Calcium transport activity of rat cardiac sarcoplasmic reticulum isolated in the presence of dithiothreitol.
We examined the effect of dithiothreitol (DTT) on the isolation and calcium transport properties of rat cardiac sarcoplasmic reticulum (SR). DTT at a concentration of 4 mM was included in the homogenization/initial isolation and the high-salt (KCl) buffers used in the preparation of the SR fraction. The inclusion of DTT in the SR isolation media did not affect the total amount of SR protein isolated from ventricular tissue. However, SR isolated in the presence of DTT exhibited a significantly increased (p less than 0.05) rate of calcium transport at free calcium concentrations ranging from 0.1 microM up to 2.0 microM, and a 26% increase in the apparent affinity of the SR calcium transport system for CA2+. DTT included in the calcium-transport reaction medium did not significantly alter the rate of SR calcium transport in SR preparations isolated in the absence or presence of DTT. These results demonstrate that functionally important sulfhydryl groups of the SR calcium transport pump may be oxidized during the isolation of the membrane fraction and that such oxidation can be reduced by including DTT in the isolation buffers.
Black
S C
SC
Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
McNeill
J H
JH
Katz
S
S
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology*; D066298:In Vitro Techniques; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D011919:Rats, Inbred Strains; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D002118:Calcium; D004229:Dithiothreitol