Paul D. Buckley

Structure and biological activity of nitrogen and oxygen coordinated nicotinic acid complexes of chromium

Abstract A series of N-coordinated M(II)(nic)2(H2O)4≠ complexes, where M = Co, Ni, Mn or Cr and the O-coordinated species Cr(III)(nic)2(H2O)4+ and Cr(III)(nic)(H2O)5+2 have been prepared. A single crystal X-ray analysis of the complex Cr(II)(nic)2(H2O)4 shows that the chromium ion is in the +2 oxidation state and that two nicotinic acid ligands are coordinated in a trans arrangement via the pyridine ring nitrogen atoms. The four equatorial positions are occupied by water molecules. Biological activity measurements with a yeast bioassay system however show that none of the N-coordinated complexes possess any glucose tolerance factor (GTF-type) activity. On the other hand, the O-coordinated chromium(III)-dinicotinic acid complex is biologically active which suggests that a trans arrangement of pyridine nitrogen atoms resembles that part of the GTF structure which is recognised by the receptors or enzymes which are involved in the expression of the biological effect.

Chromium(III) complexes and their relationship to the glucose tolerance factor. Part II. Structure and biological activity of amino acid complexes

Abstract A number of octahedral chromium complexes with amino acids are ligands have been prepared and their structures assigned on the basis of their chromatographic and spectral properties. These include complexes with the general structure Cr(AA) 2 (H 2 O) 2 where the amino acids glycine, glutamic acid and glutamine act as bidentate ligands. The analogous compound with cysteine as ligand is stable at low pH, but at high pH a terdentate cysteine complex, Cr(cysteine) 2 − , is formed. These complexes, as well as a solution of monodentate glycine aquo complexes, and Cr-nicotinic acid-glycine and Cr-nicotinic acid-cysteine complexes of undetermined structure, have been assayed for glucose tolerance factor activity using a yeast assay. Only Cr(glutamine) 2 - (H 2 O) 2 + , Cr-nicotinic acid-glycine and the mixture of complexes Cr(glycine) n (H 2 O) 6-n +3 showed significant activity. It is proposed that a trans arrangement of the non-coordinated nitrogen atoms in the ligands of these complexes can mimic the structural features of the glucose tolerance factor which are essential for biological activity.

The relationship of chromium to the glucose tolerance factor. II

After incubation with CrCl3 X 6H2O (or 51CrCl3 X 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 brewers yeast can no longer be regarded as a chromium complex.

Separation of biologically active chromium-containing complexes from yeast extracts and other sources of glucose tolerance factor (GTF) activity

A procedure has been developed, based on ion-exchange chromatography, that readily allows the separation of eleven apparently homogeneous chromium-containing fractions from a brewers yeast extract. Four of the fractions are amphoteric and show no glucose tolerance factor (GTF) activity, three are classified as negative (two of which are biologically inactive, while the third one shows a slight degree of GTF activity), whereas the four cationic chromium-containing fractions all show varying degrees of GTF activity. Application of the separation procedure to other biological sources of GTF activity resulted in a spectrum of cationic fractions, over the pH range 1.75 to 12, which suggests that GTF cannot be a single species. The cationic chromium-containing fraction from pork kidney powder and fraction P-3 from yeast appear to contain the most GTF-active material and P-3 shows saturation kinetics as expected for a biologically significant substance.

Glucose tolerance factor potentiation of insulin action in adipocytes from rats raised on a torula yeast diet cannot be attributed to a deficiency of chromium or glucose tolerance factor activity in the diet

The nature of the dietary component responsible for adipocytes having the ability to respond to Glucose Tolerance Factor (GTF) was investigated. Rats were raised on either a control diet or one of three diets differing only in the protein source (torula yeast, brewers yeast, or casein). Only in adipocytes from rats fed the torula yeast diet did a GTF fraction prepared from brewers yeast potentiate the action of suboptimal concentrations of insulin in the incorporation of label fromd-[1-14C]-glucose andd-[U-14C]-glucose into CO2 and fatty acids. It was concluded that this potentiation was not the result of a deficiency of GTF activity in torula yeast, because a GTF fraction prepared from torula yeast had similar insulin potentiating activity. Differences in response among diets were not owing to differences in levels of amino acids or owing to concentrations of 22 (Al, As, B, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mo, Na, Ni, P, Pb S, Se, Si, Sn, Sr, Zn) of the 23 trace elements investigated. The level of Mn, was low in all diets, but particularly low in the torula yeast diet. Mn deficiencies have previously been implicated in perturbations of glucose metabolism, so that it is possible that this deficiency may be responsible for the effects attributed to the torula yeast diet.

Computing in Stereochemistry - 2D or 3D Representations?

This paper reports part of a preliminary phase of a study examining students responses to a computer-based (CB) stereochemistry task and to different representations of organic molecules. Six themes in the way students used the CB tutorial have been identified: manipulation of models, multiple representations, use of colour, simplicity, familiarity and types of isomerism. It has been found that 3D animations and the colourful molecular images encouraged students to practise the use of different representations. Students’ preferences for either two-dimensional (2D) skeletal formulae or three-dimensional (3D) ball and stick representations depended on the focus of the exercise and also the type of isomerism they were investigating. The study has demonstrated that a suitably-designed, computer based task encourages students to make use of the molecular representation that, for them, best suits the problem.

Effect of chromium on the rate of carbon dioxide evolution fromSaccharomyces cerevisiae

Abstract The rate of carbon dioxide evolution by whole yeast cells was measured under conditions of both chromium starvation and availability. Preincubation experiments show that there is no evidence for the view that Saccharomyces cerevisiae can synthesize a biologically important form of chromium from simple chromium(III) salts. Chromium enters the yeast cell, possibly by way of the glucose transport system, and exerts a slight inhibitory effect on the fermentation rate but does not affect the rate of cell growth. Glucose tolerance factor fractions show a stimulatory effect in the yeast fermentation assay irrespective of whether the yeast is chromium depleted. The evidence shows that chromium is not an essential trace element for normal yeast metabolism and hence the activity of glucose tolerance factor preparations is unlikely to be due to a chromium complex as is commonly assumed. Some other as yet unidentified compound or compounds must be responsible for the observed increase in the rate of carbon dioxide evolution.

Kinetics of inhibition and hysteresis of sheep liver cytoplasmic aldehyde dehydrogenase with glyoxylic acid: further evidence relating to the two-site model for aldehyde oxidation

Despite the fact that it is an aldehyde, glyoxylic acid is not a substrate for sheep liver cytoplasmic aldehyde dehydrogenase; instead it functions as an inhibitor of both the esterase and dehydrogenase activities. From a consideration of the inhibition patterns it is concluded that glyoxylic acid does not bind in the catalytic propionaldehyde-binding domain, thus confirming the two-site model as proposed previously. Since the corresponding neutral methyl ester is a substrate it is suggested that the catalytic binding domain must contain a negatively charged group which prevents the binding of glyoxylic acid. Steady-state and pre-steady-state kinetic studies indicate that glyoxylic acid inhibits the dehydrogenase activity by converting the enzyme into a dead-end form which cannot undergo the catalytically essential conformational change. Incubation of the enzyme with NAD+ and glyoxylic acid for 10 min before the addition of propionaldehyde gave rise to hysteresis effects which can be explained on the basis of a slow isomerization of the enzyme X NAD+ X glyoxylic acid complex.

Identification of peptides from autolysates 0f Saccharomyces cerevisiae that exhibit glucose tolerance factor activity in a yeast assay

1. Cationic fractions were isolated from a low chromium (less than 0.2 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 systems as an assay for GTF.

A two-site model for the esterase and dehydrogenase activities of sheep liver aldehyde dehydrogenase

Although aldehyde dehydrogenase (ALDH) from sheep liver cytosol has a broad specificity, it will not oxidize the aldehyde group of glyoxylic acid which is in fact an inhibitor of the enzyme. The inhibition pattern is non-linear but competitive at high propionaldehyde concentrations (2-20 mM); however, a simple non-competitive pattern is observed at low (less than 100 microM) propionaldehyde concentrations (Ki = 1.6 mM). The esterase activity was unaffected by glyoxylic acid in the absence of NAD+ but a simple competitive inhibition pattern (Ki = 2.5 mM) was observed with respect to 4-nitrophenyl acetate in the presence of NAD+. The data require a two-site model in which ester and aldehyde binding sites are distinct but with a second propionaldehyde molecule, and glyoxylic acid, binding at or near the ester binding site. Consistent with this model is the fact that chloral hydrate was a non-competitive inhibitor of the esterase activity in the presence of NAD+ but a competitive inhibitor in its absence. The enzyme exhibited hysteretic behavior governed by the protonated form of an ionizable group with an apparent pKa of 7.55.