N-terminal amino acid sequence of C hordein

The C hordein (prolamin storage protein) fraction of barley endosperm has been purified and the N-terminal sequence of amino acids determined for 30 residues. No sequence was obtained for the B hordein fraction because the N-terminus was blocked.     

Ent-kaurene,occurrence and metabolism in Hordeum distichon

Barley grains contain hydrocarbons, including a material indistinguishable from ent-kaurene by GLC, and which after appropriate chemical conversions contain material behaving like ent-kauran-16,17-diol, ent-kaurene norketone and ent-17-nor-kaurane on TLC and GLC. The presence of ent-kaurene was confirmed by conversion to ent-kauran-16-ol and, following formation of acetate-[³H], recrystallization to constant specific activity with unlabelled carrier. In the initial ca. 15 hr of germination, preceding the rise in endogenous gibberellins, the level of ent-kaurene falls. Exogenous ent-kaurene-[¹⁴C] was not metabolized by intact barley grains. ent-Kauran-16,17-epoxide was formed non-enzymically by boiled extracts. Unboiled homogenates also formed ent-kauran-17-ol and ent-kauran-16,17-diol. The diol appeared to be formed from the epoxide, but the ent-kauran-17-ol was not. No recognized gibberellin precursors were detected. Nevertheless, endogenous ent-kaurene may be the stored biosynthetic precursor of gibberellins in germinating barley grains.     

Further characterization and quantitative determination of 5-methoxy-N-methyltryptamine in Phalaris arundinacea

The 100 MHz PMR spectrum of 5-methoxy-N-methyltryptamine (5MMT) isolated from Phalaris arundinacea is elucidated, acid-induced fluorescence characteristics of 5-methoxy- and 5-hydroxytryptamine alkaloids on Avicel and silica gel TLC are described, and the concentration of 5MMT in P. arundinacea is determined by fluorometric TLC scanning.     

Very-long-chain iso and anteiso branched fatty acids in N-acylphosphatidylethanolamines from a natural cyanobacterial mat of Calothrix sp.

A combination of TLC, ESI-MS/MS and GC-MS was used to identify unusual molecular species of N-acylphosphatidylethanolamines containing very-long-chain anteiso branched fatty acids (VLCFAs) from Calothrix sp. collected in Antarctica and determine their component VLCFA up to 33-methyltetratriacontanoic acid as picolinyl ester derivatives using GC-MS.     

Purification and properties of a β-N-acetylaminoglucohydrolase from malted barley

β-N-Acetylaminoglucohydrolase (β-2-acetylamino-2-deoxy-D-glucoside acetylaminodeoxyglucohydrolase, EC 3.2.1.30) was extracted from malted barley and purified. The partially purified preparation was free from α-and β-glucosidase, α- and β-galactosidase, α-mannosidase and β-mannosidase. This preparation was free from α-mannosidase only after affinity chromatography with p-amino-N-acetyl-β-D-glucosaminidine coupled to Sepharose. The enzyme was active between pH 3 and 6.5 and had a pH optimum at pH 5. A MW of 92000 was obtained by sodium dodecyl sulfate-acrylamide gel electrophoresis and a sedimentation coefficient of 4.65 was obtained from sedimentation velocity experiments. β-N-Acetylaminoglucohydrolase had a K_m of 2.5 × 10^?4 M using the p-nitrophenyl N-acetyl β-D-glucosaminidine as the substrate.     

Amino acid conjugates as metabolites of the plant growth regulator dihydrojasmonic acid in barley (Hordeum vulgare)

The biotransformation of [2-¹⁴C](±)9, 10-dihydrojasmonic acid (DJA) was studied in excised shoots of 6-day-old barley seedlings after 72 h. From the ethyl acetate extract, some minor metabolites were isolated and purified by DEAE-Sephadex A-25 chromatography, thin-layer chromatography (TLC), C₁₈-cartridges, and high-performance liquid chromatography (HPLC). The structural identification of these metabolites was performed by gas chromatography-mass spectrometry (GC-MS), circular dichroism (CD), and amino acid analysis, and the following amino acid conjugates were found:N-[(?)9,10-dihydrojasmonoyl]valine,N-[(?)9,10-dihydrojasmonoyl]isoleucine,N-[9,10-dihydrojasmonoyl]leucine,N-[11-hydroxy-9,10-dihydrojasmonoyl]valine,N-[11-hydroxy-9,10-dihydrojasmonoyl]isoleucine,N-[12-hydroxy-9,10-dihydrojasmonoyl]isoleucine; and the cucurbic acid-related compoundsN-{[3-hydroxy-2(4-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine andN-{[3-hydroxy-2(5-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine. The results suggest conjugation with isoleucine and valine, as well as preferential hydroxylation at position C-11 or hydrogenation at position C-6, as being important steps in the metabolism of (±)DJA in barley shoots.     

Amino acid conjugates as metabolites of the plant growth regulator dihydrojasmonic acid in barley (Hordeum vulgare)

The biotransformation of [2-¹⁴C](±)9, 10-dihydrojasmonic acid (DJA) was studied in excised shoots of 6-day-old barley seedlings after 72 h. From the ethyl acetate extract, some minor metabolites were isolated and purified by DEAE-Sephadex A-25 chromatography, thin-layer chromatography (TLC), C₁₈-cartridges, and high-performance liquid chromatography (HPLC). The structural identification of these metabolites was performed by gas chromatography-mass spectrometry (GC-MS), circular dichroism (CD), and amino acid analysis, and the following amino acid conjugates were found:N-[(–)9,10-dihydrojasmonoyl]valine,N-[(–)9,10-dihydrojasmonoyl]isoleucine,N-[9,10-dihydrojasmonoyl]leucine,N-[11-hydroxy-9,10-dihydrojasmonoyl]valine,N-[11-hydroxy-9,10-dihydrojasmonoyl]isoleucine,N-[12-hydroxy-9,10-dihydrojasmonoyl]isoleucine; and the cucurbic acid-related compoundsN-{[3-hydroxy-2(4-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine andN-{[3-hydroxy-2(5-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine. The results suggest conjugation with isoleucine and valine, as well as preferential hydroxylation at position C-11 or hydrogenation at position C-6, as being important steps in the metabolism of (±)DJA in barley shoots.     

Indolethylamine N-methyltransferases of Phalaris tuberosa, purification and properties

Two S-adenosylmethionine-dependent indolethylamine N-methyltransferase activities were purified from soluble extracts of Phalaris tuberosa by fractionation with (NH₄)₂SO₄, and chromatography on DEAE-Sephadex. One enzyme methylated the primary indolethylamines and the other methylated the secondary indolethylamines in the plant. These two enzymes were similar in catalytic and bulk physical properties and could not be separated during purification or by A 1.5 Agarose or hydroxylapatite chromatography. The products of enzymic reactions were identified by TLC.     

Reduction of N-acetyl methionine sulfoxide in plants

An enzymic activity which catalyzes the reduction of N-acetyl-methionine sulfoxide to l-N-acetyl-methionine has been observed in a wide variety of plant tissues. Its activity depended on the presence of dithiotreithol in the incubation medium. l-Methionine-sulfoxide was essentially inactive as a substrate. Of all the physiological reductants tested, only thioredoxin partially replaced dithiothreithol. When fractions obtained by gradient centrifugation of gently disrupted barley protoplasts were assayed for the reductase, the activity was largely associated with chloroplasts although approximately 15% was found in the cytosolic compartment. The enzyme, isolated from spinach chloroplasts, had a broad pH optima between 7.0 and 8.0, and its K_m for N-acetyl methionine sulfoxide is 0.4 millimolar. The possible participation of this ubiquitous enzyme in enzyme regulation is discussed.     

Photosynthetic responses of a wheat (Asakaze)–barley (Manas) 7H addition line to salt stress

The photosynthetic responses to salt stress were examined in a wheat (Triticum aestivum L. cv. Asakaze)–barley (Hordeum vulgare L. cv. Manas) 7H addition line having elevated salt tolerance and compared to the parental wheat genotype. For this purpose, increasing NaCl concentrations up to 300 mM were applied and followed by a 7-day recovery period. Up to moderate salt stress (200 mM NaCl), forcible stomatal closure, parallel with a reduction in the net assimilation rate (P _N), was only observed in wheat, but not in the 7H addition line or barley. Since the photosynthetic electron transport processes of wheat were not affected by NaCl, the impairment in P _N could largely be accounted for the salt-induced decline in stomatal conductance (g _s), accompanied by depressed intercellular CO₂ concentration and carboxylation efficiency. Both, P _N and nonstomatal limitation factors (L_ns) were practically unaffected by moderate salt stress in barley and in the 7H addition line due to the sustained g _s, which might be an efficient strategy to maintain the efficient photosynthetic activity and biomass production. At 300 mM NaCl, both P _N and g _s decreased significantly in all the genotypes, but the changes in P _N and L_ns in the 7H addition line were more favourable similar to those in wheat. The downregulation of photosynthetic electron transport processes around PSII, accompanied by increases in the quantum yield of regulated energy dissipation and of the donor side limitation of PSI without damage to PSII, was observed in the addition line and barley during severe stress. Incomplete recovery of P _N was observed in the 7H addition line as a result of declined PSII activity probably caused by enhanced cyclic electron flow around PSI. These results suggest that the better photosynthetic tolerance to moderate salt stress of barley can be manifested in the 7H addition line which may be a suitable candidate for improving salt tolerance of wheat.     

Characterisation of the endospermal trypsin inhibitor of barley

A trypsin inhibitor was isolated from grains of two row barley (cv. Proctor). The purified protein was identical with the corresponding inhibitor of a six row barley (cv. Pirkka); both proteins showed, a Pi of 7.4. The N-terminal amino acid was phenylalanine and an arginine residue was involved in the active site. Effects of substrate concentration showed that the inhibition was noncompetitive with a K_i of about 0.9 × 10^?7M. An enzyme-inhibitor complex was demonstrated by disc electrophoresis.     

E. coli tRNAPhe modified at the 3-(3-amino-3-carboxypropyl)uridine with a photoaffinity label is fully functional for aminoacylation and for ribosomal interaction

E. coli tRNA^Phe was modified at its 3-(3-amino-3-carboxypropyl)uridine residue with the N-hydroxysuccinimide ester of N-4-azido-2-nitrophenyl)glycine. Exclusive modification of this base was shown by two-dimensional TLC analysis of the T₁ oligonucleotide and nucleoside products of nuclease digestion. The fully modified tRNA could be aminoacylated to the same level as control tRNA. The aminoacylated tRNA was as active as control tRNA in non-enzymatic binding to the P site of ribosomes, and in EFTu-dependent binding to the rirobosomal A site. The functional activity of this photolabile modified tRNA allows it to be used to probe the A and P binding sites on ribosomes and on other proteins that interact with tRNA. Crosslinking to the ribosomal P site has been shown.     

Thin-layer chromatographic separation of pineal gland derivatives of serotonin-14C

A two-dimensional TLC method has been developed which separates N-acetylserotonin, hydroxytryptophol, hydroxyindoleacetic acid, methoxyindoleacetic acid, methoxytryptophol, and melatonin.     

The telocentric tandem repeat at the p-arm is not conserved in Mus musculus subspecies

Mouse chromosomes, with the exception of the Y chromosome, are telocentric. The telomere at the p-arm is separated from the centromere by the tL1 sequence and TLC tandem repeats. A previous report showed that the TLC array was also conserved in other strains of the subgenus Mus. These results suggest that the TLC arrays promote the stable evolutionary maintenance of a telocentric karyotype in the subgenus Mus. In this study, we investigated the degree of conservation of TLC arrays among a variety of wild-derived inbred strains, all of which are descendants of wild mice captured in several areas of the world. Genomic PCR analysis indicates that the sequential order of telomere-tL1 is highly conserved in all strains, whereas tL1-TLC is not. Next, Southern blot analysis of DNAs isolated from a panel of mouse subspecies showed both Mus musculus domesticus and Mus musculus castaneus subspecies possess TLC arrays. Unexpectedly, this repeat appears to be lost in almost all Mus musculus musculus and Mus musculus molossinus subspecies, which show a clear geographic divide. These results indicate that either other unknown sequences were replaced by the TLC repeat or almost all M. m. musculus and M. m. molossinus subspecies do not have any sequence between the telomere and minor satellites. Our observation suggests that the TLC array might be evolutionarily unstable and not essential for murine chromosomal conformation. This is the first example of the subspecies-specific large genome alterations in mice.     

Rubipodanin A,the First Natural N-Desmonomethyl Rubiaceae-Type Cyclopeptide from Rubia podantha,Indicating an Important Role of the N 9-Methyl Group in the Conformation and Bioactivity

One new cyclic hexapeptide named rubipodanin A (1), which is the first identified natural N-desmonomethyl Rubiaceae-type cyclopeptide, together with six known Rubiaceae-type cyclopeptides (2–7) were obtained using the TLC cyclopeptide protosite detection method with ninhydrin from the roots and rhizomes of Rubia podantha. The cyclopeptide structures were elucidated by extensive spectroscopic analysis, including 1D-NMR, 2D-NMR, IR, UV and MS. The solution conformation and biological activities of 1 and RA-V (4) were evaluated, and the results demonstrated that the N ⁹-methyl group plays a vital role in the maintenance of the conformation and bioactivity.     

An S-adenosyl-l-methionine; coniine methyltransferase from Conium maculatum

Evidence is presented for a cell free system from Conium maculatum which catalyses the transfer of a methyl group from S-adenoysl-l-methionine to coniine with the formation of N-methyl coniine. Maximum enzyme activity which occurred in the unripe fruits was enhanced by dithiothreitol, and evidence for the role of sulphydryl groups of the enzyme was obtained from inhibition with p-CMB, iodoacetamide and N-methyl maleimide. A divalent metal cation dependency was not detected.     

The N-permethylation of chitosan and the preparation of N-trimethyl chitosan iodide

Chitosan was N-permethylated by reaction with formaldehyde and sodium borohydride under controlled conditions (pH 4·0, 15°C, reaction times 12 and 8 h, respectively). The N-permethylated chitosan was reacted with methyl iodide at 35°C and N-trimethyl chitosan iodide with a quaternary nitrogen degree of 60% was obtained. This material may have uses as an antibiotic and an ion exchange material.     

N-(ϱ-coumaryl)-tryptamine and N-ferulyl tryptamine in kernels of Zea mays

N-(?-coumaryl)tryptamine and N-ferulyltryptamine were isolated from aqueous acetone extracts of ground kernels of Zea mays by successive column chromatography on partially sulfonated styrenedivinylbenzene copolymer resin, lipophilic Sephadex and preparative TLC. Identification of these compounds was made by GCMS of their trimethylsilyl derivatives and the trimethylsilyl derivatives of their acid hydrolysis products.     

A novel method for the methylation of heterocyclic amino groups. Conversion of guanosine into its 2-N-methyl- and 2-N,2-N-dimethyl derivatives

The heterocyclic amino-compounds 11a, 13a, 13b, and 17 reacted with formaldehyde and p-thiocresol (14) in alcoholic solution to give the corresponding N-methylphenylthiomethyl derivatives (16, 15a, 15b, and 18a, respectively) in satisfactory to good yields. The reactions were catalyzed by acetic acid. 2-N-Methylguanosine (6a) was obtained in good yield by treatment of 15b with sodium borohydride followed by acidic hydrolysis, or alternatively by Raney nickel desulfurization of 15a followed by ammonolysis of the product. Sodium borohydride reduction of 18a gave 21 in good yield. 2-N,2-N-Dimethylguanosine (6b) was obtained from 19a in three steps.     

Radiotracer evidence implicating phosphoryl and phosphatidyl bases as intermediates in betaine synthesis by water-stressed barley leaves

In barley, glycine betaine is a metabolic end product accumulated by wilted leaves; betaine accumulation involves acceleration of de novo synthesis from serine, via ethanolamine, N-methylethanolamines, choline, and betaine aldehyde (Hanson, Scott 1980 Plant Physiol 66: 342-348). Because in animals and microorganisms the N-methylation of ethanolamine involves phosphatide intermediates, and because in barley, wilting markedly increases the rate of methylation of ethanolamine to choline, the labeling of phosphatides was followed after supplying [¹⁴C]ethanolamine to attached leaf blades of turgid and wilted barley plants. The kinetics of labeling of phosphatidylcholine and betaine showed that phosphatidylcholine became labeled 2.5-fold faster in wilted than in turgid leaves, and that after short incubations, phosphatidylcholine was always more heavily labeled than betaine. In pulse-chase experiments with wilted leaves, label from [¹⁴C]ethanolamine continued to accumulate in betaine as it was being lost from phosphatidylcholine. When [¹⁴C]monomethylethanolamine was supplied to wilted leaves, phosphatidylcholine was initially more heavily labeled than betaine. These results are qualitatively consistent with a precursor-to-product relationship between phosphatidylcholine and betaine.