Abstract
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.
Highlights
Erythrosin B was used to photo-oxidize the sarcoplasmic reticulum Ca2؉-ATPase
Because Lys-515, which is the point of attachment of FITC to the enzyme, is neither unusually reactive toward FITC (Murphy, 1988) nor essential for ATP hydrolysis (Maruyama et al, 1989), it has been suggested that it is the fluorescein moiety of FITC that directs the reaction toward the ATP site
In this study we use erythrosin B, a halogenated derivative of fluorescein that is considered to bind to enzyme nucleotide binding sites with high affinity and specificity (Lundblad and Noyes, 1984; Neslund et al, 1984), to photo-oxidize residues on sarcoplasmic reticulum (SR) Ca2ϩ-ATPase, and we examine its influence on the partial reactions of the hydrolytic cycle
Summary
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. Many amino acids have been labeled, identified, and located within the primary structure using a variety of probes for the nucleotide binding site These experiments have almost always involved probes that bind covalently to the enzyme, such as FITC (Mitchinson et al, 1982), 8-azido-ATP (Lacapere et al, 1993), pyridoxal 5Ј-phosphate (Yamagata et al, 1993), oxidized ATP (Mignaco et al, 1990), or chemical reagents that cause radical structural changes, such as glutaraldehyde (McIntosh, 1992) or dithiothreitol (Daiho and Kanazawa, 1994). Kinetic parameters such as those involved in enzyme phosphorylation and dephosphorylation could not always be assessed with the labeled enzyme, and the precise functional role of the labeled amino acids remains uncertain. Erythrosin is capable of promoting selective photo-oxidation of amino acids, which can be useful in the identification of groups involved in catalysis
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.