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PubMed1988

Estimation of lipid concentrations on thin-layer plates by densitometry of transparent copies.

A relatively simple procedure for the quantitative estimation of phospholipids resolved on thin-layer plates has been developed. After resolution in an appropriate solvent, the lipids are visualized by staining with iodine, ninhydrin, or molybdate and then photocopied onto transparent sheets with a standard office copy machine. The density of each spot on the photocopy, measured with a simple silicon cell area densitometer, is a direct function of each lipid applied to the plate over at least a six- to eightfold range in concentration. Under controlled conditions the staining and photocopying steps are quite reproducible. Known concentrations of the choline, ethanolamine, serine, and inositol derivatives of L-alpha-phosphatidic acid applied either separately or as mixtures can be determined essentially quantitatively (100 +/- 5%) by this procedure following their resolution on the thin-layer plates.

Animals↗
PubMed1985

N-cyclo-[Leu5]enkephalin: a rational approach for the synthesis of conformationally restricted cyclic pentapeptides.

The enkephalins are neuropeptides belonging to the class of endogenous opioids. The conformationally restricted analog, N-cyclo-[Leu5]enkephalin, was recently synthesized. Since the synthesis of cyclic pentapeptides which lack proline and contain amino acids with bulky side chains is problematic, the synthesis, purification, and analytical characterization of N-cyclo-[Leu5]enkephalin is described in detail. This conformationally restricted cyclic pentapeptide was prepared from H-Gly-Phe-Leu-[O-(2,6-dichlorobenzyl)-Tyr]-Gly-NHNH2, which was synthesized by the solid-phase method of peptide synthesis. Cyclization was accomplished through an azide intermediate at high dilution, using high-pressure liquid chromatography to monitor the reaction. The desired cyclic monomer was isolated and purified by semipreparative HPLC. The structure of the purified cyclic product was confirmed by multiple chemical techniques including amino acid analysis, lack of an amino terminus (as assessed by reaction with ninhydrin and Edman reagent), and mass spectroscopy.

Amino Acids↗
PubMed1987

The purification and characterization of subunits alpha, beta, and gamma from the rabbit reticulocyte eukaryotic initiation factor 2.

Eukaryotic initiation factor 2 (eIF-2) contains three nonidentical subunits, alpha, beta, and gamma. The simultaneous purification of all three subunits was achieved by reverse-phase HPLC using a 0.1% trifluoroacetic acid-acetonitrile binary solvent system. The order of the eluted subunits, beta, alpha, and gamma, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After hydrolysis in 6 N HCl, picomole level amino acid composition analysis was achieved by the ninhydrin reaction on a Beckman 6300 system. Using second-derivative spectroscopic analysis, Trp was detected in all three subunits. All three subunits were subjected to amino-terminal sequence analysis. The amino-terminal of eIF-2 alpha from amino acid positions 1 to 23 inclusive was determined. The order of eight amino acids from the amino-terminal of eIF-2 gamma was also determined. This characterization and partial determination of the primary sequence of these subunits permit the utilization of molecular biology techniques in order to elucidate the complete primary structure. Additionally, the partial amino acid sequence data permitted the designation of synthetic gene probes as well as the identification of eIF-2 alpha and gamma cDNA and/or genomic clones.

Amino Acid Sequence↗
PubMed1988

Inhibition of porphyrin biosynthesis by exogenous 5-aminolevulinic acid in an aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114.

Exogenously administered 5-aminolevulinic acid (ALA) inhibited the formation of bacteriochlorophyll a (Bchl a) in a dose-dependent manner in the aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh 114, under dark growth conditions. The ALA concentration required for half-inhibition after 24-h growth was estimated to be about 3.0 mM. Porphyrin and Bchl precursors were not found in either the cells or the growth medium. The same inhibition was also observed with cytochrome c formation. When ALA was incubated with intact cells, a large amount of ALA was converted to an unknown metabolite. The pH optimum of the conversion was 7.8. The metabolite did not react with Ehrlich's reagent, but did so with ninhydrin, giving a yellow color. Based on analyses by several techniques including mass spectrometry, ir spectrometry, and paper electrophoresis, it was identified as 4-hydroxy-5-aminovaleric acid (HAVA). Authentic HAVA prepared from ALA was a competitive inhibitor of the enzyme, porphobilinogen synthase of Erythrobacter. The Ki value for authentic HAVA was calculated to be 2.4 mM from a Dixon plot and the HAVA concentration required for half-inhibition was 17 mM. It is concluded that in Erythrobacter cells, exogenous ALA is converted to the metabolite, HAVA, which is responsible for the inhibition of porphobilinogen synthase as well as that of Bchl a and cytochrome formation.

Aminolevulinic Acid↗
PubMed1991

Deamidation and succinimide formation by gamma-N-methylasparagine: potential pitfalls of amino acid analysis.

The biological function of the post-translationally methylated amino acid gamma-N-methylasparagine (gamma-NMA) in proteins is unknown. We are examining the premise that amide methylation protects against deamidation. The free amino acids Asn, gamma-NMA, Gln, and delta-N-methylglutamine (delta-NMG) were incubated at elevated temperature and a variety of pH conditions to assay for deamidation. Gln disappears 12- to 14-fold more rapidly than delta-NMG, and Asn hydrolyzes to Asp and NH3 as expected. However, the gamma-NMA deamidation rate is severely overestimated by simply measuring the disappearance of starting material because gamma-NMA undergoes a cyclization reaction in preference to deamidation. At pH 1 the predominant gamma-NMA reaction is formation of stable 3-amino-N-methylsuccinimide (NMS) and this occurs greater than 10-fold faster than Asn deamidation. At pH 4.0, 7.4, and 9.0 NMS is readily formed but it is unstable and partitions between the parent compound, gamma-NMA, and a second species, alpha-N-methylasparagine. At pH 7.4 and 9.0 gamma-NMA disappears 4-fold slower than Asn but the methyl amide hydrolysis rate is diminished by as much as 13-fold. The Asn incubations over the pH range 1-9 yield scant evidence of a succinimide intermediate. It is concluded that the amide methylation provides a unique reaction pathway and stabilization for the N-methylsuccinimide species. Amino acid analysis by o-phthalaldehyde postcolumn reaction fails to detect isoasparagine, alpha-N-methylasparagine, and NMS. Amino acid analysis by precolumn derivatization with phenyl isothiocyanate destroys NMS and therefore cannot quantitate this compound. The ninhydrin postcolumn derivatization method is able to detect and quantitate all of these amino acid species.

Amino Acids↗
PubMed1976

Proteolysis of canine apolipoprotein by acid proteases in canine liver lysosomes.

Canine liver lysosomes were purified by sucrose discontinuous density gradient centrifugation and then ruptured by sonication to obtain the soluble fraction. This soluble lysosomal fraction, which contained a 25-fold increase in acid phosphatase activity per mg of total protein when compared with the original homogenate, was incubated with a subfraction (1.110 less than d less than 1.210 g/cm3, HDL3) of canine high density lipoproteins (HDL) at pH 3.8. HDL3 proteolysis by lysosomal proteases, measured as the release of peptides and amino acids by the ninhydrin reaction, followed hyperbolic curves with straight lines (r = 0.99) obtained on Lineweaver-Burk plots. Km calculated from the Lineweaver-Burk plot was 635 mug of HDL3 protein per 0.5 ml of incubation mixture. Optimum HDL3 proteolysis was observed from pH 3.8 to 4.5. Incubation with the other subcellular organelle fractions did not result in HDL3 proteolysis. To evaluate the effects of enzyme inhibitors, iodoacetate, p-chloromercuribenzoate (both specific for the endopeptidase, cathepsin B (EC 3.4.22.1)) and pepstatin (specific for the endopeptidase, cathepsin D (EC 3.4.23.5) were tested. Iodoacetate and p-chloromercuribenzoate inhibited HDL3 proteolysis 100% and bovine serum albumin proteolysis 65%. Pepstatin inhibited HDL3 proteolysis 45% and bovine serum albumin proteolysis 70%. The in vitro data presented support the hypothesis that hepatic lysosomes play an important role in HDL3 catabolism in the dog. Furthermore, results obtained from enzyme inhibition studies suggest that a specific lysosomal endopeptidase, cathepsin B, may play the key role in HDL3 proteolysis.

Acid Phosphatase↗
PubMed1987

Lipid analysis of a novel type of cell secretion in the exocrine pancreas: the pancreasomes.

A novel type of cell secretion termed 'microvesicular secretion' has been described recently in the exocrine pancreas. According to this process, microvesicles are released by acinocytes in the pancreas acinar lumen. These microvesicles, identified as 'pancreasomes', were characterized by the presence of a major glycoprotein component originating in the exocrine acinar cell. In the present work, phospholipids of pancreasomes have been identified. Five classes of phospholipid were found: phosphatidylcholines, phosphatidylethanolamines, lysophosphatidylcholines, sphingomyelins and another minor class of ninhydrin-positive phospholipid (phosphatidylserines or lysophosphatidylethanolamines). The ratios of neutral lipids to phospholipids were particularly high (3:1), as estimated by GLC of their fatty acid content. Analysis of fatty acid composition of pancreasomes lipids revealed a very high proportion of two saturated fatty acids, palmitic (40%) and stearic (24%), whereas two main unsaturated fatty acids, oleic (17%) and linoleic (8%), were found in smaller proportions. Differential scanning calorimeter studies on washed pancreasomes indicated that there was no lipid phase transition in their membrane, despite the absence of cholesterol. Our observations show that pancreasomes have an unusual lipid composition and confirm our previous conclusion based on protein analysis that the release of pancreasomes occurs according to an hitherto undescribed type of secretion, in which a glycoprotein is released associated with specific domains of the luminal plasma membrane.

Animals↗
PubMed1975

Purification of an inhibitor of DNA photolyase with fluorescent spectra similar to those of the enzyme.

1. The binding of DNA photolyase, the enzyme which uncouples cyclobutadipyrimidines in DNA upon illumination, to its substrate was enhanced by a substance isolated from acidified autolysates of baker's yeast. 2. This substance, referred to as activator, was partially purified by chloroform extraction, ion-exchange chromatography on Dowex 50 and DEAE-cellulose, and gel filtration on Sephadex G-10. Upon lyophilization and storage at -20 degrees C, it was converted to a potent inhibitor of enzyme binding to substrate. 3. The inhibitor appeared as a single band in two thin-layer chromatography systems and was detected by ultraviolet absorbance, fluorescence, and ninhydrin staining. 4. The similarity between the fluorescence spectra of the inhibitor and the enzyme suggested that the inhibitor was structurally analogous to a chromophore in the enzyme and that the activator from which the inhibitor was derived may be the active chromophore of the enzyme.

Biological Assay↗
PubMed1971

Number and types of peptide chains in thyroglobulin: tryptic peptides of noniodinated hog thyroglobulin.

In order to identify the number and types of peptide chains in thyroglobulin, noniodinated 19-S thyroglobulin obtained from goitrogen-treated hogs was exhaustively digested with trypsin (EC 3.4.4.4) after reduction and S-carboxymethylation. The digestion mixture was preliminarily separated into 30 fractions on Sephadex G-100 or G-15 and SE-Sephadex columns. The number of various tryptic peptides contained in each fraction was determined on peptide maps, where spots were detected with ninhydrin for total peptides and with each specific reagent for arginine, histidine or tyrosine-containing peptides. The number of total peptides observed in most of the fractions was estimated to be half the number of lysine plus arginine residues found in each fraction per mole of thyroglobulin, and the number of specific peptides was also close to half the number of each specific amino acid. These findings imply that thyroglobulin has 2-fold symmetry in the structure at the level of tryptic fragments and thus probably at the level of intact peptide chains.

Amino Acids↗
PubMed1976

Primary sequence of the beta-chain of Badger haemoglobin.

Badger (Meles meles) haemoglobin was purified by paper electrophoresis and converted into globin. Chain separation was carried out on a CM-cellulose column in the presence of 8 M urea. The beta-chain was aminoethylated, purified by gel filtration and submitted to tryptic digestion. A fingerprint obtained with the enzymic digests showed 17 distinct ninhydrin-positive spots from which 20 pure peptides were isolated by further electrochromatographic separations. These peptides were sequenced using Dansyl-Edman and Ptc-Edman degradation techniques. The presence of amide residues was confirmed after aminopeptidase M hydrolysis. Taking human haemoglobin beta-chain as a model, the covalent structure could be completely resolved without the help of any further overlapping technique. The following substitutions were noted (badger/human, position): Ala/Pro5, Ser/Ala13, Tyr/Phe41, Asp/Glu43, Ser/Ala70, Glu/Asp73, Lys/Ala76, Asn/His77, Lys/Thr87, Lys/Arg104 and Gln/Pro125. A comparison with other haemoglobin beta-chains already sequenced shows a greater similarity with dog haemoglobin, the only example of beta-chain of known structure in the order of Carnivores.

Amino Acid Sequence↗
PubMed1985

Natural occurrence of guanidinooxypropylamine in Wistaria floribunda and the sword bean Canavalia gladiata.

We found a new guanidinooxyamine in Wistaria floribunda seeds and seedlings of the sword bean, Canavalia gladiata. This amine was not only ninhydrin-positive but also gave a positive alkaline nitroprusside-ferricyanide reaction. It was characterized as gamma-guanidinooxypropylamine [H2N (NH=)CNHO (CH2)3NH2] by comparison with the authentic compound on column and thin-layer chromatograms visualized with specific reagents and by reductive cleavage. Evidence for the occurrence of another unusual guanidino amine, homoagmatine in W. floribunda seeds was also presented.

Chromatography, High Pressure Liquid↗
PubMed1983

On the mechanism of covalent binding of butylated hydroxytoluene to microsomal protein.

The structures of cysteine conjugates of 3,5-di-tert-butyl-4-hydroxytoluene (BHT) and the binding sites of BHT metabolites on microsomal protein were investigated by 13C nuclear magnetic resonance (13C-NMR) and gas-liquid chromatography/mass spectrometry. The cysteine conjugates of 2,6-di-tert-butyl-4-hydroxymethylphenol (BHT-alcohol) and 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (quinone methide), which are metabolites of BHT found in rat liver and specifically reacts with thiol compounds, were prepared as alcoholic aqueous solutions. The molecular structure of the cysteine conjugate of BHT-alcohol agreed completely with that of quinone methide in 13C-NMR spectra or mass spectra. These spectra of both conjugates further showed that the conjugates are due to thioether binding between the 4-methyl group of metabolites and the sulfhydryl group of cysteine. When [14C]BHT-bound microsomes prepared in vitro were enzymatically hydrolyzed with Pronase E, the major radioactive material that eluted with methanol from a column of Amberlite XAD-2 and gave a positive ninhydrin reaction was identified as a cysteine conjugate of BHT by comparing its Rf values on TLC and mass spectrum. On the basis of the results, it was apparent that the binding site of activated substituents of BHT on protein was mainly the sulfhydryl group of cysteine residue.

Animals↗
PubMed1985

Peptide-containing fractions in depression.

A mixture of peptides and glycoproteins has been found in benzoic acid-precipitable material from urines of psychomotorically agitated and retarded endogenous depressive patients. This complex mixture of compounds is fractionated on a Sephadex G-25 gel, from which the different peaks are further separated on Biogel P2. The G-25 elution profiles ultraviolet absorbance, 280 nm) from depressive patients deviated from the normal pattern. The increase in hydrolyzable ninhydrin-colorable material of the P2 fractionation step encountered in psychotic depression was several-fold that of the normal population. Neurochemically active peptide-containing fractions were found. As explanation of these findings, it is probable that a genetically determined peptidase insufficiency is present, causing a peptide overflow when the secretion outstrips the breakdown. This model could easily combine more psychodynamic models with the genetic-biological models. The variability of the peptide patterns could possibly reflect the considerable clinical variability of the syndrome. Furthermore, the presence of a group of active compounds with different neuropharmacological activities might reflect the composite nature of the depressive syndrome.

Adult↗
PubMed1975

Isolation of an endogenous compound from the brain with pharmacological properties similar to morphine.

This investigation was carried out to test the hypothesis that the brain contains a substance that functions as an endogenous mediator at central morphine receptor sites. The extraction and purification of a low molecular weight substance are described. This substance inhibits neurally evoked contractions of the mouse vas deferens and guinea pig myenteric plexus. The inhibitory action of this substance was antagonized by the 3 narcotic antagonists naloxone, naltrexone and MR 1302 at nanomolar concentrations. The narcotic antagonists alone had no effect on the assay tissues. The purified substance had no effect on the guinea pig or rabbit vas deferens, tissues which do not possess morphine receptor sites. The pharmacological activity of the substance is destroyed by carboxypeptidase and leucine aminopeptidase, it is hydrophilic and behaves as an ampholyte in ion exchange systems. These observations and the production of ninhydrin positive products on acid hydrolysis indicate the presence of a small molecular weight (less than 700) peptide. The morphine-like substance was unevenly distributed in the brain, the highest concentrations occurring in the striatum, midbrain, pons and medulla. No activity could be detected in extracts of cerebellum, liver or lung. It is suggested that the compound isolated in this investigation forms part of a central pain suppressive system and may also have a wider neurochemical role in the brain.

Animals↗
PubMed1977

Amino acids in the synaptic vesicle fraction from calf brain: content, uptake and metabolism.

A synaptic vesicle fraction was prepared from calf brain cortex, containing 10 identified amino acids and two unidentified ninhydrin-positive compounds, one of which is apparently a peptide. The most plentiful amino acids were taurine (1.8 nmol/g original tissue), glutamic acid (1.8), serine (0.9), aspartic acid (0.8) and GABA (0.8); the others identified were cysteic acid (or cysteinesulphinic acid), glutamine, alanine, glycine and lysine. The unknown peptide occurred in a high concentration (about 16 alanine equivalents/g), and contained mainly aspartic acid and serine. Cysteic acid (or cysteinesulphinic acid) also occurred in relatively high amounts, but its peak contained acid-labile impurities. The influx of [14C]glutamate into the vesicles took place by means of non-saturable migration, while two saturable systems having very similar properties were dominant only at low glutamate concentrations. Influx constants for these quantitatively low uptake systems were Km, 34 and 92 micrometer, and Vmax, 33 and 49 nmol/min/g obtained by v versus v/S plot. Almost the same values were also obtained by a 1/v versus 1/S plot. GAD and GABA-T activities in the vesicles were only 1/200th of those in the synaptosomes.

4-Aminobutyrate Transaminase↗
PubMed1976

In vitro acylation of the xi-amino group of L-lysine in calf thymus histones by the carcinogen, beta-propiolactone.

We had previously reported that the carcinogen, beta-propiolactone (BPL) reacted in vitro with histones in whole mouse skin chromatin and that among the histone classes BPL was preferentially bound to the lysine-rich histones H1 and H1 degrees. In order to determine if in vitro reaction of BPL with calf thymus histones resulted in binding of BPL to L-lysine, we synthesized the model compounds XI-N-(3-hydroxypropionyl)lysine (HPL) and xi-N-(I-carboxyethyl)lysine (CEL) from BPL and L-lysine. The alpha-amino group of L-lysine was protected from reaction with BPL by the formation of a copper chelate. Structures were assigned on the basis of infrared spectra, pKa values and chemical analyses. BPL was reacted in vitro with calf thymus histones and the BPL-reacted calf thymus histones and control calf thymus histones were digested with trypsin followed by pronase. The respective digests were each chromatograhed on a column of AA-15 cation-exchange resin. The elution profiles of the two digests were very similar except for the appearance of a new ninhydrin-positive peak (NNPP) in the eluate of the trypsin-pronase digest of BPL-reacted calf thymus histones. When compounds HPL and CEL were added to the trypsin-pronase digest of control calf thymus histones and the mixture chromatographed on AA-15, both compounds were resolved from the other peptide (or amino acid) peaks. HPL was eluted in the same fractions as NNPP, HPL and NNPP exhibited identical RF values on silica gel TLC with acidic, alkaline and neutral solvents. CEL was not identified as a product of the reaction between BPL and calf thymus histones.

Animals↗
PubMed1975

An enzymic method for the determination of the glycine/taurine ratio of conjugated bile acids in bile.

A method is described in which the ratio of the glycine- to taurine-conjugated bile acids (G/T ratio) in bile is determined. After pretreatment of the bile for removal of the lipids, the bile acids are deconjugated enzymically with choloylglycine hydrolase (EC 3.5.1.24). The amino acids taurine and glycine are liberated in this way, after appropriate isolation, taurine and/or glycine are then determined with ninhydrin, enabling the establishment of the G/T ratio. A nearly complete hydrolysis was obtained for 6 conjugated bile acids, while the recovery of these acids when added to hog or ox bile was quantitative. The mean G/T ratio for hog bile, ox bile and human B-bile was 6.3, 2.5 and 2.0, respectively. The amount of total, free and conjugated bile acids can be determined by this method, combined with the 3alpha-hydroxysteroid dehydrogenase technique for bile acid determination described by Iwata and Yamasaki [1]. A high G/T ratio was observed in 3 cases of Crohn's disease in the small bowel, but the extent of deconjugation in B-bile was lower than in duodenal fluid. The determination of the G/T ratio can be complementary to our knowledge of the metabolism of bile salts in certain gastro-intestinal disorders.

Amidohydrolases↗
PubMed1976

Identification of beta-aminoisobutyric acid in uremic serum.

An unidentified ninhydrin-positive substance found in uremic sera but not found in normal sera was isolated by gel-filtration through Sephadex G-75 followed by high voltage paper electrophoresis (pH 3.5), and identified as beta-aminoisobutyric acid using paper chromatography and automated amino acid analyzer. The quantitative determination of beta-aminoisobutyric acid in serum revealed that the level of beta-aminoisobutyric acid in uremic sera was much higher than that of normal sera. Gas chromatographic determination of the enantiomorphs of beta-aminoisobutyric acid showed that uremic sera contain R- and S-isomers of the amino acid, but with the R-isomer as the dominating form.

Aminoisobutyric Acids↗