Synthesis of quinolinate from D-aspartate in the mammalian liver-Escherichia coli quinolinate synthetase system.

Two proteins (A and B) from $\text{Escherichia coli}$ are required for the synthesis of the NAD precursor quinolinate from aspartate and dihydroxyacetone phosphate. Mammalian liver contains a FAD linked protein which replaces $\text{E. coli}$ B protein for quinolinate synthesis. D-aspartic acid but not L-aspartic acid is a substrate for quinolinic acid synthesis in a system composed of the B protein replacing activity of mammalian liver and $\text{E. coli}$ A protein. In contrast the $\text{E. coli}$ B protein-$\text{E. coli}$ A protein quinolinate synthetase system requires L-aspartic acid as substrate. The previous report that L-aspartate was a substrate in the liver-$\text{E. coli}$ system was due to contamination of commercially available [¹⁴C]L-aspartate with [¹⁴C]D-aspartate. These and other observations suggest that liver B protein is D-aspartate oxidase and $\text{E. coli}$ B protein is L-aspartate oxidase.     

The effect of inorganic phosphate on calcium influx into rat heart mitochondria

The effect of inorganic phosphate on the accumulation of Ca²⁺ by heart mitochondria has been reinvestigated. Inorganic phosphate has no effect on the initial rate of Ca²⁺ uptake supported by respiration on either ascorbate $\text{plus}$ tetramethylenephenylene diamine, pyruvate $\text{plus}$ malate, or glutamate $\text{plus}$ malate, although it does increase the final amount of Ca²⁺ accumulated; evidence suggests that the latter phenomenon requires phosphate influx via the phosphate carrier. It is concluded that the earlier reports that phosphate augments the initial rate of Ca²⁺ influx reflects an effect of phosphate on succinate oxidation, which was employed in the previous studies, rather than an Ca²⁺ transport itself.     

Replacement of the B protein requirement of the E. coli quinolinate synthetase system by chemically-generated iminoaspartate

Two proteins (A and B) from $\text{Escherichia}\text{,}\text{coli}$ are required for $\text{in}\text{,}\text{vitro}$ synthesis of the NAD⁺ precursor, quinolinate, from L-aspartate and dihydroxyacetone phosphate. The requirement for B protein and L-aspartate in this system can be replaced by millimolar concentrations of oxaloacetate and ammonia if they are added together. This finding supports the concept that the B protein (L-aspartate oxidase) functions to form iminoaspartate which is condensed with dihydroxyacetone phosphate by the A protein to form quinolinate.     

Inhibition of mouse brain synaptosomal gamma-aminobutyric acid transport by pyridoxal 5'-phosphate

Crude lysates from a strain of enterotoxigenic $\text{E. coli}$ have been shown to catalyse the incorporation of [³²P] from [adenylate-³²P] NAD⁺ into an 11,000 dalton protein in rat liver membranes. [³²P] incorporation paralleled adenylate cyclase activation and the results suggest that the mechanism of action of the heat-labile $\text{E. coli}$ enterotoxin may involve ADP-ribosylation of an intracellular acceptor protein.     

Uncoupler and anaerobic resistant transport of phosphate in Escherichia coli

Active transport of inorganic phosphate into whole cells of a strain (AB3311) derived from $\text{Escherichia coli}$ K12 was found to be partially resistant to 50 μM carbonyl cyanide $\text{m}$-chlorophenyl hydrazone (CCCP), a powerful uncoupler of oxidative phosphorylation. The presence of 10 mM dithiothreitol (DTT) before the addition of CCCP completely prevented the inhibition of phosphate uptake caused by the uncoupler. The addition of DTT to the CCCP-inhibited system restored phosphate uptake to the control rate even when added 5 min after the phosphate transport assay was started. This uncoupler resistant transport is insensitive to anaerobiosis, or the addition of 10 mM KCN which reduces oxygen consumption to less than 1% that of aerobic controls. Additional studies of transport in a mutant (CBT302) deficient in membranebound Ca²⁺-, Mg²⁺-ATPase activity also demonstrated the retention of appreciable inorganic phosphate uptake under anaerobic conditions.     

The roles of cytochrome b5 in a reconstituted N-demethylase system containing cytochrome P-450.

Compound 102804 isolated from $\text{Bacillus cereus}$ has been found to be a potent inhibitor of the N⁵-methyltetrahydrofolate-homocysteine transmethylase isolated from $\text{Escherichia coli}$ B. This inhibition was noted when 102804 was added to the enzyme reaction mixture after the reaction started or concurrently with the preparation of the mixture. Chemically inactivated 102804 has no activity as an inhibitor of this enzyme system.     

Occurrence and induction of spermidine-N1-acetyltransferase in Escherichia coli

Spermidine acetylase activity was detected in extracts prepared from $\text{Escherichia coli}$ and there was a marked increase in activity over the early period of growth. This increase reached a maximum 3 h after inoculation and was followed by an increase in ornithine decarboxylase activity. The acetylase was also able to use spermine as a substrate, but not putrescine. With spermidine and acetyl-CoA as substrate, the product formed was exclusively N¹-acetyl-spermidine. This is the first evidence for the occurrence in bacteria of spermidine-N¹-acetyltransferase, an enzyme which has previously been described in mammalian cells. These results suggest that acetylation of spermidine may be involved in the growth of $\text{Escherichia coli}$ and in the regulation of its polyamine content.     

Occurrence in mammalian liver of a protein which replaces the B protein of E. coli quinolinate synthetase.

$\text{Escherichia coli}$ contains two proteins (A and B) which together convert dihydroxyacetone phosphate and aspartate to quinolinic acid, a precursor of NAD. Although mammalian liver homogenate does not catalyze this reaction it contains a protein which will replace the B protein of the $\text{E. coli}$ system. The behavior of the liver protein on Sephadex G-75 suggests it is much smaller than the $\text{E. coli}$ B protein. Liver B protein also appears to contain tightly bound FAD while FAD is easily removed from the $\text{E. coli}$ B protein. The pH optimum for the hybrid system $\text{E. coli}$ A protein-liver B protein is 9.0 while in the pure $\text{E. coli}$ system the optimum is pH 8.0. The hybrid system is inhibited by NAD to the same extent as the pure $\text{E. coli}$ system.     

Dependence of Escherichiacoli pyridine nucleotide transhydrogenase on phospholipids and its sensitivity to N-ethylmaleimide

A partially purified preparation of pyridine nucleotide transhydrogenase (E.C. 1.6.1.1.) (energy-independent) has been obtained from membranes of $\text{Escherichia}\text{coli}$ by means of deoxycholate extraction and DEAE-cellulose chromatography in the presence of Triton X-100. The enzyme was lipid-depleted by treating with cholate and ammonium sulfate. The preparation was reactivated by various phospholipids, in particular, bacterial cardiolipin and phosphatidyl glycerol. Phosphatidyl ethanolamine, the major phospholipid in the outer membrane of $\text{E.}\text{coli}$, was relatively ineffective in stimulating activity. The membrane-bound pyridine nucleotide transhydrogenase is slowly inhibited by N-ethylmaleimide. Protection against inhibition was achieved with NAD⁺ and NADP⁺, but NADPH served to accelerate the rate of inhibition.     

Lack of involvement of lipoic acid in membrane-associated energy transduction in Escherichia coli.

Oxidative phosphorylation, active transport of proline, aerobic- and ATP-driven proton translocation and transhydrogenation of NADP⁺ by NADH, occurred in lipoic acid-deficient cells or vesicles of a lipoic acid auxotroph of $\text{E. coli}$, W1485 lip 2. Addition of lipoic acid had little effect on these processes. Tributyltin chloride, which has been proposed to inhibit oxidative phosphorylation by reaction with lipoic acid (Cain $\text{et al.}$, Biochem. J. (1977) $\text{166}$, 593), was an effective inhibitor of aerobic and ATP-dependent proton translocation and transhydrogenation in lipoic acid-deficient vesicles from this organism. Our results do not support the proposal of Partis $\text{et al.}$ (FEBS Lett. (1977) $\text{75}$, 47) that lipoic acid is involved in the energy transducing processes associated with the membrane of $\text{E. coli}$.     

Cupric ion resistance as a new genetic marker of a temperature sensitive R plasmid, Rtsl in Escherichia coli.

A temperature sensitive kanamycin (Km) resistant R plasmid, Rtsl, was found to confer cupric ion (Cu²⁺) resistance on its hosts in $\text{Escherichia}\text{coli}$. At conjugal transfer, two kinds of segregants were obtained from Rtsl, i.e. Cu²⁺ resistant, Km sensitive and Km resistant, Cu²⁺ sensitive plasmids. Protein T existed in $\text{E.}\text{coli}$ cells harboring Rtsl or the Cu^rKm^s-plasmid. The inhibitory effect on the host cell growth at 43°C was observed with Rtsl⁺ or the Km^rCu^s-plasmid⁺ cells. A relationship between these Rtsl derivatives and Rtsl in $\text{Proteus}\text{mirabilis}$ which has been studied was discussed.     

Orthophosphate and histone dependent polyphosphate kinase from E. coli

A polyphosphate kinase has been purified over 100-fold from an extract of $\text{E.}\text{coli}$ K-12. It requires both orthophosphate and a basic protein (histone or protamine) for maximum activity. Because its activity is stimulated by histone, polyphosphate kinase may easily lead to an error in the determination of protein kinase in the cell extract. Our data suggest that the stimulatory effect of orthophosphate on polyphosphate kinase may be important in the regulation of phosphate metabolism in the microorganism.     

Influence of protease inhibitors and energy metabolism on intracellular protein breakdown in starving Escherichia coli

It has been reported that certain inhibitors of serine proteases block intracellular protein breakdown in $\text{E. coli}$ subjected to nutritional deprivation. We show here that the protease inhibitors p-toluene sulfonyl fluoride and pentamidine isethionate inhibit protein breakdown in $\text{E. coli}$ deprived of glucose, but not in bacteria starved for inorganic phosphate or ammonia. Furthermore, we find that the protease inhibitors cause a drastic decline in cellular ATP levels when glucose is omitted from the incubation medium. It is concluded that these protease inhibitors influence protein breakdown by interfering with cellular energy production, rather than by interacting with a specific serine protease.     

Peptide chain initiation with chemically formylated Met-tRNAs from E. coli and yeast

Chemically formylated Met-tRNA_m^Met and Met-tRNA_f^Met species from $\text{E.}$$\text{coli}$ and yeast were tested for their capacity to serve as chain-initiators in a cell-free system from $\text{E.}$$\text{coli}$. In the presence of R 17 mRNA, initiation factors and $\text{E.}$$\text{coli}$ ribosomes, all four Met-tRNAs could form functional initiation complexes as measured by ribosomal binding kinetics, fMet-puromycin formation and synthesis of a dipeptide fMet-Ala. Unformylated Met-tRNA_f^Met from $\text{E.}$$\text{coli}$ displayed significantly less activity as a peptide chain-initiator than the formylated Met-tRNA_m^Met species from $\text{E.}$$\text{coli}$ and yeast. Although the latter tRNAs were less effective initiators than the “physiological” initiator tRNAs, the data seem to indicate that a blocked α-amino group represents the major token of identification by which Met-tRNA is admitted to function in $\text{E.}$$\text{coli}$ peptide chain initiation.     

Demethylation of O6-methylguanine in a synthetic DNA polymer by an inducible activity in Escherichiacoli

$\text{O}$⁶-Methyl[8-³H]deoxyguanosine in a synthetic DNA polymer, poly(dC, dG, m⁶dG), is demethylated by cell-free extracts of $\text{Escherichia}\text{coli}\text{B}\text{r}$ adapted by exposure to $\text{N}$-methyl-$\text{N}$′-nitro-$\text{N}$-nitrosoguanidine, as shown by the appearance of ³H-labeled deoxyguanosine in hydrolysates of the recovered DNA. The demethylating activity could not be detected in extracts of nonadapted $\text{E}\text{.}\text{coli}$. These results provide direct evidence that a previously described inducible repair activity in $\text{E}\text{.}\text{coli}$ acts by demethylating $\text{O}$⁶-methylguanine at the DNA level.     

Isolation of an acyl-CoA carboxylase from the tapeworm spirometra mansonoides

An acyl-CoA carboxylase, which catalyzes the carboxylation of acetylpropionyl-, and butyryl-CoA, has been isolated from the tapeworm $\text{Spirometra}\text{mansonoides}$. The enzyme has an absolute requirement for ATP, Mg²⁺, and HCO₃${}^{\mathrm{?}}$ and, in addition, requires K⁺ for full catalytic activity. The enzyme has been purified 50-fold by a combination of calcium phosphate gel adsorption, ion-exchange column chromatography, and gel filtration. In its substrate specificity, K⁺ requirement, molecular size, and antigenic behavior, the tapeworm enzyme is similar to the acyl-CoA carboxylase of another helminth— the free-living nematode $\text{Turbatrix}\text{aceti}$.     

Correction for non-uniform phosphate labeling of E. coli and phage RNAs synthesized in vivo

$\text{E. coli}$ cells grown to phosphate starvation incorporate ³²PO₄ unequally into the α position of the four ribonucleotide triphosphates during a short period of labeling. A method for determining the relative specific activities of nucleotides in RNA molecules synthesized under these conditions and correcting sequence data is described.     

Pyridoxal 5' phosphate: a selective inhibitor of oncornaviral DNA polymerases.

Pyridoxal 5′ phosphate at concentrations < 0.5 mM inhibits $\text{polymerization}$ of deoxynucleoside triphosphate catalysed by variety of DNA polymerases isolated from type C RNA tumor viruses, as well as $\text{E.}\text{coli}$, but $\text{does}\text{not}$ affect the polymerase associated RNase H activity. Both phosphate and aldehyde groups of pyridoxal phosphate are essential for the inhibition which appears to be mediated through the reversible Schiff base.     

Origin of the labile sulfide in the iron-sulfur proteins of Escherichiacoli

A method has been devised for measuring the abundance of sulfur-34 in the hydrogen sulfide released upon the acidification of $\text{Escherichia}\text{coli}$ cells. Evidence is presented, based on the rate at which the hydrogen sulfide is released from the cells as well as the total amount released, that this hydrogen sulfide originates from the iron-sulfur proteins present in the cells. The sulfur-34 abundance in this hydrogen sulfide which was isolated from cells grown with [sulfane-³⁴S]thiocystine, a compound which can differentially label $\text{in}\text{vivo}$ the sulfur-34 abundance of cysteine and hydrogen sulfide, shows cysteine sulfur and not hydrogen sulfide to be the origin of the sulfide sulfur of iron-sulfur proteins in aerobically grown $\text{E.}\text{coli}$     

The incision and strand rejoining step in the excision repair of 5,6-dihydroxy-dihydrothymine by crude E. coli extracts

Strand resealing in the $\text{in}$$\text{vitro}$ excision repair of 5,6-dihydroxy-dihydrothymine in osmium tetroxide oxidized polyd(A-T) by crude $\text{E.}$$\text{coli}$ extracts is accomplished by polynucleotide ligase. Osmium tetroxide oxidized polyd(A-T)_serves as a chemically well defined model substrate containing damage of the kind introduced into DNA by ionizing radiation. In the first incision step of excision repair approximately one endonucleolytic nick is introduced into the polymer by extracts of $\text{E.}$$\text{coli}$ endoI^? and $\text{E.}$$\text{coli}$ endoI^?$\text{uvr}$A6^? per ring damaged thymine residue removed.