N-Carbamoyl-ß-alanine amidohydrolase from Agrobacterium tumefaciens C58: a promiscuous enzyme for the production of amino acids.

The availability of enzymes with a high promiscuity/specificity relationship permits the hydrolysis of several substrates with a view to obtaining a certain product or using one enzyme for several productive lines. N-Carbamoyl-ß-alanine amidohydrolase from Agrobacterium tumefaciens (Atßcar) has shown high versatility to hydrolyze different N-carbamoyl-, N-acetyl- and N-formyl-amino acids to produce different α, ß, γ and δ amino acids. We have calculated the promiscuity index for the enzyme, obtaining a value of 0.54, which indicates that it is a modestly promiscuous enzyme. Atßcar presented the highest probability of hydrolysis for N-carbamoyl-amino acids, being the enzyme more efficient for the production of α-amino acids. We have also demonstrated by mutagenesis, modelling, kinetic and binding experiments that W218 and A359 indirectly influence the plasticity of the enzyme due to interaction with the environment of R291, the key residue for catalytic activity.

Influence of sequential yeast mixtures on wine fermentation.

The use of Pichia fermentans in pure cultures and sequential mixtures with Saccharomyces cerevisiae has been studied to improve the aromatic compounds and characteristics of a wine. P. fermentans has proved to be a good starter strains for must fermentation in the winemaking industry. It has shown the same level of sulphur tolerance and the same growth rate as S. cerevisiae. We have demonstrated that only 2 days of must fermentation with P. fermentans in sequential mixtures are enough to increase the following compounds in the wine both qualitatively and quantitatively: acetaldehyde, ethyl acetate, 1-propanol, n-butanol, 1-hexanol, ethyl caprilate, 2,3-butanediol and glycerol. Maintaining this non-Saccharomyces strain in contact with the must for longer periods quantitatively increases the flavour composition.

Thermodynamic analysis of the binding of glutathione to glutathione S-transferase over a range of temperatures.

The binding properties of a glutathione S-transferase (EC 2.5.1.18) from Schistosoma japonicum to substrate glutathione (GSH) has been investigated by intrinsic fluorescence and isothermal titration calorimetry (ITC) at pH 6.5 over a temperature range of 15-30 degrees C. Calorimetric measurements in various buffer systems with different ionization heats suggest that protons are released during the binding of GSH at pH 6.5. We have also studied the effect of pH on the thermodynamics of GSH-GST interaction. The behaviour shown at different pHs indicates that at least three groups must participate in the exchange of protons. Fluorimetric and calorimetric measurements indicate that GSH binds to two sites in the dimer of 26-kDa glutathione S-transferase from Schistosoma japonicum (SjGST). On the other hand, noncooperativity for substrate binding to SjGST was detected over a temperature range of 15-30 degrees C. Among thermodynamic parameters, whereas DeltaG degrees remains practically invariant as a function of temperature, DeltaH and DeltaS degrees both decrease with an increase in temperature. While the binding is enthalpically favorable at all temperatures studied, at temperatures below 25 degrees C, DeltaG degrees is also favoured by entropic contributions. As the temperature increases, the entropic contributions progressively decrease, attaining a value of zero at 24.3 degrees C, and then becoming unfavorable. During this transition, the enthalpic contributions become progressively favorable, resulting in an enthalpy-entropy compensation. The temperature dependence of the enthalpy change yields the heat capacity change (DeltaCp degrees ) of -0.238 +/- 0.04 kcal per K per mol of GSH bound.

A calorimetric study of the binding of S-alkylglutathiones to glutathione S-transferase.

The binding of three competitive glutathione analogue inhibitors (S-alkylglutathione derivatives) to glutathione S-transferase from Schistosoma japonicum, SjGST, has been investigated by isothermal titration microcalorimetry at pH 6.5 over a temperature range of 15--30 degrees C. Calorimetric measurements in various buffer systems with different ionization heats suggest that no protons are exchanged during the binding of S-alkylglutathione derivatives. Thus, at pH 6.5, the protons released during the binding of substrate may be from its thiol group. Calorimetric analyses show that S-methyl-, S-butyl-, and S-octylglutathione bind to two equal and independent sites in the dimer of SjGST. The affinity of these inhibitors to SjGST is greater as the number of methylene groups in the hydrocarbon side chain increases. In all cases studied, Delta G(0) remains invariant as a function of temperature, while Delta H(b) and Delta S(0) both decrease as the temperature increases. The binding of three S-alkylglutathione derivatives to the enzyme is enthalpically favourable at all temperatures studied. The temperature dependence of the enthalpy change yields negative heat capacity changes, which become less negative as the length of the side chain increases.

Effect of dietary coconut oil on lipoprotein composition of young chick (Gallus domesticus).

1. The composition of HDL, the major lipoprotein fraction from chick serum, drastically changed after 2 weeks of coconut oil feeding. Total cholesterol and triacylglycerols significantly increased following dietary 10 or 20% coconut oil supplementation. 2. Changes in LDL composition were less profound, cholesterol being the only component that increased by coconut oil supplementation (10 or 20%). 3. IDL proteins were the only components that increased following the same dietary treatment (20%). 4. VLDL cholesterol and proteins also increased after 1-2 weeks of 20% coconut oil supplementation to the diet. 5. Of total lipoproteins, the cholesterol content strongly increased after dietary treatment, while triacylglycerols did not change significantly.

Influence of protein/lipid ratio of diet on cholesterol synthesis and esterification in eel liver.

The effect of protein/lipid ratio of diets on hepatic cholesterol has been studied in European eel and correlated with changes in the main enzymes responsible for cholesterol metabolism. The growth rates of animals were similar when dietary lipid level was 12%. However, a 25% protein/20% fat (25/20) diet produced a decrease in the weight gain when compared with that observed after feeding a 30/20 diet. At low fat level (12%), the decrease in dietary protein produced a little but significant increase in total cholesterol, mainly due to the esterified form. On the contrary, a 25/20 diet produced a lower cholesterol accumulation than that a 30/20 diet. These results suggest that a minimal protein level was required for an optimal utilization of dietary fat for cholesterol deposition in liver. No significant differences were found in 3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate kinase, mevalonate 5-phosphate kinase and mevalonate 5-pyrophosphate decarboxylase when compared the effect of 40/12 and 30/12 diets as well as that of 30/20 and 25/20 diets, suggesting that differences in hepatic cholesterol content were not due to differences in cholesterol synthesis but in the transport to the liver. Changes in the esterified cholesterol were parallel to those found in acyl-CoA: cholesterol acyltransferase, corroborating the main role of this enzyme in the regulation of hepatic cholesterol esterification.

Induction in Gallus domesticus of experimental hypercholesterolemia by saturated fat. Effects on cholesterogenic enzyme activity.

The effect of coconut oil supplementation to the diet (10 or 20%) on lipid levels in plasma and liver as well as on the cholesterogenic enzyme activity were studied in 14-day-old chicks. Treatments for 1 or 2 weeks did not interfere in the growth rate of animals nor in the liver weight. The 10% coconut oil group showed a significant increase of plasma cholesterol after 2 weeks of treatment, while after 1 week the increase was not statistically significant. The 20% coconut oil group increased plasma cholesterol from the first week. Triacylglycerol content increased after each coconut oil supplementation to the diet during the first week. Hepatic cholesterol did not change significantly after any treatment assayed. No significant difference was observed in the cholesterogenic activity, measured as hepatic 3-hydroxy-3-methylglutaryl-CoA reductase, so that this study provides a perfect model of hypercholesterolemic animals without changes in their cholesterogenic ability.

Characterization of chick serum lipoproteins isolated by density gradient ultracentrifugation.

Serum lipoproteins from 12h fasted male chicks (15-day-old) were separated into 20 fractions by isopycnic density gradient ultracentrifugation. A new procedure was described by collecting the different fractions from the bottom of tube instead of by aspiration from the meniscus of each tube. Analyses of chemical composition of serum lipoproteins have permitted to reevaluate the density limits of major classes: VHDL, d greater than 1.132 g/ml; HDL, d 1.132-1.084 g/ml; LDL, d 1.084-1.038; IDL, d 1.038-1.022; and VLDL d less than 1.022. HDL fractions clearly predominated (approx. 77% of total lipoproteins) while IDL and VLDL were present at low percentage. LDL was the fraction richest in cholesterol; triacylglycerol content clearly increased from HDL to VLDL, while protein content decreased. All the chemical components of chick serum lipoproteins were accumulated in HDL, although triacylglycerol was relatively distributed in all the lipoprotein classes.

A procedure for the simultaneous determination of lipid and protein in biomembranes and other biological samples.

Very small sample sizes frequently become the limiting factor in biochemical and biomembrane studies in which routine quantification of protein and bulk lipids are required. The procedure described here allows the simultaneous determination of protein and lipid without initial, multiple aliquots. The method is based on the quantitative precipitation of proteins from a defined hexane/isopropanol mixture. The liquid phase resulting after decanting and concentrating to dryness can then be used to assay the lipid content directly. Quantitative assay of protein can be achieved after resuspension of the pelleted material by addition of sodium dodecyl sulfate (0.1%) and deoxycholate (1%). The method is also applicable to other types of lipid- and protein-containing samples with a broad range of protein/lipid ratios and lipid compositions, as they occur, for example, in serum lipoproteins.

Effects of clofibrate on the main regulatory enzymes of cholesterogenesis.

The in vivo effect of clofibrate on the main regulatory enzymes of cholesterogenesis has been comparatively studied for the first time in chick liver and brain. 3-Hydroxy-3-methylglutaryl-CoA reductase and mevalonate 5-pyrophosphate decarboxylase from chick liver were significantly inhibited by this hypocholesterolenic drug, while mevalonate kinase and mevalonate 5-phosphate kinase were not affected. No enzyme from chick brain was significantly inhibited by the in vivo treatment. However, both liver and brain reductase activity was inhibited in vitro by clofibrate, inhibition that was progressive with increasing concentrations (1.25-5.00 mM) of drug.

A procedure for eliminating interferences in the lowry method of protein determination.

A modification of the Lowry assay for the quantitative protein measurement in the presence of interfering materials has been developed. The method is based on a precipitation with a single-phase hexane:isopropanol solvent system and later resuspension of protein pellets with sodium dodecyl sulfate and deoxycholate. The new procedure eliminates the interference caused by Triton X-100, phospholipids, or dithiothreitol providing yields higher than 95% and seems to be especially suitable for protein determination on membrane preparations in samples with small volumes and/or very low protein concentrations.

Age-related changes in the mevalonate metabolism in vivo in chick kidneys.

The mevalonate incorporation in vivo into total nonsaponifiable lipids by chick kidneys drastically increased after hatching, reaching similar levels to those previously observed in liver. Cholesterol was the major sterol formed from mevalonate from 11 days onward, while a fraction of polar nonsaponifiable lipid(s) was observed as the major compound(s) synthesized at 5-8 days. Relative percentages of squalene, squalene oxide(s) and lanosterol synthesized from mevalonate also increased between 11-18 days after hatching. Results in this paper demonstrate for the first time the accumulation of a fraction of nonsaponifiable lipid(s) identified as lanosterol derivatives and cholesterol precursors formed by kidneys from [5-14C]mevalonate in experiments carried out in vivo, as well as their evolution during postnatal period.