Further Investigations on the p-Nitrophenylphosphatase Activity of Intact Ehrlich Ascites Tumor Cells

1978 ◽  
Vol 33 (3-4) ◽  
pp. 227-230 ◽  
Author(s):  
Reiner Merz ◽  
Monika Löffler ◽  
Friedhelm Schneider
Keyword(s):  
Tumor Cells ◽  
Active Sites ◽  
Ehrlich Ascites Tumor ◽  
Ehrlich Ascites Tumor Cells ◽  
Ascites Tumor ◽  
Intact Cells ◽  
Blocking Agents ◽  
Ehrlich Ascites ◽  
Eat Cells ◽  
Hydrolysis Of

The substrate specificity and the effects of nucleotides and SH-blocking agents on the p-nitro- phenylphosphatase activity of intact Ehrlich ascites tumor cells (EAT) cells were studied, ᴅʟ-β- Glycerophosphate, o-phosphoethanolamine, cholinephosphate, glucose-6-phosphate, o-carboxyphenyl- phosphate,, phosphoenolpyruvate and AMP were not attacked by intact cells. ATP > GTP > UTP > PPi > pNPP were cleaved with decreasing velocity. A stimulation of the cleavage of p-NPP by the following nucleotides was observed with decreasing effectivity: ATP > ADP > GTP > UTP; AMP was ineffective. The phosphatase activity was not affected by malate, tartrate and glutathion disulfide. The SH blocking agents diamide and thimerosal were more effective in­hibitors of the pNPPase than of the ATPase activity, whereas the hydrolysis of ATP is more affected by the ATP analog adenylylimidodiphosphate. The present data are best compatible with a double headed enzyme: Both active sites interact with ATP, only one is active against p-NPP and sensitive against SH-blocking agents.

Shrinkage insensitivity of NKCC1 in myosin II-depleted cytoplasts from Ehrlich ascites tumor cells

2007 ◽  
Vol 292 (5) ◽  
pp. C1854-C1866 ◽  
Author(s):  
Else K. Hoffmann ◽  
Stine F. Pedersen
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Tumor Cells ◽  
P38 Mapk ◽  
Myosin Ii ◽  
Ehrlich Ascites Tumor ◽  
Ehrlich Ascites Tumor Cells ◽  
Ascites Tumor ◽  
Intact Cells ◽  
Ehrlich Ascites

Protein phosphorylation/dephosphorylation and cytoskeletal reorganization regulate the Na+-K+-2Cl− cotransporter (NKCC1) during osmotic shrinkage; however, the mechanisms involved are unclear. We show that in cytoplasts, plasma membrane vesicles detached from Ehrlich ascites tumor cells (EATC) by cytochalasin treatment, NKCC1 activity evaluated as bumetanide-sensitive 86Rb influx was increased compared with the basal level in intact cells yet could not be further increased by osmotic shrinkage. Accordingly, cytoplasts exhibited no regulatory volume increase after shrinkage. In cytoplasts, cortical F-actin organization was disrupted, and myosin II, which in shrunken EATC translocates to the cortical region, was absent. Moreover, NKCC1 activity was essentially insensitive to the myosin light chain kinase (MLCK) inhibitor ML-7, a potent blocker of shrinkage-induced NKCC1 activity in intact EATC. Cytoplast NKCC1 activity was potentiated by the Ser/Thr protein phosphatase inhibitor calyculin A, partially inhibited by the protein kinase A inhibitor H89, and blocked by the broad protein kinase inhibitor staurosporine. Cytoplasts exhibited increased protein levels of NKCC1, Ste20-related proline- and alanine-rich kinase (SPAK), and oxidative stress response kinase 1, yet they lacked the shrinkage-induced plasma membrane translocation of SPAK observed in intact cells. The basal phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was increased in cytoplasts compared with intact cells, yet in contrast to the substantial activation in shrunken intact cells, p38 MAPK could not be further activated by shrinkage of the cytoplasts. Together these findings indicate that shrinkage activation of NKCC1 in EATC is dependent on the cortical F-actin network, myosin II, and MLCK.

Multiple Effects of Amobarbital on Ehrlich Ascites Tumor Cells. Inhibition of Pyruvate Dehydrogenase

1977 ◽  
Vol 32 (11-12) ◽  
pp. 944-948 ◽  
Author(s):  
Stefan Postius ◽  
Friedhelm Schneider
Keyword(s):  
Tumor Cells ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Lipid Fraction ◽  
Energy Charge ◽  
Ehrlich Ascites Tumor ◽  
Ehrlich Ascites Tumor Cells ◽  
Ascites Tumor ◽  
Intact Cells ◽  
Ehrlich Ascites

Abstract The inhibition of the proliferation of hyperdiploid Ehrlich ascites tumor cells in suspension cultures by amobarbital is coupled to an increased glycolytic activity as shown by lactic acid pro­ duction and glucose consumption; higher concentrations of amobarbital than 1 mᴍ enhance the ATP/ADP ratio of the total cell. The actual activity of pyruvate dehydrogenase of intact cells is completely inhibited in the presence of 2 mᴍ amobarbital as was shown by the 14CO2 evolution from [1-14C] pyruvate or the incorporation of 14CO2 into the total lipid fraction of the cells from [U-14C] pyruvate or from [U-14C] lactate. The pyruvate dehydrogenase complex from Ehrlich ascites tumor cells is completely inhibited by 1 mᴍ amobarbital in vitro. The activity of α-oxoglutarate dehydrogenase is inhibited by amobarbital, too, as was shown by measuring the 14CO2 evolution from [1-14C] glutamate with intact cells. It is suggested that the inhibition of pyruvate dehydrogenase in the presence of amobarbital is the result of a direct action on the enzyme as well as the consequence of a change in the cellular redox state or its energy charge.

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