Energy requirement for biosynthesis of DNA in Escherichia coli: Role of membrane-bound energy-transducing ATPase (coupling factor)

A mutant of Escherichia coli missing energy-transducing ATPase and known to be defective in a variety of membrane functions from earlier studies (Yamamoto, T. H., Mével-Ninio, M. and Valentine, R. C. (1973) Biochim. Biophys. Acta 314, 267–275; Thipayathasana, P. and Valentine, R. C. (1974) Biochim. Biophys. Acta 347, 464–468; Mével-Ninio, M. and Yamamoto, T. (1974) Biochim. Biophys. Acta 357, 63–66) has been found to be blocked for anaerobic DNA synthesis. The rate of anaerobic DNA synthesis in the mutant, measured as radioactive adenine incorporation into the alkali-resistant fraction of whole cells, is about 1/6 the rate of DNA synthesis in the wild type culture under similar conditions. Addition of NO₃^- or O₂ restores DNA biosynthesis in the mutant. The entry of radioactive adenine is not appreciably affected in the mutant by anaerobiosis. It is concluded that coupling factor plays a role in some step(s) of DNA biosynthesis.     

Control of ribonucleotide reductase in heat- and cold-sensitive mammalian cell-cycle mutants

Two heat-sensitive (reversibly arrested in G1 phase at 39.5 degrees C, multiplying at 33 degrees C) and two cold-sensitive (reversibly arrested in G1 phase at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were tested for ribonucleotide reductase activity, using cells made permeable to nucleotides. After transfer of the heat-sensitive mutant cells to 39.5 degrees C, ribonucleotide reductase activity, similar to thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77-85), but unlike DNA polymerase alpha (Schneider, E., Müller, B. and Schindler, R. (1985) Biochim. Biophys. Acta 825, 375-383), decreased rapidly and in parallel with numbers of cells in S phase, whereas in the cold-sensitive mutant cells brought to 33 degrees C, ribonucleotide reductase activity decreased approx. 8 h later than numbers of DNA-synthesizing cells. When arrested heat- or cold-sensitive mutant cells were returned to the permissive temperature, ribonucleotide reductase activities, similar to DNA polymerase alpha and to thymidine kinase in heat-sensitive mutants, increased essentially in parallel with reentry of cells into S phase, whereas the increase in thymidine kinase activity in the cold-sensitive mutants was previously shown to occur approx. one cell-cycle time later. This indicates that ribonucleotide reductase and thymidine kinase are coordinately expressed in the heat-sensitive, but independently regulated in the cold-sensitive mutants.     

Studies on pig liver mevalonate-5-diphosphate decarboxylase

A procedure in which three sequential enzymes of cholesterol biosynthesis, mevalonate kinase (ATP: (R)-mevalonate 5-phosphotransferase, EC 2.7.1.36), phosphomevalonate kinase (ATP: (R)-5-phosphomevalonate phosphotransferase, EC 2.7.4.2) and mevalonate-5-diphosphate decarboxylase (ATP: (R)-5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33), from pig liver, could be purified in the one operation is described. Mevalonate kinase and phosphomevalonate kinase were utilized for the enzymic synthesis of mevalonate 5-diphosphate (both 1-14C-labelled and unlabelled), the substrate for mevalonate-5-diphosphate decarboxylase, using excess free ATP4-. A radioactive assay for the enzyme, based on the release of 14CO2 from [1-14C]mevalonate-5-diphosphate, was developed. The assay allowed reassessment of the metal and nucleotide specificity of the decarboxylase. ATP could be partially replaced by GTP and ITP, but no activity was observed with CTP, UTP or TTP. Apparent activation of the enzyme by ATP4- was observed as found for mevalonate kinase (C.S. Lee and W.J. O'Sullivan (1983) Biochim. Biophys. Acta 747, 215-224) and phosphomevalonate kinase (C.S. Lee and W.J. O'Sullivan (1985) Biochim. Biophys. Acta 839, 83-89). The presence of 1 mM excess free ATP4-, above that complexed as the substrate MgATP2-, decreased the Km for MgATP2- from 0.45 mM to 0.15 mM. MgADP- was shown to act as a competitive inhibitor with respect to MgATP2-.     

Steady-state kinetics of laccasse from Rhus vernicifera.

The steady-state kinetics of laccasse (monophenol, dihydroxyphenylalanine: oxygen oxidoreductase, EC 1.14.18.1) from the lacquer tree Rhus vernicifera is investigated using the respirograph method to produce Lineweaver-Burk plots of oxygen consumption rate against oxygen concentration. A ping-pong mechanisms is established. The kinetic constants obtained according to the model is in close agreement with the corresponding values obtained from earlier studies on the transient reactions between the reduced enzyme and oxygen (Andréasson, L.E., Br?ndén, R. and Reinhammar, B. (1976) Biochim. Biophys. Acta 438, 370--379) and between the oxidized enzyme and reducing substrates (Andréasson, L.E. and Reinhammar, B. (1976) Biochim. Biophys. Acta 445, 579--597).     

Interrelationships of protein and DNA syntheses during replication of mammalian cells.

During the replication of chromatin, the syntheses of the histone protein and DNA components are closely coordinated but not totally linked. The interrelationships of total protein synthesis, histone protein synthesis, DNA synthesis, and mRNA levels have been investigated in Chinese hamster ovary cells subjected to several different types of inhibitors in several different temporal combinations. The results from these studies and results reported elsewhere can be brought together into a consistent framework which combines the idea of autoregulation of histone biosynthesis as originally proposed by W. B. Butler and G. C. Mueller (Biochim. Biophys. Acta 294:481-496, 1973] with the presence of basal histone synthesis and the effects of protein synthesis on DNA synthesis. The proposed framework obviates the difficulties of Butler and Mueller's model and may have wider application in understanding the control of cell growth.     

Repartition of ATP, ADP and PO4 in isolated hepatocytes from fed and fasted rats

1. The digitonin fractionation procedure [Zuurendonk, P. F. and Tager, J. M. (1974) Biochim. Biophys. Acta, 333, 393-399] was used to determine the repartition of adenine nucleotides and inorganic phosphate in isolated hepatocytes from fed and fasted rats. 2. This repartition is not significantly modified in the presence of pyruvate or alanine or lactate + pyruvate for isolated hepatocytes from fasted rats. 3. In isolated hepatocytes from fasted rats, the mitochondrial ATP/ADP X PO4 ratio is two-fold lower than in isolated hepatocytes from fed rats. 4. The cytosolic ATP/ADP X PO4 ratio depends on the nutritional state and (or) on the added substrate for neoglucogenesis.     

Validity of the digitonin method for metabolite compartmentation in isolated hepatocytes.

1. A modification of the digitonin method of Zuurendonk & Tager (1974) (Biochim. Biophys. Acta 333, 393-399) (i.e. the 'convaentional' method) was developed that allows the fractionation of isolated hepatocytes at -5 degrees C (i.e. 'low-temperature' method). 2. With respect to compartmentation of adenine nucleotides, glutamate and citrate, the two methods yielded very similar results. 3. In contrast, the mitochondrial amounts of aspartate and malate, as revealed by the low-temperature method, were about twice as high as those found by the conventional procedure. No change in the total cellular content occurred. 4. With n-butylmalonate and glisoxepid present in the conventional digitonin medium, significantly higher amounts of malate and aspartate respectively were found in the mitochondrial pellets. The results obtained by the low-temperature method, however, were not influenced by the these inhibitors. 5. It is concluded that under the conventional conditions of cell fractionation no appreciable redistribution of adenine nucleotides, glutamate and citrate occurs.     

A spectrophotometric study of the binding of Cu(II) ions to ATP

The interaction of copper(II) with adenosine triphosphate (ATP) has been studied as a function of pH in the range pH 3-12. Our approach is the study of the effect of binding both on the ATP ultraviolet absorption spectrum and on the optical d-d transition of copper ions. The results show that Cu(II)-ATP complexes exist in a variety of forms in equilibrium, the percentage of each species varying according to the state of ionization of the intrinsic adenine, phosphate and ribose groups. These results also show a close correlation between the rate of dephosphorylation of ATP in the presence of Cu(II) ions and Cu(II) bonding to the adenine of ATP, thus supporting the hypothesis that the metal-ion/nucleic-base interactions are crucial for the observation of a metal-ion promoted dephosphorylation of ATP (D.H. Buisson and H. Sigel, Biochim. Biophys. Acta 343 (1974) 45).     

Control of DNA polymerase alpha, beta and gamma activities in heat- and cold-sensitive mammalian cell-cycle mutants

Two heat-sensitive (arrested in G1 at 39.5 degrees C) and two cold-sensitive (arrested in G1 at 33 degrees C) clonal cell-cycle mutants of the murine P-815-X2 mastocytoma line were tested for DNA polymerase alpha, beta and gamma activities. After transfer of mutant cells to the respective nonpermissive temperature, DNA polymerase alpha activities decreased more slowly than relative numbers of cells in S phase. Furthermore, numbers of DNA-synthesizing cells decreased to near-zero levels, whereas polymerase alpha activities in arrested cells were as high as 15-40% of control values. After return of arrested cells to the permissive temperature, polymerase alpha activities increased essentially in parallel with relative numbers of cells in S phase. In contrast to the changes in thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77-85), the decrease of polymerase alpha during entry of cells into proliferative quiescence thus appears to be under rather relaxed control, while after return of arrested cells to the permissive temperature the increase in polymerase alpha is tightly coupled with reentry of cells into S phase. For DNA polymerase beta and gamma activities, no obvious correlation with changes in the proliferative state of cells was detected.     

Monte Carlo studies of phospholipid lamellae. Effects of proteins, cholesterol, bilayer curvature, and lateral mobility on order parameters

In this paper we present the results of a Monte Carlo study of the effects of protein, cholesterol, bilayer curvature, and mobility on the chain order parameters of a lipid layer. The Monte Carlo method used is identical to the version developed earlier (Scott, Jr., H.L. (1977) Biochim. Biophys. Acta 469, 264–271). Simulations of protein and cholesterol effects are accomplished by insertion of a rigid stationary cylinder into the lipid matrix. The protein studies show the presence of boundary lipid (Jost, P., Griffith, O.H., Capaldi, R.H. and Vanderkooi, G. (1973) Biochim. Biophys. Acta 311, 141–152). The effect of cholesterol is dependent upon the length of the lipid hydrocarbon chains relative to the cholesterol depth of penetration. Our computer studies of bilayer curvature show the manner in which this curvature disrupts chain packing and are consistent with experimental results (Chrzeszczyk, A., Wishnia, A. and Springer, C.S. (1977) Biochim. Biophys. Acta, 470, 161–171). We also find that restricting lateral motion in chains, the simplest manner in which head group interactions can affect hydrocarbon chain order, does not measurably alter the order parameters. We argue that this provides some support for an earlier hypothesis by Scott (Scott, Jr., H.L. (1975) Biochim. Biophys. Acta 406, 329–346) regarding head group-chain interaction in monolayer experiments.     

A new form of high molecular weight DNA polymerase in the nuclei of rat ascites hepatoma cells.

A new form of high molecular weight DNA polymerase [EC 2.7.7.7] (polymerase N) was isolated from the nuclei of rat ascites hepatoma cells. Polymerase C, which was isolated previously from whole cell extract, was also isolated from the nuclei (Tsuruo, T. and Ukita, T. (1974) Biochim. Biophys. Acta 353, 146-159). Polymerase N was not found in the cytoplasmic fraction of the cell, while polymerase C existed in both the nucleus and cytoplasm. The molecular weight of polymerase N (8.7 S) was larger than that of polymerase C (7.4 S). On freezing and thawing, polymerase N was converted to polymerase C. In the nucleus the amount of polymerase N was larger than that of polymerase C. These data suggest that polymerase N, which was specifically present in the nucleus, was a complex form of polymerase C. In in vitro assay, polymerase N showed properties similar to those of polymerase C. Oligoribonucleotide was an effective initiator for the polymerization reaction by polymerase N. The DNA synthesis on single stranded fd phage DNA was greatly stimulated by the concomitant synthesis of RNA.     

Slow fluorescence quenching of type A chloroplasts. Resolution into two components.

The divalent-cation-specific ionophore A23187 is used to define two components of the slow fluorescence quenching of type a spinach chloroplasts: ionophore-reversible and ionophore-resistant quenching. Ionophore-reversible quenching predominates at relatively low light intensities and approaches saturation as light levels are increased. It is sensitive to uncouplers and to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and is dark reversible. At high light intensities the bulk (greater than 80%) of slow fluorescence quenching is ionophore-resistant. Ionophore-resistant quenching is stimulated by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at pH 7.6 and by both CCCP and methylamine at pH 9.0. It is insensitive to DCMU and is not reversed in subsequent darkness. Taken together, the two components account for all quenching observed in Type A chloroplasts. Ionophore-reversible quenching is identified with the Mg2+-mediated fluorescence quenching described by Krause (Biochim. Biophys. Acta (1974) 333, 301-313) and by Barber and Telfer (in Membrane Transport in Plants (Dainty, J., AND Zimmermann, U., eds.), pp. 281-288, Springer-Verlag, Berlin, 1974). Ionophore-resistant quenching, a first-order process requiring high light, resembles the quenching reported by Jennings et al. (Biochim. Biophys. Acta (1976) 423, 264-274). The resolution of the fluorescence quenching phenomenon into two distinct components reconciles the apparently contradictory observations of these earlier investigations.     

Control of DNA polymerase α, β and γ activities in heat- and cold-sensitive mammalian cell-cycle mutants

Two heat-sensitive (arrested in G₁ at 39.5°C) and two cold-sensitive (arrested in G₁ at 33°C) clonal cell-cycle mutants of the murine P-815-X2 mastocytoma line were tested for DNA polymerase α, β and γ activities. After transfer of mutant cells to the respective nonpermissive temperature, DNA polymerase α activities decreased more slowly than relative numbers of cells in S phase. Furthermore, numbers of DNA-synthesizing cells decreased to near-zero levels, whereas polymerase α activities in arrested cells were as high as 15–40% of control values. After return of arrested cells to the permissive temperature, polymerase α activities increased essentially in parallel with relative numbers of cells in S phase. In contrast to the changes in thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77–85), the decrease of polymerase α during entry of cells into proliferative quiescence thus appears to be under rather relaxed control, while after return of arrested cells to the permissive temperature the increase in polymerase α is tightly coupled with reentry of cells into S phase. For DNA polymerase β and γ activities, no obvious correlation with changes in the proliferative state of cells was detected.     

Calorimetric and freeze-etch study of the influence of Mg2+ on the thermotropic behaviour of phosphatidylglycerol

In the presence of Mg²⁺ ions phosphatidylglycerol shows supercooling which leads to the formation of a metastable gel phase. This contrasts with the behaviour of this negatively charged phospholipid in the presence of Ca²⁺ ions (Biochim. Biophys. Acta 339 (1974) 432). It is demonstrated that the heat content of this phospholipid is dependent on the ionic environment.     

Regulation of the amount and of the activity of phosphofructokinases and pyruvate kinases in Escherichia coli.

Two isozymes of fructose-6-phosphate kinase and two isozymes of pyruvate kinase have been detected in Escherichia coli under a wide variety of growth conditions. Their kinetic behavior has been characteriized with respect to different effectors and substrates. The conclusions reached on one hand by Malcovati and Kornberg (Biochim. Biophys. Acta (1969) 178, 420-423), on the other hand by Fraenkel, Kotlarz and Buc (J. Biol. Chem. (1973) 248, 4865-4866) have been found to be true in aerobiosis as well as in anaerobiosis. The biosynthesis of the four proteins is sensitive to the nature of the carbon sources as well as to the shift from aerobic to anaerobic conditions. Kinetics of depression after a shift to anaerobiosis have been followed and found to be of the order of the doubling time.     

An analysis of the adequacy of the asymmetric carrier model for sugar transport.

In 1972, Lieb, W. R. and Stein, W. D. (Biochim, Biophys. Acta 265, 187-207) in their review of sugar transport in human erythrocytes concluded that the conventional two-state carrier model was inconsistent with the experimental data available at that time. Since then, other papers have appeared which question the validity of the model. In this paper, we give a brief derivation of the equations describing the two-state carrier model, and analyze the predictions of the model in the classical experiments, i.e. zero-trans, infinite-cis, and equilibrium exchange. We show that the estimate of the half saturation constant of 2.8 mM for glucose at the inner face of the human red cell membrane for the infinite-cis procedure reported by Hankin, B.L., Liev, W.R. and Stein, W.D ((1972) Biochim. Biophys. Acta 288, 114-126) is unreliable. We note that all of the other experimental findings are consistent with the asymmetric carrier model.     

The Bacillus subtilis nucleoid-associated protein HPB12 strongly compacts DNA.

The HPB12 protein from the nucleoid of Bacillus subtilis was previously described, and its DNA binding properties have been reported previously (V. Salti, F. Le Hégarat, and L. Hirschbein, Biochim. Biophys. Acta 1009:161-167, 1989). The DNA-HPB12 complexes were examined by electron microscopy. They appeared as short, slightly curved rods whereas naked DNA showed no compaction. Since only a small number of complexes with an intermediate degree of folding were observed, it appears that the nucleoid-associated protein HPB12 binds cooperatively to DNA, confirming Salti et al. (V. Salti, F. Le Hégarat, and L. Hirschbein, Biochim. Biophys. Acta 1009:161-167, 1989), and gives rise to a tightly compacted DNA-protein complex. N-terminal sequencing of purified HPB12 showed that all but one of the first 26 amino acids were identical to those of the L24 ribosomal protein.     

Biological activities and spectroscopic properties of chromophoric and fluorescent analogs of adenine nucleoside and nucleotides, 2′,3′-O-(2,4,6-trinitrocyclohexadienylidene) adenosine derivatives

The ribose-modified chromophoric and fluorescent analog of ATP 2′,3′-O-(2,4,6-trinitrocyclohexadienylidene) adenosine 5′-triphosphate (TNP-ATP) has been synthesized previously (Hiratsuka, T., and Uchida, K. (1973) Biochim. Biophys. Acta 320, 635–647 and Hiratsuka, T. (1976) Biochim. Biophys. Acta 453, 293–297). In the present study, four TNP-derivatives of ATP, ADP, AMP and adenosine were synthesized and compared for several chemical, spectral and enzymatic properties. Their visible absorption and fluorescent properties were found to be quite similar. Visible absorption and fluorescence spectra of TNP-derivatives were sensitive to solvent polarity. TNP-adenosine and TNP-AMP showed considerable substrate activities with adenosine deaminase and alkaline phosphatase, respectively. TNP-ATP proved to be an excellent substitute for ATP in adenylate kinase and myosin ATPase systems. The results indicate that these analogs are useful as chromophoric and fluorescent probes for hydrophobic regions in adenine nucleoside and nucleotide requiring enzymes.     

Mouse intestinal Fe3+ uptake kinetics in vivo. The significance of brush-border membrane vesicle transport in the mechanism of mucosal Fe3+ uptake

Initial rates of mucosal uptake of Fe3+ from luminal Fe3+-nitrilotriacetate solutions by tied segments of mouse intestine in vivo have been measured. Duodenal uptake showed an approximately hyperbolic dependence of uptake on Fe3+ complex concentration (Km(app) 66 microM, Vmax 6.2 pmol/min per mg intestine) with little dependence on nitrilotriacetate:Fe3+ ratio or on added Ca2+. Duodenal uptake was greatly stimulated by hypoxic treatment of mice. Uptake rates by distal ileum were lower than by duodenum and more sensitive to added Ca2+. These results show that isolated duodenal brush-border membrane Fe3+ transport characteristics (Simpson, R.J. and Peters, T.J. (1984) Biochim. Biophys. Acta 772, 220-226) are inadequate to explain duodenal Fe3+ uptake in vivo. However, ileal uptake can be explained by the properties of isolated ileal brush-border membrane (Simpson, R.J., Raja, K.B. and Peters, T.J. (1985) Biochim. Biophys. Acta 814, 8-12).