+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

Calcium dependence of Pi phosphorylation of sarcoplasmic reticulum Ca2+-ATPase at low water content: water dependence of the E2-->E1 conversion



Calcium dependence of Pi phosphorylation of sarcoplasmic reticulum Ca2+-ATPase at low water content: water dependence of the E2-->E1 conversion



Biochimica et Biophysica Acta 1419(1): 55-63



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 Ca2+-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 Ca2+-ATPase by ATP required the presence of both water and Ca2+ in the micelles. No phosphoenzyme (EP) was detected in the presence of EGTA. Phosphorylation by Pi (inorganic phosphate) in the absence of Ca2+ 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 Ca2+. 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 Ca2+ 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 E2 into E1 is greatly reduced at low water content, so that E2-->E1 becomes the rate-limiting step of the catalytic cycle; and (2) in media of low water content, Pi can phosphorylate both E1Ca and E2. Thus, the effect of enzyme hydration is complex and involves changes in the phosphorylation reaction at the catalytic site, in the equilibrium between E2 and E1 conformers, and in their specificity for substrates.

Please choose payment method:






(PDF emailed within 1 workday: $29.90)

Accession: 045423787

Download citation: RISBibTeXText

PMID: 10366670


Related references

Calcium dependence of Pi phosphorylation of sarcoplasmic reticulum Ca2+-ATPase at low water content: Water dependence of the E2 fwdarw E1 conversion. Biochimica et Biophysica Acta 1419(1): 55-63, 1999

Calcium and proton dependence of sarcoplasmic reticulum ATPase. Biophysical Journal 44(2): 271-280, 1983

Calcium dependence during single-cycle catalysis of the sarcoplasmic reticulum ATPase. Journal of Biological Chemistry 263(24): 11786-11791, 1988

Effect of ph on calcium ion dependence of dog cardiac sarcoplasmic reticulum atpase activity. Journal of Molecular & Cellular Cardiology 17(5): 505-510, 1985

Phosphorylation of sarcoplasmic reticulum atpase by ortho phosphate in the absence of calcium gradient contribution of water activity to the enthalpy and the entropy changes. Journal of Biological Chemistry 257(9): 4993-4998, 1982

Connection between conformational mobility and temperature dependence of calcium atpase from sarcoplasmic reticulum. Biochemistry 44(12 Part 1): 1708-1714, 1979

Correlation between conformational mobility and temperature dependence of calcium atpase from sarcoplasmic reticulum. Biokhimiya 44(12): 2161-2169, 1979

Sarcoplasmic reticulum atpase kinetics magnesium dependence of the calcium induced transition. Biophysical Journal 41(2 Part 2): 168A, 1983

Dependence of thermal stability of calcium atpase from sarcoplasmic reticulum on its concentration in the membrane. Biokhimiya 45(8): 1503-1509, 1980

Temperature dependence of molecular dynamics and calcium-ATPase activity in sarcoplasmic reticulum. Cossins, A R Portland Press Proceedings; Temperature adaptation of biological membranes 1-12, 1994

The ph dependence of the calcium magnesium atpase of sarcoplasmic reticulum evidence that the calcium ion translocator bears a doubly negative charge. Journal of Membrane Biology 74(1): 25-40, 1983

Proton concentration dependence of calcium induced changes of intrinsic fluorescence in sarcoplasmic reticulum atpase. Biophysical Journal 41(2 Part 2): 170A, 1983

Dependence on membrane lipids of the effect of vanadate on calcium and ATP binding to sarcoplasmic reticulum ATPase. Zeitschrift für Naturforschung. Section C, Biosciences 39(11-12): 1137-1140, 1984

Calcium uptake by isolated sarcoplasmic reticulum examination of halothane inhibition ph dependence and calcium dependence of normal and malignant hyper thermic human muscle. Anesthesia & Analgesia 60(7): 492-498, 1981