Reaction of a carbodiimide adduct of ATP at the active site of sarcoplasmic reticulum calcium ATPase.
An adduct of a carbodiimide and ATP was synthesized from 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and the nucleotide. Despite its limited stability (t1/2 for hydrolysis of about 5 min at 25 degrees C), it was shown to react with and inactivate the calcium ATPase of sarcoplasmic reticulum in its vesicular, nonionic detergent-solubilized and purified forms. Saturation kinetics, with an ATP-EDC concentration dependence midpoint in the 10 microM range, were observed, suggesting an active-site affinity which is similar to ATP. The reaction was specific in that inactivation required reaction of about one adduct per ATPase. The modified enzyme could no longer be phosphorylated by ATP or Pi or hydrolyze p-nitrophenyl phosphate, but retained the ability to undergo the high-affinity calcium-dependent fluorescence change. It also bound trinitrophenyl-ADP and other nucleotides at least 10-fold more weakly than the unmodified ATPase. The inactivation reaction required the presence of Mg2+ and Ca2+ and was prevented by nucleotides such as ATP and ADP. For magnesium, the inactivation-enabling effect occurred with a midpoint of 3 mM. In the case of calcium, the transition resembled high-affinity binding in that it occurred cooperatively with a midpoint in the micromolar range. Higher [Mg2+] shifted this transition to higher [Ca2+]. Polyacrylamide gel electrophoresis (PAGE) demonstrated that the reaction converted the ATPase (Mr = 1.1 x 10(5)) to a species with an apparent Mr = (1.7-1.8) x 10(5). Since nonionic detergent-solubilized ATPase and purified ATPase gave similar results, intramolecular cross-linking is implicated.(ABSTRACT TRUNCATED AT 250 WORDS)
Murphy
A J
AJ
Department of Biochemistry, University of the Pacific, San Francisco, California 94115.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010710:Phosphates / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D010710:Phosphates; C066908:adenosine triphosphate-1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium