PURIFICATION, PROPERTIES AND AMINO ACID ANALYSES OF A TRIFLUOPERAZINE - RESISTANT ACTIVATOR FOR CALCIUM(11)-MAGNESIUM(11) ATPASE FROM HUMAN ERYTHROCYTE CYTOSOL
Abstract
A trifluoperazine-resistant protein activator for Ca('2+)-Mg('2+) ATPase was identified from human erythrocyte cytosol. The activity of the trifluoperazine-resistant protein activator was found to be 35% higher in young as compared to old erythrocyte cytosol. The trifluoperazine-resistant protein activator was purified to apparent homogeneity on SDS polyacrylamide gel electrophoresis by a procedure which involves ammonium sulfate precipitation, DEAE-cellulose column chromatography, heat treatment and Sephadex G-75 chromatography. The molecular weight determined by SDS polyacrylamide gel electrophoresis was about 14,000. Similar to calmodulin, the trifluoperazine-resistant protein activator for Ca('2+)-Mg('2+) ATPase also activated cAMP phosphodiesterase. When both calmodulin and trifluoperazine-resistant activator were present, cAMP phosphodiesterase activity was reduced to a level comparable to that achieved by calmodulin alone, suggesting that the action of calmodulin interfered with that of the trifluoperazine-resistant activator and that in the presence of both activators, the effect of calmodulin was predominant. For the purpose of structural analyses, a substantial improvement in the procedure for preparation was developed. The procedure using phenyl sepharose and high performance liquid chromatography allows a more rapid purification of the protein activator to homogeneity and gave higher yields. Reverse phase HPLC was used for the separation of tryptic and chymotryptic fragments of calmodulin and trifluoperazine-resistant protein activator for both analytical mapping and on a preparative scale for amino acid analyses. Similar but not identical peptide maps were obtained. The amino acid sequence analysis shows that the trifluoperazine-resistant protein activator, unlike vertebrate calmodulins which have asparaginyl residues at position 129, has aspartic acid at position 129. The properties of trifluoperazine-resistant protein activator were different from that of calmodulin. (1) The protein activator is not inhibited by the anti-calmodulin drug, trifluoperazine. (2) The elution patterns of the protein activator on hydroxylapatite columns, DEAE cellulose columns, CM-52 cation exchange columns and Phenyl sepharose columns are different from that of calmodulin. (3) The thermal stability of the trifluoperazine-resistant protein activator was less than that of calmodulin. (4) The trifluoperazine-resistant protein activator and calmodulin are separable during non-denatured polyacrylamide gel electrophoresis and anion exchange HPLC.
Degree
Ph.D.
Subject Area
Biochemistry
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