[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.