Hydrolytic enzyme substrates. 3. Phosphomonoesterase substrates

This report documents the use of a new and sensitive colorimetric method for measuring phosphomonoesterase activity. The substrates are the phosphate esters of 4-(p-nitrophenoxy)-1,2-butanediol (PNB), 4-(2,4-dinitrophenoxy)-1,2-butanediol (DNB) and 3-(p-nitrophenoxy)-1,2-propanediol (PNG). The key intermediate in the assay is the nitrophenoxy diol which is obtained by enzyme hydrolysis of its phosphate ester. Periodate oxidation of this substance in solution containing methylamine quantitatively yields its nitrophenolate ion whose concentration is determined colorimetrically. The amount of nitrophenolate ion is thus equivalent to the amount of nitrophenoxy diol whose concentration is a function of the phosphomonoesterase activity in the assay sample. The unhydrolyzed phosphomonoester is completely stable to periodate and the hydrolytic conditions used in the assay. The enzymes used to test the substrates were E. coli alkaline phosphomonoesterase and wheat germ phosphomonoesterase. These new esters were all better substrates than the glycerol phosphate esters. Their Michaelis-Menten constants were determined for E. coli phosphomonoesterase.     

Hydrolytic enzyme substrates. I. Chemical synthesis and characterization

The synthesis and characterization of a number of new phosphate, sulfate and acetate esters of 3-(p-nitrophenoxy)-1,2-propanediol (PNG); 3-(2,4-dinitrophenoxy)-1,2-propanediol (DNG); 4-(p-nitrophenoxy)-1,2-butanediol (PNB) and 4-(2,4-dinitrophenoxy)-1,2-butanediol (DNB) are described. These esters were prepared to serve as substrates for their corresponding hydrolytic enzymes. The assay system used to measure enzyme hydrolysis requires periodate oxidation of the diol formed after hydrolysis of the ester. Base treatment of the resulting aldehyde yields either p-nitrophenolate ion or the 2,4-dinitrophenolate ion depending upon the substrate. In the presence of high concentrations of methylamine and excess periodate the oxidation and elimination reactions can be carried out simultaneously at pH 7.5. The reactions leading to these results are described.     

2,3-Butanediol Metabolism in the Acetogen Acetobacterium woodii

The acetogenic bacterium Acetobacterium woodii is able to reduce CO₂ to acetate via the Wood-Ljungdahl pathway. Only recently we demonstrated that degradation of 1,2-propanediol by A. woodii was not dependent on acetogenesis, but that it is disproportionated to propanol and propionate. Here, we analyzed the metabolism of A. woodii on another diol, 2,3-butanediol. Experiments with growing and resting cells, metabolite analysis and enzymatic measurements revealed that 2,3-butanediol is oxidized in an NAD⁺-dependent manner to acetate via the intermediates acetoin, acetaldehyde, and acetyl coenzyme A. Ethanol was not detected as an end product, either in growing cultures or in cell suspensions. Apparently, all reducing equivalents originating from the oxidation of 2,3-butanediol were funneled into the Wood-Ljungdahl pathway to reduce CO₂ to another acetate. Thus, the metabolism of 2,3-butanediol requires the Wood-Ljungdahl pathway.     

Immunochemical studies on blood groups. 53. A study of various conditions of alkaline borohydride degradation on human and hog blood group substances and on known oligosaccharides

Blood group A, B, H, Le^a, Le^b, and I substances, their products of periodate oxidation and Smith degradation, and disaccharides containing 3-O-substituted reducing N-acetylhexosamines were treated with base-borohydride under three defined sets of conditions. Procedures for the assay and quantitation of the possible reduced base-degradation products, including hexenetetrol(s), 3-deoxygalactitol, galactitol, reduced chromogens, N-acetylglucosaminitol, and N-acetylgalactosaminitol are described. Extensive degradation occurred by two methods. 1 m NaBH₄ in 0.05 n NaOH at 50 ° cleaves the glycosidic linkage of the oligosaccharide chains from serine and threonine with reduction of the terminal-reducing N-acetylgalactosamine with minimal base degradation. The method is useful for isolation of complete reduced oligosaccharides from blood group substances; the structural implications of the free and oligosaccharide-bound N-acetylgalactosaminitol released are discussed.     

Enhanced 2,3-butanediol production by Klebsiella oxytoca using a two-stage agitation speed control strategy

Batch fermentative production of 2,3-butanediol by Klebsiella oxytoca was investigated using various oxygen supply methods though varying agitation speed. Based on the analysis of three kinetic parameters including specific cell growth rate (μ), specific glucose consumption rate (q_s) and specific 2,3-butanediol formation rate (q_p), a two-stage agitation speed control strategy, aimed at achieving high concentration, high yield and high productivity of 2,3-butanediol, was proposed. At the first 15 h, agitation speed was controlled at 300 rpm to obtain high μ for cell growth, subsequently agitation speed was controlled at 200 rpm to maintain high q_p for high 2,3-butanediol accumulation. Finally, the maximum concentration of 2,3-butanediol reached 95.5 g l⁻¹ with the yield of 0.478 g g⁻¹ and the productivity of 1.71 g l⁻¹ h⁻¹, which were 6.23%, 6.22% and 22.14% over the best results controlled by constant agitation speeds.     

Effect of Hydrocarbon Chain Length in 1,2-Alkanediols on Percutaneous Absorption of Metronidazole: Toward Development of a General Vehicle for Controlled Release

The objective of the present study is to investigate the effect of hydrocarbon chain length in 1,2-alkanediols on percutaneous absorption of metronidazole (MTZ). Twelve formulations (1,2-propanediol, 1,2-butanediol, 1,2-pentanediol, 1,2-hexanediol in 4% concentration, 1,2-hexanediol, and 1,2-heptanediol in 1% concentration, in the absence and presence of 1,4-cyclohexanediol, respectively) were studied in an in vitro hairless mouse skin model using Franz diffusion cell. Based on the flux values and retardation ratios (RR), a penetration retardation effect on percutaneous absorption of MTZ was observed for the formulations containing 1,2-diols having six- to seven-carbon chain in the presence of 1,4-cyclohexanediol (1,2-hexanediol with chain length of six hydrocarbons, RRs are 0.69 and 0.76 in the concentration of 4% and 1%, respectively; 1,2-heptanediol with chain length of seven hydrocarbons, RR is 0.78 in the concentration of 1%). On the other hand, no retardation effect was observed in formulations containing short alkyl chains (RRs of 1,2-propanediol, 1,2-butanediol, and 1,2-pentanediol are 0.99, 1.61, and 0.96, respectively). Instead, a penetration enhancement effect was observed for 1,2-diols having four and five carbons. In other words, effect of 1,2-alkanediols on percutaneous absorption of MTZ can be systematically modulated by simply varying number of –CH₂ groups in the hydrocarbon chain—from being a penetration enhancer to retardant. These observations shed light on mechanism of the penetration enhancement and retardation effect and provide insight into rational design of penetration enhancers and retardants. Furthermore, the combination of 1,2-alkanediols and 1,4-cyclohexanediol could become a general vehicle for controlled release of pharmaceutical and cosmetic active ingredients.

Figure

Open in a separate windowᅟKEY WORDS: 1,2-alkanediols; controlled release; hydrocarbon chain length; skin penetration     

1,2-Hydrogen shifts during the biosynthesis of patchoulenes in Pogostemon cablin

The isotope ratio in α- and γ-patchoulenes in Pogostemon cablin, that has been fed with [2-¹⁴C, 4R-³H₁]MVA, suggests that a proton loss is followed by a 1,2-alkyl shift and two 1,2-hydrogen shifts during the biosynthesis of these two sesquiterpene hydrocarbons. Whereas isotope ratios in β- and δ-patchoulene suggests that a proton loss is followed by one 1,2-hydrogen shift in β-patchoulene and two 1,2-hydrogen shifts in δ-patchoulene.     

Increase of Brain Tryptophan caused by Drugs which stimulate Serotonin Synthesis

THE concentrations of tryptophan normally present in the mammalian brain are below the Michaelis constant (K_m) of tryptophan hydroxylase^1,2, suggesting that the rate of serotonin synthesis depends more on the concentration of brain tryptophan than on the amount of enzyme. We wish to report that various treatments which have been shown to increase brain serotonin synthesis also increase the concentration of tryptophan in brain. Conversely, p-chlorophenylalanine (PCPA), which inhibits serotonin synthesis³, decreases tryptophan in brain.     

The effects of chemotactic factors on the adhesiveness of rabbit neutrophil granulocytes.

A range of chemotactic factors has been shown to affect the adhesion of rabbit peritoneal neutrophil granulocytes to cultured endothelial cells and to serum-coated glass. At chemotactically optimal concentrations, α_s-casein, β-casein, alkali denatured human serum albumin (HSA) and several synthetic formyl-peptides reduced the number of adherent neutrophils after 30 min to around 50% of control values. These effects were still observed after neutrophils, but not endothelium or serum-coated glass had been exposed to chemotactic factors and washed before use in assays. Two non-chemotactic analogues, native HSA and a non-formyl-peptide were ineffective. The dose responses for adhesion after 30 min in the presence of α_s-casein and formyl-methionyl-leucyl-phenylalanine (FMLP) were found to be inversely related to those for migration towards these substances. After incubation for 60 min in high (10^?8–10^?7 M) concentrations of FMLP, neutrophil adhesion was found to be enhanced. Neutrophil aggregation was also affected by the presence of chemotactic factors in a similar time- and dose-dependent manner to the adhesion to substratum assays. Using FMLP, it was also shown that the timing of the adhesive changes depended on the concentration of chemotactic factor present.     

Determination of urinary oxalic acid and of oxalate pool size by stable isotope dilution.

An isotope dilution procedure for oxalate based upon [1,2-¹³C₂]oxalic acid is described. For routine determinations of urinary concentration, a known quantity of sodium [1,2-¹³C]oxalate is admixed with the sample, total oxalate precipitated as the calcium salt, and converted by BF₃ catalysis to di-n-propyl esters for mass-spectrometric analysis. Selective ion monitoring provides ¹²C:¹³C ratios directly, thus precluding the necessity for quantitative recovery at any step of the rapid, single-tube assay. Following a bolus injection of sodium [1,2-¹³C]oxalate, whole body oxalate pools and their turnover rates can be determined by sequential sampling of urine. Biosynthetic rates calculated from the product of pool size and turnover are in excellent agreement with urinary excretion rates, confirming directly that urinary oxalate is a quantitative index of biosynthesis.     

Selective benzoylation of some N-acetyl-N-aryl-β-D-xylopyranosylamines

Selectivity in the benzoylation of N-acetyl-N-p-methoxyphenyl-, -p-bromophenyl-, and -p-chlorophenyl-β-D-xylopyranosylamines has been demonstrated. The structures of the products was shown by using periodate oxidation and n.m.r. spectroscopy. The relative reactivity of the hydroxyl groups was HO-3 ≈ HO-4 > HO-2. The β-D-xylopyranosylamine derivatives were shown to possess the ⁴C¹ conformation.     

Detection of Antigen-binding Cells by Combined Rosette Formation and Autoradiography

DETERMINATION of the frequency of chromosome aberrations in cultured blood lymphocytes may provide a means of measuring ionizing radiation doses, at least after whole body exposure. Much work has been done with human blood irradiated in vitro^1,2, but before these results can be applied to radiation exposure in vivo, the difference between in vitro and in vivo exposure must be shown to be quantitatively negligible.     

Catabolism of Naphthalenesulfonic Acids by Pseudomonas sp. A3 and Pseudomonas sp. C22

Naphthalene and two naphthalenesulfonic acids were degraded by Pseudomonas sp. A3 and Pseudomonas sp. C22 by the same enzymes. Gentisate is a major metabolite. Catabolic activities for naphthalene, 1-naphthalenesulfonic acid, and 2-naphthalenesulfonic acid are induced by growth with naphthalene, 1-naphthalenesulfonic acid, 2-naphthalenesulfonic acid, methylnaphthalene, or salicylate. Gentisate is also an inducer in strain A3. Inhibition kinetics show that naphthalene and substituted naphthalenes are hydroxylated by the same naphthalene dioxygenase. Substrates with nondissociable substituents such as CH₃, OCH₃, Cl, or NO₂ are hydroxylated in the 7,8-position, and 4-substituted salicylates are accumulated. If CO₂H, CH₂CO₂H, or SO₃H are substituents, hydroxylation occurs with high regioselectivity in the 1,2-position. Thus, 1,2-dihydroxy-1,2-dihydronaphthalene-2-carboxylic acids are formed quantitatively from the corresponding naphthalenecarboxylic acids. Utilization of naphthalenesulfonic acids proceeds by the same regioselective 1,2-dioxygenation which labilizes the C—SO₃⁻ bond and eliminates sulfite.     

A sensitive and reproducible fluorescent-based HPLC assay to measure the activity of acid as well as neutral beta-glucocerebrosidases

The activity of lysosomal acid β-glucocerebrosidase (AGC, EC 3.2.1.45), which hydrolyzes the O-glycosidic linkage between d-glucose and ceramide of glucosylceramide (GlcCer), is a marker for the diagnosis of Gaucher disease because the disease is caused by dysfunction of AGC due to mutations in the gene. The activity of AGC is potently inhibited by conduritol B epoxide (CBE), whereas CBE-insensitive nonlysosomal neutral β-glucocerebrosidase (NGC) activities have been found in various vertebrates, including humans. We report here a new reliable method to determine AGC as well as NGC activities using normal-phase high-performance liquid chromatography (HPLC) and NBD (4-nitrobenzo-2-oxa-1,3-diazole)- or BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-labeled GlcCer as a substrate. The reaction products of the enzymes, C6-NBD-ceramide and C12-BODIPY-ceramide, were clearly separated from the corresponding substrates on a normal-phase column within 5 min using a different solvent system. Reaction products could be detected quantitatively at concentrations ranging from 50 fmol to 50 pmol for C6-NBD-ceramide and from 10 fmol to 5 pmol for C12-BODIPY-ceramide. V_max/K_m values of human fibroblast AGC for fluorescent GlcCer were much higher than those for 4-methylumbelliferyl-β-d-glucoside (4MU-Glc), which is used prevalently for Gaucher disease diagnosis. As a result, AGC activity was detected quantitatively using fluorescent GlcCer, but not 4MU-Glc, using 5 μl of human serum or 1 × 10⁴ cultured human fibroblasts. The current method clearly showed the decrease of AGC activities in fibroblasts and serum from the patient with Gaucher disease compared with normal individuals, suggesting that the method is applicable for the diagnosis of Gaucher disease. Furthermore, this method was found to be useful for measuring the activities of nonlysosomal NGC of various cells and tissues in the presence of CBE.     

Occurrence of multiple forms of alcohol dehydrogenase in Penicillium supplemented with 2,3-butanediol

The NAD-dependent oxidation of ethanol, 2,3-butanediol, and other primary and secondary alcohols, catalyzed by alcohol dehydrogenases derived from Penicillium charlesii, was investigated. Alcohol dehydrogenase, ADH-I, was purified to homogeneity in a yield of 54%. The enzyme utilizes several primary alcohols as substrates, with K_m values of the order of 10^?4m. A K_m value of 60 mm was obtained for R,R,-2,3-butanediol. The stereospecificity of the oxidation of 2-butanol was investigated, and S-(+)-2-butanol was found to be oxidized 2.4 times faster than was R-(?)-2-butanol. The reduction of 2-butanone was shown to produce S-(+)-2-butanol and R-(?)-butanol in a ratio of 7:3. ADH-I is the primary isozyme of alcohol dehydrogenase present in cultures utilizing glucose as the sole carbon source. The level of alcohol dehydrogenase activity increased 7.6-fold in mycelia from cultures grown with glucose and 2,3-butanediol (0.5%) as carbon sources compared with the activity in cultures grown on only glucose. Two additional forms of alcohol dehydrogenase, ADH-II and ADH-III, were present in the cultures supplemented with 2,3-butanediol. These forms of alcohol dehydrogenase catalyze the oxidation of ethanol and 2,3-butanediol. These data suggest that P. charlesii carries out an oxidation of 2,3-butanediol which may constitute the first reaction in the degradation of 2,3-butanediol as well as the last reaction in the mixed-acid fermentation. Alcohol dehydrogenase activities in P. charlesii may be encoded by multiple genes, one which is expressed constitutively and others whose expression is inducible by 2,3-butanediol.     

A simple method for detecting steroid aggregation and estimating solubility in aqueous solutions

Steroids are generally sparingly soluble in water. Thus, for in vitro studies of steroid metabolism or enzymology it is common practice to solubilize steroids by the addition of a small amount (2–10%, v/v) of an organic cosolvent. Methanol, ethanol, and 1,2-propanediol, singly or in combination, have been widely used (1). Effects of organic solvents on the kinetic parameters, K_m and V_max, of steroid-metabolizing enzymes with various substrates have been demonstrated (2,3), and the results are consistent with the conclusion that organic solvent influences on catalytic activity reflect, in part, effects on the aggregation state and solubility of steroid substrates.Light-scattering measurements have been applied extensively in studies of macromolecular structure (4) and micelle formation by a large variety of amphiphilic substances [reviewed in Ref. (5)]. Jones and Gordon (6) used a commercial instrument, designed specifically for light-scattering measurements, to characterize micelle formation in aqueous solutions by Δ5-3-ketosteroids containing various substituents at the 17β position. They showed that turbidity versus concentration plots were of the form seen in studies of micelle formation (5) and that steroids can exist in solution in monomeric or micellar forms, their aggregation state being a function of the polarity of the steroid solute and the composition of the solvent.To estimate solubility quantitatively ³H- or ¹⁴C-labeled steroids have been used in conjunction with centrifugation (3), dialysis (7), or filtration (8). These techniques allow for accurate estimates of solubility, but one may encounter problems due to nonspecific absorption on membranes or the unavailability of the labeled steroid of interest.We have observed that steroid aggregation and solubility can be estimated easily and with high sensitivity with a commercially available fluorometer. In this report the method is described and examples demonstrating the reproducibility and sensitivity of the technique are presented.     

The crystal structure of 6-epi-desacetyllaurenobiolide,a germacra-1(10),4-diene-12,8α-olide from Montanoa grandiflora

A chloroform extract of Montanoa grandiflora afforded a novel 6β-hydroxy-germacradien-8,12-olide. Its structure was shown to be 6-epi-desacetyllaurenobiolide by spectral studies, chemical transformations and single crystal X-ray diffraction. The X-ray data demonstrate that the ten-membered ring exists in the crystal in the highly unusual [₁₅D⁵,₁D¹⁴] conformation, in which the methyl group at C-4 is α-oriented and the methyl group at C-10 is β-oriented. The two double bonds are approximately parallel rather than crossed.     

Engineering Cupriavidus necator H16 for the autotrophic production of (R)-1,3-butanediol

Butanediols are widely used in the synthesis of polymers, specialty chemicals and important chemical intermediates. Optically pure R-form of 1,3-butanediol (1,3-BDO) is required for the synthesis of several industrial compounds and as a key intermediate of β-lactam antibiotic production. The (R)-1,3-BDO can only be produced by application of a biocatalytic process. Cupriavidus necator H16 is an established production host for biosynthesis of biodegradable polymer poly-3-hydroxybutryate (PHB) via acetyl-CoA intermediate. Therefore, the utilisation of acetyl-CoA or its upstream precursors offers a promising strategy for engineering biosynthesis of value-added products such as (R)-1,3-BDO in this bacterium. Notably, C. necator H16 is known for its natural capacity to fix carbon dioxide (CO₂) using hydrogen as an electron donor. Here, we report engineering of this facultative lithoautotrophic bacterium for heterotrophic and autotrophic production of (R)-1,3-BDO. Implementation of (R)-3-hydroxybutyraldehyde-CoA- and pyruvate-dependent biosynthetic pathways in combination with abolishing PHB biosynthesis and reducing flux through the tricarboxylic acid cycle enabled to engineer strain, which produced 2.97 g/L of (R)-1,3-BDO and achieved production rate of nearly 0.4 Cmol Cmol⁻¹ h⁻¹ autotrophically. This is first report of (R)-1,3-BDO production from CO₂.     

Nuclear metabolism. II. Further studies on epoxide hydrolase activity

Apparent K_m- and V_max-values of nuclear styrene 7,8-oxide hydrolase were determined at different protein concentrations. In the protein concentrations range used no significant differences in the apparent K_m-values were observed. The influence of the incubation with different modifiers (i.e. SKF-525A, metyrapone, 1,2-epoxy-3,3,3 trichloropropane, cyclohexene oxide) at two different concentrations on this enzyme activity was also determined. Cyclohexene oxide and 1,2-epoxy-3,3,3-trichloropropane, two well known inhibitors of the microsomal epoxide hydrolase(s) caused a marked inhibition, metyrapone had a strong activating effect whereas SKF-525A had no effect. In vivo pretreatment with phenobarbital significantly induced the nuclear epoxide hydrolase whereas β-naphthoflavone caused a lower degree of induction. This pattern is quantitatively different but qualitatively very similar to the microsomal one. Moreover a toxifying to detoxifying enzymatic activity balance is attempted for the metabolization of the alkenic double bond of styrene, taking into account the ratio between the styrene monooxygenase (toxifying enzyme) and the styrene 7,8-oxide hydrolase (detoxifying enzyme) after the above mentioned pretreatments, both in the microsomal and nuclear fractions.