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.     

Isolation and partial characterization of the ADP-ribosylated nuclear proteins from Ehrlich ascites tumor cells

The (ADP-ribose)_n protein conjugates formed by incubation of Ehrlich ascites tumor cell nuclei with 1 mM (³H)NAD were isolated by chromatography on boronate cellulose columns with a yield of >85%. Possible contamination by glycoproteins was excluded by rechromatography after specific release of the (ADP-ribose)_n residues from their acceptors. Dodecyl sulfate gel electrophoresis revealed numerous protein bands which coincided with the (³H)ADP-ribose bands obtained by fluorography of the gels. 40% of the acceptor proteins were identified as the nucleosomal core histones. Most of these histones, however, appeared in the non-histone fraction because of extensive modification by poly(ADP-ribose). Drastic changes in properties were also seen in the true non-histone proteins which comprised 60% of the total conjugated protein. Besides several prominent acceptor proteins (M_r = 12,000; 31,000; 125,000) numerous proteins were detected indicating a considerable heterogeneity of non-histone acceptors.     

Nucleosidediphosphate kinase from Ehrlich ascites tumor cells

A phosphate-incorporating protein has been highly purified from the cytosol of Ehrlich ascites tumor cells (EAT cells). The nitrocellulose membrane method was used to follow the progress of the purification by quantitation of the [32P]phosphorylated form of the protein. The purified protein was identified as an NDP-kinase since it exhibited NDP-kinase activity and had enzyme characteristics in common with other NDP-kinases from various mammalian cells. The purified NDP-kinase was found to have a molecular weight of approximately 76,000 daltons. Moreover, the enzyme appears to consist of two distinct polypeptides (18,000 and 20,000 daltons). This enzyme contained 19 amino acids, with high levels of glycine (9.8%) and lysine (9.0%). The enzyme rapidly formed a [32P]phosphoenzyme when incubated with [gamma-32P]ATP in the presence of Mg2+ (1 mM) at the optimum pH of 7.5 even at low temperature (below 4 degrees C). This phosphoenzyme is an enzyme-bound, high-energy-phosphate intermediate, because ATP was formed from it on incubation with ADP in the presence of Mg2+ (1 mM). This finding suggests that the phosphoenzyme functions as an intermediate in NDP-kinase action.     

Evidence for monovalent phosphate transport in Ehrlich ascites tumor cells

In an effort to determine whether the Na+-dependent Pi transport system of Ehrlich ascites tumor cells exhibits specificity for H2PO4- or HPO4(-2), Pi fluxes were determined by measuring 32Pi-Pi self-exchange. Three experimental approaches were employed. First, the effect of pH on steady-state Pi transport at 0.5 and 5 mM was studied. Second, the relationship between Pi transport and Pi concentration (0.25-9.2 mM) at pH 5.6 and 7.9 was determined. Third, the dependence of Pi transport on [H2PO4-] (0.05-4.2 mM) at constant [HPO4(-2)] (0.5 mM), and the converse, [HPO4(-2)] (0.06-4.5 mM) at constant [H2PO4-] (0.5 mM), was evaluated. Ks (apparent half-saturation constant) and Jmax (maximal transport rate) were calculated by two methods: weighted linear regression (WLR) and a nonparametric procedure. The dependence of Pi flux on pH indicates that optimum transport occurs at pH 6.9. Pi transport decreases as pH is reduced when extracellular Pi is either 0.5 or 5 mM. However, at pH 7.9, Pi flux is reduced only in 0.5 mM Pi. At pH 5.6, H2PO4- comprises 93% of the total Pi present, and the calculated Ks is 0.055 +/- 0.026 mM (WLR). This is the same as the Ks determined from the initial phase of the flux vs. [H2PO4-] relationship (0.056 +/- 0.020 mM). However, at pH 7.9 (where 94% of Pi is HPO4(-2)), the measured Ks is 0.58 +/- 0.11 mM (WLR), which is ten times higher than at pH 5.6. This value is also five times greater than the Ks calculated from the flux vs. [HPO4(-20)] curve (0.106 +/- 0.16 mM). Kinetic parameters calculated by the nonparametric method, though somewhat different, gave similar relative results. Taken together, these results support two conclusions: (1) H2PO4- is the substrate for the Na+-dependent Pi transport system of the Ehrlich cell, and (2) H+ can inhibit Pi transport.     

Computer simulation study of hexokinase II from Ehrlich ascites cells.

A study of the mechanism of hexokinase II from ascites cells the effects of its binding to mitochondrial membranes has been carried out by computer simulation. This is based on experimental data of Kosow and Rose and of Gumaa and McLean, and the theoretical methods of cleveland. For the soluble enzyme the mechanism is random with ternary produce-inhibition complexes; when bound to mitochondria, the mechanism becomes ordered-on, random-off, as the binding of ATP to the free enzymes becomes negligibly slow. The requirements of experimental data for mechanistic studies are discussed.     

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.     

Structural characteristics of interferons from mouse Ehrlich ascites tumor cells.

An improved procedure for the isolation of interferons produced by mouse Ehrlich ascites tumor cells infected with Newcastle disease virus provides interferons of three size classes (33,000, 26,000, and 20,000 daltons) with specific activities between 2 and 3 x 10(9) units/mg of protein and a yield of 11 to 20%. The tryptic peptide maps of the two larger species are very similar; that of the smallest species is different, at least in part. The amino acid compositions of the three species are very close. Their NH2-terminal amino acids are identical and so are the amino acids released by carboxypeptidase A treatment. These data are consistent with the possibility that the differences in size between the three species may be due, at least in part, to unequal glycosylation.     

Interaction between daunorubicin and chromatin from Ehrlich ascites tumor cells.

Interaction of daunorubicin with chromatin from Ehrlich ascites tumor cells has been studied by spectrofluorimetry. Daunorubicin interacts with chromatin and displays at least two types of binding. The number of binding sites is reduced when compared to deoxyribonucleic acid. There is no difference in the overall structure of chromatins extracted from cells sensitive or resistant to daunorubicin.     

Evidence that luminal ER proteins are sorted from secreted proteins in a post-ER compartment.

Several soluble proteins that reside in the lumen of the ER contain a specific C-terminal sequence (KDEL) which prevents their secretion. This sequence may be recognized by a receptor that either immobilizes the proteins in the ER, or sorts them from other proteins at a later point in the secretory pathway and returns them to their normal location. To distinguish these possibilities, I have attached an ER retention signal to the lysosomal protein cathepsin D. The oligosaccharide side chains of this protein are normally modified sequentially by two enzymes to form mannose-6-phosphate residues; these enzymes do not act in the ER, but are thought to be located in separate compartments within (or near) the Golgi apparatus. Cathepsin D bearing the ER signal accumulates within the ER, but continues to be modified by the first of the mannose-6-phosphate forming enzymes. Modification is strongly temperature-dependent, which is also a feature of ER-to-Golgi transport. These results support the idea that luminal ER proteins are continuously retrieved from a post-ER compartment, and that this compartment contains N-acetylglucosaminyl-1-phosphotransferase activity.     

Particulate forms of phenylalanyl-tRNA synthetase from Ehrlich ascites cells

Over 80% of the phenylalanyl-tRNA synthetase activity in Ehrlich ascites cell homogenates was found to be associated with the high speed particulate fraction. This enzyme activity occurred in two principle forms: activity bound to the ribosomes, and activity as part of a complex sedimenting at approximately 25S in a sucrose density gradient. The ribosome-associated enzyme was shown to be bound to the 60S ribosomal subunit. Exposure of the ribosomes to RNA resulted in removal of synthetase activity from the ribosomes and the concomitant appearance of activity in a complex sedimenting at 25S.     

Evidence for the occurrence of the malate-citrate shuttle in intact Ehrlich ascites tumor cells

A possible activity of the malate-citrate shuttle has been investigated in Ehrlich ascites cells by testing the effects of 1,2,3-benzenetricarboxylic acid, an inhibitor of the malate-citrate exchange, and (?)-hydroxycitrate, an inhibitor of the citrate cleavage enzyme, on the glucose-dependent oxidation-reduction rates of pyridine nucleotides and cytochrome b as well as on ATP levels of glycolyzing cells. Moreover, to quantitate such an activity, the effects of these two inhibitors have been compared with those induced under the same experimental conditions by aminooxyacetate, an inhibitor of the malate-aspartate shuttle which is known to operate in this strain of ascites tumor. Both benzenetricarboxylic acid and hydroxycitrate are able to increase the reduction of pyridine nucleotides, which follows glucose addition to whole cells, to about the same extent. A much more pronounced effect is elicited by aminooxyacetate under the same condition. When n-butylmalonate is added to slow down the flux of glycolytic reducing equivalents to the respiratory chain via the malate-aspartate shuttle, benzenetricar-boxylic acid or hydroxycitrate promotes an ATP-driven reversal of electron transfer. Indeed, the glucose-induced reduction of cytochrome b becomes sensitive to oligomycin and the ATP level is raised significantly with respect to the value of uninhibited cells. It is concluded that the malate-citrate shuttle operates in Ehrlich ascites cells, although with a substantially lower activity with respect to the malate-aspartate shuttle.     

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.     

Long-distance interactions between Ehrlich ascites tumour cells.

In previous work, electron micrographs were made of adjacent surfaces of aldehyde-fixed, Ehrlich ascites tumour cells cultured on coverslips, after reacting some of their negatively charged surface sites with colloidal iron hydroxide (CIH) particles. It was observed that microvilli from one cell were aligned with intermicrovillus regions on another, where the density of the adsorbed CIH particles was significantly lower than in adjacent regions. Alignment, which was considered to represent interactions between the two peripheral cellular regions, took place when these regions were apparently separated by more than 200 nm, in an environment of physiologic ionic strength ( 0·145 m NaCl).In this communication we attempt to find feasible mechanisms for the alignment phenomenon in physical terms, in cases where the observed separation of 200 nm is correct, and in cases where the distances are overestimated due to preparative artifacts.It is concluded, that at distances of separation in excess of 200 nm, one feasible mechanism for alignment is that net negatively charged macromolecules diffusing out of cells in the region of their microvilli, electrostatically repel CIH-binding anionic sites in the lipid-rich “fluid” matrix of the periphery of the opposed cell, causing gaps in their distribution. The role of electrostatic and electrodynamic (van der Waals') forces in causing alignment is also discussed in terms of distance of separation.This communication is concerned with the interpretation in terms of various interactions, of electron micrographs showing evidence of alignment between microvilli from one cell with specific areas of another.     

Evidence for the oxidation of glycolytic NADH by the malate-aspartate shuttle in Ehrlich ascites tumor cells.

Actively glycolyzing Ehrlich-Lettré ascites tumor cells have been tested for their ability to reoxidize into the mitochondria reduced nicotinamide adenine dinucleotide by the malate-aspartate shuttle. The aspartate transaminase inhibitor aminooxyacetate has been used as a tool for evaluating it. Measurements of the free cytosolic $\text{(NADH)}\text{(}\text{NAD}{}^{\mathrm{+}}\text{)}$ ratio indicate that it increases gradually in the inhibitor-treated cells up to a value about ninefold higher than in the controls after 30 min of glycolytic activity. Fructose 1, 6-diphosphate and dihydroxyacetone phosphate reach a steady-state level after 5 min of incubation in the untreated cells, whereas they accumulate in large amounts in the inhibited cells. Correspondingly, a decrease in 3-phosphoglycerate concentration is observed. On the other hand, the rate of glucose utilization is affected slightly only during long observation times. From these results it may be established that in Ehrlich ascites cells reducing equivalents generated in the cytosol during aerobic glycolysis strongly influence the NAD redox state when their intramitochondrial translocation is prevented by the inactivation of the malate-aspartate shuttle.