Erythrosin isothiocyanate selectively labels lysine464 within an ATP-protectable binding site on the Ca-ATPase in skeletal sarcoplasmic reticulum membranes.
Conditions that permit the selective modification of an ATP-protectable site on the Ca-ATPase in skeletal sarcoplasmic reticulum (SR) membranes using erythrosin isothiocyanate (Er-ITC) have been identified. The major labeling site for Er-ITC has been identified using reversed-phase HPLC and positive FAB mass spectrometry after exhaustive tryptic digestion of the Er-ITC-modified Ca-ATPase. An ATP-protectable peptide corresponding to M452NVFNTEVRNLSK464VER467 is modified by Er-ITC, the average mass of which is 2830.1 +/- 0.3 Da. The exclusive modification of lysine residues indicates Lys464 as the site of Er-ITC modification. Derivatization with Er-ITC diminishes the secondary activation of steady-state ATPase activity and the rate of dephosphorylation by millimolar concentrations of ATP. In contrast, in the presence of micromolar ATP concentrations Er-ITC modification of the Ca-ATPase does not affect (i) the apparent affinity of ATP, (ii) the maximal extent of phosphoenzyme formation by ATP, (iii) the rate of steady-state ATP hydrolysis, or (iv) the rate of dephosphorylation of the Ca-ATPase. Furthermore, ATP utilization by the Ca-ATPase is unaffected by detergent solubilization, irrespective of Er-ITC modification, indicating that the secondary activation of ATP hydrolysis involves a single Ca-ATPase polypeptide chain. Therefore, Er-ITC does not interfere with the normal structural transitions associated with phosphoenzyme decay. Rather, these results indicate that Er-ITC bound to Lys464 interferes with either ATP binding to a low-affinity site or the associated structural transitions that modulate the rate of enzyme dephosphorylation.
Huang
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Department of Biochemistry, Cell and Molecular Biology, University of Kansas, Lawrence 66045-2106, USA.
Negash
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Squier
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D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D004923:Erythrosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D007425:Intracellular Membranes / Q000201:enzymology; D017879:Isothiocyanates / Q000378:metabolism* / Q000494:pharmacology; D008239:Lysine / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D018656:Muscle Fibers, Fast-Twitch / Q000201:enzymology; D010455:Peptides / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012995:Solubility
D017879:Isothiocyanates; D010455:Peptides; D010750:Phosphoproteins; C022232:erythrosine isothiocyanate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008239:Lysine; D004923:Erythrosine