Isolation of a specific granulopoiesis inhibitor from calf spleen and identification as glutathione

Abstract

A small peptide was isolated from calf spleen, specifically inhibiting murine granulopoiesis in vitro. Purification involved ultrafiltration, anion-exchange chromatography with a FPLC system and reversed-phase chromatography on HPLC. Determination of the amino acid composition, following acid hydrolysis and phenyl isothiocyanate and dabsyl chloride derivatization, revealed the amino acids glutamic acid, cysteine and glycine. Although the N-terminal amino group was not blocked, peptide sequencing with common techniques was not possible.Comparison of the isolated peptide with the well-known tripeptide glutathione by HPLC and fast atom bombardment (FAB)/tandem mass spectrometry showed the identity of both substances. Moreover, glutathione was found to be a specific granulopoiesis inhibitor in vitro at 10-100 nM, a so far unknown property of this well-known peptide.The term chalones was reintroduced by Bullough for physiological substances specifically inhibiting cell proliferation by a negative feedback mechanism [l]. Chalones were thought to exert a cell and tissue-specific but species-unspecific effect. Paukovits and Laerum [2] described a synthetic pentapeptide (called hemoregulatory peptide HPSb, pGluGlu-Asp-Cys-Lys) meeting these properties. Their reduced monomer is a specific in vitro and in vivo inhibitor of granulopoiesis [3], whereas the dimer stimulates granulocyte/ macrophage colony formation in vitro [4].Recently we have reported a similar specific in vitro granulopoiesis inhibitor, partially purified from calf spleen [S]. In this paper we describe the purification to homogeneity and the identification of this peptide as glutathione. MATERIALS AND METHODS Preparation of an acetone precipitateThe preparation has been described in detail by Kastner [6]. In short, calf spleen was extracted with ethanol (70%, by vol.) and centrifuged. The clear supernatant was added to acetone. The lyophilized extract was called the 'acetone precipitate'. UltrafiltrationUltrafiltration was performed in a special multichamber system (UFLA-None, Firma Schiitt, Gottingen), developed by Dr A. Kinawi of this institute. We used seven membranes with decreasing cut-off values, i.e. XM I00 A, XM 50 Abbreviations. DABITC, 4-dimethylaminoazobenzene-4'-isothiocyanate; FAB, fast atom bombardment; GSH, reduced glutathione; pGlu, pyroglutamic acid. _~was performed with a flow rate of 6 ml/h for 22 h at 4°C. The ultrafiltrates were lyophilized and stored at -20 "C. Fast protein liquid chromatographyFPLC was performed with equipment from Pharmacia, Freiburg. We used an anion-exchange column (MonoQ) with a NaCl gradient of 0-0.4 M for 20 min then 0.4-1 M for 2 min. The flow rate was 2 ml/min with a fraction size of 400 pl. Detection was at 254 nm. No buffer was used to avoid any interference with biological testing. Purijication vvith high-perjormance liquid chromatographyHPLC was performed on a Beckman system with a reversed-phase column 5 pm, Merck, Darmstadt). Solvent A was 0.1 % trifluoroacetic acid in water, solvent B was 0.1 % trifl...

MS/MS applications in biological problems

“…When it was compared to the well known tripeptide glutathione, FAB / MS / MS showed beyond any doubt the identity of the two substances (187).…”

“…A small peptide, isolated from calf spleen and specifically inhibiting granulopoiesis in vitro, could not be sequenced by Edman degradation, although the N-terminal group was not blocked. When it was compared to the well known tripeptide glutathione, FAB / MS / MS showed beyond any doubt the identity of the two substances (187).…”