PURIFICATION AND FUNCTIONAL-CHARACTERIZATION OF A LOW-MOLECULAR-MASS NA-ATPASE INHIBITOR PROTEIN FROM RAT-BRAIN CYTOSOL(, K+)

A number of low-molecular mass (12-13 kDa) Na+, K+-ATPase inhibitor pr oteins have been purified from rat brain cytosol by gel filtration fol lowed by FPLC fractionation on a Mono Q anion-exchange column. Eight p eaks 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 (I-50) was found was in the nanomolar range. The inhibitor seems to bind to Na+, K+-ATPase at a site distal from th e ATP-binding site. The binding to the ATPase is non-competitive. The CD analysis suggests an unordered secondary structural element. It als o inhibits p-nitrophenyl phosphatase activity from rat brain with comp arable I-50 value to that for Na+, K+-ATPase. The protein does not con tain any Trp as evident from Trp fluorescence and amino acid analysis. Amino acid analysis shows that glycine and serine, derivatives of tyr osine and phenylalanine are the predominant amino acids. The data sugg ests that it is a negatively charged protein in which the contribution of the hydrophobic part is 27%.