Spectral and catalytical properties of the sarcoplasmic reticulum Ca-ATPase labeled with N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl)-carbodiimide.
N-Cyclohexyl-N'-(dimethylamino)-carbodiimide (NCD-4) labels three sites in the sarcoplasmic reticulum Ca-ATPase which can be resolved by their spectral properties and by their effects on the catalytical activity of the enzyme. One site is not protectable by Ca2+ ions or by dicyclohexylcarbodiimide and is not essential for catalytical activity. Two Ca2+-protectable sites, whose modification leads to a biphasic inhibition of Ca-ATPase activity, have fluorescence emission maxima at 407 nm and 425 nm. The Ca-ATPase modified by NCD-4 hydrolyses ATP but does not translocate Ca2+ nor does it undergo the conformational changes associated with Ca2+ binding in the native enzyme. High concentrations of Ca2+ induce slow biphasic fluorescence quenching in the Ca-ATPase labeled selectively at the 407-nm site but the signals are largely abolished by modification of the 425-nm site. Both vanadate ions and ATP reverse this Ca2+-induced fluorescence quenching. It is proposed that NCD-4 labels the two high-affinity Ca2+-binding sites of the Sarcoplasmic reticulum Ca-ATPase and that the conformational changes in the modified enzyme may reflect interactions between the two sites.
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D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002234:Carbodiimides / Q000494:pharmacology*; D002384:Catalysis; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D002234:Carbodiimides; C038088:N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide; D000252:Calcium-Transporting ATPases; D002118:Calcium