James F. Richards

Ornithine decarboxylase activity in tissues of prolactin-treated rats.

Summary Treatment with prolactin caused increases of 3–100 fold in ornithine decarboxylase (EC 4.1.1.17) activity of kidney, adrenal gland and liver of female rats. The magnitude of the effect varied with age. In young male rats, activity of the enzyme was also stimulated markedly in spleen, as well as those tissues mentioned above, and there were smaller effects in thymus and heart.

Biochemical response of lymphoma cells to mitogenic stimulation by prolactin

A previous study showed that cultured Nb 2 node rat lymphoma cells stopped replicating when transferred to medium supplemented with horse serum instead of fetal calf serum; resumption of growth could be induced by the addition of prolactin or other lactogens. The present study shows that in the absence of prolactin cells accumulated early in the G1 phase from which, on stimulation by the hormone, they proceeded through the cell cycle in a synchronized fashion. Ornithine decarboxylase and S-adenosyl methionine decarboxylase activities were closely related to the proliferative status of the cells. In stationary cultures the enzyme activity was barely detectable; following the addition of prolactin, the levels increased over 100-fold and displayed well-defined changes as the cells proceeded through the cell cycle. The results suggest the lymphoma cells are very useful for studying biochemical events resulting from the interaction of a mitogenic polypeptide hormone and its target cell.

Ornithine decarboxylase and thymidine kinase activity in tissues of prolactin-treated rats: Effect of hypophysectomy☆

Abstract The activities of ornithine decarboxylase and thymidine kinase were determined in tissues of young intact and hypophysectomized rats at various times after treatment with prolactin. In both types of animals, ornithine decarboxylase activity increased in liver, kidney, spleen and adrenal of prolactin treated rats. Thymidine kinase activity increased only in liver and spleen of intact rats. Increase in the kinase activity was smaller, and occurred later than the change in ornithine decarboxylase. In hypophysectomized animals, thymidine kinase activity increased in spleen, but not in liver, following prolactin treatment.

Ornithine decarboxylase activity in lymphoid tissues of rats: effects of glucocorticoids.

Abstract The activity of ornithine decarboxylase was measured in several tissues of young female rats after treatment with cortisone acetate or dexamethasone. The expected increase in activity observed in liver and kidney was in marked contrast to the profound decrease found in thymus and spleen. Initially high activity in thymus was decreased to very low levels, sometimes below the limit of the assay procedure, 5 hours after treatment with dexamethasone or cortisone. There was also a large decrease in activity of the enzyme in spleen of hormone-treated rats. In both tissues, the marked effect was still evident 12 hours after treatment.

Multiple species of ornithine decarboxylase in rat tissues: Effects of dexamethasone

Abstract Several species of ornithine decarboxylase were separated by chromatography of rat thymus and kidney extracts on DEAE-Sepharose CL-6B. One major and one minor species were absent from thymus of rats two hours after hormone treatment but otherwise, the elution profile was identical to thymus from control animals. The elution patterns of ODC activity in kidneys of rats treated 2.5 or 5 hours before sacrifice with dexamethasone differ from that of control kidney and from each other. Enzyme from kidneys early after hormone treatment is eluted earlier than enzyme from control tissue, while at 5 hours, the enzyme is eluted much later than in the control. This suggests that the hormone-induced activity is subsequently modified.

Multiple ionic forms of ornithine decarboxylase differ in degree of phosphorylation

Two major ionic forms of ornithine decarboxylase were separated by column chromatography of extracts of kidneys from androgen-treated male CD-1 mice on DEAE-Sepharose CL-6B, and purified individually to apparent homogeneity. On SDS-PAGE, a single major protein band of Mr 50000 was present in each. When incubated with casein kinase II, purified from rat liver cytosol, only one form of the enzyme, which represented 20% of the total ornithine decarboxylase in the tissue, became phosphorylated. The major form, which was eluted later from the column, could be phosphorylated only after treatment with alkaline phosphatase, indicating that the phosphatase removed enzyme-bound phosphate already attached at the casein kinase II phosphorylation site. Evidence for the occurrence of a phosphorylated form of the enzyme in kidneys of dexamethasone-treated rats is also presented.

Inhibition of hormone-stimulated ornithine decarboxylase activity by lithium chloride

Effects of Li+ on hormone-stimulated ornithine decarboxylase (ODC) activity were determined in kidney and liver of rats treated with dexamethasone or prolactin (PRL) and also in cultured, PRL-stimulated Nb2 lymphoma cells. In both systems, LiCl led to rapid and marked decreases in ODC activity. The inhibitory effect of Li+ in exponentially growing Nb2 lymphoma cell cultures, measured at 45 min, was dose-dependent, ranging from 10% at 0.1 mM LiCl to 95% at 10 mM LiCl. Surprisingly, on continued incubation with 10 mM LiCl, the lymphoma cells partially overcame the inhibition, showing ODC activities which reached a maximal value of ca 50% of the control at 4.5 h. The inhibition by Li+ could not be reduced by adding myo-inositol to the culture medium. LiCl did not inhibit ODC activity when added to cell-free extracts of rat tissues and Nb2 lymphoma cells indicating it did not act directly on the enzyme; however, there is evidence that, in intact cells, Li+ enhances the rate of inactivation of the enzyme.

The effect of growth hormone on DNA synthesis in rat spleen

Abstract The synthesis of DNA in the spleen of immature rats was increased following the administration of growth hormone. The activity of both the thymidine kinase and DNA polymerase enzymes was also increased by this treatment. The rate of DNA synthesis and the specific activity of the polymerase were increased by a factor of 4–5, while the thymidine kinase activity was increased by a factor of 25–30.

Changes in antizyme-ornithine decarboxylase complexes in tissues of hormone-treated rats

The presence of antizyme-ornithine decarboxylase complex in thymus and kidney of rats was demonstrated using the method of Y Murakami et al. [(1985) Biochem. J. 225, 689-697]. A very small amount of complex was found in kidney of control rats, accounting for only 1-3% of total enzyme in the tissue, while in thymus, approximately one-third of the total ornithine decarboxylase in thymus occurred as an antizyme-enzyme complex. After treatment with dexamethasone, both free ornithine decarboxylase and antizyme-ornithine decarboxylase decreased in thymus, the free enzyme activity decreasing more rapidly. In kidney, the concentration of the antizyme-ornithine decarboxylase complex increased after dexamethasone treatment, but only after the induction of free enzyme activity had reached its peak and begun to decrease. The pattern of the changes in amount of antizyme-ornithine decarboxylase complex after prolactin treatment differed from those observed in the dexamethasone-treated animals. In both kidney and thymus, the concentration of antizyme-ornithine decarboxylase complex increased concurrently with the induction of free enzyme activity. Both free and complexed ornithine decarboxylase had increased at 2.5 h after prolactin treatment and continued to increase to maximum specific activities at similar rates. In thymus, the amount of ornithine decarboxylase present as a complex reached 70% of the total in the tissue. In both thymus and kidney, the concentration of antizyme-ornithine decarboxylase complex decreased more slowly than did free enzyme activity. Free antizyme was observed only in thymus of dexamethasone-treated animals. The amount of measurable inhibitor was decreased if cycloheximide was given with dexamethasone.

An inhibitor of ornithine decarboxylase in lactogen-deprived Nb2 node rat lymphoma cells

A previous study has shown that the activity of ornithine decarboxylase in cultured Nb2 node rat lymphoma cells falls to undetectable levels when cells become quiescent following incubation in lactogen (prolactin)-deficient medium. In the present study, it was found that addition of extracts of the lactogen-deprived, quiescent cells to extracts of log-phase cells markedly reduced the ornithine decarboxylase activity of the latter, the inhibitory activity being proportional to the amount of quiescent cell extract added. Evidence is presented that the ornithine decarboxylase-inhibitory activity in the quiescent cell extracts is due to an antizyme-like, polypeptide factor with an Mr of approx. 28,000. The activity of the inhibitor appears to be directed rather specifically against ornithine decarboxylase, since the activities of S-adenosylmethionine decarboxylase, thymidine kinase and uridine kinase were not affected. The Nb2 cell ornithine decarboxylase inhibitor may have an important role in modulating the cellular levels of ornithine decarboxylase as they change in response to the withdrawal and restoration of extracellular mitogenic lactogens.