guanosine-diphosphate and Carcinoma--Ehrlich-Tumor

Cell swelling following hypoosmotic stress leads to the activation of volume-sensitive ion channels that allow a K+ and Cl- efflux accompanied by water loss. A Ca2+-insensitive K+ channel (I(K,vol)) has been described in Ehrlich cells that can be activated by hypotonicity and leukotriene D4 and is inhibited by clofilium. We have studied the activation and deactivation by osmotic stimuli of this channel. A G-protein appears to be involved in these processes since GTP-gamma-S accelerates deactivation, while GDP-beta-S blocks the channel in the open state, a result mimicked by pertussis toxin (PTX). In addition, PTX accelerates the onset of I(K,vol). We propose that I(K,vol) is tonically inhibited by a PTX-sensitive G-protein.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cations; Cell Size; Chlorides; Egtazic Acid; Electric Conductivity; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Hypotonic Solutions; Ion Channels; Mice; Pertussis Toxin; Potassium; Quaternary Ammonium Compounds; Thionucleotides; Tumor Cells, Cultured; Virulence Factors, Bordetella

A particulate fraction consisting of heavy organelles such as nuclei and mitochondria was prepared from Ehrlich ascites tumor cells. From this fraction we have purified a GTP-binding protein with a molecular mass of 33 kDa (MTG33) by guanidine hydrochloride extraction followed by four steps of column chromatography. The Kd value of MTG33 for GTP was 17 nM. [alpha-32P]GTP-binding to MTG33 was inhibited by GTP and GDP, but not appreciably by ATP, CTP, UTP, or GMP. MTG33 hydrolyzed GTP to GDP at a rate of 4.5 mmol/min/mol protein. Subcellular fractionation analysis of mouse liver revealed that the heavy mitochondrial fraction contained the highest level of MTG33. Furthermore, dual immunofluorescence examination indicated that the staining of NIH 3T3 cells with anti-MTG33 antibody is coincident with the distribution of mitochondrial succinate dehydrogenase. Of the mouse organs examined, the heart contained the highest level of MTG33. These results strongly suggest that MTG33 is a GTP-binding protein located in mitochondria.

Topics: 3T3 Cells; Animals; Binding Sites; Carcinoma, Ehrlich Tumor; Cell Nucleus; Fluorescent Antibody Technique; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Male; Mice; Mitochondria; Mitochondria, Liver; Molecular Weight; Subcellular Fractions

A major site of regulation of polypeptide chain initiation is the binding of Met-tRNA to 40 S ribosomal subunits which is mediated by eukaryotic initiation factor 2 (eIF-2). The formation of ternary complex, eIF-2.GTP.Met-tRNA, is potently inhibited by GDP. Measurement of the parameters for guanine nucleotide binding to eIF-2 is critical to understanding the control of protein synthesis by fluctuations in cellular energy levels. We have compared the dissociation constants (Kd) of eIF-2.GDP and eIF-2.GTP and find that GDP has a 400-fold higher affinity for GDP than GTP. The Kd for GDP is almost an order of magnitude less than has been reported previously. The difference between the Kd values for the two nucleotides is the result of a faster rate constant for GTP release, the rate constants for binding being approximately equal. This combination of rate constants and low levels of contaminating GDP in preparations of GTP can explain the apparently unstable nature of eIF-2.GTP observed by others. Mg2+ stabilizes binary complexes slowing the rates of release of nucleotide from both eIF-2.GDP and eIF-2.GTP. The competition between GTP and GDP for binding to eIF-2.guanine nucleotide exchange factor complex has been measured. A 10-fold higher GTP concentration than GDP is required to reduce [32P] GDP binding to eIF-2.guanine nucleotide exchange factor complex by 50%. The relevance of this competition to the regulation of protein synthesis by energy levels is discussed.

Topics: Animals; Carcinoma, Ehrlich Tumor; Eukaryotic Initiation Factor-2; Guanine Nucleotide Exchange Factors; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Magnesium; Peptide Initiation Factors; Proteins; RNA, Transfer, Met; Temperature

Guanine nucleotide exchange factor (GEF) is a multisubunit protein involved in the initiation of translation. Although numerous models have been proposed for its mechanism of action, none have been definitive. An assay dependent on GEF activity was developed using highly purified eukaryotic initiation factor 2 (eIF-2) and GEF from Ehrlich cells. GEF was considered in terms of an enzyme whose catalytic function was the exchange of eIF-2-bound [alpha-32P]GDP for unlabeled nucleotide. The turnover number of GEF at 37 degrees C, calculated on the basis of enzyme kinetic methods is 0.027 s, which is consistent with in vivo rates of protein synthesis. Moreover, kinetic data support an enzyme-substituted mechanism as the mode of GEF function. This mechanism proposes the existence of a GEF.eIF-2.GDP complex and excludes the possibility of two guanine nucleotide binding sites on eIF-2. An analogous mechanism has been recently reported for elongation factor Ts, suggesting the importance of this mechanism to protein synthesis. The mechanism of inhibition of GEF function by eIF-2 alpha phosphorylation has also been investigated. It has been generally assumed that the mechanism by which eIF-2(P) traps GEF is an excessively stable complex, from which GEF is released very slowly. Data presented here, however, reveal that eIF-2(P).GDP is a competitive inhibitor of GEF (rather than an irreversible inhibitor) competing with eIF-2.GDP for binding to GEF. Even though the eIF-2(P).GDP.GEF complex dissociates too rapidly to measure, GEF is trapped because it has at least 150-fold greater affinity for eIF-2(P).GDP than for eIF-2.GDP. The implications of competitive inhibition with respect to the mechanism of reversal of inhibition by an eIF-2(P) phosphatase are discussed.

Topics: Animals; Binding, Competitive; Carcinoma, Ehrlich Tumor; Catalysis; Eukaryotic Initiation Factor-2; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Kinetics; Peptide Initiation Factors; Phosphorylation; Proteins

Previously, we have shown that phosphorylation of the eukaryotic initiation factor eIF-2 alpha increases under several physiological stresses in which protein synthesis is inhibited in Ehrlich ascites tumor cells. As phosphorylated eIF-2 [eIF-2(alpha P)] is a potent inhibitor of guanine nucleotide exchange factor (GEF), it seemed likely that it was responsible for the inhibition. We have assayed GEF activity levels in extracts prepared from Ehrlich cells exposed to three such stresses, namely heat shock, serum deprivation and glutamine deprivation. Activity was estimated by the ability of GEF to enhance the release of [alpha-32P]GDP from purified eIF-2 [a modification of the reticulocyte lysate assay of Matts, R. L. & London, I. M. (1984) J. Biol. Chem. 259, 6708]. GEF activity was reduced from control values in extracts of heat-shocked cells and serum-deprived cells, concomitant with increased eIF-2 alpha phosphorylation. Inhibition of GEF activity in heat-shocked and serum-deprived cells was reversed to control levels by increasing the concentration of purified eIF-2.GDP added as substrate in the GEF assay. Since we have shown elsewhere that eIF-2(alpha P).GDP inhibits GEF by competition with eIF-2.GDP, the complete reversal of inhibition of GEF activity in heat-shocked and serum-deprived cells indicates that inhibition is due solely to phosphorylation of eIF-2 alpha. In glutamine-deprived cells phosphorylation of eIF-2 alpha was increased modestly and GEF activity was reduced but GEF activity could not be fully reversed by addition of eIF-2.GDP, suggesting that GEF may also be regulated in other ways. There are greater amounts of GEF relative to eIF-2 in Ehrlich cells (approximately 50%) compared with rabbit reticulocytes (approximately 20%). This explains the efficient rates of protein synthesis in control Ehrlich cells even though they have 30% of their eIF-2 phosphorylated which is enough to inhibit GEF and initiation in reticulocytes completely but only enough to trap approximately 60% of the GEF in Ehrlich cells.

Topics: Animals; Carcinoma, Ehrlich Tumor; Eukaryotic Initiation Factor-2; Glutamine; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Peptide Initiation Factors; Proteins; Rabbits; Temperature

The physiological correlation between NDP-kinase and the enzyme-associated guanine nucleotide binding proteins (G1 and G2) has been studied in vitro. It was found that incubation of the phosphoenzyme (enzyme-bound high-energy phosphate intermediate) of NDP-kinases with one of the nucleoside 5'-diphosphates (NDPs) in the presence of divalent cations (Mg2+ and Ca2+) results in the formation of nucleoside 5'-triphosphates (NTPs) within 40 sec even at low temperatures (below 4 degrees C) without strict base-specificity; and high-energy phosphates on the phosphoenzyme can transfer preferentially to GDP on the guanine nucleotide binding proteins (G1, G2 and r-p21 protein) in the presence of 0.25 mM Ca2+ or 1 mM Mg2+ even if any other NDPs are present in the reaction mixtures. These observations suggest that NDP-kinase may be responsible for the phosphate-transfer between GDP on the guanine nucleotide binding proteins and its phosphoenzyme.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cations, Divalent; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Nucleoside-Diphosphate Kinase; Phosphoproteins; Phosphotransferases; Proto-Oncogene Proteins; Ribonucleotides

The physiological correlation between nucleoside-diphosphate kinases (NDP-kinases) and the 21-kDa guanine nucleotide-binding proteins (G1 and G2) which are copurified with the enzymes from the cell membrane fractions of Ehrlich ascites tumor cells has been biochemically investigated in vitro. We found that: incubation of the phosphoenzyme (enzyme-bound high-energy phosphate intermediate) of NDP-kinases (F-I and F-II) with one of the nucleoside 5'-diphosphates in the presence of 1 mM Mg2+ or 0.25 mM Ca2+ results in the rapid formation of nucleoside 5'-triphosphates without strict base specificity; GDP on the guanine nucleotide-binding proteins (G1, G2 and recombinant v-rasH p21) acts as a phosphate acceptor for the high-energy phosphates of the phosphoenzyme in the presence of 0.25 mM Ca2+; and [32P]GTP is preferentially formed from the 32P-labelled phosphoenzyme F-I and GDP-bound G1 or GDP-bound recombinant v-rasH p21 protein, even if any other nucleoside 5'-diphosphates are present in the reaction mixture. Although [32P]GTP formed was bound with the guanine nucleotide-binding proteins, it was immediately hydrolyzed by the proteins themselves in the presence of 5 mM Mg2+, but not in the presence of 0.25 mM Ca2+. Available evidence suggests that NDP-kinase may be responsible for the activation of the guanine nucleotide-binding proteins (G1, G2 and p21 proteins) through phosphate transfer by the enzyme.

Topics: Adenosine Diphosphate; Animals; Calcium; Carcinoma, Ehrlich Tumor; Cell Membrane; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Magnesium; Mice; Nucleoside-Diphosphate Kinase; Nucleotides; Phosphates; Phosphotransferases

Eukaryotic cell polypeptide chain initiation factor eIF-2 forms ternary complexes with GTP and initiator Met-tRNAf. These complexes can be destabilized in vitro by the addition of salt-washed 40S ribosomal subunits. Our evidence suggests that this destabilization is mediated by GDP generated by premature hydrolysis of the GTP molecule present in the ternary complex. With complexes formed by using a partially purified preparation of eIF-2 from Ehrlich ascites tumor cells, it is possible to reverse the 40S subunit induced inhibition by creating conditions which eliminate free GDP from the system. This reversal probably occurs due to exchange of GTP for the GDP bound to the initiation factor, in a reaction catalyzed by another factor present in the eIF-2 preparation. However, if the eIF-2 has previously been phosphorylated by the reticulocyte heme-controlled repressor, the 40S subunit induced inhibition cannot be reversed by elimination of free GDP. The instability of initiation complexes containing eIF-2, together with the impairment of guanine nucleotide exchange after phosphorylation of eIF-2 [Clemens, M.J., Pain, V.M., Wong, S.-T., & Henshaw, E. C. (1982) Nature (London) 296, 93-95], may be an important aspect of the mechanism of the inhibition of translation by the heme-controlled repressor.

Topics: Animals; Carcinoma, Ehrlich Tumor; Eukaryotic Initiation Factor-2; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Mice; Peptide Chain Initiation, Translational; Peptide Initiation Factors; Phosphorylation; Proteins; Reticulocytes; Ribosomes; RNA, Transfer, Amino Acyl; RNA, Transfer, Met

Formation of the ternary complex Met-tRNAi X eukaryotic initiation factor (eIF) 2 X GTP from eIF-2 X GDP requires exchange of GDP for GTP. However, at physiological Mg2+ concentrations, GDP is released from eIF-2 exceedingly slowly (Clemens, M.J., Pain, V.M., Wong, S.T., and Henshaw, E.C. (1982) Nature (Lond.) 296, 93-95). However, GDP is released rapidly from impure eIF-2 preparations, indicating the presence of a GDP/GTP exchange factor. We have now purified this factor from Ehrlich cells and refer to it as GEF. CM-Sephadex chromatography of ribosomal salt wash separated two peaks of eIF-2 activity. GEF was found in association with eIF-2 in the first peak and co-purified with eIF-2 under low salt conditions. It was separated from eIF-2 in high salt buffers and further purified on hydroxylapatite and phosphocellulose. Gel electrophoresis of our purest preparations showed major bands at 85, 67, 52, 37, 27, and 21 kDa. Purified GEF increased the rate of exchange of [32P] GDP for unlabeled GDP 25-fold but did not function with phosphorylated eIF-2 (alpha subunit). The factor also stimulated markedly the rate of ternary complex formation using eIF-2 X GDP as substrate with GTP and Met-tRNAi but not using phosphorylated eIF-2 X GDP as substrate. eIF-2 is released from the 80 S initiation complex with hydrolysis of GTP. If eIF-2 X GDP is actually the complex released, then GEF is absolutely required for eIF-2 to cycle and it is therefore a new eukaryotic initiation factor. Furthermore, the inability of GEF to utilize eIF-2 (alpha P) X GDP explains how phosphorylation of eIF-2 can inhibit polypeptide chain initiation.

Topics: Animals; Carcinoma, Ehrlich Tumor; Eukaryotic Initiation Factor-2; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Magnesium; Mice; Molecular Weight; Peptide Initiation Factors; Phosphorylation; Proteins; RNA, Transfer, Amino Acyl

Our previous reports have presented evidence suggesting the existence in tumor cells of a second control site of glycolysis of pyruvate kinase as a competition for adenosine diphosphate between this enzyme and mitochondria, which is responsible for the Crabtree effect. Now, by using cells partially permeabilized to nucleotides and phosphorylated substrates, we provide evidence supporting the existence in hepatocytes of a partial control by adenosine triphosphate at phosphofructokinase, which is followed by the total control by adenosine triphosphate at pyruvate kinase. The partial or nonoperation of this second site in Ehrlich ascites tumor cells appears to be the cause for the characteristic aerobic glycolysis, Crabtree effect, and low Pasteur effect of these cells.

Topics: Adenosine Monophosphate; Adenosine Triphosphate; Animals; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Glucose; Glycolysis; Guanosine Diphosphate; In Vitro Techniques; Lactates; Liver; Male; NAD; Osmotic Pressure; Phosphoenolpyruvate; Phosphofructokinase-1; Pyruvate Kinase; Rats; Sonication