Chemical crosslinking and enzyme kinetics provide no evidence for a regulatory role for the 53 kDa glycoprotein of sarcoplasmic reticulum in calcium transport.
m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) was used to cross-link the protein components of rabbit skeletal muscle sarcoplasmic reticulum. Analysis of cross-linked material by SDS-polyacrylamide gel electrophoresis showed that both the (Ca(2+)-Mg2+)-ATPase and the 53 kDa glycoprotein could be cross-linked, since the amount of protein at the locations on the gel corresponding to uncross-linked material was reduced in the presence of 1.0 mM MBS. Cross-linked products of 130 kDa, 200-260 kDa and approx. 300 kDa were identified. Probing the cross-linked products with monoclonal antibodies against ATPase, 53 kDa glycoprotein and calsequestrin revealed no cross-linked products containing the ATPase and either calsequestrin or the 53 kDa glycoprotein over the range of molecular weights examined here. Possible interactions between the ATPase and calsequestrin or the 53 kDa glycoprotein were also investigated by studying the ATPase activity for the purified ATPase and for the ATPase in sarcoplasmic reticulum vesicles made permeable to Ca2+ with A23187. Effects of Ca2+ and ATP on the two systems were indistinguishable, providing no evidence for a major modulatory role of calsequestrin or the 53 kDa glycoprotein on the ATPase.
Burgess
A J
AJ
Department of Biochemistry, University of Southampton, U.K.
Matthews
I
I
Grimes
E A
EA
Mata
A M
AM
Munkonge
F M
FM
Lee
A G
AG
East
J M
JM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal; D001692:Biological Transport; D015153:Blotting, Western; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002155:Calsequestrin / Q000378:metabolism; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism*; D011506:Proteins / Q000032:analysis; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D013388:Succinimides
D000911:Antibodies, Monoclonal; D002155:Calsequestrin; D003432:Cross-Linking Reagents; D008562:Membrane Glycoproteins; D011506:Proteins; D013388:Succinimides; D000001:Calcimycin; C013097:3-maleimidobenzoyl N-hydroxysuccinimide; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium