A new mechanism by which an H+ concentration gradient drives the synthesis of adenosine triphosphate, pH jump, and adenosine triphosphate synthesis by the Ca2+-dependnet adenosine triphosphatase of sarcoplasmic reticulum

De Meis, L.; Tume, R.K.

Biochemistry 16(20): 4455-4463


ISSN/ISBN: 0006-2960
PMID: 20933
DOI: 10.1021/bi00639a020
Accession: 068518217

Download citation:  

Article/Abstract emailed within 0-6 h
Payments are secure & encrypted
Powered by Stripe
Powered by PayPal

The Ca2+-dependent ATPase purified from the sarcoplasmic reticulum of skeletal muscle synthesized ATP in the absence of a Ca2+ concentration gradient. This was attained in a 2-step procedure in which the enzyme was initially phosphorylated by Pi in the absence of Ca2+ and then the phosphate was transferred to ADP by the addition of high levels of Ca2+. The degree of enzyme phosphorylation by Pi varied with the Ca2+ concentration and pH of the assay medium. At pH 6.0, 50% inhibition was observed in the presence of 10-20 .mu.M Ca2+. At pH 5, while the level of phosphoenzyme was reduced, 50% inhibition was only observed in the presence of 1-2 mM Ca2+. The transfer of phosphate from the phosphoenzyme to ADP was dependent upon the saturation of a low-affinity Ca2+-binding site. After phosphorylation by Pi, it was possible to transfer the phosphate to ADP at progressively lower Ca2+ concentrations as the pH was raised from 6.0-8.1. Maximal ATP synthesis was attained at a Ca2+ concentration range of 1-2 mM at pH 8.1, compared with more than 40 mM at pH 6.0. When the enzyme was phosphorylated at pH 5.0 in the presence of 0.6 mM CaCl2, net synthesis of ATP was observed if at the time of ADP addition the pH was rapidly increased to 8.0. The synthesis of ATP correlated with the dephosphorylation of phosphoenzyme and study of the time course of these reactions revealed identical time constants, the half-time for each being in the range of 30-40 ms. The rapid dephosphorylation of phosphoenzyme was only observed upon the addition of both ADP and high concentrations of Ca2+, dephosphorylation by Ca2+ alone being about 100 times slower.