Isolation of rat cardiac sarcoplasmic reticulum with improved Ca2+ uptake and ryanodine binding.
The instability of the oxalate-supported Ca2+ uptake activity of rat cardiac sarcoplasmic reticulum (CSR) in ventricular homogenates most likely accounts for the low specific activity of the rate of oxalate-supported Ca2+ uptake in previously reported fractions of isolated rat CSR. We have found that CSR vesicles with improved Ca2+ transport capabilities can be isolated if 1 M KCl is used to stabilize the CSR activity and to allow the extraction of the CSR from the cellular debris. The average rate of Ca2+ uptake by the isolated rat CSR in the presence of 10 mM oxalate at 37 degrees C was 0.45 mumols/min-mg in the absence of CSR Ca2+ channel blockers and 0.87 mumols/min-mg in the presence of 10 microM ruthenium red. The Ca(2+)-dependent ATPase activity under the conditions of oxlate-supported uptake was 1.25 mumols/min-mg and 0.84 mumols/min-mg in the absence and presence of 10 microM ruthenium red, respectively. The rat CSR vesicles bound 3H-ryanodine with a Kd of 1.45 nM and a Bmax of 3.7 pmol mg. The level of phosphorylated intermediate was 0.30 nmol/mg. The values Bmax, EP and Ca(2+)-ATPase activity are from one-third to one-half of those previously reported for isolated canine CSR vesicles. These results suggest that the isolated rat CSR may be quite similar to dog CSR.
Feher
J J
JJ
Department of Physiology, Medical College of Virginia, Richmond 23298.
Davis
M D
MD
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002458:Cell Fractionation / Q000379:methods; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D010070:Oxalates / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012433:Ryanodine / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D010070:Oxalates; D012433:Ryanodine; D002118:Calcium