Search PubMed⌕ Search

Biomedical subjects

G De Luca

Publications and source records attributed to G De Luca.

At least 163 records · Page 9Linked to original sources
PubMed1976

Effect of some nucleotides on the regulation of glycosaminoglycan biosynthesis.

The effect of some nucleotides on UDP-glucose dehydrogenase (EC. 1.1.1.22) and UDP-glucose 4'-epimerase (EC 5.1.3.2) extracted from epiphysial-plate cartilage of newborn pigs was investigated. UDP-xylose acts as a co-operative allosteric inhibitor of UDP-glucose dehydrogenase, whereas it does not inhibit UDP-glucose 4'-epimerase activity: the inhibition of UDP-glucose dehydrogenase results in an increase of UDP-galactose synthesis, in agreement with the equilibrium constant of UDP-glucose 4'-epimerase reaction. Because of the presence of UDP-glucose 4'-epimerase activity in the enzyme extract, the addition of UDP-galactose induces an increase in reaction rate of UDP-glucose dehydrogenase. NADH inhibits both UDP-glucose dehydrogenase and UDP-glucose 4'-epimerase activities: in the presence of non-saturating NAD concentrations, NADH acts as a co-operative allosteric inhibitor of both enzymes. The inhibitory effect of NADH seems to be strikingly correlated with the value of NAD/NADH ratio and pH. In any case, the percentage inhibition of UDP-glucose 4'-epimerase, under the same experimental conditions, is always higher than that of UDP-glucose dehydrogenase.

Animals↗
PubMed1976

[Histochemical demonstration of uridine diphospho-glucoso-4'-epimerase activity].

In the byosinthesis of glycosaminoglycans, UDP-glucose is utilized by two enzymes: UDP-glucose dehydrogenase which produces UDP-glucuronic acid (chondroitin sulphate precursor), and UDP-glucose 4'-epimerase which produces UDP-galactose (keratan sulphate precursor). The mechanisms regulating these two reactions have particular interest mainly considering that many connective tissues can modify its glycosaminoglycan production with aging; it is well-known that cartilage of young animals synthesizes almost exclusively chondroitin sulphate while cartilage of old animals synthesizes both chrondroitin sulphate and keratan sulphate. The kinetic parameters of both enzymes utilizing UDP-glucose have been recently investigated and some mechanisms responsible for UDP-glucose utilization in glycosaminoglycan biosynthesis have been evidenced. Under histoenzymological viewpoint, we have confirmed the inhibiting effect of UDP-xilose on UDP-glucose dehydrogenase and the possible role of such nucleotide in aging processes of cartilage. In order to study this problem even by a histoenzymological approach, an original method for histochemical determination of UDP-glucose 4'-epimerase activity in connective tissue cells was developed. This method seem to be more sensitive than that described by other authors. In standard conditions the sections of the frozen tissue were incubated in Tris-HCL buffer, pH 8.8 (Tris concentration 0.025 M), containing 0.5 mM UDP-galactose, 2 mM NAD, 0.6mM NBT and an excess of UDP-glucose dehydrogenase (about 300 mU). Control experiments in the absence of UDP-galactose, UDP-glucose dehydrogenase and in the absence of both UDP-galactose an- UDP-glucose dehydrogenase were also carried out. Under our experimental conditions, UDP-glucose 4'-epimerase present in the cells epimerizes UDP-galactose (added in the incubation mixture) to UDP-glucose which can bo oxidized by the excess of UDP-glucose dehydrogenase to UDP-glucuronic acid with a consequent NADH formation. The NADH formed is able to reduce and precipitate NBT. As a control of experimental sistem, we have determined the increase in O.D. at 525 nm of a reaction solution that was incubated directly in the spectrophotometer cuvette, at 37 degrees C with UDP-galactose 0.2 mM, NAD 2 mM, NBT 0.6 mM, 200 mU of UDP-glucose, dehydrogenase, 400mU of UDP-glucose 4'-epimerase and Tris HCL buffer pH 8.8 to a final volume of 1 ml. Histoenzymological and biochemical results demonstrate that this method is specific for and sensitive to UDP-glucose 4'-epimerase activity.

Animals↗
PubMed1975

Biosynthesis of glycosaminoglycans: uridine diphosphate glucose 4'-epimerase from cornea and epiphysial-plate cartilage.

UDP-glucose 4'-epimerase (EC 5.1.3.2.) was extracted from newborn-pig epiphysial-plate cartilage and whole bovine cornea. The formation of radioactive UDP-galactose from UDP[U-14C]glucose was demonstrated by radioautography after separation of the sugar nucleotides by paper chromatography or t.l.c. The pH optimum and the Km values for UDP-glucose, UDP-galactose and NAD+ were determined in both tissues. UDP-galactose and UDP-glucuronic acid formation after incubation with different UDP-glucose concentrations was followed; the same experiment was carried out using different UDP-galactose concentrations and following the formation of UDP-glucose and UDP-glucuronic acid. At equilibrium, the ratio UDP-glucose/UDP-galactose reaches a value of about 3.5. The results obtained seem to indicate that UDP-glucose 4'-epimerase activity is strongly dependent on that of UDP-glucose dehydrogenase. The physiological meaning of UDP-glucose 4'-epimerase in glycosaminoglycan biosynthesis in the two tissues under study is discussed on the basis of the Km values of UDP-glucose 4'-epimerase and UDP-glucose dehydrogenase and on the basis of the rate of UDP-glucose and UDP-galactose utilization.

Animals↗
PubMed1973

Uridine diphosphate glucose dehydrogenase from cornea and epiphysial-plate cartilage.

1. UDP-glucose dehydrogenase (EC 1.1.1.22) was extracted from epiphysial-plate cartilage of newborn pigs and from whole bovine corneas. 2. Formation of UDP-glucuronic acid was demonstrated by radioautography after separation of the sugar nucleotides by paper chromatography or t.l.c.: in these conditions a radioactive glucuronic acid spot also appears. 3. UDP-xylose prevented the formation in the incubation mixture of both UDP-glucuronic acid and free glucuronic acid. 4. In both tissues the dependence of the enzyme activity on pH and the K(m) values for UDP-glucose and NAD(+) were determined. 5. Inhibition by UDP-xylose with respect to UDP-glucose was investigated. The plots of 1/v versus 1/[UDP-glucose], and of percentage inhibition versus UDP-xylose concentration and the Hill coefficient showed that a co-operative effect existed between UDP-xylose-binding sites. 6. The physiological meaning of the different affinities of cartilage and cornea enzymes for UDP-xylose is discussed and related to the different glycosaminoglycan contents of the two connective tissues studied.

Alcohol Oxidoreductases↗