A tyrosine-specific protein kinase from Ehrlich ascites tumor cells

A protein tyrosine kinase that phosphorylates both alpha and beta subunits of inactivated (Na+,K+)-ATPase from dog kidney was purified about 500-fold from Ehrlich ascites tumor cell membranes. The enzyme required divalent cations Mn2+, Mg2+, or Fe2+ but was inhibited by Cu2+ or Zn2+. The purified enzyme phosphorylated the beta subunit about five times faster than the alpha subunit of the (Na+,K+)-ATPase. The random polymer poly(Glu80Tyr20) was an excellent substrate while casein was only marginally phosphorylated. In contrast, the purified transforming gene product of Rous sarcoma virus phosphorylated all three substrates and the (Na+,K+)-ATPase was preferentially phosphorylated on the alpha subunit. The transforming gene product of Fujinami sarcoma visue and EGF receptor kinase from A431 cells phosphorylated (Na+,K+)-ATPase poorly whereas casein was an excellent substrate. The molecular weight of the partially purified protein tyrosine kinase from Ehrlich ascites tumor cells determined by gel filtration was about 60,000. One of two major phosphorylated phosphopeptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis had an Mr of 60 kDa, thus suggesting that it might be the autophosphorylated protein tyrosine kinase. A phosphatase that hydrolyzes phosphorylated histones or poly(Glu80Tyr20) was partially purified from the same membrane.     

DNA ligase from mouse Ehrlich ascites tumor cells

The molecular (Mr = 120,000; s20, w = 5S) and catalytic properties (Km (ATP) = 3 microM; Km (nicked DNA) = 0.2 microM; Km (Mg2+) = 3 mM) of DNA ligase from mouse Ehrlich ascites tumor cells are similar to those of the enzymes from calf thymus and rodent liver. The activity level of DNA ligase from the tumor cells is about 10-fold higher than that from mouse liver. Immunochemical titration of DNA ligase with antibodies against the calf thymus enzyme showed that the higher level of DNA ligase activity in the tumor cells is due to an increase in enzyme quantity and not to elevation of the catalytic efficiency of the enzyme molecule. These results suggest that there is little apparent difference between the qualities of DNA ligases from the tumor cells and normal tissues of rodents and calf.     

Purification of interferon from mouse Ehrlich ascites tumor cells

Interferon production was induced in mouse Ehrlich ascites tumor cells by infection with Newcastle disease virus. The interferon produced was purified by precipitation with ammonium sulfate, chromatography on carboxymethyl-Sephadex, treatment with blue dextran and polyethylene glycol, gel filtration on Bio-Gel P-60 and Bio-Gel P-200, chromatography on phosphocellulose, isoelectric focusing, and chromatography on octyl-Sepharose. The specific activity of the product was 1.6 x 10(9) NIH mouse interferon reference standard units/mg of protein. Electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate indicated that the apparent molecular weight of the interferon-active material ranged from 25,000 to 35,000. As revealed by staining the gels with Coomassie brilliant blue, the interferon activity co-migrated with the major, broad protein band. Minor, stainable bands of proteins were free of interferon activity and their apparent molecular weight was smaller than 12,000.     

Investigations on the function of creatine kinase in Ehrlich ascites tumor cells

1. Growth and viability of in vitro cultured Ehrlich ascites tumor cells are not significantly impaired by exogenous creatine up to 40mM. Retardation of cell growth by higher concentrations depends on cell density. 2. Ehrlich cells grown in the presence of high concentrations of creatine accumulate creatine phosphate to high levels (up to 23 nmol/10(6) cells in the presence of 40mM creatine). 3. A nearly complete interruption of glycolytic ATP production or inhibition of the oxidative ATP synthesis reduces the maximal creatine to about 40-50% of controls. 4. Studies on the intracellular distribution of creatine kinase have shown, that the enzyme is only associated with the mitochondrial fraction. Titration of isolated mitochondria with digitonin revealed that the activity is located in the inter-membrane space and partly bound to the outer site of the inner membrane. 5. By growth of Ehrlich cells in creatine-free medium it is possible to obtain "creatine phosphate-depleted" cells (creatine phosphate less than 10% of controls). The growth of creatine phosphate-depleted cells as compared to controls is significantly reduced under energetic stress situations. The protein synthesis of these cells after an energetic stress (lack of glucose and oxygen) is significantly reduced as compared to creatine phosphate containing cells. 6. It is concluded that in these cells creatine kinase/creatine phosphate is a thermodynamic buffer system and not part of an energy shuttle as is postulated for muscle cells.     

Interferon induction of polyamine-dependent protein kinase activity in Ehrlich ascites tumor cells

Treatment of Ehrlich ascites tumor cell cultures $\text{in}\text{vitro}$ with interferon induces a protein kinase activity that is activated by the polyamines, spermidine and spermine. Putrescine antagonizes the activation. The protein kinase yields a phosphorylated endogenous polypeptide of M_r 68,000–70,000. The polyamine-dependent protein kinase activity cofractionates with a double-stranded RNA-dependent protein kinase activity during affinity chromatography on poly (I) ·poly (C) - agarose or by chromatography on phosphocellulose. The double-stranded RNA-dependent protein kinase also phosphorylates an endogenous polypeptide of M_r 68,000–70,000. Unsuccessful attempts to discriminate between these two protein kinase activities on the bases of their respective capacities to be activated by either double-stranded RNA or spermidine/spermine, suggest that a single protein kinase enzyme may be activated by these strikingly dissimilar modifiers.     

Synergistic effect of ammonium and potassium ions on pyruvate kinase from Ehrlich ascites tumor cells

The effect of NH4+ on M2-pyruvate kinase isolated from Ehrlich ascites tumor cells was investigated. The enzyme is activated by NH4+ more efficiently than by K+. Moreover, a synergistic interaction of the two cations is observed since NH4+ increases the affinity of the enzyme for K+. The affinity of the enzyme for phosphoenolpyruvate is also increased in the presence of NH4+, and in these conditions the activating effect of fructose-1,6-bisphosphate is reduced. It is proposed that NH4+ be considered a specific allosteric activator of the tumor enzyme.     

Mannose toxicity in Ehrlich ascites tumor cells

Mannosephosphate isomerase (MPI) showed a higher activity than hexokinase (HKM) in its ability to phosphorylate mannose in the spleen, thymus, brain, liver, striated muscles, kidneys, and testes from BALB/c mice. This led to a HKM/MPI ratio of less than 1 in all the organs and tissues mentioned. In contrast, Ehrlich ascites tumor cells obtained from the peritoneum of BALB/c mice had low MPI activity (half of the HKM activity and, therefore, a ratio of 2). Mannose, which is nontoxic to nontumor cells at a concentration of 0.1 M, induced marked in vitro mortality of the tumor cells. Incubation of Ehrlich ascites tumor cells with mannose resulted in a high accumulation of mannose-6-phosphate and a marked depletion of ATP which did not appear when the cells were incubated with glucose. These facts may explain the selective mortality caused by mannose in the tumor cells studied.     

DNA polymerase in nucleoli isolated from Ehrlich ascites tumor cells

DNA replication was investigated in nucleoli isolated from Ehrlich ascites tumor cells. DNA synthesis was dependent on the presence of the four deoxynucleoside triphosphates and magnesium, but was reduced in the presence of ATP. The pH optimum for DNA replication was 8.5 to 9.0 N-Ethyl-maleimide reduced the reaction significantly. DNA synthesis occurred on nucleolar chromatin and was stimulated by treatment of the nucleoli with a small amount of DNase I. Addition of exogenous DNA to the reaction mixture significantly stimulated [³H]dTMP incorporation.     

Release of free fatty acids from Ehrlich ascites tumor cells

Ehrlich ascites tumor cells release free fatty acids (FFA) during in vitro incubation in media that contain albumin. The released FFA are derived by lipolysis from endogenous lipid esters. Addition of glucose to the incubation medium greatly decreases the quantity of fatty acid released by the cells. Cyanide, which inhibits endogenous lipid oxidation but not lipolysis, increases the quantity of fatty acid released to media containing albumin and causes free fatty acid to accumulate in the cells in the absence of exogenous albumin. The release of fatty acid, either preformed or derived by lipolysis during prolonged incubations, occurs under conditions of net fatty acid uptake from the incubation medium. Net release of fatty acid from the cell occurs only when fatty acid-extracted albumin is present in the extracellular medium; extrapolation of the data suggests that net release will not occur under physiological conditions. It is postulated that free fatty acid uptake and release are independent processes, the direction of net fatty acid movement being determined by the relationship between cellular free fatty acid concentration (regulating efflux) and the molar ratio of free fatty acid to albumin in the extracellular medium (regulating uptake).     

Isolation and reconstitution of furosemide-binding proteins from Ehrlich ascites tumor cells

Summary Furosemide-binding proteins were isolated from cholate-solubilized membranes of Ehrlich ascites tumor cells by affinity chromatography, using furosemide as ligand. Solubilized proteins retarded by the affinity material were eluted by furosemide. In reducing and denaturing gels, the major proteins eluted by furosemide were 100 and 45 kDa. In nonreducing, nondenaturing gels, homodimers of both polypeptides were found, whereas no oligomeric proteins containing both polypeptides were seen. It is concluded that the furosemide gel binds two distinct dimeric proteins. The isolated proteins were reconstituted into phospholipid vesicles and the K⁺ transport activity of these vesicles was assayed by measurement of⁸⁶Rb⁺ uptake against a large opposing K⁺ gradient. The reconstituted system was found to contain a K⁺ transporting protein, which is sensitive to Ba²⁺ like the K⁺ channel previously demonstrated to be activated in intact cells after cell swelling.