Degradation of (+/-)-synephrine by Arthrobacter synephrinum. Oxidation of 3,4-dihydroxyphenylacetate to 2-hydroxy-5-carboxymethyl-muconate semialdehyde.

1. Cell-free extracts of Arthrobacter synephrinum catalyse the oxidation of 3,4-dihydroxy-phenylacetate. 2. The product of oxidation was characterized as 2-hydroxy-5-carboxymethylmuconate semialdehyde from its chemical behaviour as well as from nuclear-magnetic-resonance spectra. 3. A 3,4-dihydroxyphenylacetate 2,3-dioxygenase (EC 1.13.11.15) was partially purified from A. synephrinum. 4. The enzyme had a Km of 25 micrometer towards its substrate and exhibited typical Michaelis-Menten kinetics. 5. The enzyme also catalysed the oxidation of 3,4-dihydroxymandelate and 3,4-dihydroxyphenylpropionate, at reaction rates of 0.5 and 0.04 respectively of that for 3,4-dihydroxyphenylacetate. 6. The enzyme was sensitive to treatment with thiol-specific reagents. 7. The molecular weight of the enzyme as determined by Sephadex G-200 chromatography was approx. 282000.     

Action of beta-galactosidase on novel synthetic macromolecular substrates. A processive enzymic reaction controlled by coulombic interactions

Macromolecular beta-galactosidase substrates were prepared by attaching o-nitrophenyl-beta-galactoside to carboxymethyldextran with positively charged linking groups. Almost all of the substituents were susceptible to enzymic hydrolysis by two distinct pathways. Under some conditions, there was random reaction to give a soluble product. In other conditions, in the initial stages of the reaction, most of the substituents of some, but not all, of the substrate polymers were hydrolyzed to give a product which precipitated as a second aqueous phase. Kinetics of hydrolysis were studied with respect to charge and molecular weight of both the enzyme and substrate. Factors that caused a decrease in Km favored formation of the second phase product. The reaction has similarities to the processive catalytic reactions found in naturally occurring enzyme systems with polymeric charged substrates.     

Metabolism of DL-(+/-)-phenylalanine by Aspergillus niger.

A fungus capable of degrading DL-phenylalanine was isolated from the soil and identified as Aspergillus niger. It was found to metabolize DL-phenylalanine by a new pathway involving 4-hydroxymandelic acid. D-Amino acid oxidase and L-phenylalanine: 2-oxoglutaric acid aminotransferase initiated the degradation of D- and L-phenylalanine, respectively. Both phenylpyruvate oxidase and phenylpyruvate decarboxylase activities could be demonstrated in the cell-free system. Phenylacetate hydroxylase, which required reduced nicotinamide adenine dinucleotide phosphate, converted phenylacetic acid to 2- and 4-hydroxyphenylacetic acid. Although 4-hydroxyphenylacetate was converted to 4-hydroxymandelate, 2-hydroxyphenylacetate was not utilized until the onset of sporulation. During sporulation, it was converted rapidly into homogentisate and oxidized to ring-cleaved products. 4-Hydroxymandelate was degraded to protocatechuate via 4-hydroxybenzoylformate, 4-hydroxybenzaldehyde, and 4-hydroxybenzoate.     

(+/-)8-Amino-5,6,7,8-tetrahydroisoquinolines as novel antinociceptive agents

Several amine-substituted 8-amino-5,6,7,8-tetrahydroisoquinolines were examined as conformationally-constrained analogs of the nicotinic cholinergic (nACh) 3-(aminomethyl)pyridines. Although these ligands failed to bind at nACh receptors, the N-ethyl-N-methyl analog 3d was found to be at least equipotent with nicotine in rodent tests of antinociception. The mechanism of action of 3d is currently unknown.     

Metabolism of (+/-)-N-(n-propyl) amphetamine by Cunninghamella echinulata.

(+/-)-N-(n-propyl) amphetamine (I), a secondary amine, was readily metabolized by Cumminghamella echinulata. The products included known C- and N-oxygenated mammalian metabolites as well as N-acetylamphetamine and were identified by gas chromatography and mass spectrometry.     

alpha,beta-Dibenzyl-gamma-butyrolactone lignan alcohols: total synthesis of (+/-)-7'-hydroxyenterolactone, (+/-)-7'-hydroxymatairesinol and (+/-)-8-hydroxyenterolactone

Two trans-alpha,beta-dibenzyl-gamma-butyrolactone lignans carrying a hydroxyl group at the beta-benzylic carbon atom and a alpha-hydroxy alpha,beta-dibenzyl-gamma-butyrolactone lignan were synthesized in racemic form using the tandem conjugate addition reaction to construct the basic lignan skeleton. Subsequent reaction steps involved either a catalytic reduction of the regenerated keto group to the alcohol, or a hydrogenolysis to benzylic methylene followed by lactone enolate formation and oxidation to give the alpha-hydroxybutyrolactones. These procedures were applied for the synthesis of 7'-hydroxyenterolactones and 7'-hydroxymatairesinols, and 8-hydroxyenterolactones, respectively. The diastereomeric mixtures of these compounds were separated either by HPLC techniques or column chromatography and the structures were elucidated using NMR spectroscopy.     

Metabolism of (+/-)-N-(n-propyl) amphetamine by Cunninghamella echinulata.

(+/-)-N-(n-propyl) amphetamine (I), a secondary amine, was readily metabolized by Cumminghamella echinulata. The products included known C- and N-oxygenated mammalian metabolites as well as N-acetylamphetamine and were identified by gas chromatography and mass spectrometry.     

A novel enzymic determination of maltose

A novel enzymic determination of maltose with four enzymes (a new enzyme, maltose 1-epimerase [EC 5.1.3.-], maltose phosphorylase [EC 2.4.1.8], beta-phosphoglucomutase [EC 5.4.2.6], and glucose-6-phosphate dehydrogenase [EC 1.1.1.49]) is described. Maltose was rapidly and quantitatively determined within about 2 min by means of maltose 1-epimerase. The standard curve was linear up to 1.5 micromol/mL. The within-run and between-run studies gave precision (CV) values of < 2.0% and < 3.0%, respectively. No significant interferences by mono- and disaccharides were observed with the proposed method under this study. There was a good correlation (r = 0.997) between the results obtained by the enzymic and HPLC methods. This method fulfills the need for an accurate, specific and simple assay of maltose, and it is less time consuming than HPLC and enzymic methods previously reported.     

A novel Arthrobacter beta-galactosidase with homology to eucaryotic beta-galactosidases.

An Arthrobacter beta-galactosidase has homology with the lysosomal acid beta-galactosidases from humans and mice and with a Xanthomonas manihotis enzyme. Phylogenetic analysis of the deduced amino acid sequence showed an unusual pattern, with this procaryotic enzyme clustering within the animal clade. The gene encodes a subunit of 52 kDa, and the enzyme appears to be active as a dimer. The enzyme hydrolyzed substrates with either a beta-1,4 or a beta-1,3 linkage.     

A vector method of representation of enzymic reactions: 1. Parametric classification of types of enzymic reaction

A new “parametric” classification of the types of single-substrate enzymic reactions is proposed that includes 15 different types: seven inhibited, seven activated and one initial (uninhibited, i=0 and nonactivated, a=0). The new classification takes into account both the “parametricity” of these reactions and the mechanisms of their action. It unites all the types in an original symmetric system which vividly demonstrates the interconnection between separate (strictly definite) types of inhibited and activated enzymic reactions. The proposed classification permits the revision and some corrections to traditional “competitive” terminology as well as the application of these ideas and mathematical approaches pertinent to the calculation of reactions of enzyme inhibition for data analysis of enzyme activation.     

First synthesis of (+/-)-robinlin

The first synthesis of (+/-)-robinlin (1), a novel homo-monoterpene with strong bioactivity in the brine shrimp lethality test, was achieved by starting from 3-isobutyloxy-2,6,6-trimethyl-2-cyclohexen-1-one (2).     

Total synthesis of (+/-)-syn-copalol

The labdane diterpene derivative, syn-copalol [(+)-5] is the alcohol part of syn-copalyl diphosphate [(+)-4]. In this paper, racemic (+/-)-5 was synthesized from a known racemic lactone in 8 steps. The current and our previous syntheses provide all four copalol derivatives [(+)-3, (-)-3 and (+/-)-5] which are required for the biosynthetic study of polycyclic diterpenes.     

A new assay method for hydrogenase based on an enzymic electrode reaction. The enzymic electric cell method.

A new assay method for hydrogenase [EC 1.12.2.1] based on the enzymic electrode reaction of H2-H+ equilibrium has been established. The method is based on the experimental fact that the short-circuit current of the electric cell composed of an electrode with hydrogenase and methylviologen as the mediator of H2-H+ equilibrium and a saturated calomel electrode as the counter electrode, is practically proportional to the amount of hydrogenase in the cell. The new method is referred to as the "enzymic electric cell method." This technique has applications not only to routine activity assay but also to the direct determination of the time course of enzyme denaturation, which has not previously been possible.     

The synthesis and nicotinic binding activity of (+/-)-epiquinamide and (+/-)-C(1)-epiepiquinamide

The synthesis of (+/-)-epiquinamide 1 and (+/-)-C(1)-epiepiquinamide 2 based on the use of a Curtius rearrangement to introduce the C(1) amino residue is reported. In a competition binding assay for [(3)H]epibatidine binding to rat brain membranes neither (+/-)-1 nor (+/-)-2 showed any significant level of nicotinic activity.     

Utilization of l-threonine by a species of Arthrobacter. A novel catabolic role for `aminoacetone synthase''

1. A species of Arthrobacter (designated Arthrobacter 9759) was isolated from soil by its ability to grow aerobically on l-threonine as sole source of carbon atoms, nitrogen atoms and energy; the organism also grew well on other sources of carbon atoms including glycine, but no growth was obtainable on aminoacetone or dl-1-aminopropan-2-ol. 2. During growth on threonine, (14)C from l-[U-(14)C]threonine was rapidly incorporated into glycine and citrate, and thereafter into serine, alanine, aspartate and glutamate. 3. With extracts of threonine-grown cells supplied with l-[U-(14)C]threonine, evidence was obtained of the NAD and CoA-dependent catabolism of l-threonine to produce acetyl-CoA plus glycine. Short-term incorporation studies in which [2-(14)C]acetate and [2-(14)C]glycine were supplied (a) to cultures growing on threonine, and (b) to extracts of threonine-grown cells, showed that the acetyl-CoA was metabolized via the tricarboxylic acid cycle and glyoxylate cycle whereas the glycine was converted into pyruvate via the folate-dependent ;serine pathway'. 4. The threonine-grown organism contained ;biosynthetic' threonine dehydratase and a potent NAD-linked l-threonine dehydrogenase but possessed no l-threonine aldolase activity. 5. Evidence was obtained that the acetyl-CoA and glycine produced from l-threonine had their immediate origin in the alpha-amino-beta-oxobutyrate formed by the threonine dehydrogenase; the CoA-dependent cleavage of this compound was catalysed by an alpha-amino-beta-oxobutyrate CoA-ligase, which was identified with ;aminoacetone synthase'. A continuous spectrophotometric assay of this enzyme was developed, and it was found to be inducibly synthesized only during growth on threonine and not during growth on acetate plus glycine. 6. By using a reconstituted mixture of separately purified l-threonine dehydrogenase and alpha-amino-beta-oxobutyrate CoA-ligase (i.e. ;aminoacetone synthase'), l-[U-(14)C]threonine was broken down to [(14)C]glycine plus [(14)C]acetyl-CoA (trapped as [(14)C]citrate). 7. There was no evidence of aminoacetone metabolism by Arthrobacter 9759 even though a small amount of this amino ketone appeared in the culture medium during growth on threonine.     

Syntheses of (+/-)-methyl 6'alpha-demethyl-6'alpha-cyanoabscisate and (+/-)-methyl 6'alpha-demethyl-6'alpha-methoxycarbonylabscisate

New abscisic acid analogs possessing a cyano or methoxycarbonyl group at the 6'alpha-position of methyl abscisate were synthesized by regioselective hydrocyanation. These compounds had weak activity in the rice second leaf sheath elongation test.     

(+/-)-3'-O, 4'-O-dicynnamoyl-cis-khellactone, a derivative of (+/-)-praeruptorin A, reverses P-glycoprotein mediated multidrug resistance in cancer cells

P-glycoprotein (Pgp) is an ATP-driven membrane exporter for a broad spectrum of hydrophobic xenobiotics. Pgp-overexpression is a common cause of multidrug resistance (MDR) in cancer cells and could lead to chemotherapeutic failure. Through an extensive herbal drug screening program we previously showed that (+/-)-praeruptorin A (PA), a naturally existing pyranocumarin isolated from the dried root of Peucedanum praeruptorum Dunn., re-sensitizes Pgp-mediated MDR (Pgp-MDR) cancer cells to cancer drugs. A number of PA derivatives were synthesized and one of these, (+/-)-3'-O, 4'-O-dicynnamoyl-cis-khellactone (DCK), was more potent than PA or verapamil in the reversal of Pgp-MDR. In Pgp-MDR cells DCK increased cellular accumulation of doxorubicin without affecting the expression level of Pgp. In Pgp-enriched membrane fractions DCK moderately stimulated basal Pgp-ATPase activity, suggesting some transport substrate-like function. However, DCK also inhibited Pgp-ATPase activity stimulated by the standard substrates verapamil or progesterone with decreased V(max)s but K(m)s were relatively unchanged, suggesting a primarily non-competitive mode of inhibition. While the binding of substrates to active Pgp would increase the reactivity of the Pgp-specific antibody UIC2, DCK decreased UIC2 reactivity. These results suggest that DCK could bind simultaneously with substrates to Pgp but perhaps at an allosteric site and thus affect Pgp-substrate interactions.