Chemical modification and fluorescence labeling study of Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum using iodoacetamide and its N-substituted derivatives.
Sarcoplasmic reticulum membrane vesicles from rabbit skeletal muscle were treated with iodoacetamide (IAA) at pH 7.0 and 30 degrees C. At 1.0 mM IAA, 1 mol of IAA per mol of ATPase peptide was bound in 1 h. Under these conditions, IAA was attached specifically to the B-tryptic fragment portion of the peptide. The binding of IAA did not affect the Ca2+-transporting activity of ATPase. Three fluorescent derivatives of iodoacetamide, 5-(2-acetamidoethyl)aminonaphthalene-1-sulfonate (IAEDANS), 5-iodoacetamido fluorescein (IAF), and 5-iodoacetamido eosin (IAE), were also tested for reactivity toward sarcoplasmic reticulum ATPase at 30 degrees C and pH 7.0. In 1 h at 50 microM concentration, each of these fluorescent labels modified ATPase to a labeling density of 1 mol per mol of ATPase. Neither IAEDANS nor IAF at this labeling density affected Ca2+-transporting activity, but IAE reduced it to 20% of the untreated control. The target site of IAEDANS at this labeling density was located exclusively on the B-fragment portion, as was the case with IAA, but IAF label was found on both A1 and B fragments after limited tryptic digestion. IAEDANS was used as a B-fragment portion-directed conformational probe of Ca2+-transport ATPase, and an increase in fluorescence intensity accompanying E1Ca-P formation was detected. The fluorescence enhancement was abolished when E1Ca-P X ADP beta S was formed by adding ADP beta S to preformed E1Ca-P. This suggests that the conformation of ATPase in the neighborhood of the IAEDANS binding site may be altered in response to the dissociation of ADP from the phosphorylated intermediate.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes; D007460:Iodoacetamide / Q000031:analogs & derivatives / Q000378:metabolism*; D007461:Iodoacetates / Q000378:metabolism*; D009282:Naphthalenesulfonates / Q000378:metabolism; D009711:Nucleotides / Q000494:pharmacology; D010446:Peptide Fragments; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology; D014357:Trypsin
D005452:Fluoresceins; D005456:Fluorescent Dyes; D007461:Iodoacetates; D009282:Naphthalenesulfonates; D009711:Nucleotides; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D013873:Thionucleotides; C030812:adenosine 5'-O-(2-thiodiphosphate); D000244:Adenosine Diphosphate; C015296:5-iodoacetamidofluorescein; C006421:1,5-I-AEDANS; D014357:Trypsin; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium; D007460:Iodoacetamide