Degradation of RNA in Nuclei From Ehrlich Ascites Cells

1975 ◽  
Vol 53 (1) ◽  
pp. 79-90 ◽  
Author(s):  
E. Ann Speers ◽  
Richard G. von Tigerstrom
Keyword(s):  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Nuclear Fraction ◽  
Whole Cells ◽  
Cell Extracts ◽  
Ehrlich Ascites ◽  
Ehrlich Ascites Cells ◽  
Nuclear Rna ◽  
Rnase Ii ◽  
Major Acid

Nuclei were prepared from Ehrlich ascites cells in 80% yield by homogenization of the cells in an aqueous solution containing Triton N-101 and washing of the nuclear fraction by centrifugation and resuspension. Compared to the enzyme activities present in cell extracts, approximately 47% exo-RNase I, 15% alkaline RNase II, 9% acid RNase II and 7% acid phosphatase were associated with the nuclear fraction after isolation. Exo-RNase I and alkaline RNase II were rapidly lost from nuclei during incubation at 37 °C. The degradation of newly synthesized RNA in nuclei incubated at 37 °C was followed by polyacrylamide gel electrophoresis and by characterization of acid-soluble degradation products. The rate of hydrolysis of the nuclear RNA was rapid during the initial stages of incubation and then proceeded at a much reduced rate. Nucleoside 5′-phosphates were the major acid-soluble degradation products, in agreement with the presence of exo-RNase I. Although a considerable amount of alkaline RNase II was associated with the nuclear fraction, extensive endonucleolytic cleavage of the nuclear RNA was not apparent. Compared to the processing of nuclear RNA in whole cells, however, the degradation in isolated nuclei was relatively non-specific.

Multiple Molecular Forms Of Plasminogen Activator Produced By Cultured Bovine Endothelial Cells

1981 ◽  
Author(s):  
Eugene G Levin ◽  
David J Loskutoff
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Molecular Forms ◽  
Cell Extracts ◽  
Fresh Serum ◽  
Vessel Patency ◽  
Bovine Endothelial Cells ◽  
Overlay Technique

The production of plasminogen activator (PA) by the vascular endothelium has been implicated in the maintenance of vessel patency. Cultured bovine aortic endothelial (BAE) cells were employed to study and compare the cell associated and secreted forms of PA. Samples were fractionated by polyacrylamide gel electrophoresis in the presence of SDS. PA activity in the gel was localized by the fibrin-overlay technique. Cellular PAs were found to be membrane associated and to consist of a major form of Mr 48,000 (C48) and minor forms of 53,000 (C53), 74,000 (C74), and 100,000 (C100). Incubation of the cell extracts at 37°C resulted in the appearance of two additional forms of Mr 41,000 and 33,000 suggesting that these forms were degradation products. Serum-free conditioned medium (CM) contained secreted PAs of Mr 52,000 (S52), 55,000 (S55), 74,000 (S74) and 100,000 (S100). In addition, a broad zone of fibrinolytic activity was observed in the region between Mr 80,000 and 95,000. Cellular PAs have isoelectric points of pH 8.5-8.6 and 7.5 while secreted PAs demonstrate activity at pH 8.6, 8.5, 8.0, and 7.5. The forms showed differential sensitivities to DFP with S74 and C74 being inactivated by 1mM DFP within one Hr at 37°C while S52 and C48 were still partially active after treatment with 10mM for two Hrs. S100 was completely retractile to treatment with 40 mM DFP. The addition of fresh serum to confluent cultures resulted in the disappearance of C48 and C74, and of S52 and S74, but caused an increase in C100 and S100. These studies indicate that several forms of PA are produced by endothelial cells and suggest that the production of each may be independently regulated.

scholarly journals The role of actin in temperature-dependent gel-sol transformation of extracts of Ehrlich ascites tumor cells.

1979 ◽  
Vol 80 (2) ◽  
pp. 465-480 ◽  
Author(s):  
M Ishiura ◽  
Y Okada
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Dnase I ◽  
Ehrlich Ascites Tumor ◽  
Muscle Actin ◽  
Ascites Tumor ◽  
Temperature Dependent ◽  
Cell Extracts ◽  
Ehrlich Ascites

Ehrlich ascites tumor cell extracts form a gel when warmed to 25 degrees C at pH 7.0 in sucrose solution, and the gel rapidly becomes a sol when cooled to 0 degrees C. This gel-sol transformation was studied quantitatively by determining the volume or the total protein of pellets of gel obtained by low-speed centrifugation. The gelation depended on nucleotide triphosphates, Mg2+, KCl, and a reducing agent. Gelation was inhibited reversibly by 0.5 microM free Ca2+, and 25--50 ng/ml of either cytochalasin B or D, but it was not affected by 10 mM colchicine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the gel was composed of six major proteins with mol wt greater than 300,000, 270,000, 89,000, 51,000, 48,000, and 42,000 daltons. The last component was identified as cell actin because it had the same molecular weight as muscle actin and bound with muscle myosin and tropomyosin. The role of actin in gelation was studied by use of actin-inhibitors. Gelation was inhibited by a chemically modified subfragment-1 of myosin, which binds with F-actin even in the presence of ATP, and by bovine pancreatic DNase I, which tightly binds with G-actin. Muscle G-actin neutralized the inhibitory effect of DNase I when added at an equimolar ratio to the latter, and it also restored gelation after its inhibition by DNase I. These findings suggest that gelation depends on actin. However, the extracts showed temperature-dependent, cytochalasin-sensitive, and Ca2+-regulated gelation as did the original extracts when the cell actin in the extracts was replaced by muscle actin, suggesting that components other than cell actin might be responsible for these characteristics of the gelation.

Changes in Ribonucleic Acid Degrading Enzymes in Ehrlich Ascites Cells During Growth and After Actinomycin D Treatment

1972 ◽  
Vol 50 (3) ◽  
pp. 244-252 ◽  
Author(s):  
Richard G. von Tigerstrom
Keyword(s):  
Acid Phosphatase ◽  
Tumor Cells ◽  
Enzyme Activities ◽  
Actinomycin D ◽  
Inhibitor Activity ◽  
Rnase Inhibitor ◽  
Ehrlich Ascites ◽  
Untreated Tumor ◽  
Ehrlich Ascites Cells ◽  
Rnase Ii

Ehrlich ascites cells were treated with actinomycin D before transplantation. These cells, collected after 7 days, show increased alkaline RNase II, acid RNase II, and phosphodiesterase II activities and low RNase inhibitor activity as compared to untreated tumor cells collected 7 days after transplantation. RNase I, phosphodiesterase I, and acid phosphatase activities were unchanged. Smaller increases in the same enzyme activities, but no change in RNase inhibitor activity, were observed when untreated cells collected after 4 days were compared to untreated cells collected after 7 days of growth. Ehrlich ascites cells collected after 4 days synthesized protein and RNA at a slightly faster rate than those collected after 7 days. Actinomycin D treated cells synthesized protein at a rate identical to that of the control cells; net synthesis of RNA, however, was significantly reduced. One possible reason for this may be the higher RNase and phosphodiesterase activities in actinomycin D treated cells.

scholarly journals Hydrolysis of Pork Muscle Sarcoplasmic Proteins byLactobacillus curvatus and Lactobacillus sake

1999 ◽  
Vol 65 (2) ◽  
pp. 578-584 ◽  
Author(s):  
Silvina Fadda ◽  
Yolanda Sanz ◽  
Graciela Vignolo ◽  
M.-Concepción Aristoy ◽  
Guillermo Oliver ◽  
...  
Keyword(s):  
Amino Acids ◽  
Free Amino Acids ◽  
Free Amino ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Lactobacillus Curvatus ◽  
Whole Cells ◽  
Sarcoplasmic Proteins ◽  
Lactobacillus Sake ◽  
Cell Extracts

ABSTRACT Lactobacillus curvatus CECT 904 and Lactobacillus sake CECT 4808 were selected on the basis of their proteolytic activities against synthetic substrates. Further, the effects of whole cells, cell extracts, and a combination of both enzymatic sources on muscle sarcoplasmic proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography analyses. Strains of both species displayed proteinase activities on five sarcoplasmic proteins. The inoculation of whole cells caused a degradation of peptides, whereas the addition of cell extracts resulted in the generation of both hydrophilic and hydrophobic peptides. This phenomenon was remarkably more pronounced when L. curvatus was involved. Whole cells also consumed a great amount of free amino acids, while the addition of intracellular enzymes contributed to their generation.L. sake accounted for a greater release of free amino acids. In general, cell viability and also proteolytic events were promoted when cell suspensions were provided with cell extracts as an extra source of enzymes.

Heterogeneity of alkaline ribonuclease in the mouse and Ehrlich ascites cells

1977 ◽  
Vol 55 (11) ◽  
pp. 1171-1179 ◽  
Author(s):  
Richard G. von Tigerstrom ◽  
Janet M. Manchak
Keyword(s):  
Culture Medium ◽  
Specific Activity ◽  
Divalent Metal Ions ◽  
Heat Treated ◽  
Ehrlich Ascites ◽  
Ehrlich Ascites Cells ◽  
Alkaline Ribonuclease ◽  
Rnase Ii

The specific activity of alkaline RNase II was 600 to 1800 times higher in mouse pancreas than in mouse liver, serum, ascites fluid, and Ehrlich ascites cells grown intraperitoneally. Ehrlich ascites cells grown in cell culture medium had a much lower alkaline RNase II activity than cells grown intraperitoneally. Chromatography on CM-52 cellulose of acid- and heat-treated preparations showed a considerable heterogeneity of the mouse enzymes. Depending on the source of the extract, two to six forms of alkaline RNase were eluted. Pancreatic extract contained two RNase forms. These also seemed to be present as minor components in preparations from other sources except Ehrlich ascites cells grown in vitro. Ehrlich ascites cells grown in vivo contained forms of the RNase which were not present in other extracts. Possible reasons for this heterogeneity were investigated. In addition to their stability to acid and heat the different RNase forms were similar in that they were much more active at alkaline pH than at acidic pH, they did not require divalent metal ions for activity, and they degraded RNA 'endonucleolytically.' Also, native DNA, denatured DNA, and poly A were poor substrates compared with RNA. Some differences seemed to exist, however, with respect to their abilities to degrade poly U and poly C and their sensitivities to the endogenous RNase inhibitor.

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