Purification and functional characterization of a low-molecular-mass Na+, K+-ATPase inhibitor protein from rat brain cytosol.
A number of low-molecular mass (12-13 kDa) Na+, K+-ATPase inhibitor proteins have been purified from rat brain cytosol by gel filtration followed by FPLC fractionation on a Mono Q anion-exchange column. Eight peaks were obtained using 0.1 M NaCl eluent of which one peak was found to be the most potent inhibitor of Na+, K+-ATPase. The molcular mass of the inhibitor was about 13 kDa on 16.5% SDS/PAGE. The concentration at which 50% inhibition (I50) was found was in the nanomolar range. The inhibitor seems to bind to Na+, K+-ATPase at a site distal from the ATP-binding site. The binding to the ATPase is non-competitive. The CD analysis suggests an unordered secondary structural element. It also inhibits p-nitrophenyl phosphatase activity from rat brain with comparable I50 value to that for Na+, K+-ATPase. The protein does not contain any Trp as evident from Trp fluorescence and amino acid analysis. Amino acid analysis shows that glycine and serine, derivatives of tyrosine and phenylalanine are the predominant amino acids. The data suggests that it is a negatively charged protein in which the contribution of the hydrophobic part is 27%.
Bhattacharyya
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Department of Chemistry, Bose Institute, Calcutta, India.
Sen
P C
PC
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001921:Brain / Q000201:enzymology; D001923:Brain Chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003600:Cytosol / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D004791:Enzyme Inhibitors; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase