micelles can be studied in low-water environments that have the potential of restricting conformational mobility in specific steps of the reaction cycle. Sarcoplasmic reticulum Ca 2+-ATPase was incorporated into a reverse-micelle system (TPT) composed of toluene, phospholipids, Triton X-100 and varying amounts of water (0.5–7%, v/v). Phosphorylation of the Ca 2+-ATPase by ATP required the presence of both water and Ca 2+ in the micelles. No phosphoenzyme (EP) was detected in the presence of EGTA. Phosphorylation by Pi (inorganic phosphate) in the absence of Ca 2+ was observed at water content below that necessary for phosphorylation by ATP. In contrast to what is observed in a totally aqueous medium, EP formed by Pi was partially resistant to dephosphorylation by Ca 2+. However, the addition of non-radioactive Pi to the EP already formed caused a rapid decrease in radiolabelled enzymes, as expected for the isotopic dilution, indicating the existence of an equilibrium (E+Pi↔EP). Phosphorylation by Pi also occurred in TPT containing millimolar Ca 2+ concentrations in a range of water concentrations (2–5% v/v). The substrates p-nitrophenyl phosphate, acetyl phosphate, ATP and GTP increased the EP level under these conditions. These results suggest that: (1) the rate of conversion of the ATPase conformer E 2 into E 1 is greatly reduced at low water content, so that E 2→E 1 becomes the rate-limiting step of the catalytic cycle; and (2) in media of low water content, Pi can phosphorylate both E 1Ca and E 2. Thus, the effect of enzyme hydration is complex and involves changes in the phosphorylation reaction at the catalytic site, in the equilibrium between E 2 and E 1 conformers, and in their specificity for substrates.