| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1988: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1987: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
Recently, possible involvement of the endogenous digitalis-like substance in the mechanism of essential hypertension has been suggested. In this work the purification and identification of this digitalis-like substance in the urine of DOCA (deoxycorticosterone acetate) treated hypertensive rats were attempted. This substance was purified using methods of an ultrafiltration (Amicon YM-10), a CM Sepharose column chromatography, a Sephadex G-10 column chromatography, an anti-digoxin antibody affinity column chromatography, a reversed phase HPLC and a molecular sieve HPLC. The purified material was recognized as a single substance by a TLC chromatography. This substance dose-dependently inhibited Na^+, K^+-STPase and binding of ^<125>I-digoxin to anti-digoxin antibody, and elevated the blood pressure with intravenous injection. This substance lost the inhibitory activity of Na^+, K^+-ATPase by the acid hydrolysis (6N HCl, 110 C, 20 h) and treatments with proteolytic enzymes such as pronase, prolidase, aminopeptidase P and lysine endopeptidase. The amino acid analysis of this substance gave 14 species of amino acids. Now, the amino acid sequence of this substance is under the investigation by protein sequencer. Existence of only a minute amount of this substance in the urine and a blockade of the N-terminal of this peptide might prevent rapid identification of this substance. A similar substance was observed in rat brain extract.
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