Differential effects of stimulus termination on excitation and desensitization of folic acid receptors and guanylate cyclase in Dictyostelium discoideum

The response of guanylate cyclase to addition of extracellular stimuli is well documented. Here we report for the first time the response of guanylate cyclase to removal of stimuli. Three methods were employed to terminate rapidly a stimulus of folic acid. (1) Addition of a highly active folate deaminase preparation, or (2) 12-fold dilution of the stimulated cell suspension, or (3) addition of an excess concentration of a non-agonistic derivative of folic acid, i.e., 2-deaminofolic acid, which chases the folate agonist from its cell-surface receptors. Accumulation of cGMP terminated instantaneously upon addition of deaminase, but degradation of the synthesized cGMP was not observed until 10–12 s after stimulation. Also in a cGMP phosphodiesterase-lacking ‘streamer’ mutant an instantaneous termination of further cGMP accumulation was observed upon stimulus removal. This suggests that the termination of cGMP accumulation is due to inactivation of guanylate cyclase instead of a steady state of cGMP synthesis and degradation. Further accumulation of cGMP was approx. 75% reduced upon dilution of a cell suspension after stimulation with both agonists. Stimulation by 300 nM folic acid or by 30 nM N¹⁰-methylfolic acid (a more potent agonist) yielded identical results. However, upon addition of deaminofolic acid the accumulation of cGMP continued normally if the cells had been stimulated with N¹⁰-methylfolic acid, but only slightly in the case of a folic acid stimulus. The effect of stimulus duration on desensitization was monitored; it was observed that 50% desensitization was induced by stimulation for 1 s, while 4 s was sufficient for maximal desensitization. Short stimuli were observed to elicit high levels of desensitization without much excitation of guanylate cyclase. A desensitization-like process was observed at the level of the folate-binding chemotactic receptors as well. Relationships between the cGMP response data and folic acid receptor kinetics are discussed.     

Subunit-subunit interactions in TF1 as revealed by ligand binding to isolated and integrated α and β subunits

The binding of 3′-O-(1-naphthoyl)adenosinetriphosphate (1-naphthoyl-ATP), ATP and ADP to TF₁ and to the isolated α and β subunits was investigated by measuring changes of intrinsic protein fluorescence and of fluorescence anisotropy of 1-naphthoyl-ATP upon binding. The following results were obtained. (1) The isolated α and β subunits bind 1 mol 1-naphthoyl-ATP with a dissociation constant (K_D(1-naphthoyl-ATP)) of 4.6 μM and 1.9 μM, respectively. (2) The K_D(ATP) for α and β subunits is 8 μM and 11 μM, respectively. (3) The K_D(ADP) for α and β subunits is 38 μM μM and 7 μM, respectively. (4) TF₁ binds 2 mol 1-naphthoyl-ATP per mol enzyme with K_D = 170 nM. (5) The rate constant for 1-naphthoyl-ATP binding to α and β subunit is more than 5 · 10⁴ M⁻¹s⁻¹. (6) The rate constant for 1-naphthoyl-ATP binding to TF₁ is 6.6 · 10³ M⁻¹ · s⁻¹ (monophasic reaction); the rate constant for its dissociation in the presence of ATP is biphasic with a fast first phase (k^A₋₁ = 3 · 10⁻³s⁻¹) and a slower second phase (k^A₋₂ < 0.2 · 10⁻³s⁻¹). From the appearance of a second peak in the fluorescence emission spectrum of 1-naphthoyl-ATP upon binding it is concluded that the binding sites in TF₁ are located in an environment more hydrophobic than the binding sites on isolated α and β subunits. The differences in kinetic and thermodynamic parameters for ligand binding to isolated versus integrated α and β subunits, respectively, are explained by interactions between these subunits in the enzyme complex.     

Anion inhibition studies of two new β-carbonic anhydrases from the bacterial pathogen Legionella pneumophila

We investigated the cloning, catalytic activity and anion inhibition of the β-class carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Legionella pneumophila. Two such enzymes, lpCA1 and lpCA2, were found in the genome of this pathogen. These enzymes were determined to be efficient catalysts for CO₂ hydration, with k_cat values in the range of (3.4–8.3) × 10⁵ s⁻¹ and k_cat/K_M values of (4.7–8.5) × 10⁷ M⁻¹ s⁻¹. A set of inorganic anions and small molecules was investigated to identify inhibitors of these enzymes. Perchlorate and tetrafluoroborate were not acting as inhibitors (K_I >200 mM), whereas sulfate was a very weak inhibitor for both lpCA1 and lpCA2 (K_I values of 77.9–96.5 mM). The most potent lpCA1 inhibitors were cyanide, azide, hydrogen sulfide, diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K_I values ranging from 6 to 94 μM. The most potent lpCA2 inhibitors were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, with K_I values ranging from 2 to 13 μM. As these enzymes seem to be involved in regulation of phagosome pH during Legionella infection, inhibition of these targets may lead to antibacterial agents with a novel mechanism of action.     

Cloning,characterization and anion inhibition studies of a γ-carbonic anhydrase from the Antarctic bacterium Colwellia psychrerythraea

We have cloned, purified and characterized the γ-carbonic anhydrase (CA, EC 4.2.1.1) present in the genome of the Antarctic bacterium Colwellia psychrerythraea, which is an obligate psychrophile. The enzyme shows a significant catalytic activity for the physiologic reaction of CO₂ hydration to bicarbonate and protons, with the following kinetic parameters: k_cat of 6.0 × 10⁵ s⁻¹ and a k_cat/K_m of 4.7 × 10⁶ M⁻¹ × s⁻¹. This activity was inhibited by the sulfonamide CA inhibitor (CAI) acetazolamide, with a K_I of 502 nM. A range of anions was also investigated for their inhibitory action against the new enzyme CpsCA. Perchlorate, tetrafluoroborate, fluoride and bromide were not inhibitory, whereas cyanate, thiocyanate, cyanide, hydrogensulfide, carbonate and bicarbonate showed K_Is in the range of 1.4–4.4 mM. Diethyldithiocarbamate was a better inhibitor (K_I of 0.58 mM) whereas sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid were the most effective inhibitors detected, with K_Is ranging between 8 and 38 μM. The present study may shed some more light regarding the role that γ-CAs play in the life cycle of psychrophilic bacteria as the Antarctic one investigated here.     

Anion inhibition study of the β-carbonic anhydrase (CahB1) from the cyanobacterium Coleofasciculus chthonoplastes (ex-Microcoleus chthonoplastes)

We investigated the catalytic activity and inhibition of the β-class carbonic anhydrase (CA, EC 4.2.1.1) CahB1, from the relict cyanobacterium Coleofasciculus chthonoplastes (previously denominated Microcoleus chthonoplastes). The enzyme showed good activity as a catalyst for the CO₂ hydration, with a k_cat of 2.4 × 10⁵ s⁻¹ and a k_cat/K_m of 6.3 × 10⁷ M⁻¹ s⁻¹. A range of inorganic anions and small molecules were investigated as inhibitors of CahB1. Perchlorate and tetrafluoroborate did not inhibit the enzyme (K_Is >200 mM) whereas selenate and selenocyanide were ineffective inhibitors too, with K_Is of 29.9–48.61 mM. The halides, pseudohalides, carbonate, bicarbonate, trithiocarbonate and a range of heavy metal ions-containing anions were submillimolar–millimolar inhibitors (K_Is in the range of 0.15–0.90 mM). The best CahB1 inhibitors were N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K_Is in the range of 8–75 μM, whereas acetazolamide inhibited the enzyme with a K_I of 76 nM. This is the first kinetic and inhibition study of a cyanobacterial CA. As these enzymes are widespread in many cyanobacteria, being crucial for the carbon concentrating mechanism which assures substrate to RubisCO for the CO₂ fixation by these organisms, a detailed kinetic/inhibition study may be essential for a better understanding of this superfamily of metalloenzymes and for potential biotechnological applications in biomimetic CO₂ capture processes.     

Tissue kallikrein processes small proenkephalin peptides

Tissue kallikrein may play a role in processing precursor polypeptide hormones. We investigated whether hydrolysis of natural enkephalin precursors, peptide F and bovine adrenal medulla docosapeptide (BAM-22P), by hog pancreatic kallikrein is consistent with this concept. Incubation of peptide F with this tissue kallikrein resulted in the release of Met⁵-enkephalin and Met⁵-Lys⁶-enkephalin. Met⁵-Lys⁶-enkephalin was the main peptide released, indicating that the major cleavage site was between two lysine residues. At 37°C and pH 8.5, the K_M values for formation of Met⁵-enkephalin and Met⁵-Lys⁶-enkephalin were 129 and 191 μM, respectively. Corresponding k_cat values were 0.001 and 0.03 s⁻¹ and k_cat/K_M ratios were 8 and 1.6·10² M⁻¹ · s⁻¹, respectively. Cleavage of peptide F at acidic pH (5.5) was negligible. When BAM-22P was used as a substrate, Met⁵-Arg⁶-enkephalin was released, thus indicating cleavage between two arginine residues. At pH 8.5, K_M was 64 μM, k_cat was 4.5 s⁻¹, and the k_cat/K_M ratio was 7 · 10⁴ M⁻¹ · s⁻¹. At 5.5, the pH of the secretory granules, K_M, k_cat and k_cat/K_M were 184 μM, 1.9 s⁻¹ and 10⁴ M⁻¹ · s⁻¹, respectively. It is unlikely that peptide F could be a substrate for kallikrein in vivo; however, tissue kallikrein could aid in processing proenkephalin precursors such as BAM-22P by cleaving Arg-Arg peptide bonds.     

Cloning,characterization and anion inhibition study of the δ-class carbonic anhydrase (TweCA) from the marine diatom Thalassiosira weissflogii

We investigated the catalytic activity and inhibition of the δ-class carbonic anhydrase (CA, EC 4.2.1.1) from the marine diatom Thalassiosira weissflogii, TweCA. The enzyme, obtained by cloning the synthetic gene, was an efficient catalyst for the CO₂ hydration, its physiological reaction, with a k_cat of 1.3 × 10⁵ s⁻¹ and a k_cat/K_M of 3.3 × 10⁷ M⁻¹ s⁻¹. A range of inorganic anions and small molecules were investigated as inhibitors of TweCA. Chloride and sulfate did not inhibit the enzyme (K_Is >200 mM) whereas other halides and pseudohalides were submillimolar–millimolar inhibitors (K_Is in the range of 0.93–8.3 mM). The best TweCA inhibitors were hydrogen sulfide, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K_Is in the range of 9–90 μM, whereas acetazolamide inhibited the enzyme with a K_I of 83 nM. This is the first kinetic and inhibition study of a δ-class CA. However, these enzymes are widespread in the marine phytoplankton, being present in haptophytes, dinoflagellates, diatoms, and chlorophytic prasinophytes, contributing to the CO₂ fixation by sea organisms. A phylogenetic analysis with all five genetic families of CAs showed that α- and δ-CAs are evolutionarily more related to each other with respect to the γ-CAs, although these three families clustered all together. On the contrary, the β- and ζ-CAs are also related to each other but phylogenetically much more distant from the α-, γ and δ-CA cluster. Thus, the study of δ-CAs is essential for better understanding this superfamily of metalloenzymes and their potential biotechnological applications in biomimetic CO₂ capture processes, as these enzymes are part of the carbon concentrating mechanism used by many photosynthetic organisms.     

The Functional Roles of the His247 and His281 Residues in Folate and Proton Translocation Mediated by the Human Proton-coupled Folate Transporter SLC46A1

This report addresses the functional role of His residues in the proton-coupled folate transporter (PCFT; SLC46A1), which mediates intestinal folate absorption. Of ten His residues, only H247A and H281A mutations altered function. The folic acid influx K_t at pH 5.5 for H247A was ↓8.4-fold. Although wild type (WT)-PCFT K_i values varied among the folates, K_i values were much lower and comparable for H247-A, -R, -Q, or -E mutants. Homology modeling localized His²⁴⁷ to the large loop separating transmembrane domains 6 and 7 at the cytoplasmic entrance of the translocation pathway in hydrogen-bond distance to Ser¹⁷². The folic acid influx K_t for S172A-PCFT was decreased similar to H247A. His²⁸¹ faces the extracellular region in the seventh transmembrane domain. H281A-PCFT results in loss-of-function due to ∼12-fold↑ in the folic acid influx K_t. When the pH was decreased from 5.5 to 4.5, the WT-PCFT folic acid influx K_t was unchanged, but the K_t decreased 4-fold for H281A. In electrophysiological studies in Xenopus oocytes, both WT-PCFT- and H281A-PCFT-mediated folic acid uptake produced current and acidification, and both exhibited a low level of folate-independent proton transport (slippage). Slippage was markedly increased for the H247A-PCFT mutant. The data suggest that disruption of the His²⁴⁷ to Ser¹⁷² interaction results in a PCFT conformational alteration causing a loss of selectivity, increased substrate access to a high affinity binding pocket, and proton transport in the absence of a folate gradient. The His²⁸¹ residue is not essential for proton coupling but plays an important role in PCFT protonation, which, in turn, augments folate binding to the carrier.     

Role of silicon in diatom metabolism

1. In silicic acid-starved cells of the diatom Nitzschia alba, ⁶⁸Ge(OH)₄ is transported against a concentration gradient, leading to intracellular concentrations of germanic acid up to 3500 times greater than the exogenous concentrations. The accumulated substrate is osmotically active, as determined by its efflux into germanic acid-free medium.
2. Metabolic energy is required for Ge(OH)₄ transport, since uptake is completely inhibited by 1 mM DNP, 5×10^-2 M sodium azide or 1 mM iodacetamide, and is strongly inhibited by CCCP and antimycin A. Inhibition of protein synthesis with 20 μg/ml cycloheximide does not affect the initial velocity of transport, but strongly reduces the steady state intracellular concentration.
3. A double reciprocal plot of uptake velocity versus substrate concentration yields a biphasic curve. The kinetic data are consistent with the interpretation that N. alba has two transport systems for germanic acid; a high affinity-low capacity (K _s=0.36 μM; V _max 1.2 μmoles/10⁸ cells/min) system and a low affinity-high capacity (K _s=5 μM; V _max 6.2 μmoles/10⁸ cells/min) system.
4. The implications of these findings for silicic acid transport and metabolism in N. alba are discussed.

     

Cloning,expression and some properties of α-carbonic anhydrase from Helicobacter pylori

The α-carbonic anhydrase gene from Helicobacter pylori strain 26695 has been cloned and sequenced. The full-length protein appears to be toxic to Escherichia coli, so we prepared a modified form of the gene lacking a part that presumably encodes a cleavable signal peptide. This truncated gene could be expressed in E. coli yielding an active enzyme comprising 229 amino acid residues. The amino acid sequence shows 36% identity with that of the enzyme from Neisseria gonorrhoeae and 28% with that of human carbonic anhydrase II. The H. pylori enzyme was purified by sulfonamide affinity chromatography and its circular dichroism spectrum and denaturation profile in guanidine hydrochloride have been measured. Kinetic parameters for CO₂ hydration catalyzed by the H. pylori enzyme at pH 8.9 and 25°C are k_cat=2.4×10⁵ s⁻¹, K_M=17 mM and k_cat/K_M=1.4×10⁷ M⁻¹ s⁻¹. The pH dependence of k_cat/K_M fits with a simple titration curve with pK_a=7.5. Thiocyanate yields an uncompetitive inhibition pattern at pH 9 indicating that the maximal rate of CO₂ hydration is limited by proton transfer between a zinc-bound water molecule and the reaction medium in analogy to other forms of the enzyme. The 4-nitrophenyl acetate hydrolase activity of the H. pylori enzyme is quite low with an apparent catalytic second-order rate constant, k_enz, of 24 M⁻¹ s⁻¹ at pH 8.8 and 25°C. However, with 2-nitrophenyl acetate as substrate a k_enz value of 665 M⁻¹ s⁻¹ was obtained under similar conditions.     

Regulatory sulfhydryl groups, conformational change, and allosteric inhibition in rabbit muscle fructose diphosphatase

The 16 sulfhydryl groups of native, homogeneous rabbit muscle fructose diphosphatase can all react with 5,5′-dithiobis-(2-nitrobenzoic acid). High concentrations of substrate (1–2 mm) decrease the reaction rate of the sulfhydryl groups, while concentrations up to 70 μm have no effect. After titration of the four most rapidly reacting sulfhydryl groups there is a marked desensitization toward the allosteric inhibitor AMP. In the presence of 30 μm AMP only 4–5 sulfhydryl groups/tetramer react with 5,5′-dithiobis-(2-nitrobenzoic acid), and the enzyme again becomes desensitized toward AMP inhibition. Together with a 3.5-fold increase in the I₅₀ for AMP inhibition, the K_m for Mg²⁺ or Mn²⁺ ions is also increased. In the presence of 7 mm MgCl₂ or 0.28 mm MnCl₂ only 6–8 sulfhydryl groups are modified. The rapid reaction of 4 sulfhydryl groups again results in desensitization. There is neither a protection by the substrate against inactivation, nor a protection by the allosteric inhibitor against desensitization. It is concluded that AMP and the divalent cations induce conformational changes in the protein molecule making 11–12 or 8–10 sulfhydryl groups inert for 5,5′-dithiobis-(2-nitrobenzoic acid), respectively. The K_m for fructose-1,6-diphosphate is not changed after the modification of 4–5 sulfhydryl groups.     

Inhibition of intestinal absorption of 5-methyltetrahydrofolate by fluoxetine

Thein vitro uptake of 5-methyltetrahydrofolate (5-MeTHF) by rat and human intestine is dose-dependently inhibited by the antidepressant drug fluoxetine (FLX). In rat jejunum rings, 0.2 mM FLX inhibited the uptake of 5-MeTHF (0.25 μM) by 32% (15 min) and 49% (45 min). In brush border membrane vesicles (BBMV) from rat jejunum, 0.2 mM FLX inhibited the folate uptake at the overshoot (90 s) by 40 %. Similar inhibition was observed with human Caco-2 cells and duodenal biopsies. FLX action is exerted on the active transport component of the folate uptake, since the drug has no effect when the passive diffusion component becomes prominent by high substrate concentration, or by 0-4 ºC incubation or by addition of the folate transport inhibitor DIDS (1mM). The kinetic analysis with rat BBMV suggests a non-competitive inhibition of the 5-MeTHF transport by FLX, with apparent values for K_M = 0.89 μM, Vmax = 1.89 pmol/mg prot./10 s, and K_I = 0.21 mM. After 21 days of treatment with FLX (10 mg/kg/day), the folate uptake by jejunum rings or by BBMV from the treated rats was diminished, and the folate levels in erythrocytes and serum were also decreased.     

Characterization of an intradiol dioxygenase involved in the biodegradation of the chlorophenoxy herbicides 2,4-D and 2,4,5-T

Hydroxyquinol 1,2-dioxygenase, an intradiol dioxygenase, which catalyzes the cleaving of the aromatic ring of hydroxyquinol, a key intermediate of 2,4-D and 2,4,5-T degradation, was purified from Nocardioides simplex 3E cells grown on 2,4-D as the sole carbon source. This enzyme exhibits a highly restricted substrate specificity and is able to cleave hydroxyquinol (K_m for hydroxyquinol as a substrate was 1.2 μM, V_max 55 U/mg, K_cat 57 s⁻¹ and K_cat/K_m 47.5 μM s⁻¹), 6-chloro- and 5-chlorohydroxyquinol. Different substituted catechols and hydroquinones are not substrates for this enzyme. This enzyme appears to be a dimer with two identical 37-kDa subunits. Protein and iron analyses indicate an iron stoichiometry of 1 iron/65 kDa homodimer, α₂ Fe. Both the electronic absorption spectrum which shows a broad absorption band with a maximum at 450 nm and the electron paramagnetic resonance spectra are consistent with a high-spin iron(III) ion in a rhombic environment typical of the active site of intradiol cleaving enzymes.     

The response to ribulose bisphosphate4− (RuBP4−) and RuBP-Mg2− in catalysis by structurally divergent RuBP carboxylase/oxygenases

Free ribulose bisphosphate (RuBP^4?) rather than its magnesium complex (RuBP-Mg^2?) was the apparent substrate for spinach ribulose bisphosphate carboxylase/oxygenase. The apparent K_m for total RuBP (pH 8.0 at 30° C) increased with increasing Mg²⁺ concentrations from 11.6 μM at 13.33 mM Mg²⁺ to 32.6 μM at 40.33 mM Mg²⁺. Similarly the apparent K_m for RuBP-Mg^2? complex increased with increasing Mg²⁺ from 9.4 μM at 13.33 mM Mg²⁺ to 29.7 μM at 40.33 mM Mg²⁺. However, the K_m values for uncomplexed RuBP^4? were independent of the (saturating) concentration of Mg²⁺ (K_m=2.2 μM). The V_max did not vary with the changing concentrations of Mg²⁺. In contrast, the K_m for total RuBP remained constant with varying Mg²⁺ concentrations (K_m=59.5 μM) for the enzyme from R. rubrum. The apparent K_m for the RuBP-Mg^2? complex decreased with increasing Mg²⁺ concentrations from 16.0 μM at 7.5 mM Mg²⁺ to 5.9 μM at 27.5 mM Mg²⁺. The initial velocity for the C. vinosum enzyme was also found to be independent of the (saturating) concentration of Mg²⁺ when total RuBP was varied in the assay. Thus the response to total RuBP by these two bacterial enzymes, which markedly differ in structure, was closely similar.     

Effect of insulin on folic-acid transport in cultured fibroblasts

Uptake of folic acid was measured in secondary cultures of skin fibroblasts from fetal rats. The cultures were made quiescent by 24 hours preincubation in medium containing 1% serum and subsequent 3 hours preincubation in phosphate buffered saline. The uptake of ³H-folic acid was linear with time during 15 seconds and reached a plateau level at 2–3 minutes. There was no further increase in the intracellular radioactivity until the end of the experiments at 10 minutes. The uptake of folic acid in fibroblasts was not concentrative and proceeded until equilibration with the extracellular concentration. Intracellular metabolic conversion of folic acid was not significant during the time of the experiments (up to 10 minutes). Insulin caused a two-fold increase in the initial rate of folate uptake as determined from the 15 second uptake values. The dose response curves for the insulin effect showed that 85% of the maximal effect was exerted by 1 m?M insulin. A lag period of 7–10 minutes was observed after the addition of insulin and before the effect on folic acid uptake was manifested. Thereafter the effect increased with the time of preincubation with insulin. The concentration dependence of folate uptake yielded non homogeneous curves. At low concentrations of substrate, saturable components were observed while at high concentration (above 5 × 10^?6 M) a linear component was observed. Insulin increased the slope of the linear component and the V_max of the saturable component while the K_m remained unaltered.     

Folate-dependent enzymes in cultured Chinese hamster ovary cells: induction of 5-methyltetrahydrofolate homocysteine cobalamin methyltransferase by folate and methionine.

The effects of media vitamin B₁₂(CNB₁₂), l-methionine, folic acid, dl-5-methyltetrahydrofolate (5-MeH₄folate), homocysteine, and other nutrients on four one-carbon enzymes in cultured Chinese hamster ovary (CHO) cells were examined. Excess 10 mm methionine elevates the amount of B₁₂ methyltransferase 1.8 – 2.3-fold at media folate concentrations of 0.2 – 2.0 μm. Conversely, excess 100 μm folic acid increases the amount of B₁₂ holoenzyme by 2.4 – 3.0-fold when the medium contains 0.01 – 0.1 mm methionine. These increases in B₁₂ methyltransferase promoted by 100 μm media folate and 10 mm methionine are inhibited by cycloheximide. 5-MeH₄folate will support growth and induce methyltransferase synthesis more efficiently than folic acid.Upon transfer to methionine-free media, wild-type CHO cells will survive and can be repeatedly subcultured in the absence of exogenous methionine, provided it is supplemented with 1.0 μm CNB₁₂, 0.1 mm homocysteine, and 100 μm folic acid or 10 μm dl-5-MeH₄folate. No growth occurs if homocysteine is omitted, but a requirement for added CNB₁₂ does not become evident until the cells have undergone at least two or three divisions. Survival upon transfer from 0.1 mm methionine-containing to methionine-free media is dependent upon the B₁₂ holomethyltransferase content of the cells used as an inoculum. Inoculum cells must have been previously grown in media supplemented with 1.0 μm CNB₁₂ to stabilize and convert apo- to holomethyltransferase, and 100 μm folate (or 10 μm dl-5-MeH₄folate) to induce maximal enzyme-protein synthesis. Transfer to methionine-deficient medium does not result in more than a 20–25% increase in the cellular B₁₂ enzyme content over the level already induced by 100 μm folate in 0.1 mm methionine-supplemented media. A mutant auxotroph CHO AUXB1 with a triple growth requirement for glycine + adenosine + thymidine (McBurney, M. W., and Whitmore, G. F. (1974) Cell, 2, 173) cannot survive in media lacking exogenous methionine. High concentrations of media folic acid or dl-5-MeH₄folate fail to induce elevated amounts of B₁₂ methyltransferase in this mutant. Excess 10 mm medium methionine does, however, elevate its B₁₂ enzyme as in the parent CHO cells. An additional mutant AUXB3 that requires glycine + adenosine (McBurney, M. W., and Whitmore, G. F. (1974) Cell, 2, 173) barely survives in methionine-deficient media. It has a folate-induced B₁₂ enzyme level intermediate between wild-type CHO cells and AUXB1. The level of B₁₂ methyltransferase induced by high media folate concentrations is a critical determinant of CHO cell survival in methionine-free media.     

Calcium regulated chloride permeabilities in primary cultures of rabbit colonocytes

To determine if calcium-dependent secretagogues directly act on epithelial cells to elicit CI⁻ secretion, their effects on CI⁻ transport and intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were determined in primary cultures of rabbit distal colonic crypt cells. The Cl⁻ sensitive fluorescent probe, 6-methoxyquinolyl acetoethyl ester, MQAE and the Ca²⁺-sensitive fluorescent probe, fura-2AM were used to assess Cl⁻ transport and [Ca²⁺]_i, respectively. Basal Cl⁻ transport (0.274 ± 0.09 mM/sec) was inhibited significantly by the Cl⁻ channel blocker diphenylamine-2-carboxylate (DPC, 50 μM, 0.068 ± 0.02 mM/sec; P < 0.001) and the Na⁺/K⁺/2Cl⁻ cotransport inhibitor furosemide (1 μM, 0.137 ± 0.04 mM/sec; P < 0.01). Ion substitution studies using different halides revealed the basal influx to be I⁻ > F⁻ ≥ Cl⁻ > Br⁻. DPC inhibited I⁻ influx by ∼50%, F⁻ influx by 80%, Cl⁻ influx by 85%, and Br⁻ influx by 90%. Furosemide significantly inhibited influx of Br⁻ (84%) and Cl⁻ (81%) but not of F⁻ and I⁻. The effects of agents known to alter biological response by increasing [Ca²⁺]_i in other epithelial systems were used to stimulate Cl⁻ transport. Cl⁻ influx in mM/second was stimulated by 1 μM histamine (0.58 ± 0.05), 10 μM neurotensin (2.07 ± 0.32), 1 μM serotonin (1.63 ± 0.28), and 0.1 μM of the Ca²⁺ ionophore A23187 (2.05 ± 0.40). The Cl⁻ permeability stimulated by neurotensin, serotonin, and A23187 was partially blocked by DPC or furosemide added alone or in combination. Histamine-induced Cl⁻ influx was significantly inhibited by only furosemide. Indomethacin blocked histamine-stimulated Cl⁻ permeability but had no effect on the actions of the other agents. These studies, focusing on isolated colonocytes without the contribution of submucosal elements, reveal that (1) histamine stimulates Cl⁻ transport by activating the Na⁺/K⁺/2Cl⁻ cotransporter via a cyclooxygenase-dependent pathway; (2) neurotensin, serotonin, and A23187 activate both Cl⁻ channels and the cotransporter, and their actions are cyclooxygenase-independent. © 1996 Wiley-Liss, Inc.     

Block of voltage-dependent K+ channels in insect muscle by the diacylhydrazine insecticide RH-5849, 4-aminopyridine,and quinidine

In calcium-free saline, voltage-clamped ventral longitudinal muscles of housefly larvae have maintained (I_K) and transient (I_A) voltage-dependent K⁺ currents. With 500 ms conditioning pulses, inactivation of I_A had a midpoint at ?53 mV and changed e-fold in 3.46 mV. I_A inactivated completely at ?40 mV, with a time constant of 71 ms, allowing the effects of various K⁺ channel blockers to be studied on I_K in isolation. RH-5849 (1,2-dibenzoyl-1-tert-butylhydrazine), a novel insect growth regulator, induces a lethal premature molt in insect larvae by mimicking the action of the molting hormone at ecdysone receptors. RH-5849 also causes acute neurotoxicity in some insects by selectively blocking of I_K in nerve and muscle. While most channel blockers have a Hill coefficient near 1, consistent with a simple one molecule per channel block mechanism, RH-5849 and the analog RH-1266 were found in the present study to block I_K channels in insect muscle with a Hill coefficient of 1.5. The lC₅₀ (concentration that caused 50% block) for block of I_K was 59 μM for RH-5849 and 40 μM for RH-1266. While tetraethylammonium blocked I_K by only 20% at 100 mM, 4-aminopyridine blocked the current with an lC₅₀ of 1.2 mM and a Hill coefficient of 0.97. Quinidine was the most potent blocker of I_K in this study, with an lC₅₀ of 20 μM. Block of I_K by either RH-5849 or 4-aminopyridine was independent of test pulse potential, but block by quinidine increased with depolarization. Block of I_K by RH-5849 and quinidine was time dependent, suggesting an open channel block mechanism, but the time course was too fast relative to channel activation for kinetic analysis. The lC₅₀ for block of I_K by RH-5849 decreased with temperature, with a Q₁₀ of 0.52. I_A was also blocked by RH-5849, but was less sensitive than I_K. The lC₅₀ for block of I_A by RH-5849 was 775 μM, 13-fold higher than the lC₅₀ for block of I_K. © 1992 Wiley-Liss, Inc.     

Enzymatic production of α-dehydrobiotin from biotin

The enzymatic production of α-dehydrobiotin (α-DHB), an antibiotic, from biotinyl-CoA using acyl-CoA oxidase and from biotin using a coupling system of biotinyl-CoA synthetase and acyl-CoA oxidase was developed. Acyl-CoA oxidase was found to show activity for biotinyl-CoA. K_m and V_max values of acyl-CoA oxidase for biotinyl-CoA were 75 μM and 3.92 μmol min⁻¹ mg⁻¹, respectively. Optimum reaction conditions for the α-DHB production from biotin were examined. The maximum production of α-DHB (4.29 μmol ml⁻¹) was obtained, when the reaction was carried out at 30°C for 36 h in a mixture consisting of 100 mM potassium phosphate buffer (pH 8.0), 20 mM biotin, 20 mM ATP, 60 mM CoA, 20 mM MgCl₂, 2 units of biotinyl-CoA synthetase, 90 units of acyl-CoA oxidase and 25 units of catalase in a total volume of 0.6 ml under aerobic conditions. The product was purified from 14 ml of the reaction mixture and 10 mg of crystals with white needle form were obtained. From NMR, mass spectra and other physical analyses, this compound was identified as (+)-trans-α-DHB.