Possible Energy-Dependency of Protocollagen Proline Hydroxylase Reaction

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Protocollagen proline hydroxylase (P. p.hydroxylase) plays a role in the regulatory steps of collagen biosynthesis. The enzyme was purified upto the homogeneity from chick embryo extract. Labelled protocollagen was prepared by the incubation of 12-day-old rat fetuses with 4-3 H-proline, and the assay of the enzyme activity was performed according to the method of Hutton et al. P. p.hydroxylase activity was markedly enhanced by the addition of ATP or GTP, provided the incubation medium was omitted by dithiothreitol (DTT) and bovine serum albumin. Among the nucleoside triphosphates, GTP was the most effective followed by ATP and ITP, while the pyrimidine nucleotides were less effective. Nucleoside diphosphates showed no effect on the stimulation, while those inhibited the enhancement by nucleoside triphosphates competitively. AMPPCP, AMPPNP or AMPCPP were inactive in enhancing the P. p.hydroxylase activity. Cyclic AMP or dATP were also inactive. DTT enhanced the hydroxylase activity, and once stimulated by DTT, GTP or ATP was not effective in increasing the activity. Kinetic studies revealed that the enhancement was mainly due to the increase of Vmax, both in nucleotide- and DTT-stimulation. These results suggest that the P. p.hydroxylase reaction requires γ-phosphate of a purine nucleotide, so it is likely that the reaction may use the energy yielded from the hydrolysis of NTP to NDP and Pi. On the other hand, this process should be related with an oxidation-reduction reaction of sulfhydryl groups. Tentatively we should like to present two models for the enzyme molecule to explain the enhancing effects of NTP and DTT. To clarify whether the mechanism is, further experiments are now in progress.
Japan Society of Clinical Chemistryの論文

Possible Energy-Dependency of Protocollagen Proline Hydroxylase Reaction

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