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Peptide Histidine Isoleucinamide (PHI)-(1–27)-Gly as a New Major Form of PHI in the Rat Small Intestine^*

ANNICK CAUVIN, ANDRÉ VANDERMEERS, MARIE-CLAIRE VANDERMEERSPIRET, JEAN RATHE, PATRICK ROBBERECHT and JEAN CHRISTOPHE

Department of Biochemistry and Nutrition, Medical School, Université Libre de Bruxelles B-1000 Brussels, Belgium

Address all correspondence and requests for reprints to: Dr. Jean Christophe, Department of Biochemistry and Nutrition, Medical School, Universite Libre de Bruxelles, Boulevard de Waterloo 115, B-1000 Brussels, Belgium.

Abstract

Three immunoreactive peptide histidine isoleucinamide (PHI) forms (I, II, and III) from a rat small intestinal extract were separated on a Fractogel column, using a specific RIA. Peak III was identified as rat PHI-(1–27)-NH2 based on its coelution with a synthetic standard and its amino acid sequence. Peak I was tentatively considered as PHI extended with the connecting peptide preexisting between PHI and vasoactive intestinal peptide in their common precursor, based on its apparent mol wt. Peak II was the most abundant form (based on immunoassay) and has not been described previously. It was purified to homogeneity by using a RIA throughout the first three chromatographic steps, then a fast RRA (on rat liver membranes) during the last three purification steps. This new PHI variant was identified as rat PHI-(1–27)-Gly, as judged by full sequencing amino acid analysis after C-terminal digestion by carboxypeptidase-Y and by coelution with synthetic rat PHI- (1–27)-Gly.

Footnotes

^* This work was supported by Grant 3.4504.81 from the Fund for Medical Scientific Research (Belgium), a Concerted Action from the Ministry of Scientific Politics (Belgium), Grant ROI-DK17010-12 from the NIH and Grant STJ-0001-1-B from the E.E.C.

Recipient of an IRSIA doctoral fellowship. 1 The following abbreviations are used: PHI, peptide histidine isoleucinamide-(1–27); PHM, peptide histidine methioninamide-(1–27); VIP, vasoactive intestinal peptide; PHV, peptide histidine valine-(1–42); CP, connecting peptide; PAM, peptidylglycine a-amidating monooxygenase; Pth, phenylthiohydantoin; PTC, phenylthiocarbamyl; Fmoc, 9-fluorenylmethoxycarbonyl; PFP, pentafluorophenyl; HOBT, 1-hydroxybenzotriazole; DHBT, 3,4-dihydro-3-hydroxy-4-oxo-l,2,3- benzotriazine.

Received March 20, 1989.

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