A procedure for the separation and quantitation of tryptophan and amino sugars on the amino acid analyzer

Tryptophan, 5-methyl tryptophan, glucosamine, and galactosamine can be separated from each other and hydrolysis products including lysinoalanine by chromatography on a 6 × 260-mm column of W-3H resin. The column is developed at 70°C for 20 min with pH 3.95 (0.4 Na⁺) buffer, followed by pH 6.4 (1 Na⁺) buffer for 55 min using a Beckman 119 CL amino acid analyzer. The recovery of the internal standards, 5-methyl tryptophan and galactosamine, can then be used to correct for tryptophan and glucosamine losses, respectively. The procedure uses the column and buffers normally employed for protein hydrolysate analysis and does not require additional resin columns, special buffers, or flow rate changes.     

Note on the lysinoalanine content of alkali-treated barley

The concentration of lysinoalanine in a sample of barley treated with 35 g NaOH kg^?1 was found to be 1.16 g kg^?1 compared with zero concentration in control samples. Although such a level in the diet might result in a reduced growth rate and produce kidney lesions in the rat, the nutritional risk to farm stock, including non-ruminant animals, would appear to be small if allowance is made for reduced concentrations of important amino acids when formulating diets.     

Limits and Sites of Lysinoalanine Formation in Lysozyme, α-Lactalbumin and αs1- and β-Caseins by Alkali Treatment

Lysinoalanine was determined by gas chromatography. Lysinoalanine formation in lysozyme depended on alkali concentration, pH, temperature and exposure time. The upper limits of lysinoalanine formation in lysozyme and α-lactalbumin were related to the number of lysine residues with a cystine disulfide bond in the adjacent position rather than the individual contents of these residues. In cases of α_sl- and β-caseins, the upper limits were related to the number of phosphoserine residues, regardless of their sequential relationship to lysine residues. Gel electrophoresis suggested that intermolecular cross-linking took place in the α-lactalbumin and caseins.     

Effect of Emodin on the Mutagenicity of 3-Amino-1-methyl-5H-pyrido[4,3-b]indol toward Salmonella

Mass spectra of N-trifluoroacetyl n-butyl ester (BTFA) of ornithinoalanine (OAL) and lanthionine (LAN) were compared with those of the BTFA derivatives of lysinoalanine (LAL) and the related amino acids. A difference of m/z 14, corresponding to one methylene group, was found in each pair of characteristic fragments between BTFA-OAL and BTFA-LAL. A temperature-programmed gas-liquid chromatography and gas chromatography-mass spectrometry were developed to analyze BTFA-LAN, BTFA-OAL and BTFA-LAL. More LAL and LAN were formed in α-lactalbumin than lysozyme by high-temperature treatment in water. OAL was detected in lysozyme treated at 100° and 120°C in alkali solution, but not in α-lactalbumin.     

N-ε-fructosyllysine and N-ε-carboxymethyllysine,but not lysinoalanine,are available for absorption after simulated gastrointestinal digestion

Food processing leads to a variety of chemical modifications of amino acids in food proteins. Recent studies have shown that some modified amino acids resulting from glycation reactions can pass the intestinal barrier when they are bound in dipeptides. In this study, we investigated as to what extent modified amino acids are released from post-translationally modified casein during simulated gastrointestinal digestion. Casein was enriched with N-ε-fructoselysine, N-ε-carboxymethyllysine, and lysinoalanine, in different degrees of modification. The casein samples were subjected to a two-step proteolysis procedure, simulating gastrointestinal digestion. The digestibility of modified casein as measured by analytical size-exclusion chromatography (SEC) decreased with increasing degree of modification especially after enrichment of fructoselysine and lysinoalanine. Semi-preparative SEC of digested casein samples revealed that fructoselysine and carboxymethyllysine are released bound in peptides smaller than 1,000 Da, which is comparable to native amino acids. The glycation compounds should, therefore, be available for absorption. Lysinoalanine as a crosslinking amino acid, however, is mostly released into longer peptides of at least 30–40 amino acids which should strongly impair its absorption availability.     

Analysis of hog thyroglobulin. Identification of galactosamine and absence of lysinoalanine, a potential product of tyrosine coupling

A small peak in the amino acid analysis of hog thyroglobulin was observed in the region reported for lysinoalanine and galactosamine. Since galactosamine had been previously reported absent in hog thyroglobulin, the possibility that this peak was lysinoalanine, a potential product of the coupling of two iodotyrosines, was investigated. Tests, however, showed that the material was galactosamine and that hog thyroglobulin contains no significant amount of lysinoalanine. Approximately 5 moles of galactosamine were found per mole of thyroglobulin.     

Semi-microbiological synthesis of an active lysinoalanine-bridged analog of glucagon-like-peptide-1

Some modified glucagon-like-peptide-1 (GLP-1) analogs are highly important for treating type 2 diabetes. Here we investigated whether GLP-1 analogs expressed in Lactococcus lactis could be substrates for modification and export by the nisin dehydratase and transporter enzyme. Subsequently we introduced a lysinoalanine by coupling a formed dehydroalanine with a lysine and investigated the structure and activity of the formed lysinoalanine-bridged GLP-1 analog. Our data show: (i) GLP-1 fused to the nisin leader peptide is very well exported via the nisin transporter NisT, (ii) production of leader-GLP-1 via NisT is higher than via the SEC system, (iii) leader-GLP-1 exported via NisT was more efficiently dehydrated by the nisin dehydratase NisB than when exported via the SEC system, (iv) individual serines and threonines in GLP-1 are dehydrated by NisB to a significantly different extent, (v) an introduced Ser30 is well dehydrated and can be coupled to Lys34 to form a lysinoalanine-bridged GLP-1 analog, (vi) a lysinoalanine(30-34) variant's conformation shifts in the presence of 25% trifluoroethanol towards a higher alpha helix content than observed for wild type GLP-1 under identical condition, (vii) a lysinoalanine(30-34) GLP-1 variant has retained significant activity. Taken together the data extend knowledge on the substrate specificities of NisT and NisB and their combined activity relative to export via the Sec system, and demonstrate that introducing a lysinoalanine bridge is an option for modifying therapeutic peptides.     

Isolation and partial characterization of a 51Cr complex from Brewers' yeast

A butanol: H₂O extract of Brewers' yeast grown in a medium which contained ⁵¹Cr was analyzed by gel-filtration chromatography. A single radioactive peak was eluted at an elution volume which suggested a molecular weight of approximately 400–600 daltons. Subsequent examination of pooled radioactive fractions obtained from gel-filtration chromatography demonstrated that the ⁵¹Cr complex was eluted in a single peak from both cation- and anion-exchange resins. The elution characteristics of the ⁵¹Cr complex indicated that the compound is a single anionic species. The ⁵¹Cr complex was purified by a combination of gel-filtration chromatography and ion-exchange chromatography and subsequently analyzed by thin-layer chromatography. The results indicated that the ⁵¹Cr complex from Brewers' yeast is a peptide that contains at least six amino acids. When a partially purified preparation of the ⁵¹Cr complex from yeast was administered orally to rats, the absorption and retention of ⁵¹Cr was significantly greater than that in rats given ⁵¹Cr in the form of CrCl₃·6H₂O. These experiments demonstrate that chromium is associated with a single metal-binding peptide in Brewers' yeast and indicate that the chromium in this complex is absorbed and retained more efficiently than chromium salts.     

Cytokinins: A Rapid Extraction and Purification Method

A method is described for the isolation, purification and quantitation of free cytokinin bases and ribosides using ethyl acetate at pH 7.7 for the extraction. The extraction is almost complete (97.7%) as determined by using N⁶-(Δ²-isopentenyl)adenine-8-¹⁴C. The subsequent fast purification by chromatography on a standardized silica gel column in chloroform-methanol (7:3 v/v) is followed by thin layer chromatography (silica gel 60 F²⁵⁴) in chloroform-acetic acid (8:2 v/v). The recovery of N⁶-(Δ²-isopentenyl)adenine-8-¹⁴C after this two step purification was 78–82%. The efficiency of the method was determined by applying this procedure to N⁶-(Δ²-isopentenyl)adenine and N⁶(Δ²-isopentenyl)adenosine. Using gas liquid chromatography the recovery for N⁶-(Δ²-isopentenyl)adenosine was determined to be 61% and compared to 43% for N⁶-(Δ²-isopentenyl)adenosine, showing the suitability of the described method for gas liquid chromatography.     

Properties of a Zn2+-glycerophosphocholine cholinephosphodiesterase from bovine brain membranes

A Zn²⁺-glycerophosphocholine cholinephosphodiesterase was purified with a specific activity of 4.6 μmole/min·mg protein from bovine brain membranes by procedures involving PI-PLC solubilization, concanavalin A affinity chromatography, CM-sephadex chromatography and Sephadex G-150 chromatography. Based on molecular weight determination gel chromatography and SDS polyacrylamide gel electrophoresis, the phosphodiesterase activity appears to be a dimeric protein (110 kDa) composed of two subunits with a molecular weight of approximately 54 kDa. The Km value for p-nitrophenylphosphocholine and the optimum pH were found to be 16 μM and pH 10.5, respectively. The phosphodiesterase was inhibited by Cu²⁺, but not the other divalent metal ions. The activity of the apoenzyme was remarkably activated by Co²⁺ or Zn²⁺, but not Mn²⁺ or Mg²⁺. In addition, the inactivation of the enzyme in glycine buffer was prevented by Mn²⁺ or Zn²⁺, but not Co²⁺ or Mg². In a separate experiment, comparing properties of the purified and membrane-bound phosphodiesterases, the forms of two enzymes were quite similar except in stability. Both enzymes were more stable at pH 7.4 than pH 5 or 10. However, the membrane-bound enzyme was more stable than the soluble enzyme at all three pHs. These data suggest that the activity of the phosphodiesterase may be stabilized in-vivo.     

The Methylated Purines of Tetrahymena pyriformis Transfer Ribonucleic Acid*

SYNOPSIS. By phenol extraction and DEAE cellulose column chromatography, tRNA was isolated from Tetrahymena pyriformis strain GL. Following acid hydrolysis of the tRNA, the methylated purine content was determined by Dowex 50 column chromatography and paper chromatography. The most abundant methylated guanine derivative was found to be N²-DMG. Also present were 1-MG, N²-MG, and 7-MG. The most abundant methylated adenine was found to be 1-MA; no 2-MA was detected. Small amounts of the N⁶-methyladenines were detected.     

Metabolism of O-Ethyl S,S-Diphenyl Phosphorodithioate (Hinosan®) by Mycelial Cells of Pyricularia oryzae

Metabolism of organophosphorus fungicide Hinosan^® (O-ethyl S, S-diphenyl phosphorodithioate) by mycelia of P. oryzae, rice blast fungus, was studied using ³²P–, ³⁵S– and non-labeled compounds, by ion exchange chromatography, paper chromatography, thin-layer chromatography and gas chromatography, and identifying the metabolites and their derivatives with authentic compounds.

The main metabolic pathway is hydrolysis of one P-S linkage followed by the other P-S linkage or ethyl ester linkage and finally yielding phosphoric acid. A part of the fungicide metabolizes to hydroxylated intermediate metabolite, O-ethyl S-p-hydroxyphenyl S-phenyl phosphorodithioate. No significant difference in rate and mode of metabolism was found in this experiment between susceptible and resistant clones against the fungicide.     

Structure and function of the Bacillus thuringiensis protein crystal

The structural chemistry of the Bacillus thuringiensis parasporal protein crystal is discussed in terms of purification techniques, removal of contaminating proteases, crystal subunit size, crystal shape, interchain crosslinks, the ultimate toxin, and lysinoalanine. The alkaline pH cleavage of disulfide bonds is stressed in relationship to this role in crystal solubilization and toxin formation. The future implication s of plasmid-coded crystal formation and B. thuringiensis var. israelensis (effective against mosquitoes and black flies) are also discussed.     

Metabolism of [14C]indole-3-acetic acid by the cortical and stelar tissues of Zea mays L. roots

Reverse-phase high-performance liquid chromatography was used to analyse ¹⁴C-labelled metabolites of indole-3-acetic acid (IAA) formed in the cortical and stelar tissues of Zea mays roots. After a 2-h incubation in [¹⁴C]IAA, stelar segments had metabolised between 1–6% of the methanol-extractable radioactivity compared with 91–92% by the cortical segments. The pattern of metabolites produced by cortical segments was similar to that produced by intact segments bathed in aqueous solutions of [¹⁴C]IAA. In contrast, when IAA was supplied in agar blocks to stelar tissue protruding from the basal ends of segments, negligible metabolism was evident. On the basis of its retention characteristics both before and after methylation, the major metabolite of [¹⁴C]IAA in Zea mays root segments was tentatively identified by high-performance liquid chromatography as oxindole-3-acetic acid.Abbreviations HPLC High-performance liquid chromatography - IAA Indole-3-acetic acid     

A Novel Chromatographic Method for the Preparation of High Density Lipoproteins

High density lipoproteins (HDL) were isolated by a procedure employing polyanion precipitation and column chromatography. The product was free of low density lipoproteins (LDL) but serum albumin (HSA) was still present. The remaining HSA was removed by an immuno-adsorbent column. The HDL isolated by our method was compared to another HDL preparation isolated from the same plasma sample by the combination of ultra centrifugation and gel chromatography.¹ It was found to have approximately the same lipid and protein composition as the HDL isolated by conventional techniques.¹ Minor differences included a higher phospholipid and apoprotein E content and lower triglyceride and ApoC II content of the HDL isolated by column chromatography. The method described here is considerably less tedious than earlier techniques, can be scaled up without substantial increase in labor and results in an approximately 30% higher yield than the method described by Rudel et al.¹     

S-adenosylhomocysteine is bound to pineal hydroxyindole o-methyltransferase

A substance with maximal absorbance at 260 nm was co-chromatographed with hydroxyindole O-methyltransferase (EC 2.1.1.4) by immunoaffinity chromatography. The co-chromatographed substance was separated from the transmethylase by stepwise elution and was identified as S-adenosylhomocysteine by spectrophotometrical analysis, and by thin-layer chromatography. Identity of S-adenosylhomocysteine was confirmed by determination of demethylated product by using a mixture of [carboxyl-¹⁴C]S-adenosylmethionine and [methyl-³H]S-adenosylmethionine as a substrate. The immunoaffinity chromatography provides direct evidence for a presence of the enzyme-product complex $\text{in vivo}$ and $\text{in vitro}$. At low concentration of S-adenosylmethionine enzymatic activity was inhibited by the co-purified S-adenosylhomocysteine. The endogenous S-adenosylhomocysteine bound to the enzyme probably regulates the melatonin biosynthesis $\text{in vivo}$.     

Digestibility of extruded proteins and metabolic transit of N ε -carboxymethyllysine in rats

Milk proteins are frequently used as supplements in fortified foods. However, processing produces chemical changes which likely affect the nutritional advantage. This study was intended to explore the possible difference in digestibility between extruded and non-extruded caseins and how the dietary N ^ε -carboxymethyllysine (CML) is metabolised. Normal rats were randomized into either an extruded protein diet (EP) or the same with unextruded proteins (UEP), for two periods of 2 weeks at 7 to 9 and 11 to 13 weeks of age. However, no difference in protein digestibility was detected between the two diets, either in young or in adult animals, despite a 9.4-fold higher level of CML and an 8.5-fold higher level of lysinoalanine in the EP than in the UEP. No diet-related changes were observed in plasma CML, either protein bound or free. Amounts of 38 and 48 % of the orally absorbed CML were excreted in urine and faeces, respectively, in UEP-fed rats. Lower rates of excretion were found in the EP-fed rats (23 and 37 %, respectively). A second animal study using a single oral dose of free CML (400 μg/rat) was set up to measure the systemic concentration of CML every hour from 0 to 4 h. It revealed that protein-bound CML was not affected by the oral dose of CML, and the highest free CML level found in the circulation was 600 ng/mL. Extruded proteins, therefore, appear to be well digested, and CML rapidly eliminated. Since its elimination is, however, incomplete, the question of its biodistribution and metabolism remains open.     

Characterization of a nicotinic acetylcholine receptor from rabbit skeletal muscle and reconstitution in planar phospholipid bilayers.

The nicotinic acetylcholine receptor from rabbit skeletal muscle was isolated by affinity chromatography and characterized by ¹²⁵I- α -Bungarotoxin binding and gel filtration chromatography. Quantal conductance events were observed when this material was added to planar phospholipid bilayers. These changes were stimulated by carbamylcholine and antagonized by curare, Butx, dithiothreitol and concanavalin A. The receptor preparation was found to bind 0.2 nMoles ¹²⁵I- α-Bungarotoxin per mg protein and the molecular weight was estimated to be 390,000.     

The synthesis of unlabeled and 32P-labeled phosphocitrate and analytical systems for its identification

A method for the synthesis of phosphocitrate is described using 2-cyanoethyl phosphate to phosphorylate triethyl citrate. Following alkaline hydrolysis of the coupled intermediate, phosphocitrate was purified by ion-exchange chromatography on an AG 1-X8 (HCO₃^?) column. The method was also used to prepare [³²P]phosphocitrate. Phosphocitrate was characterized by ¹H NMR, ³¹P NMR, and ¹³C NMR spectroscopy. In addition methods for thin-layer chromatography and enzyme assay are detailed for the detection of phosphocitrate.     

Elemental sulfur production during mixotrophic growth on hydrogen and thiosulfate of thermophilic hydrogen-oxidizing bacteria

In this study we measured the exogenous production and the intracellular content of elemental sulfur (S⁰) in the thermophilic sulfur-oxidizing bacteriaHydrogenobacter spp. andBacillus schlegelii during mixolithoautotrophic growth on hydrogen and thiosulfate. Under these conditions, all strains produced and released white-yellow hydrophilic S⁰ particles into the growth medium. Hydrophilic S⁰ was separated from cells by a differential low-speed centrifugation procedure. The S⁰ pellets were dried, and the S⁰ was purified by column chromatography and by thin-layer chromatography (TLC). The S⁰ TLC-band could be stained with triphenyltetrazolium chloride and piperidine procedure. Determination of intracellular S⁰ content was performed from fresh cells absolutely free of exogenous S⁰ particles. quantitation of S⁰ was performed by high-performance liquid chromatography, colorimetric thiocyanate procedure, and by UV-spectra analyses. All the strains studied, in particularB. schlegelii strains, released significant quantities of S⁰ into the growth media. In contrast, the intracellular S⁰ content was very low. Significant rhodanese activity in the presence of thiosulfate was measured with toluenepermeabilized cells and cell-free extracts ofB. schlegelii (type strain) andHydrogenobacter spp. (strain T3).