Volume 104, number 1 FEBS LE’ITERS August 1979 GROWTH-MODULATING HUMAN PLASMA TRXPEPTIDE: RELATIONSHIP BETWEEN MOLECULAR STRUCTURE AND DNA SYNTHESIS IN HEPATOMA CELLS Loren PICKART and M. Michael THALER* department of Pedinttics and the Liver Center, University of Califo~~~ San Raneisco, CA 94143, USA Received 29 May 1979 1. Introduction H-glycylhistidyllysine-OH (GHL) is a tripeptide found at approximately 200 ng/ml in human plasma in association with the albumin and o-globulin frac- tions [ 1,2 J. When added to culture medium at 1S-100 ng/ml, synthetic GHL promotes the pro- liferation of hepatoma cells ]2,3], lymphocytes [4] and T-strain mycoplasma [5]; maintains the viability of normal hepatocytes [2,3], eosinophils [6], and macrophages [7]; inhibits the growth of glial cells [8]; and supports the growth and differentiation of neurons ]8] and ascaris larvae [9]. At higher concentration, the tripeptide inhibits L929 cell growth (500 ng/ml) [lo] and maintains the viability of mast cells (20 pg/ml) during degranulation tests [ 1I]. In general, GHL acts to reduce or eliminate the amount of serum required for culture of the various cell types or orga- nisms [2-4,6-9,111. Although the mechanism of action of GHL is unknown, the tripeptide is co-iso- lated with copper and iron [ 12,131, and acts syner- gistically with these transition metals to stimulate the growth and metabolism of hepatoma cells maintained in grows-l~it~g amounts of serum [ 12,131. In this paper we describe the effects of 9 synthetic analogs of GHL on DNA synthesis in hepatoma cell culture. The results indicate that 2 structural features are involved in the bioactivities of GHL: (1) the histi- dyllysyl linkage and (2) a glycyl residue in either terminal position. * Address reprint request to: M. Michael Thaler, Department zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFED of Pediatrics U-587, University of California, San Francisco, CA 94143, USA Elsevierf2Vorth-Hof Biomedi~aI Press 2, Materials and methods Synthetic peptides were obtained from the follow- ing suppliers: Gly-His-Lys from Fierce Chemical Co., Rickford, IL; Gly-His-(e-Gly-His)-Lys from Peninsula Laboratories, San Carlos, CA; Gly-His, Gly- Lys and His-Lys from Sigma Chemical Co., St. Louis, MO; His-Lys-Gly and His-Gly-Lys from Dr C. J. Lote, synthetized by methods detailed elsewhere [ 141; Gly-Lys-His, Gly-His-Orn, and Gly-His-Lys-His were prepared in our laboratory by solid phase synthesis 131. The hepatoma cell line (HTC,) was obtained from the UCSF Cell Culture Facility; fetal calf serum, Swim’s S-77 medium, Eagle’s basal medium from GIBCO, Berkeley, CA; [methyZ-3H]thymidine, 28 Ci/mM, from International Chemical and Nuclear, Irvine, CA; plastic 25 cm2 T-flasks from Falcon Plastics, Oxnard, CA. 2.1. Purification zyxwvutsrqponmlkjihgfedcbaZYXWVU of peptides All peptides were repurified by thin-layer silica gel chromatography, using as solvent CHCl3fMeOH/l7% NQOH : 2/2/l by volume, and eluted from the plate by methods previously published [2]. The peptide- containing solutions were passed through a Dowex 50-X4 column (1 X 5 cm) at pH 7.0 in 0.1 M sodium phosphate. Double glass distilled water (10 column volumes) was passed through the column and peptide eluted with 5 ml 0.1 NaOH. The basic eluate was immediately neutralized with 1 N HCl, the solution lyophilized, then dissolved in 1 ml of 1% acetic acid. This solution was desalted on a Sephadex G-10 column (1 X 100 cm) eluted with 1% acetic acid, peptide 119