Reaction mechanism of Ca2+-dependent adenosine triphosphatase of sarcoplasmic reticulum. ATP hydrolysis with CaATP as a substrate and role of divalent cation

ATP hydrolysis with CaATP as a substrate was characterized at 0 degrees C and pH 7.0 using purified ATPase preparations of sarcoplasmic reticulum and compared with that with MgATP as a substrate. The maximal rate of enzyme phosphorylation and the Km value for the phosphorylation were 8 to 10 times less for CaATP than for MgATP. Each substrate appeared to act as a competitive inhibitor with respect to the other in enzyme phosphorylation. The phosphoenzyme formed from CaATP turned over slowly because the conversion rate of the ADP-sensitive (E1P) to ADP-insensitive (E2P) phosphoenzyme was very slow. E2Ps, formed from both CaATP and MgATP, were similar in that KCl, MgCl2, or ATP accelerated their decomposition. Their sensitivity to KCl and/or ATP was retained even after a long incubation with excess EDTA. When the enzyme had been phosphorylated from CaATP, calcium remained bound to the enzyme even in the presence of excess EDTA. The observed parallelism between the amount and behavior of the enzyme-bound calcium and those of E2P strongly suggests that 1 mol of E2P has 1 mol of tightly bound calcium. During steady state ATP hydrolysis with CaATP as a substrate, a significant amount of the enzyme-ATP complex accumulated as a reaction intermediate because of slow dissociation of CaATP from the CaATP-enzyme complex and slow enzyme phosphorylation from the CaATP-enzyme complex. These results indicate that Mg2+ is not essential for the turnover of the calcium pump ATPase. It was proposed that the metal component of the substrate basically determines affinity of the substrate to the enzyme and the catalytic mechanism of subsequent reaction steps.     

Hormonal regulation of translatable mRNA of glucose-6-phosphate dehydrogenase in primary cultures of adult rat hepatocytes

The quantity of translatable mRNA of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) in primary cultures of adult rat hepatocytes subjected to different hormonal conditions was determined with a reticulocyte-lysate, cell-free system. The level of glucose-6-phosphate dehydrogenase mRNA was about 5-fold higher in the presence of insulin than in its absence. This increase of glucose-6-phosphate dehydrogenase mRNA reached a maximum 12 h after the addition of insulin. The maximum level of induction of glucose-6-phosphate dehydrogenase mRNA required 10(-8) M insulin. Glucagon and triiodothyronine had no effect on the glucose-6-phosphate dehydrogenase mRNA level. The increase of glucose-6-phosphate dehydrogenase activity correlated with the increase in level of mRNA of this enzyme. This suggests that the changes in glucose-6-phosphate dehydrogenase activity in response to the above hormonal changes are primarily due to changes in the amount of mRNA coding for this enzyme.     

Steady-state kinetics and spectral properties of Corynebacterium sarcosine oxidase

The overall reaction kinetics of Corynebacterium sarcosine oxidase were investigated and the reaction was shown to follow a ping-pong, bi-bi mechanism with two substrates, sarcosine and molecular oxygen. Sarcosine analogs, such as acetate, propionate and methoxyacetate, were competitive inhibitors of the reaction. Acetate caused characteristic alterations in optical and circular dichroic spectra, indicating that the microenvironment of the substrate-binding region of the enzyme increased in hydrophobicity on binding with the substrate analog. The dissociation constants of the analogs calculated from the spectral changes were in agreement with the kinetic inhibition constants. Inorganic metallic ions were also inhibitory. Of interest was the finding that the inhibition by Hg2+ was proportional to the square of its concentration, which suggests that at least two sulfhydryl groups are related to the catalytic activity of the enzyme.     

Mutations resistant to bromodeoxyuridine in mouse lymphoma cells selected by repeated exposure to EMS characteristics of phenotypic instability and reversion to HAT resistance by 5-azacytidine

Mutations induced by repeated EMS treatments were investigated by using mouse L5178Y cells. The frequency of TG^r mutations increased linearly with the number of EMS treatments whereas the yield of BrdUrd^r mutations showed a curvilinear dose-response curve. The BrdUrd^r frequency was roughly proportional to the square of the TG^r frequency and the results were compatible with the hypothesis that BrdUrd^r cells were induced by two mutational events within a cell. Most of the BrdUrd^r colonies isolated after 6 EMS treatments, however, were unstable. When BrdUrd^r colonies that had arisen in BrdUrd medium after 2 weeks' incubation were isolated in normal medium, the descendant cells showed a nearly normal level of thymidine incorporation and low plating efficiencies of about 1% in BrdUrd medium. In contrast, after isolation of the same colonies in BrdUrd medium, a low level of thymidine incorporation and high plating efficiencies in BrdUrd medium were observed in the descendant cells.

Reverse selection from BrdUrd^r to HAT^r was accomplished with frequencies of 10⁻⁶−10⁻³ for the descendants grown in BrdUrd medium, and AzaCyd treatment drastically increased the reversion frequency to nearly 10⁻¹. Further re-revertants from HAT^r to BrdUrd^r were also found with frequencies of 10⁻³−10⁻² without treatment. These results indicate that the initial BrdUrd^r cells did not result from inactivation of the thymidine-kinase gene but that the mode of gene expression was altered in some way.     

Isolation and characterization of vitamin A-sensitive Chinese hamster lung cell lines

Retinyl acetate (RA)-sensitive variants (RAs-2 and RAs-3) of V79 cell line were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The variants were stable and showed a 3- to 4-fold increase in sensitivity to RA compared to parental V79 cells. The RAs-2 clone was also sensitive to retinol and retinol palmitate. The RA-sensitivity behaves as a recessive trait in all hybrids of RAs-2 and V79. A number of physiological parameters were indistinguishable in V79 and RAs-2 cells, including the extent of uptake of [3H]retinol, the release of K+ from the cells induced by RA, and the levels of retinol and retinoic acid binding proteins. However, one possible correlation with the RA-sensitive phenotype was observed: Gomori acid-phosphatase staining of RA-treated RAs-2 and V79 cells indicated that lysosomal membrane of RAs-2 cells was more labile than those of the parental V79 cells.     

Tyrosine phosphorylation of specific proteins after mitogen stimulation of chicken embryo fibroblasts.

We found that stimulation of density-inhibited chicken embryo fibroblasts with serum, epidermal growth factor (EGF), platelet-derived growth factor, (PDGF), or multiplication-stimulating activity (MSA) leads to an increase in tyrosine phosphorylation of proteins in the region of Mr 40,000 (40K) to 42K. The increase in tyrosine phosphorylation after serum or EGF stimulation was transient, reaching a maximum at about 5 min and then declining. By fine-resolution analysis of proteins separated on sodium dodecyl sulfate-polyacrylamide gels, we found that after EGF stimulation, the major increase in phosphotyrosine content was in a 42K Mr protein, with a smaller increase in a 40K Mr protein. The increased phosphorylation in the 40K to 42K Mr region accounted for almost all of the increase in phosphotyrosine observed in these cells. These phosphotyrosine-containing proteins were different from the major phosphotyrosine-containing protein of Rous sarcoma virus-transformed chicken embryo fibroblasts, which migrates at an approximate Mr of 36K. Increased tyrosine phosphorylation of proteins of similar Mr was found in 3T3 cells treated with EGF, but not in NR-6 cells, which lack detectable EGF receptors. It is possible that the 40K to 42K Mr phosphotyrosine-containing proteins are involved in the integration of the biological response to a number of different growth factors.     

Termination of early gestation with vaginal prostaglandin F2α tablets

The abortifacient activity of prostaglandin F_2α was investigated by placing one or two 50 mg tablets of prostaglandin F_2α in THAM salt into the vagina of nine women less than 4 weeks pregnant at intervals of 2 to 4 hours for a 24 hour period. Serum levels of HCG, estradiol (E₂), progesterone and 17α-hydroxyprogesterone were measured by radioimmunoassay prior to starting therapy and at frequent intervals thereafter for 48 hours. All but two patients had significant side-effects, mainly diarrhea and vomiting, indicating that systemic absorption took place. Although bleeding was induced in 8 of 9 women, only 3 had complete abortions. A D&C was performed on all patients 48 hours after starting therapy. A significant fall in HCG levels was noted only in the patients who aborted. Only 3 of the 9 women had significant changes in steroid levels. A fall in progesterone and 17α-hydroxyprogesterone occurred in the 3 women who aborted and took place following the fall in HCG. Estradiol levels remained in the same range in all subjects. These findings indicate that prostaglandin F_2α when administered in this vehicle and this dosage is relatively ineffective as an abortifacient. When effective, its action would appear to be due to contractions of uterine muscle and not secondarily to luteolysis.