Postprandial endothelial function: The effect of various types of fat and of red wine intake on flow-mediated dilation in healthy volunteers

Background: Current evidence suggests that postprandial lipid composition may have either atherogenic or anti-atherogenic properties. This study investigated the acute postprandial effect of three different types of fat (saturated, mono-unsaturated, poly-unsaturated) and of red wine upon endothelial function of healthy subjects.Methods: We fed 21 healthy young subjects four meals containing 1000  kcal and 50 g fat each. Three of the meals contained different fat sources: yellow cheese (saturated fat-meal A), olive oil (monounsaturated fat-meal B) and margarine (polyunsaturated fat-meal C). The fourth  meal contained the same amount of saturated fat as the first one together with 250 cc of red wine (meal D). We measured serum lipoproteins, with the use of standard laboratory techniques. Βrachial artery flow-mediated vasodilation (FMD), an index of endothelial function, was also assessed with B-Mode vascular ultrasound. All measurements were performed before, 2 and 4 h after each mealResults:  All four meals significantly raised plasma triglycerides. Meal A was associated with a decrease in FMD  2 and 4 hours after its intake (16.5±5% vs. 13±4% vs. 12.3±6% , p=0.030) while with meal D FMD profoundly decreased 2 hours postprandially  with a rapid return to baseline levels at 4 hours  (14.3±9.5 vs. 8.6±5.5 vs. 12.6±7.5 , p=0.214). This type of response was associated with a concomitant increase of basal brachial artery diameter at 2 hours postpandially (3.7±0.6 mm vs 4.2±0.7 mm vs 3.9±0.7mm at 0, 2 and 4 hours respectively , p=0.000). Meals B and C did not significantly affect FMD. Conclusions. Saturated fat acutely decreases FMD while mono-unsaturated and poly-unsaturated fat do not have any harmful effect on endothelium. The concomitant intake of red wine with saturated fat is associated with a delayed counteracting effect on lipid-induced postprandial endothelial dysfunction

The binding of beta-glycerophosphate to glycogen phosphorylase b in the crystal.

The binding of beta-glycerophosphate (glycerol-2-P) to glycogen phosphorylase b in the crystal has been studied by X-ray diffraction at 3 A resolution. Glycerol-2-P binds to the allosteric effector site in a position close to that of AMP, glucose-6-P, UDP-Glc, and phosphate. In this position, glycerol-2-P is stabilized through interactions of its phosphate moiety with the guanidinium groups of Arg 309 and Arg 310 which undergo conformational changes, and the hydroxyl group of Tyr 75, while the same residues and solvent are involved in van der Waals interactions with the remaining part of the molecule. Kinetic experiments indicate that glycerol-2-P partially competes with both the activator (AMP) and the inhibitor (glucose 6-phosphate) of phosphorylase b. A comparison of the positions of glycerol-2-P, AMP, glucose 6-phosphate, UDP-Glc, and Pi at the allosteric site is presented

Crystallization of Pig Skeletal Phosphorylase-B - Purification, Physical and Catalytic Characterization

Journal URL: http://www.sciencedirect.com/science/journal/0167483

Uridine(5')diphospho(1)-alpha-D-glucose. A binding study to glycogen phosphorylase b in the crystal.

UDP-glucose is an R-state inhibitor of glycogen phosphorylase b, competitive with the substrate, glucose 1-phosphate and noncompetitive with the allosteric activator, AMP. Diffusion of 100 mM UDP-glucose into crystals of phosphorylase b resulted in a difference Fourier synthesis at 0.3-nm resolution that showed two peaks: (a) binding at the allosteric site and (b) binding at the catalytic site. At the allosteric site the whole of the UDP-glucose molecule can be located. It is in a well defined folded conformation with its uracil portion in a similar position to that observed for the adenine of AMP. The uracil and the glucose moieties stack against the aromatic side chains of Tyr-75 and Phe-196, respectively. The phosphates of the pyrophosphate component interact with Arg-242, Arg-309 and Arg-310. At the catalytic site, the glucose-1-P component of UDP-glucose is firmly bound in a position similar to that observed for glucose 1-phosphate. The pyrophosphate is also well located with the glucose phosphate interacting with the main-chain NH groups at the start of the glycine-loop alpha helix and the uridine phosphate interacting through a water molecule with the 5'-phosphate of the cofactor pyridoxal phosphate and with the side chains of residues Tyr-573, Lys-574 and probably Arg-569. However the position of the uridine cannot be located although analysis by thin-layer chromatography showed that no degradation had taken place. Binding of UDP-glucose to the catalytic site promotes extensive conformational changes. The loop 279-288 which links the catalytic site to the nucleoside inhibitor site is displaced and becomes mobile. Concomitant movements of residues His-571, Arg-569, and the loop 378-383, together with the major loop displacement, result in an open channel to the catalytic site. Comparison with other structural results shows that these changes form an essential feature of the T to R transition. They allow formation of the phosphate recognition site at the catalytic site and destroy the nucleoside inhibitor site. Kinetic experiments demonstrate that UDP-glucose activates the enzyme in the presence of high concentrations of the weak activator IMP, because of its ability to decrease the affinity of IMP for the inhibitor site

Pyridoxal phosphate site in glycogen phosphorylase b: structure in native enzyme and in three derivatives with modified cofactors.

The detailed environment of the essential cofactor pyridoxal 5'-phosphate in glycogen phosphorylase b, resulting from crystallographic refinement at 1.9-A resolution, is described. The pyridoxal ring is buried in a nonpolar site containing three aromatic rings while the 5'-phosphate group is highly solvated and makes only three direct contacts to the protein. The pyridine nitrogen interacts via a water with protein atoms [main chain carbonyl oxygen (Asn-133) and OH of tyrosine (Tyr-90)]. The crystal structures of three active derivatives of phosphorylase reconstituted with 5'-deoxypyridoxal 5'-methylenephosphonate (PDMP), 6-fluoropyridoxal 5'-phosphate (6-FPLP), and pyridoxal (PL) in place of the natural cofactor have been determined at 2.5-A resolution. The results for PDMP-phosphorylase show a closer proximity of the phosphonate group to the NZ atom of a lysine (Lys-574) than that observed in the native enzyme, consistent with 31P NMR studies that have shown a change in ionization state of the phosphonate group compared to the native cofactor phosphate. The replacement of the polar 5'-ester linkage by a CH2 group results in a small shift of a water and its hydrogen-bonded tyrosine (Tyr-648). In 6-FPLP-phosphorylase the fluorine is accommodated with no significant change in structure. It is suggested that substitution of the electronegative fluorine at the 6-position may result in lower activity of 6-FPLP-phosphorylase through a strengthening of hydrogen-bonded interactions to the pyridine nitrogen N1.(ABSTRACT TRUNCATED AT 250 WORDS