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Nucleoside phosphotransferase in animal tissues. Tissue distribution and kinetic properties
Amphibian, avian and mammal tissues contain a nucleoside phosphotransferase clearly different from those previously described in vegetables and bacteria.
Whatever the animal source, the enzyme showed many similar characteristics as far as substrate specificity, dependence upon Mg2+ instability at 37 °C, and the protecting effect of nucleotides were concerned. Moreover, when submitted to gel filtration, the enzyme behaved in all cases as a dissociable high molecular weight protein, whose degree of association was controlled by nucleotides.
In amphibian and avian tissues multiple forms of the enzyme seem to be present which differ for the substrate concentration at half-maximal velocity (S0.5); the concentration of nucleotide effector which affords half-maximal protection at 37 °C (P0.5); and the Hill coefficient for monophosphate donor. Within each single species, the higher the interaction coefficient was, the lower S0.5 and P0.5 values were.
In mammalian tissues one form of nucleoside phosphotransferase seems to prevail where cooperative interactions are almost absent and whose S0.5 as well as P0.5 values do not vary significantly from one tissue to another
Morphological and biochemical effects of glucocorticoids in chick embryo hepatocytes during development
The administration in ovo of hydrocortisone-21-phosphate caused, in chick embryo liver, a reduction of the number of hepatocytes which can be isolated from 1 mg dry weight of liver and a marked increase of their size. Moreover, the treatment diminished the incorporation of thymidine into acid-insoluble fraction in these cells whilst it augmented the content of protein, RNA, DNA and the level of thymidine kinase/cell. These effects were highest at 8-10 days, then declined with the age, disappearing after 18th day of incubation. Similar effects were obtained by injecting other glucocorticoids or ACTH. Combined treatment with metopirone abolished the effects found with ACTH, but did not modify the action of hydrocortisone. These findings suggest that glucocorticoids interfere with the proliferative cycle of hepatocytes by inhibiting the mitotic phase and favouring the production of abnormally large cells
Biochemical aspects of chick embryo retina development: the effects of glucocorticoids
In chick embryo retina during development, DNA synthesis and the activities of DNA polymerase, thymidine kinase, thymidylate synthetase, and ornithine decarboxylase (ODC) declined in parallel from day 7 to 12. The administration in ovo of hydrocortisone reduced significantly, particularly at 8-10 days of incubation, both DNA synthesis and the four enzyme activities tested. The effect was dose dependent, reaching the maximum with 50-100 nmol of hydrocortisone, 8-16 h after treatment. The highest inhibition was found for ODC activity (70%), followed by thymidine kinase activity (62%) and DNA synthesis (45%), whereas activities of DNA polymerase and thymidylate synthetase were reduced only by 30%. The inhibitory effect was exerted by all the glucocorticoids tested, with dexamethasone and hydrocortisone being the most efficacious. The results support the view that glucocorticoids reduce the proliferative events in chick embryo retina, particularly at 8-10 days of embryonic life
High-performance liquid chromatographic method for the determination of insulin synthesis in biological systems
Purification of a fetal bovine serum factor that inhibits DNA synthesis in chick embryo retinas
The inhibitory effect of D-glucosamine on thymidine kinase in chick embryo retinas and HeLa cells
D-Glucosamine markedly inhibits thymidine incorporation into the TCA-insoluble fraction and thymidine kinase activity in HeLa cells. Both the inhibitory effects are also observed in isolated retinas of chick embryos. In this case the inhibitory effects are age-dependent and the magnitude of the responses decreases with embryonic development. In addition the time of exposure to D-glucosamine which is necessary to reveal the inhibitory effect on thymidine kinase increases with the age of the embryos
Regulatory properties of nucleoside phosphotransferase from mucosa of chicken intestine
Nucleoside phosphotransferase from chicken intestinal mucosa is an associated multisubunit protein which can dissociate into components of lower molecular weight. The associated and the dissociated forms have the same substrate specificity but the first (A) shows a higher V(max) and a lower S(0.5) value than the second (B), whichever phosphate donor or nucleoside acceptor was employed. Moreover with form A the interaction coefficient in the Hill plots; as measured with a phosphate donor, varied: they always showed a higher result (about 2) than with form B (about 1). A brief preincubation at 37°C of form A modified the values both of the inactivation constant and of the enzyme kinetic parameters, which became similar to those reported for form B. Form B of nucleoside phosphotransferase is, unlike form A, only moderately sensitive to the regulatory effects of nucleoside diphosphates
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