A new isochorismate synthase specifically involved in menaquinone (vitamin K2) biosynthesis encoded by the menF gene

Abstract A new gene ( menF ) encoding an isochorismate synthase specifically involved in menaquinone (vitamin K₂) biosynthesis has been cloned and sequenced. Overexpression of the encoded polypeptide under the influence of a T7 promoter showed an increase in specific activity of 2200-fold. Treatment with protamine sulfate resulted in another 3.5-fold increase in specific activity (7700-fold compared to the parent strain). The relative molecular mass of the overexpressed protein was M _r 49 000, which is in full agreement with the DNA sequence predicted molecular mass of 48777 Da. Purified enzyme converted chorismate to isochorismate with the product of the reaction shown to be isochorismate by its thermal conversion to salicylic acid. The fluorescence spectrum generated by the formed salicylic acid was identical to that of authentic salicylic acid. The 5' end of the flanking menD gene has also been redefined.     

Tyrosine Hydroxylase Phosphorylation in Bovine Adrenal Chromaffin Cells: The Role of Intracellular Ca2+ in the Histamine H1 Receptor-Stimulated Phosphorylation of Ser8, Ser19, Ser31, and Ser40

Abstract: Tyrosine hydroxylase (TOH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated by phosphorylation. Activation of histaminergic H₁ receptors on cultured bovine adrenal chromaffin cells stimulated a rapid increase in TOH phosphorylation (within 5 s) that was sustained for at least 5 min. The initial increase in TOH phosphorylation (up to 1 min) was essentially unchanged by the removal of extracellular Ca²⁺. In contrast, the H₁-mediated response was abolished by preloading the cells with BAPTA acetoxymethyl ester (50 µ M ) and significantly reduced by prior exposure to caffeine (10 m M for 10 min) to deplete intracellular Ca²⁺. Trypticphosphopeptide analysis by HPLC revealed that the H₁ response in the presence or absence of extracellular Ca²⁺ resulted in a major increase in the phosphorylation of Ser¹⁹ with smaller increases in that of Ser⁴⁰ and Ser³¹. In contrast, although a brief stimulation with nicotine (30 µ M for 60 s) also resulted in a major increase in Ser¹⁹ phosphorylation, this response was abolished in the absence of extracellular Ca²⁺. These data indicate that the mobilization of intracellular Ca²⁺ plays a crucial role in supporting H₁-mediated TOH phosphorylation and may thus have a potentially important role in regulating catecholamine synthesis.     

Alkalinization Prolongs Recovery from Glutamate-Induced Increases in Intracellular Ca2+ Concentration by Enhancing Ca2+ Efflux Through the Mitochondrial Na+/Ca2+ Exchanger in Cultured Rat Forebrain Neurons

Abstract: Increasing extracellular pH from 7.4 to 8.5 caused a dramatic increase in the time required to recover from a glutamate (3 µ M , for 15 s)-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in indo-1-loaded cultured cortical neurons. Recovery time in pH 7.4 HEPES-buffered saline solution (HBSS) was 126 ± 30 s, whereas recovery time was 216 ± 19 s when the pH was increased to 8.5. Removal of extracellular Ca²⁺ did not inhibit the prolongation of recovery caused by increasing pH. Extracellular alkalinization caused rapid intracellular alkalinization following glutamate exposure, suggesting that pH 8.5 HBSS may delay Ca²⁺ recovery by affecting intraneuronal Ca²⁺ buffering mechanisms, rather than an exclusively extracellular effect. The effect of pH 8.5 HBSS on Ca²⁺ recovery was similar to the effect of the mitochondrial uncoupler carbonyl cyanide p -(trifluoromethoxyphenyl)hydrazone (FCCP; 750 n M ). However, pH 8.5 HBSS did not have a quantitative effect on mitochondrial membrane potential comparable to that of FCCP in neurons loaded with a potential-sensitive fluorescent indicator, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1). We found that the effect of pH 8.5 HBSS on Ca²⁺ recovery was completely inhibited by the mitochondrial Na⁺/Ca²⁺ exchange inhibitor CGP-37157 (25 µ M ). This suggests that increased mitochondrial Ca²⁺ efflux via the mitochondrial Na²⁺/Ca²⁺ exchanger is responsible for the prolongation of [Ca²⁺]_i recovery caused by alkaline pH following glutamate exposure.     

Acyl-coenzyme A: isopenicillin N acyltransferase from Penicillium chrysogenum: effect of amino acid substitutions at Ser227, Ser230 and Ser309 on proenzyme cleavage and activity

Abstract Using a high level Escherichia coli expression system for the Penicillium chrysogenum penDE gene, we have produced acyl-coenzyme A: isopenicillin N acyltransferase (AT) enzymes containing amino acid substitutions at three conserved Ser residues. Chosen for study based on amino acid sequence homologies to other proteins, Ser²²⁷, Ser²³⁰ and Ser³⁰⁹ were changed to Cys or Ala to assess amino acid side chain involvement in proenzyme cleavage and AT enzyme mechanism. Substitutions at Ser²³⁰ had no effect on proenzyme cleavage, acyl-coenzyme A: IPN acyltransferase (IAT) or acyl-coenzyme A: 6-aminopenicillanic acid acyltransferase (AAT) activities. While Ser²²⁷→Cys had no effect, Ser²²⁷→Ala produced uncleaved proenzyme lacking both AAT and IAT activities, suggesting that the presence of a nucleophilic side chain at this residue is required for proenzyme cleavage and AT activity. Substitution of Ser³⁰⁹→Cys did not appreciably prevent proenzyme cleavage, IAT or AAT activity. Recombinant AT (recAT) proenzyme containing Ser³⁰⁹→Ala was cleaved; however, IAT and AAT activities were not observed. This separation of proenzyme cleavage from IAT and AAT activities has not been previously observed, and suggests that Ser³⁰⁹ is involved in substrate acylation.     

Excretion of ions (Cd2+, Li+, Na+ and Cl−) by Tamarix aphylla

Excretion of minerals by the NaCl-resistant and comparatively cadmium-resistant tree Tamarix aphylla (L.) Karst, was investigated. Cd²⁺ was excreted by plants exposed for 1–10 days to 9 or 45 μ M Cd²⁺ solutions. Excretion of this toxic ion increased considerably with time but was less than 5% of the quantities that had been accumulated in the shoots. Excretion of Na⁺ and Cl⁻ was positively correlated with NaCl concentration (1.5, 10, 50 m M ) of the medium. The Na⁺/Cl⁻ ratios of the excrete were positively correlated with the concentration of the treatment solution. Ca²⁺ excretion decreased with increasing NaCl concentrations of the solution. Excretion of K⁺ and Mg²⁺ was only little affected by NaCl. Excretion of Li⁺ occurred whenever this element was supplied in the uptake solution; daily excretion rates of Li⁺ increased with time. The ecological significance of excretion is discussed in relation to the low selectivity of the mechanism in T. aphylla .     

Cloning and Characterization of an ATPase Gene from Pneumocystis carinii which Closely Resembles Fungal H+ ATPases

ABSTRACT. A gene encoding a P-type cation translocating ATPase was cloned from a genomic library of rat-derived Pneumocystis carinii. The nucleotide sequence of the gene contains a 2781 base-pair open reading frame that is predicted to encode a 101, 401 dalton protein composed of 927 amino acids. The P. carinii ATPase protein (pcal) is 69–75% identical when compared with eight proton pumps from six fungal species. The Pneumocystis ATPase is less than 34% identical to ATPase proteins from protozoans, vertebrates or the Ca⁺⁺ ATPases of yeast. The P. carinii ATPase contains 115 of 121 residues previously identified as characteristic of H⁺ ATPases. Alignment of the Pneumocystis and fungal proton pumps reveals five homologous domains specific for fungal H⁺ ATPases.     

Passive uptake of K+(86Rb+) in sunflower roots at low external K+ concentrations

Passive fluxes of K⁺ (⁸⁶Rb) into roots of sunflower ( Helianthus annuus L. cv. Uniflorus) were determined at low K⁺ concentration (0.1 and 1.0 mM K⁺) in the ambient solution. Metabolic uptake of K⁺ was inhibited by 10⁻⁴M 2,4-dinitrophenol (DNP). K⁺ (⁸⁶Rb) fluxes were studied both continuously and by time differentiation of uptake. In high K⁺ roots passive uptake was directly proportional to the K⁺ concentration of the uptake solution, indicating free diffusion. This assumption was supported by the fact that passive Rb⁺ uptake was not affected by high K⁺ concentrations. In low K⁺ roots the passive uptake of K⁺ was higher than in high K⁺ roots. The increase was possibly due to carrier-mediated K⁺ transport. As K⁺ effluxes were quantitatively similar to influxes, it is suggested that passive K⁺ fluxes represent exchange diffusion without relation to net K⁺ transport.     

N 5, N10-Methylenetetrahydromethanopterin reductase from Methanosarcina barkeri

N ⁵ N ¹⁰-Methylenetetrahydromethanopterin reductase was purified 13-fold to apparent homogeneity from methanol grown Methanosarcina barkeri . The colourless enzyme was found to be composed of four identical subunits of apparent molecular mass 36 kDa. It catalysed the reduction of methylenetetrahydromethanopterin ( K _m=15 μM) to methyltetrahydromethanopterin with reduced coenzyme F₄₂₀ ( K _m=12 μM) at a specific rate ( V _max) of 2200 μmol min⁻¹· mg protein⁻¹ ( K _cat=1320 s⁻¹). With respect to coenzyme specificity, molecular properties and catalytic mechanism the enzyme was found to be similar to CH₂=H₄MPT reductase of Methanobacterium thermoautotrophicum which phylogenetically is only distantly related to M. barkeri .     

Nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II and analyses of the deduced protein sequence

Abstract The complete nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II (EII^Man) was determined. The gene consisted of 2052 base pairs encoding a protein of 683 amino acid residues; the molecular mass of the protein subunit was calculated to be 72570 Da. The N-terminal hydrophilic domain of EII^Man showed 39.7% homology with a C-terminal hydrophilic domain of Escherichia coli glucose-specific enzyme II (EII^Glc). Similar homology was shown between the C-terminal sequence of EII^Man and the E. coli glucose-specific enzyme III (EIII^Glc), or the EIII-like domain of Streptococcus mutans sucrose-specific enzyme II. Sequence comparison with other EIIs showed that EII^Man contained residues His-602 and Cys-28 which were homologous to the potential phosphorylation sites of EIII^Glc, or EIII-like domains, and hydrophilic domains (IIB) of several EIIs, respectively.     

l-Methionine-dl-sulfoximine resistant Het+ Nif+ and Het− Nif− strains of Nostoc muscorum assimilating methylamine as ammonium nitrogen source

Abstract The Het⁺ Nif⁺ and Het⁻ Nif⁻ strains of Nostoc muscorum sensitive to growth inhibition by methylamine (MA), overcame the MA inhibition as a result of their mutation to l -methionine- dl -sulfoximide (MSX)-resistant phenotype, which enabled them to assimilate MA like an ammonium nitrogen source. The MSX-resistant Het⁺ Nif⁺ strain synthesized the inhibitor-resistant transferase-defective glutamine synthetase (GS), which unlike parental GS underwent MA-dependent in vivo activation. These results suggest the involvement of GS enzyme in control of MA assimilation in cyanobacteria.     

Sucrose and ouabain effects on the kinetic properties of a membrane-bound (Na++ K++ Mg2+) ATPase in sugar beet roots

Kinetic studies of a microsomal (Na⁺+ K⁺+ Mg²⁺)ATPase from sugar beet roots ( Beta vulgaris L. cv. Monohill) show that sucrose influences the MgATPase in different ways depending on the presence of K⁺ and/or Na⁺ 1) In the presence of the substrate MgATP and Na⁺ the effect of sucrose follows simple Michaelis-Menten kinetics. 2) In the presence of substrate together with K⁺ or (K⁺+ Na⁺), sucrose has little effect on the ATPase activity. 3) In the presence of Na⁺, onabain acts as an uncompetitive inhibitor with respect to MgATP. 4) In the presence of K⁺ or (K⁺+ Na⁺), the inhibition by ouabain is somewhat depressed and shows non-linearity when 1/v is plotted versus 1/MgATP. 5) Sucrose and Na⁺ activate in a competitive way, so that a successive increase of the Na⁺ level decreases the activation by sucrose. Both K_m and V-values are thereby changed. 6) The sucrose activation in the presence of Na⁺ is also influenced by ouabain. It is, therefore, suggested that Na⁺ may regulate the interference between the Na⁺/K⁺ pump and a sucrose sensitive system.     

Demonstration of an Na+/H+ exchanger in mouse keratinocytes measured by the novel pH-sensitive fluorochrome SNARF-calcein

In many cell types cytoplasmic alkalization is an early marker for cell activation. An amiloride-sensitive Na⁺/H⁺ exchanger is an important regulator of this process. However, in keratinocytes the existence of a Na⁺/H⁺ exchanger nor a proliferation-associated increase in intracellular pH (pH_i) has been demonstrated.
The aim of this study was to investigate whether or not keratinocytes, derived from the BALB/MK cell line, contain a Na⁺/H⁺ exchanger and whether cytoplasmic alkalization is proliferation-associated in these cells. This mouse keratinocyte cell line can easily be switched between a proliferative and a quiescent state under defined culture conditions. The novel pH-sensitive dye seminaphthorhodafluor (SNARF)-calcein proved to be very suitable for flow cytometric pH_i measurements in BALB/MK cells. Initial measurements of the pH_i using a cocktail of the established fluorochromes 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and SNARF-1 failed because of the differential uptake and binding kinetics of these pH-sensitive dyes.
Using SNARF-calcein we were able to show proliferation to be associated with increased pH_i. However, culture conditions were critical for these measurements. Our data indicate that the Na⁺/H⁺ exchanger is involved in this process, since acid load and pH_i-recovery experiments showed the alkalization to be amiloride-sensitive.     

Ca2+ dependence and La3+ interference of ultraviolet radiationinduced K+ efflux from rose cells

A low fluence of ultraviolet radiation (UV) causes cultured cells of Rosa damascena Mill cv. Gloire de Guilan to lose intracellular K⁺. This effect required the presence of Ca²⁺ in the medium. A reduction in the concentration of free Ca²⁺ to 10⁻⁵ M with ethyleneglycol-bis-(β-aminoethyl-ether)-N.N.N',N'-tetraacetic acid (EGTA) buffer inhibited the UV-stimulated efflux; this was correlated with a discharge of the membrane potential and a stimulation of the leakage of K⁺ from unirradiated cells. All the same effects were seen with La³⁺ at 0.2 m M. At 0.02 m M La³⁺, the UV-stimulated efflux of K⁺ was blocked without concomitant effects on the membrane potential or K⁺ efflux from control cells. It is suggested that removal of Ca²⁺ blocks or masks the UV-induced leakage of K⁺ by destabilizing the plasma membrane. In addition, La³⁺ may specifically inhibit the UV-stimulated opening of K⁺ or anion channels.     

Hydrolysis of glucuronide-based substrates mediated by tungsten, Cu2+, Fe2+, and Zn2+

A variety of metal microprojectiles are currently used for carrying foreign DNA into living cells via particle-acceleration techniques. While developing a microprojectile-mediated protocol for transforming cells of sugarbeet ( Beta vulgaris L.), formation of a blue precipitate was observed with the indigoqenic substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-gluc) in the absence of gusA DNA encoding β-D-glucuronidase (GUS). Tungsten microcarriers, but not gold or silicon carbide, proved capable of catalyzing the cleavage of the glucuronide residue from three histochemical substrates evaluated: X-gluc, salmon X-gluc and magenta X-gluc. Indigo-stained sugarbeet cells were observed following bombardment with tungsten in the absence of DNA. Addition of oxidative catalysts to tungsten microcarriers during substrate incubation had no apparent effect on the metal-mediated catalysis. Treatment of microcarriers with Proteinase K and heat ruled out the presence of enzymes. Microbiological evaluation indicated the absence of contaminating microbes. Similarly, metal-catalyzed hydrolysis of the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronic acid (4-MUG) was observed in the presence of tungsten spheres but not with gold or silicon carbide particles. With this substrate, hydrolysis also occurred with millimolar concentrations of Cu²⁺, Fe²⁺ and Zn²⁺ ions. Consequently, careful monitoring of DNA-minus controls and avoidance of millimolar concentrations of Cu²⁺, Fe²⁺ and Zn²⁺ ions are recommended in microprojectile bombardment experiments where transient assays for gusA expression are performed.