Effects of adenyl-5'-imidodiphosphate and vanadate Ion on the intermolecular cross-linking of Ca2(+)-ATPase in the sarcoplasmic reticulum membrane with N,N'-(1,4-phenylene)bismaleimide.
The functional significance of the molecular interaction of Ca2(+)-ATPase in the sarcoplasmic reticulum (SR) membrane was examined using intermolecular cross-linking of Ca2(+)-ATPase with N,N'-(1,4-phenylene)bismaleimide (PBM). When SR vesicles were allowed to react with 1 mM PBM at pH 7 and 23 degrees C for various intervals and subjected to SDS-PAGE, the amount of the major band of monomeric ATPase decreased with a half life of about 20 min. Higher orders of oligomers were concurrently formed without accumulation of any particular species of oligomer. When SR vesicles were allowed to react with 1 mM PBM in the presence of 1 mM adenyl-5'-imidodiphosphate (AMP-PNP), the rate of oligomerization was markedly reduced and the amount of dimeric Ca2(+)-ATPase increased with time. After 1 h, more than 40% of the Ca2(+)-ATPase had accumulated in the dimeric form. When 1 mol of fluorescein isothiocyanate (FITC) was bound per mol of ATPase, the effects of AMP-PNP on the cross-linking with PBM were completely abolished. When SR vesicles were treated with PBM in the presence of 0.1 mM vanadate in Ca2+ free medium, the oligomerization of the Ca2(+)-ATPase by PBM was strongly inhibited. The vanadate effect on the cross-link formation was completely removed by the presence of Ca2+ and AMP-PNP in the reaction medium. When SR vesicles were pretreated with PBM in the presence of AMP-PNP and digested with trypsin for a short time, the dimeric ATPase was degraded to a peptide with an apparent molecular mass of about 170 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
Yamasaki
K
K
Department of Biology, Faculty of Science, Osaka University.
Yamamoto
T
T
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology*; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology; D008301:Maleimides / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology*
D008301:Maleimides; D009711:Nucleotides; D010446:Peptide Fragments; D000266:Adenylyl Imidodiphosphate; D014638:Vanadates; D000255:Adenosine Triphosphate; C011328:N,N'-4-phenylenedimaleimide; D014357:Trypsin; D000252:Calcium-Transporting ATPases