The Partial Purification and Properties of Uridine Kinase from Ehrlich Ascites Tumor Cells

1973 ◽  
Vol 51 (4) ◽  
pp. 379-389 ◽  
Author(s):  
Gerald Krystal ◽  
Peter G. Scholefield
Keyword(s):  
Molecular Weight ◽  
Tumor Cells ◽  
Weight Fraction ◽  
Ehrlich Ascites Tumor ◽  
Partial Purification ◽  
Heat Inactivation ◽  
Ehrlich Ascites Tumor Cells ◽  
Ascites Tumor ◽  
Uridine Kinase ◽  
Ehrlich Ascites

In the partial purification of uridine kinase (ATP:uridine 5′-phosphotransferase, EC 2.7.1.48) from Ehrlich ascites tumor cells, two active fractions were obtained by chromatography on Sepharose 6B. Their molecular weights, as determined by a gel filtration and sucrose density centrifugation, were approximately 120 000 and 30 000. The larger molecular weight species was less stable on incubation at 60° and slightly less sensitive to CTP inhibition than the lower molecular weight fraction. Both fractions were stabilized against heat inactivation by ATP and CTP.The two fractions displayed similar apparent Km values for uridine and ATP, similar specificities for substrates and inhibitors, and similar divalent cation requirements.In the partial purification of uridine kinase from mouse intestine, only the larger molecular weight fraction was obtained by chromatography on Sepharose 6B.In addition, some preliminary evidence for a monomer–polymer relationship between the two molecular weight species is presented.

scholarly journals Effects of fixation and postfixation treatments on volume of injured cells;.

1975 ◽  
Vol 23 (4) ◽  
pp. 251-270 ◽  
Author(s):  
A Penttila ◽  
E M McDowell ◽  
B F Trump
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Tumor Cells ◽  
Phosphate Buffer ◽  
Sulfonic Acid ◽  
Osmium Tetroxide ◽  
Ehrlich Ascites Tumor ◽  
Ehrlich Ascites Tumor Cells ◽  
Ascites Tumor ◽  
Ehrlich Ascites

The effects of fixation with glutaraldehyde (GA), formaldehyde (FA), glutaraldehyde-formaldehyde (GA-FA), flutaraldehyde-osmium tetroxide (GA-Os04) and osmium tetroxide (OsO4) on cel volume were studied in control, p-chloromercuribenzene sulfonic acid (PCMBS)-treated and hypotonically-treated Ehrlich ascites tumor cells. Among the variables investigated were concentration of the tixative agent, osmolity of the buffer, total osmolaity of the fixation solution, osmolaltity of the postfixation buffer and the time of fixation and postfixation treatment; in addition, the effects of adding calcium and high molecular weight compounds to the fixative solution were studied. When the effects of standard fixatives on control, PCMBS- and hypotonically-treated cells were compared, marked differences were apparent in the behavior of control and injured cells. Control cells retained near prefixation volume in 3% GA and 3% GA-1% OsO4, swelled in 4% FA or 1% OsO4 and phosphate buffer (tkrp), whereas tpcmbs (310 mosM KRP)- and hypotonically-treated cells (103 mos M KRP) shrank in all aldehyde fixatives but swelled in 1% OsO4. Reducing the buffer osmolality had similar effects on control and injured cells although, there were variations in degree...

Partial Purification and Characterization of RNA Polymerase from Nuclei of Ehrlich Ascites Tumor Cells

1973 ◽  
Vol 51 (5) ◽  
pp. 586-596 ◽  
Author(s):  
D. G. R. Blair ◽  
A. K. Mukherjee
Keyword(s):  
Rna Polymerase ◽  
Tumor Cells ◽  
Reaction Medium ◽  
Ehrlich Ascites Tumor ◽  
Partial Purification ◽  
Ehrlich Ascites Tumor Cells ◽  
Ascites Tumor ◽  
Absolute Requirement ◽  
Ehrlich Ascites ◽  
Calf Thymus

DNA-dependent RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase) was partially purified from nuclei of Ehrlich ascites tumor cells by the techniques of sonic disruption in the presence of 0.3 M ammonium sulfate, ultracentrifugation, salt fractionation, and dialysis. The purified enzyme had an absolute requirement for DNA and was inhibited by actinomycin D and other known inhibitors of DNA-dependent RNA polymerase, but not by rifamycin or rifampicin. The four ribonucleoside triphosphates and Mn2+ ions were also required for optimal activity. Optimal concentrations for Mn2+ and Mg2+ ions and (NH4)2SO4 were 2.4 mM, 6.0 mM, and 0.05 M, respectively. All the native and heat-denatured DNA's that were tested served as templates, but the relative activities with the two forms of DNA depended upon the composition of the reaction medium. Of the natural RNA's and 11 synthetic homo- and heteropolyribonucleotides that were tested, the RNA's, poly(A), poly(A,U), and poly(A,C) were slightly active, but less than 5% as efficient as calf thymus native DNA, with 3H-UTP as the labeled substrate.

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