Properties and characterization of a highly purified sarcoplasmic reticulum Ca2+-ATPase from dog cardiac and rabbit skeletal muscle

Sarcoplasmic reticulum (SR) Ca2+-ATPase was purified from dog cardiac and rabbit skeletal muscle using Triton X-100 at optimal ratios of 0.5 for cardiac and 0.5 to 1.0 for skeletal SR. The yields of Ca2+-ATPase were 4 to 5 and 1 to 2.2 mg/100 mg of cardiac and skeletal SR protein, respectively. The enzyme activities were 547 +/- 67 mumol ADP/mg/h for cardiac and 1192 +/- 172 mumol ADP/mg/h for skeletal Ca2+-ATPase. Removal of excess Triton X-100 increased the enzyme activities to 719 +/- 70 and 1473 +/- 206 mumol ADP/mg/h, respectively. The residual content of Triton X-100 for cardiac and skeletal Ca2+-ATPase was 20 and 5 mol/mol of enzyme, respectively. Maximum levels of phosphoenzyme were 4.4 +/- 0.2 and 5.6 +/- 0.6 nmol/mg in each case. A single protein band of 100 kDa was obtained for each purified Ca2+-ATPase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The preparations were stable at -80 degrees C for 5 months in the presence of 1 mM Ca2+. The phospholipid content of the purified enzyme was 2-fold greater than that of native cardiac and skeletal SR microsomes. Repeated washing of the purified enzyme preparation did not alter the phospholipid content or the specific activities.     

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.     

Whole body autoradiographic distribution of exogenously administered renin in mice

The distribution of exogenously administered renin was investigated using whole body autoradiography. Purified renin from mouse submaxillary gland (SR) was labeled with radioactive iodine (125I). This labeled renin (125I-SR) and Na125I were administered into the tail vein of male ddY mice, in doses of 10.2 and 16.4 mu Ci/30 g body weight, respectively. Mice were killed by an overdose of ether, and autoradiography was performed on whole body sections. To separate free 125I liberated from 125I-SR, sections were treated with perchloric acid. A major accumulation of 125I-SR, acid-insoluble, was evident in the renal cortex, whereas the hepatic accumulation of 125I-SR was minor. Radioactivity in the thyroid and submaxillary glands, in the stomach, and in urine was also apparent, but disappeared after acid treatment, except in the thyroid glands. Radioactivity in the brain, intestinal content, spleen, and adrenal glands was nil. These autoradiograms provide the first evidence that exogenously administered renin is mainly distributed in the renal cortex.     

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.     

Evidence for the presence and structure of asparagine-linked oligosaccharide units in the core protein of proteodermatan sulphate.

Hormonally produced changes in the synthesis and secretion of the serum copper-containing protein caeruloplasmin were studied in primary cultures of rat liver parenchymal cells isolated by the collagenase-perfusion technique. A rabbit antibody directed against rat caeruloplasmin was used to immunoprecipitate labelled caeruloplasmin. Isolated liver cells synthesized and secreted caeruloplasmin over a period of 3 days. Synthesis and secretion of this protein was enhanced when cells were treated with dexamethasone. The accumulation of copper was also moderately enhanced with glucocorticoid treatment. Inclusion of adrenaline in the culture medium resulted in elevated incorporation of copper into newly synthesized caeruloplasmin as well as an increase in 64Cu-labelled caeruloplasmin in the culture medium. However, adrenaline did not seem to increase the secretion of 3H-labelled protein, despite the elevation in secreted 64Cu-caeruloplasmin. This may be due to a large increase in the intracellular pool of 64Cu caused by enhanced accumulation of this metal when adrenaline is included in the incubation medium. Enhanced copper accumulation was also seen when cells were treated with glucagon. Adrenaline-stimulated accumulation of 64Cu could be inhibited by including phenoxybenzamine, an alpha-adrenergic blocker, in the culture medium. Elevation of extracellular copper caused enhancement in the detection of labelled caeruloplasmin in the medium of cultured cells, probably owing to the ability of this metal to stabilize the protein.     

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.