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Volume 271, Number 31, Issue of August 2, 1996 pp. 18423-18430
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Effects of Photo-oxidizing Analogs of Fluorescein on the Sarcoplasmic Reticulum Ca²⁺-ATPase
FUNCTIONAL CONSEQUENCES FOR SUBSTRATE HYDROLYSIS AND EFFECTS ON THE PARTIAL REACTIONS OF THE HYDROLYTIC CYCLE

(Received for publication, May 22, 1995, and in revised form, March 25, 1996)

From the Departamento de Bioquímica Médica, ICB/CCS, Universidade Federal do Rio de Janeiro, Cidade Universitária, CEP 21941-590, Rio de Janeiro, RJ, Brasil

Erythrosin B was used to photo-oxidize the sarcoplasmic reticulum Ca²⁺-ATPase. The ATPase activity is rapidly and irreversibly inhibited by photo-oxidation with erythrosin. This inhibition is protected by the presence of ATP during the photo-oxidation period. After photo-oxidation, the steady-state phosphorylation by ATP remains almost unchanged, whereas phosphorylation by inorganic phosphate is impaired. The pseudo-first order rate constants for phosphorylation by 15 µM ATP at 25 °C are strongly inhibited when starting from either a Ca²⁺-bound or a Ca²⁺-free enzyme form, decreasing from 145 to 23 s¹ for the Ca²⁺-bound form and from 50 to 18 s¹ for the Ca²⁺-free form. Concurrently, the rate constants for dephosphorylation are also severely inhibited, changing from a fast double exponential to a very slow single exponential decay in the reverse direction and from a moderately slow single to a very slow single exponential decay in the forward direction. Ca²⁺ binding data show that the phosphorylated intermediate formed by the photo-oxidized enzyme contains two occluded Ca²⁺, and TNP-ATP fluorescence measurements indicate that it accumulates in a E₁-P·Ca₂-like conformation. Protection by ADP against glutaraldehyde-induced cross-linking indicates that ADP binding to Ca²⁺-ATPase is not impaired by photo-oxidation nor by free erythrosin. These data support the view that an ADP-insensitive, Ca²⁺-bound, slowly interconverting phosphoenzyme is formed. Thus, photo-oxidation with erythrosin B leads to impairment of phosphoryl transfer reactions and related conformational changes.

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