Impact of structural information on understanding lipolytic function.
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Binding Sites↗
Biomedical subjects
C J van Bemmel
Publications and source records attributed to C J van Bemmel.
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.
Amino Acid Sequence↗
Hepatic de novo fatty acid synthesis in the rat.
Generally, the rate of hepatic de novo fatty acid (FA) synthesis (lipogenesis) in vivo is determined by measuring the amount of newly synthesized FA present in the liver 1 h or less after injection of label (3H2O) for FA synthesis (1-hour value of labelled FA). Since this value may well be affected by momentary conditions, our objective was to investigate whether the amount of labelled FA present in the liver 24 h after injection of 3H2O can be used as a parameter of lipogenesis (24-hour value of labelled FA). To this end, effects of the amounts of dietary fat, sucrose and linoleic acid and the effect of meal feeding versus d libitum feeding on this 24-hour value were investigated. The 24-hour value decreased with the dietary fat level and was higher in rats fed a diet in which starch was partly replaced by sucrose [20% of metabolizable energy (E%)]. This is in accordance with literature data on the 1-hour value of labelled FA. No effect of meal feeding versus ad libitum feeding on the 24-hour value of labelled FA was found. Furthermore, no significant effect of the dietary linoleic acid level (1-10 E%) on the 24-hour value of labelled FA was found, although when lipogenesis was stimulated by feeding a diet containing 20 E% sucrose, the 24-hour value tended to be higher at 1 E% linoleic acid than at 2.5 E% linoleic acid or higher.
Animals↗