Binding of a denatured heme protein and ATP to erythroid spectrin.
Spectrin is a large, worm-like cytoskeletal protein that is abundant in all cell types. The denatured heme enzyme, horseradish peroxidase showed significant decrease in the reactivation yield, after 30 min of refolding, in presence of increasing concentrations of spectrin from that in the absence. This indicated that spectrin could bind denatured HRP and inhibit their refolding. In presence of 1 mM ATP and 10 mM MgCl(2) the spectrin binding of denatured HRP is abolished. This activity of decreasing the reactivation yield was found to be ATP-dependent and the denatured enzyme after 30 min refolding in the presence of spectrin, pretreated with Mg/ATP, showed about 40% increase in the reactivation yield compared to the same in absence of spectrin. Fluorescence spectroscopic studies indicated binding of ATP to native spectrin showing concentration-dependent quenching of tryptophan fluorescence by ATP. The apparent dissociation constant of binding of ATP to spectrin was estimated to be 1.1 mM. A high affinity binding of spectrin with denatured HRP has been characterized (K(d) = 16 nM). Since these properties are similar to those of established molecular chaperone proteins, these data indicate that spectrin might have a chaperone-like function in erythrocytes.
Chakrabarti
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A
Biophysics Division, Saha Institute of Nuclear Physics, 37 Belgachia Road, Calcutta, 700037, India. abhijit@biop.saha.ernet.in
Bhattacharya
S
S
Ray
S
S
Bhattacharyya
M
M
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000737:chemistry; D006041:Goats; D006420:Hemeproteins / Q000737:chemistry / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011489:Protein Denaturation / Q000187:drug effects / Q000502:physiology; D017510:Protein Folding; D013049:Spectrin / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D006420:Hemeproteins; D018832:Molecular Chaperones; D013049:Spectrin; D014364:Tryptophan; D000255:Adenosine Triphosphate; D006735:Horseradish Peroxidase