Colour reactions given by sugars and diphenylamine-aniline spray reagents on paper chromatograms

Bailey, R. W.; Bourne, E. J.

doi:10.1016/s0021-9673(01)98394-3

Cited by 201 publications

(48 citation statements)

References 11 publications

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“…Solvent systems contained acetone-water (90:10 v/v) or ethyl acetate-acetic acid-methanol-water (60:20:10:10 v/v). Reducing sugars were stained with aniline-diphenylamine-phosphoric acid [9] as well as with urea-HC1 [11]; ketoses were stained with anthron reagent [ 10]. Radioautography was done by exposing 'Orsay-T4' X-ray films (Agfa, Belgium) for I00 hr to a total radioactivity of 5-50 nCi.…”

Section: Methodsmentioning

confidence: 99%

Comparative investigations on the metabolism of formaldehyde in the presence of ribose‐5‐phosphate in cell‐free extracts of yeasts grown on methanol

et al. 1974

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Section: Methodsmentioning

confidence: 99%

Comparative investigations on the metabolism of formaldehyde in the presence of ribose‐5‐phosphate in cell‐free extracts of yeasts grown on methanol

et al. 1974

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“…After neutralisation with Dowex 50 W cationic-exchange resin, an aliquot was separated by thin-layer chromatography using Sigma silica gel TLC plates in a solvent system of acetonitrile/water (85: 15, by vol.). Mannose was used as the standard and was detected by spraying with the diphenylamine/aniline/phosphoric acid reagent [7].…”

Section: Characterisation Of Productmentioning

confidence: 99%

Protein O‐mannosylation in Candida albicans

Weston¹,

Nassau²,

Henly³

et al. 1993

European Journal of Biochemistry

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A protein-0-D-mannosyltransferase (PMT) assay was optimised using a microsomal membrane preparation from Candida albicans and a peptide acceptor, YNPTSV.[14C]Mannose was transferred from dolichyl phosphate [14C]mannose to the threonine or serine residues of the peptide. During the assay, the peptide was highly susceptible to proteolysis. A blocked peptide Ac-YNPTSV-NH, was resistent to proteolysis and was apparently a better acceptor for 0-mannosylation. This peptide had a K,,, value of 4.3 mM in the assay. A number of other peptides were tested with altered sequences. Maximum incorporation of [14C]mannose was obtained with a pentapeptide YATAV (K, 2.2 mM) which was further improved by blocking both ends: Ac-YATAV-NH, (K, 0.25 mM). Finally, and unexpectedly, an improvement was noted if the acetyl group on the N terminus was replaced by a biotin residue. Biotin-YATAV-NH, had a K, of 0.075 mM. The biotin residue may be important in increasing the lipophilicity of the peptide and thus aid its adhesion to the Candida membranes. The simplest peptide that could act as an efficient mannose acceptor was Ac-ATA-NH,, whilst no incorporation was observed with Ac-GTG-NH,.Both Candida and Saccharomyces species contain carbohydrate chains which are 0-linked to the hydroxyl group of threonine and serine residues of polypeptide chains, giving rise to mannoproteins. The sequence of reactions for the formation of 0-linked mannoproteins has been described previously [l -31 and involves the transfer of mannose from a dolichyl phosphate mannose (Dol-P-Man) to the protein which is catalysed by an enzyme, protein O-mannosyltransferase (PMT), that is believed to be unique to fungi. Several attempts to purify the enzyme from Saccharomyces cerevisiae [4, 51 have been partially successful but it has proved difficult because the enzyme is membrane-bound. Little is known about the enzyme or its assay requirements, although some information is available on the use of different peptide acceptors for PMT in Saccharomyces [3, 4, 61.PMT has been even less well studied in Candida albicans; hence, we have developed an assay for the enzyme which utilises microsomal membranes as enzyme source and exogenously added peptide as the mannose acceptor. We were interested to study the peptide sequence requirements for mannosylation by the enzyme and have investigated a number of variables. In particular, we have examined the effects of peptide length, preference for a serine or threonine residue and the influence of the amino acids immediately flanking the 0-mannosylation site.Correspondence to A. Weston, Chemotherapy Department, Microbiology, Division, Glaxo Group Research Ltd, Greenford Road, Greenford, Middlesex, England, UB6 OHE Abbreviations. PMT, protein 0-D-mannosyltransferase; Dol-PMan, dolichyl phosphate mannose ; Me,SO, dimethylsulphoxide ; Abu, 2-aminobutyric acid.Enzymes. Protein 0-D-mannosyltransferase (EC 2.4.1.109). MATERIALS AND METHODSAll chemicals were analar grade unless otherwise stated. GDP-['4C]mannose (specific activity 272 Ci/mol) ...

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“…The same result was obtained in tests on Kieselgel-60 plates with the basic solvent propan-2-ol/l ;d (viv) ammonia (2/l, v/v) and with the neutral solvent ethanol/water (80/20, v/v). When the incubation with periodate had proceeded for about 16 h, no fluorcscent products could be detected in the reaction mixture.…”

Section: Discovcrj and Puvif Ication Of' Methunopterinmentioning

confidence: 99%

Structural Elements of Methanopterin, a Novel Pterin

Keltjens

Huberts

Laarhoven

et al. 1983

European Journal of Biochemistry

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During short -time la be1 i ng ex per imcn t s , cells of M~tlzario huctcriunz tlzcrn?ouirto trophicwm incorporate a substantial part of I4CO2 in a yellow fluorescent compound (called YFC) [Daniels. L. & Zeikus,-J. G. (1978) J . Bacterinl. 136,[75][76][77][78][79][80][81][82][83][84]. As the compound was present only in small amounts, its more abundant, metabolic precursor was identified, extracted and purified by column chromatography.The chromophore of this compound is 2-amino-4-hydroxypteridinc (pterin) as indicated by its ultravioletvisible-light absorption and fluorescence properties. Decomposition studies revealed the presence of a number of structural elements, viz. glutamic acid. phosphate and a hcxosamine. 'H-NMR and "C-NMR spectra pointed to the presencc of additional, as yet unidentified, elements. The compound is a complex. novel pterin derivative, which we have called methanopterin.Most methanogenic bacteria are capable of reducing carbon dioxide to methane with hydrogen as the electron donor [I]. In this process a number of unique cocnzyines are involved. Firstly, coenzyme M (2-mercaptoethanesulfonic acid) and its methylated derivative, 2-methylincrcaptoethanesulfonic acid, were described [2]. These Short-term labeling experiments revealed the presence of another Factor, named after its most striking feature, yellow fluorescent coinpound (YFC) [I I]. I4CO2 incorporation ir,to this compound proceeds rapidly : thus, it might be an intcrmediate formed in the early steps of reduction but almost nothing is known about its structure.Here we wish to report the purification and the characterization of the structural elemcnts of a novel coFxtor from Mrthai~nhuc~terium tlirrnzouutotroplii~i~~i. The compound is the metabolic counterpart of the yellow fluorescent compound, a s could be shown from introductory experiments with ccllfree extracts.The compound was found to be : I 2-amino-4-hydroxypteridine (pterin) containing a complex sidechain substituted at C-6 and presumably C-7 of the ptcrin moiety. Therefore, we introduce the trivial name 'methanoptcrin'.

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Colour reactions given by sugars and diphenylamine-aniline spray reagents on paper chromatograms

Cited by 201 publications

References 11 publications

Comparative investigations on the metabolism of formaldehyde in the presence of ribose‐5‐phosphate in cell‐free extracts of yeasts grown on methanol

Comparative investigations on the metabolism of formaldehyde in the presence of ribose‐5‐phosphate in cell‐free extracts of yeasts grown on methanol

Protein O‐mannosylation in Candida albicans

Structural Elements of Methanopterin, a Novel Pterin

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