Reaction of a carbodiimide adduct of ATP at the active site of sarcoplasmic reticulum calcium ATPase
| Autor: | Alexander J. Murphy |
|---|---|
| Rok vydání: | 1990 |
| Předmět: |
Time Factors
Stereochemistry ATPase Fluorescence spectrometry chemistry.chemical_element Calcium-Transporting ATPases Calcium Biochemistry Phosphates chemistry.chemical_compound Adenosine Triphosphate Animals Nucleotide Enzyme kinetics Carbodiimide chemistry.chemical_classification Binding Sites biology Chemistry Muscles Active site Calcium ATPase Kinetics Sarcoplasmic Reticulum Spectrometry Fluorescence biology.protein Rabbits |
| Zdroj: | Biochemistry. 29:11236-11242 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi00503a012 |
| Popis: | 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 (t 1/2 for hydrolysis of about 5 min at 25 o 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 μM 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 P i 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 Mg 2+ and Ca 2+ and was prevented by nucleotides such as ATP and ADP. For magnesium, the inactivation-enabling effect occurred with a midpoint of 3mM. In the case of calcium, the transition resembled high-affinity binding in that it occurred cooperatively with a midpoint in the micromolar range. Higher [Mg 2+ ] shifted this transition to higher [Ca 2+ ]. Polyacrylamide gel electrophoresis (PAGE) demonstrated that the reaction converted the ATPase (M r =1.1×10 5 ) to a species with an apparent M r =(1.7−1.8)×10 5 . Since nonionic detergent-solubilized ATPase and purified ATPase gave similar results, intramolecular cross-linking is implicated. Comparison with control ATPase of the effect of limited trypsin proteolysis; which normally produces three fragments (A 1 ,A 2 , and B), showed only the A 2 fragment was obtained from the cross-linked ATPase; a cross-link between the A 1 and B fragments is thereby suggested. Trypsinization before and after ATP-EDC treatment gave simila |
| Databáze: | OpenAIRE |
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