The isolation of glucose tolerance factors from brewer's yeast and their relation to chromium

A new dietary factor, the glucose tolerance factor (GTF), was reported in 1957 that improved impaired glucose tolerance in rats. Most studies on GTF have used brewer's yeast as the starting material, and it has been postulated that the active material is a low-mol wt organic complex containing Cr³⁺. It seemed thus important to isolate an active GTF from chromium-rich yeast (228 ppm Cr) obtained by incubation with chromium and to compare each fraction with corresponding ones from untreated yeast (0.48 ppm Cr). We developed an isolation and purification procedure by fractionation of yeast extract on an anion and cation exchange resin, and tested the GTF activity (glucose oxidation) on rat adipocytes. PIXE (proton-induced X-ray emission) was used to measure the chromium content of the individual fraction. Individual fractions with GTF activity did not differ between Cr-rich and Cr-deficient yeast, and there was no relationship between Cr content and GTF activity. This does not support the hypothesis that chromium is an obligatory constituent of the GTF, assuming that GTF is a unique substance.     

The relationship of chromium to the glucose tolerance factor. II

After incubation with CrCl₃·6H₂O (or⁵¹CrCl₃·6H₂O) for 25 days, a sterile growth medium, whole yeast cells harvested after growth on a similar chromium-containing medium for the same period, and the spent growth medium remaining after removal of the yeast were each subjected to the separation procedure reported previously [S.J. Haylock, P.D. Buckley and L.F. Blackwell, J. Inorg. Biochem., in press]. The results obtained showed that most of the eleven chromium-containing fractions isolated previously were artifacts formed as a result of direct reaction between the chromium and components of the medium. An anionic complex (which was the major chromium-containing fraction isolated) was identified as a chromium-glucose complex, but one possessing no biological activity. The biologically active chromium-containing fractions (P-3 and P-4) that were only present after yeast had been grown in the medium were further purified, however, during the purification steps, the biological activity was cleanly separated from the chromium material for both P-3 and P-4. Fraction P-4 was subsequently shown to consist of approximately 90% tyramine, but pure tyramine was not active in the yeast bioassay. Although the structure of the glucose tolerance factor-active component in fraction P-3 could not be determined due to the presence of high concentrations of salt that could not be separated on gel filtration columns, the results show that the glucose tolerance factor from brewer's yeast can no longer be regarded as a chromium complex.     

Effects of brewer's yeast on glucose tolerance and serum lipids in Chinese adults

This study was designed to investigate the supplemental effects of brewer's yeast on serum glucose and lipids in Chinese adults. Twenty-two participants (8 males and 14 females) were recruited from Pingtung city. Mean age of the group was 51 yr, and fasting values of total cholesterol and glucose were from 3.21 to 6.90 and 4.3 to 6.2 mmol/L, respectively. Before supplementation, a 75 g oral glucose load was administered after an overnight (9 h) fast. Blood was drawn before and at 30, 60, 90, and 120 min after the glucose load. The subjects were randomly assigned into either brewer's yeast or torula yeast group and consumed 10 g yeast powder daily for 12 wk. Brewer's yeast demonstrated a beneficial effect on decreasing serum triacylglycerol values (p<0.05). The brewer's yeast supplemented group had an increment at 0 min (p<0.01) and significant decrements at 60 (p<0.05) and 90 (p<0.01) min of oral glucose tolerance test (OGTT). However, after 12-wk supplementation, torula yeast increased glucose values at both 0 and 30 min (p<0.05) after a glucose load. Brewer's yeast and torula yeast addition significantly altered glucose concentrations at 60 min after the glucose dosage (p<0.05). Brewer's yeast had significantly decreasing effects on insulin output both at 90 (p<0.05) and 120 min (p<0.01) after the glucose load. Likewise, serum insulin contents decreased at 90 min (p<0.01) after supplementation in the group given torula yeast. Brewer's yeast supplementation had beneficial effects both on serum triacylglycerol and on 60-min and 90-min glucose values of OGTT.     

Chromium uptake bySaccharomyces cerevisiae and isolation of glucose tolerance factor from yeast biomass

Fermentations with yeastSaccharomyces cerevisiae in semiaerobic and in static conditions with the addition of chromic chloride into the used molasses medium were analysed. It was proved that the addition of optimal amounts of CrCl₃ into the basal medium enhanced the kinetics of alcohol fermentations. The addition of 200 mg/l CrCl₃ into the medium stimulated both the yeast growth and the ethanol production in all experimental conditions. On the other hand, the results showed that Cr³⁺ ions were incorporated into yeast cells during fermentation. Under these conditions the accumulation of Cr³⁺ ions was performed by yeast cells during the exponential growth phase, and with enriched amounts of 30–45 (μg/g_d.m. of cells. Yeast biomass enriched with chromium ions was extracted with 01 mol/l NH₄OH assuming that the extracts had the glucose tolerance factor (GTF). Then the extracts were passed through a gel-filtration column in order to isolate and purify the GTF. The presence of GTF in the purified fractions was determined by measuring the absorbance at 260 nm. It is evident from the obtained results that the added purified fractions enhanced the rates of CO₂ production as well as the glucose utilization during alcoholic fermentation. As expected, the enhancement of both rates depended on the amounts of extracts added to the fermentation substrate. Thus, it is evident that purified extracts contained the GTF compound, and that Cr³⁺ ions were bonded to the protein molecule.     

The nucleotide sequence of asparagine tRNA from brewer's yeast

The nucleotide sequence of asparagine tRNA from brewer's yeast has been determined using postlabeling methods. The primary structure is as follows: pG-A-C-U-C-C-A-U-G-m2G-C-C-A-A-G-D-D-G-G-D-D-A-A-G-G-C-m2 2G- U-G-C-G-A-C-U-G-U-U -t6A-A-psi-C-G-C-A-A-G-A-D-m5C-G-U-G-A-G-T-psi-C-A-m1A-C-C-C-U-C-A-C-U-G -G-G-G- U -C-G-C-C-A. Its anticodon G-U-U can recognize the two codons for asparagine.     

A preliminary study of chromium distribution in chromium-rich brewer's yeast cell by NAA

The purpose of this study was to assess the chromium (Cr) distribution in chromium-rich brewer’s yeast cell. The chromium concentrations in the cell wall and protoplast fractions of the chromium-rich yeast were determined by neutron activation analysis (NAA). Moreover, the combined state of chromium and amino acid content in the Cr-rich brewer’s yeasts was analyzed and measured. The experimental results indicate that the introduction of water-soluble chromium (III) salt as a component of the culture medium for yeasts results in a substantial amount of chromium absorbed through the cell wall by the yeast, among which 80.9% are accumulated in the protoplast. It implies that, under optimal conditions, yeasts are capable of accumulating large amounts of chromium and incorporating chromium into organic compounds.     

Insulin-like activity of chromium-binding fractions from brewer's yeast

51CrCl3 was added to the incubation medium of Saccharomyces cerevisiae for up to 48 hr. After repeated freezing and thawing, lysing in 9 M urea with 1% NP-40 detergent, and dialysis against water, the lower molecular weight (Mr less than 3500) dialysate was retained on a SE53 cationic exchange column, eluted with 0.25 M NH4OH and fractionated on a Bio-gel P-2 column. The insulin-like biological activity of the fractions was measured by the 14C-glucose oxidation in isolated rat adipocytes. The biological activity that was found in two of nine fractions did not correspond to their chromium content. Moreover, identical findings were obtained when chromium was added not to the live yeast but to the yeast extract, which showed that its binding was a chemical process not requiring cellular activity. No fraction demonstrated insulin-potentiating activity on rat adipocytes.     

The isolation of lytic enzymes from Cytophaga and their application to the rupture of yeast cells

Yeast–lytic enzymes have been isolated on a pilot scale from Cytophaga species by precipitation and the light, enzyme-rich solid phase recovered by liquid-liquid separation. The enzyme complex was immobilized to soluble polymeric carbohydrates and the effectiveness of the free and immobilized enzyme for protein release and cell debris dissolution has been assessed.     

The heterogeneity of brewer's yeast old yellow enzyme

Heterogeneity of brewer's yeast old yellow enzyme (OYE) was found by anion-exchange high-performance liquid chromatography (HPLC) as well as by 13C-NMR spectroscopy of [4a-13C]FMN reconstituted into apo OYE. Though the OYE sample prepared according to the conventional procedure gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the OYE sample was found to consist of five species on anion-exchange HPLC. The 13C-NMR spectrum of the [4a-13C]FMN-reconstituted OYE gave multiple peaks corresponding to 4a-13C. This multiplicity indicates that this OYE preparation possesses heterogeneity in the environment surrounding FMN, i.e., the active site of OYE. The different species of OYE were separately obtained by preparative HPLC on an anion-exchange column. These species as well as the unresolved sample showed identical mobility on SDS-PAGE and similar but slightly different NADPH oxidase activities. This heterogeneity was shown not to have resulted from proteolytic modification during the conventional purification procedure, which includes autolysis of the yeast cells, since the enzyme extracted by mechanical destruction of the yeast cells in the presence of various protease inhibitors exhibited identical heterogeneity. The pure OYE forms obtained by preparative anion-exchange HPLC are homogeneous in the flavin environment as revealed by a single 13C-NMR signal for the [4a-13C]FMN-reconstituted species.     

Purification and partial characterization of a flocculin from brewer's yeast.

Analysis of a shear supernatant from flocculent, "fimbriated" Saccharomyces cerevisiae brewer's yeast cells revealed the presence of a protein involved in flocculation of the yeast cells and therefore designated a flocculin. The molecular mass of the flocculin was estimated to be over 300 kDa, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel permeation chromatography of the flocculin yielded an aggregate with an apparent molecular weight of > 2,000. The flocculin was found to be protease sensitive, and the sequence of its 16 N-terminal amino acids revealed at least 69% identity with the predicted N terminus of the putative protein encoded by the flocculation gene FLO1. The flocculin was isolated from flocculent S. cerevisiae cells, whereas only a low amount of flocculin, if any, could be isolated from nonflocculent cells. The flocculin was found to stimulate the flocculation ability of flocculent yeast cells without displaying lectinlike activity (that is, the ability to agglutinate yeast cells).