The panE Gene, Encoding Ketopantoate Reductase, Maps at 10 Minutes and Is Allelic to apbA in Salmonella typhimurium

In Salmonella typhimurium, precursors to the pyrimidine moiety of thiamine are synthesized de novo by the purine biosynthetic pathway or the alternative pyrimidine biosynthetic (APB) pathway. The apbA gene was the first locus defined as required for function of the APB pathway (D. M. Downs and L. Petersen, J. Bacteriol. 176:4858–4864, 1994). Recent work showed the ApbA protein catalyzes the NADPH-specific reduction of ketopantoic acid to pantoic acid. This activity had previously been associated with the pantothenate biosynthetic gene panE. Although previous reports placed panE at 87 min on the Escherichia coli chromosome, we show herein that apbA and panE are allelic and map to 10 min on both the S. typhimurium and E. coli chromosomes. Results presented here suggest that the role of ApbA in thiamine synthesis is indirect since in vivo labeling studies showed that pantoic acid, the product of the ApbA-catalyzed reaction, is not a direct precursor to thiamine via the APB pathway.     

Ketopantoate reductase activity is only encoded by ilvC in Corynebacterium glutamicum

Ketopantoate reductase catalyzes the second step of the pantothenate pathway after ketoisovalerate, common intermediate in valine, leucine and pantothenate biosynthesis. We show here that the Corynebacterium glutamicum ilvC gene is able to complement a ketopantoate reductase deficient Escherichia coli mutant. Thus ilvC, encoding acetohydroxyacid isomeroreductase, involved in the common pathway for branched-chained amino acids, also exhibits ketopantoate reductase activity. Enzymatic activity was confirmed by biochemical analysis in C. glutamicum. Furthermore, inactivation of ilvC in C. glutamicum leads to auxotrophy for pantothenate, indicating that ilvC is the only ketopantoate reductase- encoding gene in C. glutamicum.     

Metabolic Basis for the Isoleucine, Pantothenate or Methionine Requirement of ilvG Strains of Salmonella typhimurium

Salmonella typhimurium strain DU501, which was found to be deficient in acetohydroxy acid synthase II (AHAS II) and to possess elevated levels of transaminase B and biosynthetic threonine deaminase, required isoleucine, methionine, or pantothenate for growth. This strain accumulated α-ketobutyrate and, to a lesser extent, α-aminobutyrate. We found that α-ketobutyrate was a competitive substrate for ketopantoate hydroxymethyltransferase, the first enzyme in pantothenate biosynthesis. This competition with the normal substrate, α-ketoisovalerate, limited the supply of pantothenate, which resulted in a requirement for methionine. Evidence is presented to support the conclusion that the ambivalent requirement for either pantothenate or methionine is related to a decrease in succinyl coenzyme A, which is produced from pantothenate and which is an obligatory precursor of methionine biosynthesis. The autointoxification by endogenously produced α-ketobutyrate could be mimicked in wild-type S. typhimurium by exogenously supplied α-ketobutyrate or salicylate, a known inhibitor of pantothenate biosynthesis. The accumulation of α-ketobutyrate was initiated by the inability of the residual AHAS activity provided by AHAS I to efficiently remove the α-ketobutyrate produced by biosynthetic threonine deaminase. The accumulation of α-ketobutyrate was amplified by the action of transaminase B, which decreased the isoleucine pool by catalyzing the formation of α-keto-β-methylvalerate and aminobutyrate from isoleucine and α-ketobutyrate; this resulted in release of threonine deaminase from end product inhibition and unbridled production of α-ketobutyrate. Isoleucine satisfied the auxotrophic requirement of the AHAS II-deficient strain by curtailing the activity of threonine deaminase. Additional lines of evidence based on genetic and physiological experiments are presented to support the basis for the autointoxification of strain DU501 as well as other nonpolarigenic ilvG mutant strains.     

Feedback-Resistant Acetohydroxy Acid Synthase Increases Valine Production in Corynebacterium glutamicum

Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Km^r). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum ΔilvA ΔpanB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.     

Isolation and characterization of Bacillus subtilis mutants blocked in the synthesis of pantothenic acid.

We have produced and characterized by physiological and enzymatic analyses pantothenate (pan) auxotrophs of Bacillus subtilis. panB auxotrophs are deficient in ketopantoate hydroxymethyltransferase, whereas panE mutants lack ketopantoic acid reductase. The pan mutations were mapped by phage PBS1-mediated two-factor crosses and found to be located in the interval purE-tre of the genetic map of B. subtilis.     

Succinyl-Coenzyme A Synthetase and its Role in delta-Aminolevulinic Acid Biosynthesis in Euglena gracilis

Euglena gracilis cells synthesize the key tetrapyrrole precursor, δ-aminolevulinic acid (ALA), by two routes: plastid ALA is formed from glutamate via the transfer RNA-dependent five-carbon route, and ALA that serves as the precursor to mitochondrial hemes is formed by ALA synthase-catalyzed condensation of succinyl-coenzyme A and glycine. The biosynthetic source of succinyl-coenzyme A in Euglena is of interest because this species has been reported not to contain α-ketoglutarate dehydrogenase and not to use succinyl-coenzyme A as a tricarboxylic acid cycle intermediate. Instead, α-ketoglutarate is decarboxylated to form succinic semialdehyde, which is subsequently oxidized to form succinate. Desalted extract of Euglena cells catalyzed ALA formation in a reaction that required coenzyme A and GTP but did not require exogenous succinyl-coenzyme A synthetase. GTP could be replaced with ATP. Cell extract also catalyzed glycine-and α-ketoglutarate-dependent ALA formation in a reaction that required coenzyme A and GTP, was stimulated by NADP⁺, and was inhibited by NAD⁺. Succinyl-coenzyme A synthetase activity was detected in extracts of dark- and light-grown wild-type and nongreening mutant cells. In vitro succinyl-coenzyme A synthetase activity was at least 10-fold greater than ALA synthase activity. These results indicate that succinyl-coenzyme A synthetase is present in Euglena cells. Even though the enzyme may play no role in the transformation of α-ketoglutarate to succinate in the atypical tricarboxylic acid cycle, it catalyzes succinyl-coenzyme A formation from succinate for use in the biosynthesis of ALA and possibly other products.     

Effects of Dietary Pantothenic Acid on Growth,Intestinal Function,Anti-Oxidative Status and Fatty Acids Synthesis of Juvenile Blunt Snout Bream Megalobrama amblycephala

Four groups of juvenile Megalobrama amblycephala were fed three times daily with six semi-purified diets containing 3.39 (PA unsupplied diet), 10.54, 19.28, 31.04, 48.38 and 59.72 mg kg^-1 calcium D-pantothenate. The results showed that survival rate, final weight, specific growth rate, protein efficiency ratio and nitrogen retention efficiency all increased significantly (P<0.01) as dietary PA levels increased from 3.39 to 19.28 mg kg^-1, whereas the opposite was true for feed conversion ratio. Whole-body crude protein increased as dietary PA levels increased, while the opposite pattern was found for the crude lipid content. Intestinal α-amylase, lipase, protease, Na⁺-K⁺-ATPase, alkaline phosphatase and gamma-glutamyl transferase activities were all elevated in fish fed PA-supplemented diets. Hepatic catalase activities improved with increases in dietary PA, while the opposite was true for malondialdehyde contents. The liver PA concentration and coenzyme A content rose significantly (P<0.01), up to 31.04 mg kg^-1, with increasing dietary PA levels and then plateaued. The percentage of hepatic saturated fatty acids increased significantly (P<0.01) as dietary PA levels increased, while the percentages of monounsaturated fatty acids and polyunsaturated fatty acid (PUFA) decreased as dietary PA increased. Fish fed diets containing 19.28 and 31.04 mg kg^-1 PA exhibited higher (P<0.01) docosahexaenoic acid and PUFA percentages in muscle than those fed with other diets. The expression of the gene encoding pantothenate kinase was significantly up-regulated (P<0.01) in fish fed PA-supplemented diets. Hepatic Acetyl-CoA carboxylase α, fatty acid synthetase, stearoyl regulatory element-binding protein 1 and X receptor α genes all increased significantly (P<0.01) as dietary PA levels increased from 3.39 to 31.04 mg kg^-1. Based on broken-line regression analyses of weight gain, liver CoA concentrations and PA contents against dietary PA levels, the optimal dietary PA requirements of juvenile blunt snout bream were estimated to be 24.08 mg kg^-1.     

Modulation of epithelial sodium channel (ENaC) expression in mouse lung infected with Pseudomonas aeruginosa

Background

The intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the α, β and γ subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na⁺-K⁺-ATPase.

Methods

Pseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains. The mRNA expression of ENaC and Na⁺-K⁺-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection.

Results

The infection of the different mouse strains evoked regulation of α and β ENaC mRNA. Following Pseudomonas instillation, the expression of αENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of βENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of γENaC mRNA was detected although the general pattern of expression of the subunit was similar to α and β subunits. No modulation of α₁Na⁺-K⁺-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains.

Conclusions

These results show that Pseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.     

Pantoate Kinase and Phosphopantothenate Synthetase, Two Novel Enzymes Necessary for CoA Biosynthesis in the Archaea

Bacteria/eukaryotes share a common pathway for coenzyme A (CoA) biosynthesis. Although archaeal genomes harbor homologs for most of these enzymes, homologs of bacterial/eukaryotic pantothenate synthetase (PS) and pantothenate kinase (PanK) are missing. PS catalyzes the ATP-dependent condensation of pantoate and β-alanine to produce pantothenate, whereas PanK catalyzes the ATP-dependent phosphorylation of pantothenate to produce 4′-phosphopantothenate. When we examined the cell-free extracts of the hyperthermophilic archaeon Thermococcus kodakaraensis, PanK activity could not be detected. A search for putative kinase-encoding genes widely distributed in Archaea, but not present in bacteria/eukaryotes, led to four candidate genes. Among these genes, TK2141 encoded a protein with relatively low PanK activity. However, higher levels of activity were observed when pantothenate was replaced with pantoate. V_max values were 7-fold higher toward pantoate, indicating that TK2141 encoded a novel enzyme, pantoate kinase (PoK). A search for genes with a distribution similar to TK2141 led to the identification of TK1686. The protein product catalyzed the ATP-dependent conversion of phosphopantoate and β-alanine to produce 4′-phosphopantothenate and did not exhibit PS activity, indicating that TK1686 also encoded a novel enzyme, phosphopantothenate synthetase (PPS). Although the classic PS/PanK system performs condensation with β-alanine prior to phosphorylation, the PoK/PPS system performs condensation after phosphorylation of pantoate. Gene disruption of TK2141 and TK1686 led to CoA auxotrophy, indicating that both genes are necessary for CoA biosynthesis in T. kodakaraensis. Homologs of both genes are widely distributed among the Archaea, suggesting that the PoK/PPS system represents the pathway for 4′-phosphopantothenate biosynthesis in the Archaea.Coenzyme A (CoA)² and its derivative 4′-phosphopantetheine are essential cofactors in numerous metabolic pathways, including the tricarboxylic acid cycle, the β-oxidation pathway, and fatty acid and polyketide biosynthesis pathways. Acyl-CoA derivatives are key intermediates in energy metabolism due to their high energy thioester bonds and have been identified in all three domains of life.The mechanism of CoA biosynthesis in bacteria and eukaryotes has been well examined and involves common enzymatic conversions (1–3). CoA is synthesized from pantothenate via five enzymatic reactions; pantothenate kinase (PanK), 4′-phosphopantothenoylcysteine synthetase (PPCS), 4′-phosphopantothenoylcysteine decarboxylase (PPCDC), 4′- phosphopantetheine adenylyltransferase (PPAT), and dephospho-CoA kinase (DPCK). Although many animals rely on exogenous pantothenate to initiate CoA biosynthesis, microorganisms and plants can synthesize pantothenate from 2-oxoisovalerate and β-alanine. This is a three-step pathway catalyzed by ketopantoate hydroxymethyltransferase (KPHMT), ketopantoate reductase, and pantothenate synthetase (PS).In contrast to the wealth of knowledge on CoA biosynthesis in bacteria and eukaryotes, the corresponding pathway in the Archaea remains unclear (4). Sequence data indicate that the bacterial PPCS and PPCDC homologs and eukaryotic PPAT homologs are found on almost all of the archaeal genomes. The archaeal PPCS and PPCDC genes are fused in many cases, and the bifunctional protein from Methanocaldococcus jannaschii has been shown to exhibit both activities (5). The PPAT homolog from Pyrococcus abyssi has also been studied and confirmed to exhibit the expected PPAT activity (6). Bacterial KPHMT and ketopantoate reductase homologs can also be found, to a lesser extent, on the archaeal genomes. They are not found in the methanogens and Thermoplasmatales, and the fact that the structural similarity among archaeal enzymes is not higher than that toward enzymes from hyperthermophilic bacteria suggests that the archaeal KPHMT and ketopantoate reductase are a result of horizontal gene transfer from bacteria (4). In addition, there are candidate genes distantly related to bacterial/eukaryotic DPCK. However, PS homologs are not found in any of the archaeal genomes, and PanK homologs are found only in a few exceptional cases. Recently, Genschel and co-workers have taken a comparative genomics approach to predict the genes corresponding to the archaeal PS and PanK genes, and have also described the identification of a structurally novel PS from Methanosarcina mazei (4, 7).In this study, we describe the identification of the enzymes responsible for the conversion of pantoate to 4′-phosphopantothenate in Thermococcus kodakaraensis. The organism is a hyperthermophilic archaeon isolated from Kodakara Island, Japan (8, 9). The complete genome sequence is available (10), and gene disruption systems have been developed (11–13). To our surprise, the conversion of pantoate to 4′-phosphopantothenate in T. kodakaraensis is not brought about by the two classic enzyme reactions catalyzed by PS and PanK, but by two novel enzyme reactions; phosphorylation of pantoate (pantoate kinase) followed by the condensation of 4-phosphopantoate and β-alanine (4′-phosphopantothenate synthetase or 4-phosphopantoate:β-alanine ligase). Homologs of these two genes are distributed on almost all of the archaeal genomes, suggesting that the Archaea utilize different chemistry in the conversion from pantoate to 4′-phosphopantothenate.     

The Biosynthesis of delta-Aminolevulinic Acid in Higher Plants: II. Formation of C-delta-Aminolevulinic Acid from Labeled Precursors in Greening Plant Tissues

δ-Aminolevulinic acid was accumulated by greening cucumber (Cucumis sativus L. var. Alpha green) cotyledons, barley (Hordeum sativum var. Numar) leaves, and bean (Phaseolus vulgaris L. var. Red Kidney) leaves in the presence of various ¹⁴C-labeled precursors and levulinic acid, a competitive inhibitor of δ-aminolevulinic acid dehydrase. The radioactivity in the accumulated δ-aminolevulinic acid was measured.     

Construction of a Stable α-Galactosidase-Producing Baker's Yeast Strain

Molasses is widely used as a substrate for commercial yeast production. The complete hydrolysis of raffinose, which is present in beet molasses, by Saccharomyces strains requires the secretion of α-galactosidase, in addition to the secretion of invertase. Raffinose is not completely utilized by commercially available yeast strains used for baking, which are Mel⁻. In this study we integrated the yeast MEL1 gene, which codes for α-galactosidase, into a commercial mel⁰ baker's yeast strain. The Mel⁺ phenotype of the new strain was stable. The MEL1 gene was expressed when the new Mel⁺ baker's yeast was grown in molasses medium under conditions similar to those used for baker's yeast production at commercial factories. The α-galactosidase produced by this novel baker's yeast strain hydrolyzed all the melibiose that normally accumulates in the growth medium. As a consequence, additional carbohydrate was available to the yeasts for growth. The new strain also produced considerably more α-galactosidase than did a wild-type Mel⁺ strain and may prove useful for commercial production of α-galactosidase.     

Metabolism of Linoleic Acid or Mevalonate and 6-Pentyl-α-Pyrone Biosynthesis by Trichoderma Species

The understanding of the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species was achieved by using labelled linoleic acid or mevalonate as a tracer. Incubation of growing cultures of Trichoderma harzianum and T. viride with [U-¹⁴C]linoleic acid or [5-¹⁴C]sodium mevalonate revealed that both fungal strains were able to incorporate these labelled compounds (50 and 15%, respectively). Most intracellular radioactivity was found in the neutral lipid fraction. At the initial time of incubation, the radioactivity from [¹⁴C]linoleic acid was incorporated into 6-pentyl-α-pyrone more rapidly than that from [¹⁴C]mevalonate. No radioactivity incorporation was detected in 6-pentyl-α-pyrone when fungal cultures were incubated with [1-¹⁴C]linoleic acid. These results suggested that β-oxidation of linoleic acid was a probable main step in the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species.     

Diversity of Channels Generated by Different Combinations of Epithelial Sodium Channel Subunits

The epithelial sodium channel is a multimeric protein formed by three homologous subunits: α, β, and γ; each subunit contains only two transmembrane domains. The level of expression of each of the subunits is markedly different in various Na⁺ absorbing epithelia raising the possibility that channels with different subunit composition can function in vivo. We have examined the functional properties of channels formed by the association of α with β and of α with γ in the Xenopus oocyte expression system using two-microelectrode voltage clamp and patch-clamp techniques. We found that αβ channels differ from αγ channels in the following functional properties: (a) αβ channels expressed larger Na⁺ than Li⁺ currents (I_Na+/I_Li+ 1.2) whereas αγ channels expressed smaller Na⁺ than Li⁺ currents (I_Na+/I_Li+ 0.55); (b) the Michaelis Menten constants (K _m) of activation of current by increasing concentrations of external Na⁺ and Li⁺ of αβ channels were larger (K _m > 180 mM) than those of αγ channels (K _m of 35 and 50 mM, respectively); (c) single channel conductances of αβ channels (5.1 pS for Na⁺ and 4.2 pS for Li⁺) were smaller than those of αγ channels (6.5 pS for Na⁺ and 10.8 pS for Li⁺); (d) the half-inhibition constant (K _i) of amiloride was 20-fold larger for αβ channels than for αγ channels whereas the K _i of guanidinium was equal for both αβ and αγ. To identify the domains in the channel subunits involved in amiloride binding, we constructed several chimeras that contained the amino terminus of the γ subunit and the carboxy terminus of the β subunit. A stretch of 15 amino acids, immediately before the second transmembrane domain of the β subunit, was identified as the domain conferring lower amiloride affinity to the αβ channels. We provide evidence for the existence of two distinct binding sites for the amiloride molecule: one for the guanidium moiety and another for the pyrazine ring. At least two subunits α with β or γ contribute to these binding sites. Finally, we show that the most likely stoichiometry of αβ and αγ channels is 1α:1β and 1α:1γ, respectively.     

Metabolism of delta-Aminolevulinic Acid in Red and Blue-Green Algae

δ-Aminolevulinic acid was incorporated in vivo into C-phycocyanin and B-phycoerythrin in two species of the Rhodophyta (Cyanidium caldarium, Porphyridium cruentum) and three species of the Cyanophyta (Anacystis nidulans, Plectonema boryanum, Phormidium luridum). Amino acid analysis of phycocyanin-¹⁴C from C. caldarium cells which had been incubated with δ-aminolevulinate-4-¹⁴C showed that 84% of the radioactivity incorporated was present in the phycocyanobilin chromophore and less than 16% of the radioactivity cochromatographed with amino acids. These results indicate that δ-aminolevulinate is utilized predominantly via the porphyrin pathway in C. caldarium. Conversely, analysis of phycocyanin-¹⁴C prepared from cells of A. nidulans, P. boryanum, and P. luridum which had been incubated with radiolabeled δ-aminolevulinate demonstrated that 85%, 81%, and 93%, respectively, of the radioactivity incorporated cochromatographed with amino acids. The ratio of incorporated radioactivity in amino acids and phycoerythrobilin was 40:60 in P. cruentum phycoerythrin obtained from cells which had been incubated with δ-aminolevulinate-4-¹⁴C. Succinate-2-3-¹⁴C appeared to be as good a carbon source of amino acids as did C₄ and C₅ of δ-aminolevulinate. These data demonstrate a major alternate route (other than the porphyrin pathway) of δ-aminolevulinate metabolism in red and blue-green algae. The factors responsible for the extent to which δ-aminolevulinate is utilized for synthesis of porphyrins and their derivatives and routes of δ-aminolevulinate catabolism in the organisms employed are discussed.     

Construction and Characterization of Salmonella typhimurium Strains That Accumulate and Excrete α- and β- Isopropylmalate

Two Salmonella typhimurium strains, which could be used as sources for the leucine biosynthetic intermediates α- and β-isopropylmalate were constructed by a series of P22-mediated transductions. One strain, JK527 [flr-19 leuA2010 Δ(leuD-ara)798 fol-162], accumulated and excreted α-isopropylmalate, whereas the second strain, JK553 (flr-19 leuA2010 leuB698), accumulated and excreted α- and β-isopropylmalate. The yield of α-isopropylmalate isolated from the culture medium of JK527 was more than five times the amount obtained from a comparable volume of medium in which Neurospora crassa strain FLR₉₂-1-216 (normally used as the source for α- and β-isopropylmalate) was grown. Not only was the yield greater, but S. typhimurium strains are much easier to handle and grow to saturation much faster than N. crassa strains. The combination of the two regulatory mutations flr-19, which results in constitutive expression of the leucine operon, and leuA2010, which renders the first leucine-specific biosynthetic enzyme insensitive to feedback inhibition by leucine, generated limitations in the production of valine and pantothenic acid. The efficient, irreversible, and unregulated conversion of α-ketoisovaleric acid into α-isopropylmalate (α-isopropylmalate synthetase K_m for α-ketoisovaleric acid, 6 × 10⁻⁵ M) severely restricted the amount of α-ketoisovaleric acid available for conversion into valine and pantothenic acid (ketopantoate hydroxymethyltransferase K_m for α-ketoisovaleric acid, 1.1 × 10⁻³ M; transaminase B K_m for α-ketoisovaleric acid, 2 × 10⁻³ M).     

Specificity of Intersubunit General Anesthetic-binding Sites in the Transmembrane Domain of the Human α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor

GABA type A receptors (GABA_AR), the brain''s major inhibitory neurotransmitter receptors, are the targets for many general anesthetics, including volatile anesthetics, etomidate, propofol, and barbiturates. How such structurally diverse agents can act similarly as positive allosteric modulators of GABA_ARs remains unclear. Previously, photoreactive etomidate analogs identified two equivalent anesthetic-binding sites in the transmembrane domain at the β⁺-α⁻ subunit interfaces, which also contain the GABA-binding sites in the extracellular domain. Here, we used R-[³H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (R-mTFD-MPAB), a potent stereospecific barbiturate anesthetic, to photolabel expressed human α1β3γ2 GABA_ARs. Protein microsequencing revealed that R-[³H]mTFD-MPAB did not photolabel the etomidate sites at the β⁺-α⁻ subunit interfaces. Instead, it photolabeled sites at the α⁺-β⁻ and γ⁺-β⁻ subunit interfaces in the transmembrane domain. On the (+)-side, α1M3 was labeled at Ala-291 and Tyr-294 and γ2M3 at Ser-301, and on the (−)-side, β3M1 was labeled at Met-227. These residues, like those in the etomidate site, are located at subunit interfaces near the synaptic side of the transmembrane domain. The selectivity of R-etomidate for the β⁺-α⁻ interface relative to the α⁺-β⁻/γ⁺-β⁻ interfaces was >100-fold, whereas that of R-mTFD-MPAB for its sites was >50-fold. Each ligand could enhance photoincorporation of the other, demonstrating allosteric interactions between the sites. The structural heterogeneity of barbiturate, etomidate, and propofol derivatives is accommodated by varying selectivities for these two classes of sites. We hypothesize that binding at any of these homologous intersubunit sites is sufficient for anesthetic action and that this explains to some degree the puzzling structural heterogeneity of anesthetics.     

Production of Poly-β-Hydroxyalkanoic Acid by Pseudomonas cepacia

The possibility of using the nutritionally versatile bacterium Pseudomonas cepacia to produce poly-β-hydroxyalkanoic acid was evaluated. Chemostat culture showed that growth of P. cepacia became nitrogen limited when the molar carbon-to-nitrogen ratio of the medium fed into the fermentor was above 15. When grown under nitrogen limitation in batch culture with fructose as the sole source of carbon, P. cepacia accumulated poly-β-hydroxybutyric acid (PHB) in excess of 50% of the dry weight of its biomass. In batch culture, almost no PHB was produced until the onset of nitrogen limitation. After this point, PHB was produced at a linear rate of 0.12 g liter⁻¹ h⁻¹ (from a constant value of 1.6 g of cellular protein liter⁻¹). PHB produced by P. cepacia had a weight-average molecular weight of 5.37 × 10⁵ g mol⁻¹ and a polydispersivity index of 3.9. Poly(β-hydroxybutyric acid-β-hydroxyvaleric acid) copolymer was produced with a poly-β-hydroxybutyric acid-poly-β-hydroxyvaleric acid ratio of up to 30% by weight when propionic acid was added to the medium.     

Pantothenate production in Escherichia coli K12 by enhanced expression of the panE gene encoding ketopantoate reductase.

Using gene replacement and transposon Tn5 mutagenesis, an Escherichia coli ilvC panE double mutant completely lacking ketopantoate reductase activity was isolated. This E. coli double mutant was employed to isolate the E. coli panE gene by genetic complementation. The E. coli panE gene is characterized by a 912 bp coding region, which specifies a protein of 303 amino acids with a deduced molecular mass of 33.8 kD. A panE expression plasmid carrying the panE gene under the control of the tac promotor was constructed. Introduction of the panE expression plasmid into E. coli resulted in a threefold increase in ketopantoate reductase activity. It was also shown that the enhanced panE expression in E. coli K12 led to 3.5-fold increase in pantothenate excretion. Pantothenate excretion could even be more enhanced when the growth medium was supplemented with ketopantoate.     

Pleiotropic Mutations at the TUP1 Locus That Affect the Expression of Mating-Type-Dependent Functions in SACCHAROMYCES CEREVISIAE

The umr7–1 mutation, previously identified in a set of mutants that had been selected for defective UV-induced mutagenesis at CAN1, affects other cellular functions, including many of those regulated by the mating-type locus (MAT) in heterothallic Saccharomyces cerevisiae. The recessive umr7–1 allele, mapping approximately 20 cM distal to thr4 on chromosome III, causes clumpy growth in both a and α cells and has no apparent effect on a mating functions. However, α umr7 meiotic segregants fail to express several α-specific functions (e.g., high-frequency conjugation with a strains, secretion of the hormone α-factor and response to the hormone a-factor). In addition, α umr7 cells exhibit some a-specific characteristics, such as the barrier phenotype (Bar⁺) that prevents diffusion of α-factor and an increased mating frequency with α strains. The most striking property of α umr7 strains is their altered morphology, in which mitotic cells develop an asymmetric pear shape, like that of normal a cells induced to form "shmoos" by interaction with α-factor. Some a/α-specific diploid functions are also affected by umr7; instead of polar budding patterns, a/α umr7/umr7 diploids have medial budding like a/a, α/α and haploid strains. Moreover, a/α umr7/umr7 diploids have lost the ability to sporulate and are Bar⁺ like a or a/a strains. Revertant studies indicate that umr7–1 is a single point mutation. The umr7 mutant fails to complement mutants of both tup1 (selected for deoxythymidine monophosphate utilization) and cyc9 (selected for high iso-2-cytochrome c levels), and all three isolates have similar genetic and phenotypic properties. It is suggested that the product of this gene plays some common central role in the complex regulation of the expression of both MAT-dependent and MAT-independent functions.     

Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells : Effects of Na+ and Ca2+

The epithelial Na⁺ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na⁺ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na⁺ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na⁺ conductance was Li⁺ > Na⁺ >> K⁺ = N-methyl-d-glucamine⁺ (NMDG⁺). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na⁺ channel current, was found to be ∼5 and 8 pS when Na⁺ and Li⁺ were used as a charge carrier, respectively. K⁺ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P _o), was increased by membrane hyperpolarization. Both whole-cell Na⁺ current and conductance were saturated with increased extracellular Na⁺ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP _o) was significantly decreased when cytosolic Na⁺ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na⁺ conductance (with Li⁺ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca²⁺ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca²⁺ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca²⁺ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca²⁺ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca²⁺ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na⁺ and Ca²⁺.