Oxidative inactivation of purified plasma membrane Ca2+-ATPase by hydrogen peroxide and protection by calmodulin.
Calmodulin (CaM)-regulated plasma membrane Ca(2+)-ATPase (PMCA) is critical for the regulation of free intracellular Ca(2+) levels. PMCA activity and levels in neuronal membranes are decreased with aging, possibly due to oxidation-induced inactivation. In the present studies, inhibition of PMCA by H(2)O(2) was characterized in enzyme purified from human erythrocyte membranes. Basal and CaM-stimulated PMCA activities were inhibited by exposure to H(2)O(2) (25-100 microM). However, neither the concentration-dependent enhancement of PMCA activity by CaM nor the binding of CaM to H(2)O(2)-exposed PMCA was disrupted by treatment with H(2)O(2). Rates of inactivation by H(2)O(2) of basal and CaM-stimulated PMCA were nearly identical. The addition of CaM after exposure to H(2)O(2) did not protect enzyme activity, although the binding of CaM to PMCA before exposure to H(2)O(2) protected the enzyme completely, indicating a CaM-induced conformational state resistant to oxidation. H(2)O(2) quenched Trp fluorescence in PMCA, an index of conformational changes, with a rate similar to that observed for enzyme inactivation. H(2)O(2) enhanced the solvent accessibility of Trp residues in PMCA, whereas accessibility of the only Trp residue in the CaM-binding domain peptide was unaltered. Exposure of PMCA to H(2)O(2) led to aggregate formation partially reversible by dithiothreitol (DTT) but not to recovery of activity. Amino acid analysis indicated Cys modification following H(2)O(2) exposure but no Cys oxyacids. Because DTT did not reverse inactivation by H(2)O(2), it appears that the disulfide bond formation led to conformational changes that were not fully reversed when the bonds were reduced. Preincubation of PMCA with CaM protected the enzyme from undergoing this conformational change.
Zaidi
Asma
A
Department of Pharmacology and Toxicology, University of Kansas, Lawrence, Kansas 66045, USA.
Barŕon
Lorena
L
Sharov
Victor S
VS
Schöneich
Christian
C
Michaelis
Elias K
EK
Michaelis
Mary L
ML
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification; D002147:Calmodulin / Q000737:chemistry* / Q000502:physiology; D027682:Cation Transport Proteins; D002417:Cattle; D003545:Cysteine / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000201:enzymology; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000097:blood / Q000737:chemistry*; D007930:Leucine / Q000737:chemistry; D010084:Oxidation-Reduction; D053499:Plasma Membrane Calcium-Transporting ATPases; D011487:Protein Conformation / Q000187:drug effects; D012694:Serine / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000737:chemistry; D014633:Valine / Q000737:chemistry
D002147:Calmodulin; D027682:Cation Transport Proteins; D014443:Tyrosine; D012694:Serine; D006639:Histidine; D006861:Hydrogen Peroxide; D053499:Plasma Membrane Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D007930:Leucine; D014633:Valine; D003545:Cysteine; D004229:Dithiothreitol