Radiochemical synthesis of DL-canaline and the colorimetric assay of canaline

A procedure is available for the production of DL-[carboxy-14C]canaline from [14C]cyanide by reaction of ethyl N-hydroxyacetimidate and acrolein to form ethyl N-[3-oxopropoxy]acetimidate. The reaction product is converted to the nitrile and then to the hydantoin derivative of DL-canaline; alkaline hydrolysis produces the free amino acid (2-amino-4-aminooxypropionic acid). This procedure can be extended to the production of DL-[carboxy-14C]canavanine by guanidination of C-1-labeled DL-canaline with O-methylisourea. A markedly improved colorimetric assay for canaline has been achieved by a procedure involving carbamylation of canaline with cyanate to form O-ureidohomoserine (2-amino-4-ureidooxybutyric acid). Colorimetric analysis of the latter amino acid markedly enhances the sensitivity, reproducibility, and accuracy of the analysis of L-canaline from biological materials.     

The biological and biochemical properties of L-canaline, a naturally occurring structural analogue of L-ornithine.

Many leguminous plants synthesize L-canavanine and sequester this nitrogen-rich, non-protein amino acid in the seed (1,2). Arginase-mediated hydrolytic cleavage of L-canavanine, in a manner analogous to L-ornithine and urea formation from L-arginine, produces urea and L-canaline (3,4). The resulting canaline is distinctive in being the only naturally occurring amino acid which possesses the aminooxy group (Fig. 1). Canaline decomposes in several organic solvents employed for its analysis by partition and ion-exchange chromatography (5,6) but the important question of the overall stability of this substituted hydroxylamine has not been investigated.     

Fluorescein colchicine. Synthesis, purification, and biological activity

The synthesis of a fluorescent colchicine derivative permits the localization of colchicine-binding receptors in cells. Fluorescein colchicine (FC) was prepared by the addition of fluorescein isothiocyanate to deacetyl colchicine. The product, FC, was separated from the reactants by thin-layer chromatography (TLC). The purity of FC was demonstrated by TLC, UV spectral analysis, and analysis of the kinetics of photodecomposition. FC inhibited [3H] colchicine binding to purified brain tubulin. The biological activity of FC was compared to the activity of unlabeled colchicine on mitosis, motility, secretion, and myogenesis. The effects of FC were identical to those of unlabeled colchicine in all biological systems tested. The results demonstrate that FC may be substituted for colchicine in biological experiments without significant loss in specificity or effectiveness.     

Quantitative analysis of total membrane-bound sugars and amino sugars as alditol acetates by combined thin-layer chromatography,gas-liquid chromatography,and radiogas chromatography

A sensitive method (0.4 μg of hexoses routinely detectable) for quantitative determinations of sugars and amino sugars in biological material, particularly in membranes, is described. The method consists of a combination of thin-layer chromatography (tlc), gas-liquid chromatography (glc), and radiogas chromatography (rgc), using a highly thermostable phase (Silar 10 c) for the analysis of the specific alditol acetates. In this method, the losses incurred during hydrolysis and preparation for glc are assessed by comparison with the specific recoveries of added radiolabeled internal standards.     

Leukotriene C4: The major lipoxygenase metabolite of arachidonic acid in dog spleen

Slices of dog spleen converted [¹⁴C]-arachidonic acid (AA) to a polar material which conjugated with [³H]-glutatione. Nordihydroguaiaretic acid (NDGA) and 5,8,11,14, Eicosatetraynoic acid (ETYA) but not indomethacin, inhibited the conversion of [¹⁴C]-arachidonic acid by the spleen slices into the polar material indicating that it is derived through the lipoxygenase pathway. Physicochemical analysis of the polar metabolite of arachidonic acid after thin-layer chromatography and high pressure liquid chromatography revealed that it has chemical properties identical to authentic leukotriene C₄ standard (LTC₄). The biological activity of the purified material was found to be similar to the slow reacting substance of anaphylaxis (SRS-A), viz, it caused contraction of the guinea-pig ileum which was abolished by FPL-55172, a specific SRS-A receptor antagonist. These data suggest that dog spleen slices convert arachidonic acid through lipoxygenase pathway into a polar material that appears to be identical to LTC₄.     

Determination of peptidylglycine alpha-amidating monooxygenase activity in human serum by thin-layer chromatography

We developed a simple assay system for the quantitative evaluation of peptidylglycine alpha-amidating monooxygenase activity using as substrate a 125I-labeled synthetic tripeptide, 125I-D-Tyr-Val-Gly, thin-layer chromatography, and a radiochromatoscanner. The basic principle of this method is that thin-layer chromatography separates the reaction product, 125I-D-Tyr-Val-NH2, from the substrate in an assay mixture. The 125I activities of both substrate and product separated from each other on a thin-layer chromatography plate were quantified with a radiochromatoscanner and the rate of conversion of the substrate to the product was calculated from their counts. Human serum was used as an enzyme source and the values of alpha-amidation activity obtained by our method under optimal conditions were almost identical to those of the published method using ion-exchange chromatography (sulphopropyl-Sephadex C-50 column) and a gamma-counter. Our method makes it possible to estimate the 10-pmol level of the product using 10 microliters of human serum and to assay a large number of samples rapidly and easily. It is therefore thought to be very useful for screening various tissues for alpha-amidation activity.     

Mode of interaction of aminooxy compounds with sheep liver serine hydroxymethyltransferase

The interaction of aminooxy compounds such as aminooxyacetate (AAA), L-canaline, and hydroxylamine with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) was studied by absorption spectra and stopped-flow spectrophotometry and compared with the unique feature of interaction of O-amino-D-serine (OADS) with the enzyme [Baskaran, N., Prakash, V., Appu Rao, A. G., Radhakrishnan, A. N., Savithri, H. S., & Appaji Rao, N. (1989) Biochemistry (preceding paper in this issue)]. The reaction of AAA (0.5 mM) with the Schiff base of the enzyme resulted in the formation of pyridoxal 5'-phosphate (PLP) and was biphasic with rate constants of 191 and 19 s-1. The formation of the PLP-AAA oxime measured by decrease in absorbance at 388 nm on interaction of AAA with the enzyme had a rate constant of 5.2 M-1 s-1. On the other hand, the reaction of L-canaline with the enzyme was slower as measured by the disruption of enzyme-Schiff base than the reaction of OADS and AAA. In contrast, the formation of PLP as an intermediate could not be detected upon the interaction of hydroxylamine with the enzyme. The reaction of D-cycloserine with the enzyme was much slower (1.6 x 10(2) M-1 s-1) than the aminooxy compounds. These observations indicate that the aminooxy compounds that are structural analogues of serine (OADS, AAA, and canaline) formed PLP as an intermediate prior to the formation of oxime, whereas with hydroxylamine such an intermediate could not be detected.     

Abscisic acid in the leaf of Vernonia anthelmintica

Summary A growth inhibitor was isolated in crystalline form from the leaf of Vernonia anthelmintica and was found to be identical with abscisic acid by biological activity and physical measurements namely, melting point, mixed melting point, UV-absorption, mass spectra and paper and thin-layer chromatography.     

Induction of tryptophan oxygenase and tyrosine aminotransferase by metabolites of hydrocortisone

Metabolites of hydrocortisone were isolated from rat liver on a preparative scale, fractionated by column chromatography on Sephadex Lh-20 and silica gel and tested for biological activity. Apart from the well known neutral metabolites, steroid glucuronides and sulfates, we obtained metabolite fractions containing non-conjugated steroidal carboxy acids and acid metabolites of unknown structure. One of these fractions induced tyrosine aminotransferase (EC 2.6.1.5) in adrenalectomized female rats but not tryptophan oxygenase (EC 1.13.11.11), whereas another one mainly increased activity of tryptophan oxygenase. The doses necessary to significantly induce both enzymes were much lower in case of these metabolites than in the case of hydrocortisone itself. The active fractions eluting from silica gel column were analyzed by thin-layer chromatography in two different solvent systems. Absence of hydrocortisone in these fractions could be clearly demonstrated. Furthermore, the active fractions eluting from the silica gel column were characterized by treatment with an extract from Helix pomatia and/or diazomethane and subsequent analysis by thin-layer chromatography. We conclude, considering the biological activity of some synthesized derivatives of hydrocortisone, that the biologically active components are acid metabolites of hydrocortisone which are not identical to any of the known metabolites.     

A method for specific analysis of free fatty acids in biological samples by capillary gas chromatography.

The present report describes a simple method to selectively extract free fatty acids and analyze them by capillary gas-liquid chromatography. The procedure is based on the use of fumed silicon dioxide. In the presence of plasma, this material induces a rapid rise in the viscosity of the mixture and presents the ability to trap large particles such as emulsified lipids and lipoproteins. Albumin-bound fatty acids are thus left in the aqueous media. We present applications of our procedure for the analysis of free fatty acids in 0.2 ml of plasma from rat or human. By comparison with the method utilizing thin-layer chromatography for the separation of fatty acids and gas chromatography analysis, the present method has been found to be reliable and simple. The recovery of linoleic acid was 92.1 +/- 8.2%, a value which is about twice better than that obtained with the procedure using thin-layer chromatography. In particular, long-chain polyunsaturated fatty acids were better preserved. Our procedure does not require the use of organic solvents and its simplicity and reproducibility make it suitable for routine specific determination of the composition of free fatty acids in biological samples.     

Isolation of a urinary digitalis-like factor indistinguishable from digoxin

A digitalis-like factor has been purified to apparent homogeneity from human urine based on the inhibitory effect on [3H] ouabain binding to intact human erythrocytes. The purification scheme involved large scale adsorption followed by preparative, semipreparative and analytical high-performance liquid chromatography. The purified material showed a prominent digoxin-like immunoreactivity. The behaviour of the isolated substance was identical to that of authentic digoxin in three high-performance liquid chromatography and three thin-layer chromatography systems. Moreover, fast atom bombardment mass spectrum and proton nuclear magnetic resonance spectrum suggested that the purified material may be indistinguishable from digoxin.     

Micromethod for the quantitative determination of leucine from biological experimental material using a dansylation technic]

A method is described allowing quantitative determination of leucine from biological material up to 1 - 10(-11) M by means of dansylation of amino acids and their thin-layer chromatographic separation on polyamide microfoils. By selecting [14C]-U-L-leucine as an 'internal' standard, which is added to the tissue during the homogenization procedure, it is possible to correct for data scattering related to variations of the conversion rate or the yield of the dansylation reaction, and to losses in the different steps of thin-layer chromatography that are difficult to standardize.     

Induction of tryptophan oxygenase and tyrosine aminotransferase by metabolites of hydrocortisone

Metabolites of hydrocortisone were isolated from rat liver on a preparative scale, fractionated by column chromatography on Sephadex LH-20 and silica gel and tested for biological activity. Apart from the well known neutral metabolites, steroid glucuronides and sulfates, we obtained metabolite fractions containing non-conjugated steroidal carboxy acids and acid metabolites of unknown structure. One of these fractions induced tyrosine aminotransferase (EC 2.6.1.5) in adrenalectomized female rats but not trptophan oxygenase (EC 1.13.11.11), whereas another one mainly increased activity of tryptophan oxygenase. The doses necessary to significantly induce both enzymes were much lower in case of these metabolites than in the case of hydrocortisone itself. The active fractions eluting from silica gel column were analyzed by thin-layer chromatography in two different solvent systems. Absence of hydrocortisone in these fractions could be clearly demonstrated. Furthermore, the active fractions eluting from the silica gel column were characterized by treatment with an extract from Helix pomatia and/or diazomethane and subsequent analysis by thin-layer chromatography. We conclude, considering the biological activity of some synthesized derivatives of hydrocortisone, that the biologically active components are acid metabolites of hydrocortison which are not identical to any of the known metabolites.     

Diversion of ethanol metabolism by sulfhydryl amino acids. D-penicillamine-directed excretion of 2,5,5-trimethyl-D-thiazolidine-4-carboxylic acid in the urine of rats after ethanol administration.

Administration of the sulfhydryl amino acid; D-penicillamine, to rats prior to or simultaneously with ethanol gave rise to the urinary excretion of 2,5,5-trimethyl-D-thiazolidine-4-carboxylic acid (TTCA), which was detected by thin-layer chromatography and isolated as its N-acetyl derivative. The corresponding 2-methylthiazolidine-4-carboxylic acid could not be detected in the urine of rats treated with ethanol and L-cysteine under identical conditions. TTCA is formed by the condensation of the administered D-penicillamine with ethanol-derived acetaldehyde generated $\text{in vivo}$. This was verified by isolation of labeled TTCA after administration of D-penicillamine and ethanol-1-¹⁴C and quantitated by inverse isotope dilution assays. Disulfiram pretreatment of the rats doubled the excretion of TTCA in the urine. The physico-chemical properties of the isolated N-acetyl-TTCA were identical in all respects to an authentic, chemically-synthesized product, prepared by reacting D-penicillamine with acetaldehyde to form TTCA and then acetylating this product with acetic anhydride.     

Identification of transformation products arising from bacterial oxidation of codeine by Streptomyces griseus

14-Hydroxycodeine and norcodeine were rigorously identified as products arising from codeine oxidation by Streptomyces griseus ATCC 10137. Both products were routinely detected in extracted culture filtrates after growth of cells in the presence of codeine for 1 week. Under these conditions, about 4 mol% of the codeine starting material was consumed, with norcodeine and 14-hydroxycodeine representing the only identifiable transformation products (molar ratio, 4:1, respectively). Extraction of a series of culture filtrates and purification of the pooled metabolites by thin-layer and high-pressure liquid chromatography led to the isolation of both biological products, the structures of which were verified by high-resolution mass spectrometry and proton nuclear magnetic resonance spectroscopy. The identities of both biological products were further confirmed by comparison of their spectral properties with those of authentic standards. This is the first report providing structural evidence for the biological formation of 14-hydroxycodeine from codeine and of codeine oxidation by S. griseus.     

Identification of transformation products arising from bacterial oxidation of codeine by Streptomyces griseus.

14-Hydroxycodeine and norcodeine were rigorously identified as products arising from codeine oxidation by Streptomyces griseus ATCC 10137. Both products were routinely detected in extracted culture filtrates after growth of cells in the presence of codeine for 1 week. Under these conditions, about 4 mol% of the codeine starting material was consumed, with norcodeine and 14-hydroxycodeine representing the only identifiable transformation products (molar ratio, 4:1, respectively). Extraction of a series of culture filtrates and purification of the pooled metabolites by thin-layer and high-pressure liquid chromatography led to the isolation of both biological products, the structures of which were verified by high-resolution mass spectrometry and proton nuclear magnetic resonance spectroscopy. The identities of both biological products were further confirmed by comparison of their spectral properties with those of authentic standards. This is the first report providing structural evidence for the biological formation of 14-hydroxycodeine from codeine and of codeine oxidation by S. griseus.     

A Modified Method for Extraction and Identification of Abscisic Acid and Gibberellin-like Substances from the Olive (Olea europaea)

A procedure for extracting and identifying plant hormones, particularly abscisic acid (ABA) and the gibberellins (GA) was developed through modification of methods described in the literature. The procedure is particularly useful for studying more than one hormone simultaneously in a given sample, and when the supply of plant material is limited. The procedure was used to isolate ABA and GA-like substances from olive tissue (i.e., leaves, buds and inflorescences). Gibberellin-like substances were identified by their action on α-amylase release from embryoless barley half-seeds. Characterization of an acidic inhibitor extracted from olive inflorescences by thin-layer chromatography, fluorescence under ultraviolet light, gas chromatography, and physiological effects on wheat coleoptile sections indicate that this inhibitor, or at least a component of it, is very similar if not identical with at least one isomeric form of synthetic abscisic acid.     

NADP-specific isocitrate dehydrogenase in regulation of urea synthesis in rat hepatocytes.

The effect of inhibition of NADP-specific isocitrate dehydrogenase (EC 1.1.1.42) by DL-threo-alpha-methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylase) on urea synthesis was studied in isolated rat hepatocytes. alpha-Methylisocitrate substantially inhibited the rate of urea synthesis (35--84%) with substrates requiring net reductive amination of 2-oxoglutarate to glutamate for aspartate synthesis (i.e., L-serine, D-alanine, or NH4Cl + L-lactate). alpha-Methylisocitrate did not inhibit synthesis of urea from substrates not requiring reductive formation of glutamate (i.e. L-alanine, L-glutamine, L-asparagine, or NH4Cl + L-ornithine). The rate-limiting role of NADPH in urea synthesis was correlated with the decrease in NADPH content that occurred upon addition of NH4Cl or of alpha-methylisocitrate to hepatocytes incubated with lactate and pyruvate, indicating utilization of NADPH for reductive amination of 2-oxoglutarate and inhibition of NADPH generation via NADP-isocitrate dehydrogenase, respectively. Similar results were obtained with D-alanine and L-serine; however, alpha-methylisocitrate or NH4Cl did not substantially decrease NADPH content when L-alanine was the substrate. Inhibitors or ornithine--2-oxo acid transaminase (L-canaline or gabaculine) decreased the uptake of ornithine by hepatocytes and inhibited the alpha-methylisocitrate insensitive urea synthesis from ornithine and NH4Cl. Canaline did not inhibit urea synthesis from lactate, ornithine, and NH4Cl but the inhibition by alpha-methylisocitrate of urea formation from this combination was appreciably larger with canaline (approx. 82%) than without canaline (approx. 48%). Inhibition of urea synthesis from NH4Cl + lactate by alpha-methylisocitrate was partially prevented by oleate, octanoate, or 3-hydroxybutyrate. When the NADH content of hepatocytes was increased by 3-hydroxybutyrate, the addition of NH4Cl and/or alpha-methylisocitrate caused a decline in NADH (and NADPH) content, suggesting that reducing equivalents from NADH as well as from NADPH can support net reductive amination of 2-oxoglutarate when required for urea synthesis.     

Densitometric quantitation of individual phospholipids from natural sources separated by one-dimensional thin-layer chromatography

A method for the quantitation of small amounts of phospholipids derived from biological sources is described. Total phospholipid is determined by mineralization followed by the estimation of liberated phosphate by means of malachite green. The main phospholipid species are separated by one-dimensional thin-layer chromatography. The individual phospholipids are detected by charring with CuSO4/H3PO4. They may be directly quantitated by scanning the thin-layer chromatography plates with a laser densitometer.     

Isolation and characterisation of bis-phosphatidic acid and its partially deacylated derivatives from cultured BHK-cells

Bis-phosphatidic acid, semi-lyso-bis-phosphatidic acid and lyso-bis-phosphatidic acid were isolated from cultured baby hamster kidney fibroblasts (BHK-cells). They constituted about 0.02%, 0.1% and 1.6% respectively of total lipid phosphorus of fresh BHK-cell monolayers. These three phospholipids were also prepared by partial synthesis. The natural lipids were identical with the synthetic samples as determined by thin-layer chromatography and by partial degradation by controlled alkaline methanolysis, acetic acid hydrolysis and acetolysis. The three derivatives of bis-phosphatidic acid contained 60–80% of octadecenoic acid; in this respect they were different from other BHK-cell phospholipids.