It has been considered for some time that steroid hormones may exert their regulatory effects by influencing certain enzymes.' However, our present knowledge of the mechanisms of steroid action upon enzymes is limited. Estrogens have been shown to stimulate the transfer of hydrogen between di-and triphosphopyridine nucleotides,2 ' although there is disagreement as to the role of steroids in this reaction.4' Recently, steroids were found to inhibit reduced diphosphopyridine nucleotide oxidation by enzyme preparations from mammalian and microbial sources.6 I This action appears to be a general property shared by a large number of steroids.7This study demonstrates a highly specific inhibitory effect on mammalian glucose-6-phosphate dehydrogenase (G-6-PD) of very low concentrations of dehydroisoandrosterone, pregnenolone, and certain related steroids. These steroids are not inhibitors of yeast or spinach G-6-PD or of mammalian 6-phosphogluconic dehydrogenase or isocitric dehydrogenase.Materials and Methods.-Preparation of enzymes: G-6-PD was purified from human erythrocytes by a procedure described in detail elsewhere' involving ammonium sulfate fractionation followed by absorption on calcium phosphate gel, elution in a phosphate buffer, and a second series of ammonium sulfate precipitations. The final product generally had a specific activity 500-fold that of the crude hemolysate in a yield of about 40 per cent. This preparation was essentially free of hemoglobin and 6-phosphogluconic dehydrogenase activity.Crude enzyme preparations were obtained from human and rat tissues and from spinach by homogenizing freshly obtained material in isotonic potassium chloride buffered at pH 7.4 (10%, weight/volume). The homogenate was centrifuged 30 minutes at 18,000 X g to sediment debris. The supernatant fluid was employed for assays of enzyme activity.Partially purified yeast G-6-IPD and pig heart isocitric dehydrogenase were obtained from Sigma Chemical Corporation. IPartially purified 6-phosphogluconic dehydrogenase was prepared from rat liver by the method of Glock and AIcLean.9 Enzyme assays: Purified preparations of G-6-PD were assayed by the method of Kornberg and Horecker.10 In measuring G-6-PD activity in crude tissue preparations, due to the presence of 6-phosphogluconic dehydrogenase, the rate of TPNH formation exceeds the rate of glucose-6-phosphate oxidation. Accordingly, in crude preparations, G-6-PD activity was assayed by the method of Kornberg andHorecker"°determining the initial rate of TPNH formation by measuring the change of absorption at 340 m,4 with an automatic recording Cary spectrophotometer or, alternatively, by the method of Glock and AMcLean.9 In this latter method, G-6-PD activity is taken as the difference in the rate of TP'NH reduction when the reaction mixture contains both 6-phosphogluconate plus glucose-6-phosphate and in the presence of 6-phosphogluconate alone. Comparable results with 447

There are a number ofagents which, when added to cultures of murine erythroleukemia cells (MELC), markedly increase the probability of commitment to express the characteristics of terminal erythroid differentiation, including loss of proliferative capacity and increased accumulation of globin mRNA and hemoglobin. Some characteristics of inducer-mediated commitment of MELC to terminal erythroid differentiation were examined by determining the effects of dexamethasone (an inhibitor of inducer-mediated MELC differentiation) and of hemin (an inducer of globin mRNA accumulation). Previously, it was shown that exposure of MELC to hexamethylene-bisacetamide (HMBA) leads to commitment, detectable within 12 hr. MELC cultured with both HMBA and dexamethasone do not express commitment. MELC transferred from culture with HMBA and dexamethasone to cloning medium without these agents express commitment to terminal erythroid differentiation, indicating that MELC retain a "memory" for some early HMBA-mediated changes leading to commitment which occur even in the presence of the inhibitory steroid. The kinetics of commitment in experiments in which exposure to HMBA is interrupted, or dexamethasone is added to the culture with HMBA, suggest that there is a rate-limiting step early in the commitment process. The memory for this step persists for more than one cell cycle. Addition ofhemin to cultures with HMBA and dexamethasone initiates accumulation of globin mRNA but does not reverse the steroid-mediated inhibition of terminal cell division (that is, the cells retain their proliferative capacity). Inducer-mediated MELC commitment is associated with accumu-

Murine erythroleukemia cells (MELC) are virustransformed erythroid precursors that appear to be blocked at an erythroid precursor stage comparable to the erythroid colonyforming unit (CFU-e). These cells are useful in examining factors regulating terminal differentiation. Induced MELC are characterized by a coordinated program of gene expression, including commitment to terminal cell division, accumulation of globin mRNAs and corresponding hemoglobins, and accumulation of several other proteins, including the chromatin-associated protein H10. Two cloned variant cell lines, DR1O and RI, have been developed from inducer-sensitive DS19 cells by selection for inducer resistance.DRlO and RI cells fail to display commitment to terminal cell division when cultured with dimethyl sulfoxide (Me2SO), hexamethylene bisacetamide (HMBA), or butyric acid. Both cell lines are induced by all three agents to accumulate Hi1. DR1O cells are resistant to Me2SO-mediated accumulation of hemoglobin but are sensitive to HMBA-or butyric acid-mediated accumulation. RI cells are resistant to Me2SO-and HMBA-mediated accumulation of hemoglobin but are sensitive to butyric acid-mediated accumulation. Both DR1O and RI are commitment-negative MELC variants, displaying variable responses to inducers with respect to other features of terminal erythroid cell differentiation.Murine erythroleukemia cells (MELC) are virus-transformed erythroid cell precursors, approximating the erythroid colonyforming unit (CFU-e), that may be induced by dimethyl sulfoxide (Me2SO) (1), hexamethylene bisacetamide (HMBA) (2), and a variety of other agents to express characteristics of terminal erythroid cell differentiation (3). Induced differentiation of MELC line DSl9, a derivative of line 745A (2, 3), is characterized by the coordinated expression of commitment to terminal cell division (4, 5); accumulation of newly synthesized aand ,3globin mRNAs (6) and a-, 3mai-, and prminn-globins, hemoglobins major and minor (7,8), and erythrocyte surface proteins such as spectrin (9); augmentation of enzyme activities of the heme synthetic pathway (10); and increase in the level of H10, a chromatin-associated protein (11,12).Recently, we have shown that both inducer-mediated commitment to terminal cell division and increased globin gene expression involve multistep processes (12, 13). For example, in uninduced MELC there are alterations in the chromatin of the a,-and 8`mai-globin gene domains that, presumably, occurred at prior stage in the differentiation of these cells and are stably propagated. With exposure to HMBA or Me2SO, other alterations in chromatin structure occur at sites corresponding to the 5' flanking sequence of both the a,-and r4-globin genes (13)(14)(15). Whether these changes in chromatin reflect a nuclear site of action of HMBA or Me2SO or are secondary to inducermediated effects on other cellular functions is not established (16)(17)(18).

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The APS Journal Legacy Content is the corpus of 100 years of historical scientific research from the American Physiological Society research journals. This package goes back to the first issue of each of the APS journals including the American Journal of Physiology, first published in 1898. The full text scanned images of the printed pages are easily searchable. Downloads quickly in PDF format.

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