Effect of carbohydrate digestibility on appetite and its relationship to postprandial blood glucose and insulin levels.

BACKGROUND/OBJECTIVES: 'Slowly digestible' carbohydrates have been claimed to reduce appetite through their effects on postprandial glucose and insulin levels, but literature is inconsistent. The inconsistencies between studies might be explained by factors other than glycemic effects per se, for example, nutritional or physical properties. We tested this possibility by examining postprandial glucose, insulin and appetite responses to drinks differing only in rate and extent of digestibility of carbohydrates. This was accomplished by comparing different glucose polymers: maltodextrin (rapidly digestible) versus medium-chain pullulan (slowly but completely digestible) versus long-chain pullulan (indigestible). SUBJECTS/METHODS: In a randomized double-blind balanced crossover design, 35 subjects received drinks with 15 g test carbohydrate polymers. Key outcome measures were appetite scores, digestibility (in vitro test and breath hydrogen), and (in a subset) glucose and insulin levels. RESULTS: Digestibility, glucose and insulin data confirmed the rapid, slow and nondigestible nature of the test carbohydrates. Despite its low digestibility, only long-chain pullulan reduced appetite compared with the maltodextrin control, whereas the medium-chain pullulan did not. CONCLUSIONS: We conclude that glycemic responses per se have minimal effects on appetite, when tested in products differing in only carbohydrate digestibility rate and extent.

A novel class of protein from wheat which inhibits xylanases.

We have purified a novel class of protein that can inhibit the activity of endo-beta-1,4-xylanases. The inhibitor from wheat (Triticum aestivum, var. Soisson) is a glycosylated, monomeric, basic protein with a pI of 8.7-8.9, a molecular mass of 29 kDa and a unique N-terminal sequence of AGGKTGQVTVFWGRN. We have shown that the protein can inhibit the activity of two family-11 endo-beta-1, 4-xylanases, a recombinant enzyme from Aspergillus niger and an enzyme from Trichoderma viride. The inhibitory activity is heat and protease sensitive. The kinetics of the inhibition have been characterized with the A. niger enzyme using soluble wheat arabinoxylan as a substrate. The Km for soluble arabinoxylan in the absence of inhibitor is 20+/-2 mg/ml with a kcat of 103+/-6 s-1. The kinetics of the inhibition of this reaction are competitive, with a Ki value of 0.35 microM, showing that the inhibitor binds at or close to the active site of free xylanase. This report describes the first isolation of a xylanase inhibitor from any organism.

Engineering surface charges in a subtilisin.

Introduction of multiple charged amino acid residues in the subtilisin Savinase by genetic engineering allowed us to modify the electrostatic properties of this enzyme in a systematic way. The effects of these charge changes were investigated theoretically with the calculated electrostatic potential at the enzyme surface and experimentally using ion exchange chromatography. Our results indicate that the effect of introducing charged residues at the enzyme surface depends on the local electrostatic potential. The effects are purely additive for residues that are not too closely packed at the enzyme surface. Although it is generally accepted that polarization effects are relatively small, our data show that substantial charge shifts arise when the dominating effect of the overall charge is taken away. These shifts are not well quantified using current methods to calculate the electrostatic potential at the enzyme surface. Our work focuses [correction of focusses] on methods that will provide a better description of this surface potential.

Engineering surface charges in a subtilisin: the effects on electrophoretic and ion-exchange behaviour.

The introduction or removal of multiple charged amino acid residues in the subtilisin Savinase by genetic engineering allowed us to modify the electrostatic properties of this enzyme in a systematic way. The effects of these charge changes were investigated experimentally using ion-exchange chromatography and electrophoretic mobility in native gels all under identical conditions. The experiments clearly demonstrated that the overall charge of a given protein is not the only factor determining electrophoretic mobility at low or moderate ionic strengths. For a series of variants having identical overall positive charge a linear relation was observed between mobility towards the cathode and the total number of charged residues present. This effect was found to depend on the type of (chloride) salt used: calcium ions give rise to complete screening of all negative charges, whereas only partial screening is found for magnesium and sodium ions. In contrast, in the presence of sodium phosphate the overall charge of the enzyme becomes slightly negative. These data indicate that cations as well as anions may strongly perturb the overall charge of proteins depending on the type of salt and on the number of charged amino acid residues present. The ion-exchange behaviour demonstrated similar results, i.e. showing stronger enzyme adsorption with increasing numbers of surface charges on a cation-exchange column run below the isoelectric point of the proteins. However, the apparent sign reversal noted above for electrophoresis with sodium phosphate did not appear in the ion-exchange experiments. This work provides further insight into the adsorption of proteins to surface and the role played by small ions, particularly when electrostatic forces dominate the adsorption process.

The complete amino-acid sequence of the sweet protein thaumatin I.

The primary structure of the sweet-tasting protein thaumatin has been elucidated. The protein consists of a single polypeptide chain of 207 residues. The sequence of the N-terminal part of the chain was determined by sequenator analysis. As the protein contains only one methionine residue, it was possible to deduce the N-terminal sequence of the C-terminal cyanogen bromide fragment by automatic sequencing of the cyanogen-bromide-cleaved, succinylated protein. To arrive at the sequence of the whole protein tryptic and Staphylococcus protease peptides, together with chymotryptic peptides and a 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine (BNPS-skatole) fragment were also sequenced. Comparing the amino acid sequence of thaumatin with that of the other sweet-tasting protein, monellin, we have located five sets of identical tripeptides. Since immunological cross-reactivity of thaumatin antibodies with monellin has recently been described, one or more of these tripeptides might be part of a common antibody recombination site and possibly be involved in the interaction with the sweet-taste receptor.