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.
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 microM ATP at 25 degrees C are strongly inhibited when starting from either a Ca2+-bound or a Ca2+-free enzyme form, decreasing from 145 to 23 s-1 for the Ca2+-bound form and from 50 to 18 s-1 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.
Mignaco
J A
JA
Departamento de Bioquímica Médica, ICB/CCS, Universidade Federal do Rio de Janeiro, Cidade Universitária, CEP 21941-590, Rio de Janeiro, RJ, Brasil.
Barrabin
H
H
Scofano
H M
HM
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000528:radiation effects*; D004923:Erythrosine / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology; D010084:Oxidation-Reduction; D010777:Photochemistry; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D013379:Substrate Specificity
D005456:Fluorescent Dyes; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D004923:Erythrosine