Betaine counteracts urea-induced conformational changes and uncoupling of the human erythrocyte Ca2+ pump.
We have investigated the effects of the protein structure-perturbing and function-perturbing osmolyte urea, and one of its physiological counteracting solutes, the methylamine compound (carboxymethyl)trimethylammonium hydroxide (betaine), on the structure and function of the human erythrocyte plasma-membrane Ca(2+)-ATPase. Betaine per se promoted a conformational change in the purified ATPase as revealed by steady-state and time-resolved intrinsic fluorescence spectroscopy. The conformational change promoted by betaine was shown to be related to changes in the degree of compaction of the protein structure, as detected by fluorescence-quenching measurements using acrylamide and iodide, non-charged and charged quenchers, respectively. In contrast, urea promoted a biphasic increase in exposure of tryptophan residues of the purified ATPase to the aqueous medium. With the use of membrane-bound ATPase, increasing concentrations of urea up to 1.5 M promoted a twofold increase in the Ca(2+)-ATPase activity, and the simultaneous inhibition of Ca2+ accumulation indicated that ATP hydrolysis became uncoupled from Ca2+ transport. Higher urea concentrations promoted a pronounced inhibition of ATP hydrolysis. In the absence of urea, betaine decreased ATP hydrolysis without affecting Ca2+ transport, whereas it counteracted the strong inhibition of Ca(2+)-ATPase activity by urea concentrations as high as 7 M. Betaine also protected Ca2+ accumulation against inhibition with concentrations of urea up to 1.5 M, indicating that the methylamine is able to counteract the uncoupling of the ATPase observed at lower urea concentrations. These results suggest that betaine modifies the effects of urea on the erythrocyte Ca(2+)-ATPase, through specific solute-induced conformational changes that protect the energy-transduction capacity of the enzyme.
Coelho-Sampaio
T
T
Departamento de Bioquímica, Universidade Federal do Rio de Janeiro, Brazil.
Ferreira
S T
ST
Castro Júnior
E J
EJ
Vieyra
A
A
D001622:Betaine / Q000737:chemistry / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000097:blood / Q000737:chemistry* / Q000187:drug effects; D004357:Drug Synergism; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D011487:Protein Conformation / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology*
D001622:Betaine; D014508:Urea; D000252:Calcium-Transporting ATPases; D002118:Calcium