Inf. J. Biochem. Vol. 22, No. 8, pp. W-846. 1990 Printed in Great Britain. All rights reserved IDENTIFICATION OF PEPTIDES FROM AUTOLYSATES OF SACCHAROMYCES CER,!3’1,S~~E THAT EXHIBIT GLUCOSE TOLERANCE FACTOR ACTIVITY IN A YEAST ASSAY ERIN M. O’DONOGHUE, JUAN A. COOPER, TIMOTHY G. JACKSON. PETER R. SHEPHERD, PAUL D. BUCKLEYand LEONARDF. BLACKWELL Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand [ Td. 69-0991 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQ (Received 13 December 1989) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQ Abstract-l. Cationic fractions were isolated from a low chromium (co.2 ppm) commercial yeast extract in an attempt to purify the material responsible for glucose tolerance factor (GTF) activity observed in a standard yeast assay system. 2. Following previously described procedures a fraction with GTF activity but containing negligible chromium was isolated, which on further purification was found to be composed of many separate small basic peptides. 3. Much of the activity of the yeast GTF material in the yeast assay could be attributed to the presence of basic peptides and free amino acids acting as nitrogen sources for the yeast. 4. Additional activity was present in the yeast GTF sample. which was not due to a synergistic effect of the mixed amino acids and peptides although the component of the yeast extract responsible for this activity was not identified. 5. The results show that the GTF fractions isolated according to most previously published procedures are highly impure, and conclusions drawn about the nature of GTF based on these isolates must remain open to question. 6. The activity due to the presence of peptides and amino acids is a major cause of lack of specificity of the yeast system as an assay for GTF. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFE INTRODUCTION Fractions isolated from intact cells of brewer’s yeast (Saccharomy ces cerevisiae) or from partially- hydrolysed brewer’s yeast extracts (Toepfer et al., 1977; Mirsky et al., 1980; Haylock et al., 1983a) and collectively known as the glucose tolerance factor (GTF) have been the subject of numerous investi- gations over the past 30 years. The accepted view has been that GTF is a chromium complex (Mertz, 1975) but its structure has never been satisfactorily eluci- dated. Recently we isolated the chromium-containing fractions from a commercial brewer’s yeast extract (Haylock et al., 1983a) but only two of these (desig- nated P3 and P4) showed biological activity in a yeast bioassay (Haylock et al., 1983b) and hence could be possibly considered as GTF fractions by the accepted criteria. On further purification of these two bio- logically-active chromium-containing fractions, the chromium and the biological activity were completely separated (Haylock et al., 1983b). One fraction (P4) was shown to contain tyramine but authentic samples of this compound were inactive in both the yeast (Haylock et al., 1983a) and adipocyte assays (Davies et al., 1985). Hence the nature of the active material in this fraction remains unknown. Other workers have also recently prepared frac- tions from brewer’s yeast that do not contain chromium but which exhibit GTF activity in; the yeast assay (Holdsworth and Appleby, 1984), the low chromium adipocyte assay (Hwang er al., 1987; Davies et al., 1985; Held et al., 1984) and in an in vivo mouse assay system (Barseghian, 1987). Other work indicates that the active chromium complexes that were obtained from yeast extracts are not a function of living cells but rather were artefacts formed by spontaneous chemical reactions between chromium and components of the yeast extracts (Kumpulainen et al., 1978; Haylock et al., 1983b; Hwang et al., 1987). While the combined data indicate that chromium does not play an essential role in the structure of the material responsible for GTF activity, none of these investigations has identified a discrete structure that accounts for the observed biological activity. In this paper we report the utilization of the separation scheme described by Haylock et al. (1983b) to isolate larger amounts of the active cationic fractions obtained from a low chromium commercial brewer’s yeast extract. In the absence of a marker, such as was conveniently provided by chromium, purification of this material was moni- tored using a yeast bioassay system (Burkeholder and Mertz, 1967; Mirsky et al., 1980; Haylock et al., 1982). This assay system was used as a screening assay on the basis that it has been shown (Davies et al., 1985) that the fractions which are active in the more definitive adipocyte assay are also active in the yeast bioassay (although the reverse is not necessarily true). The aim of this work is to determine whether a discrete structure can be identified in yeast extracts which could account for the observed GTF activity. 841