Rapid Paper Chromatography or Carbohydrates and Related Compounds

Gordon, H. T.; Thornburg, Wayne; Werum, L.N.

doi:10.1021/ac60113a020

Cited by 388 publications

(77 citation statements)

References 11 publications

(8 reference statements)

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“…The oily product was homogeneous on silica-gel thinlayer chromatography using acetone -water (8 : 2, v/v) as tho solvent system (detection by ultraviolet and periodate-benzidine [22]). …”

Section: Methodsmentioning

confidence: 99%

Kinetic Properties of Phenylalanyl‐tRNA and Seryl‐tRNA Synthetases for Normal Substrates and Fluorescent Analogs

Hertz¹,

Zachau²

1973

European Journal of Biochemistry

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The kinetics of phenylalanyl-tRNA and seryl-tRNA formation were investigated with tRNAs and aminoacyl-tRNA synthetases from yeast.Phenylalanyl-tRNA synthetase yielded linear Lineweaver-Burk plots with tRNAPhe, phenylalanine, and 1 ,Ne-ethenoadenosine triphosphate ( EATP) as variable substrates. According to equilibrium dialysis in the absence or presence of phenylalaninyl adenosine 5'-phosphate, phenylalanyl-tRNA synthetase possesses one binding site for phenylalanine. For ATP as variable substrate, the deviation from linearity in the Lineweaver-Burk plot, observed by other investigators, was confirmed. The slope of the curve indicates the presence of more than two ATP binding sites.Seryl-tBNA spthetase yielded a linear Lineweaver-Burk plot only with EATP as variable substrate. The Lineweaver-Burk plots for serine and tRNAS-9' were non-linear ; the interpretation we favor involves positive cooperativity between amino acid binding sites and between tRNA binding sites. Hill plots of the kinetic data showed that the enzyme possesses a t least two binding sites for each of these substrates. The kinetic data for ATP could be interpreted as showing more than two binding sites with negative and positive cooperativity in binding of successive ATP molecules.The aminoalkyl adenylates, phenylalaninyl adenosine 6'-phosphate and serinyl adenosine 5'-phosphate, competitively inhibited the aminoacylation reaction with respect to amino acid.EATP functions in place of ATP in phenylalanyl-tRNA and seryl-tRNA formation although with rather Werent kinetic properties. Modified tRNAPhe and tRNASer, in which the 3'-terminal adenosine was replaced by ethenoadenosine, were prepared by a C-C-A transferase-catalyzed reaction of EATP. These modified tRNAs show kinetic properties very similar to those of the unmodified tRNAs and can therefore be used, in place of the unmodsed tRNAs, as fluorescent probes in synthetase-tRNA interaction studies.The kinetic behavior of phenylalanyl-tRNA synthetase appears to be much simpler than that of seryl-tRNA synthetaae, despite the fact that the former enzyme is twice as big and contains twice as many subunits as the latter one. The comparative simplicity of the one enzyme relative to the other correlates with previous results on interactions with substrates, which were obtained by fluorescence measurements and nuclease protection studies.

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“…The oily product was homogeneous on silica-gel thinlayer chromatography using acetone -water (8 : 2, v/v) as tho solvent system (detection by ultraviolet and periodate-benzidine [22]). …”

Section: Methodsmentioning

confidence: 99%

Kinetic Properties of Phenylalanyl‐tRNA and Seryl‐tRNA Synthetases for Normal Substrates and Fluorescent Analogs

Hertz¹,

Zachau²

1973

European Journal of Biochemistry

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“…3MM strips were developed overnight in descending mode with ethyl acetate, pyridine, 0.1 M boric acid (65:25:20), dried, cut into 1-cm zones, and analyzed for radioactivity by scintillation spectrometry. GlcNAcitol and GalNAcitol standards were visualized using a modification of the periodate-benzidine dip procedure (27). The paper strips were dipped in acetone, 0.1 M NaIO 4 (95:5), allowed to air dry for 3 min, and then dipped in acetone/acetic acid/H 2 O/o-tolidine (96:0.6:4.4:0.2 gm).…”

Section: Characterization Of Glycan Products Formed In In Vitromentioning

confidence: 99%

A Novel Epimerase That Converts GlcNAc-P-P-undecaprenol to GalNAc-P-P-undecaprenol in Escherichia coli O157

Rush

Alaimo²,

Robbiani³

et al. 2010

Journal of Biological Chemistry

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Escherichia coli strain O157 produces an O-antigen with the repeating tetrasaccharide unit ␣-D-PerNAc-␣-L-Fuc-␤-D-Glc-␣-D-GalNAc, preassembled on undecaprenyl pyrophosphate (Und-P-P). These studies were conducted to determine whether the biosynthesis of the lipid-linked repeating tetrasaccharide was initiated by the formation of GalNAc-P-P-Und by WecA. 3 H]GlcNAc-P-P-Und was reformed when membranes from strain O157 were incubated with exogenous [ 3 H]GalNAc-P-P-Und. The inability of Z3206 to complement the loss of the gne gene in the expression of the Campylobacter jejuni N-glycosylation system in E. coli indicated that it does not function as a UDP-GlcNAc/UDPGalNAc epimerase. Based on these results, GalNAc-P-P-Und is synthesized reversibly by a novel GlcNAc-P-P-Und epimerase after the formation of GlcNAc-P-P-Und by WecA in E. coli O157.The Escherichia coli cell envelope contains an inner plasma membrane, a stress-bearing peptidoglycan layer, and an asymmetric outer membrane consisting of a phospholipid inner monolayer and an outer monolayer composed of bacterial lipopolysaccharide (LPS).2 LPS contains three components, the lipid A anchor, the 3-deoxy-D-manno-oct-2-ulosonic acid-containing core, and the O-antigen region (see Refs.

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“…Aldonic and uronic acids move very slowly in this system; therefore, in order to separate the aldonic acid species the chromatogram was developed for 48 h. Gulonic acid (Sigma) and idonic acid (graciously provided by Dr. T. Murphy, Pfizer, Inc.) were included in all chromatograms and were identified by a potassium permanganate stain (33). To ensure that the hydrolysis procedure did not affect the migration of the [ 3 H]aldonic acid, both gulonic acid and idonic acid were hydrolyzed identically to the 3 H-oligosaccharides.…”

Section: Hydrolysis Of Heparan Sulfate Chains To Monosaccharides-thementioning

confidence: 99%

Heparanases Produce Distinct Populations of Heparan Sulfate Glycosaminoglycans in Chinese Hamster Ovary Cells

Bame

Robson

1997

Journal of Biological Chemistry

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Once internalized, cell-associated heparan sulfate proteoglycans are degraded to short glycosaminoglycans by the action of endoglycosidases or heparanases. We have begun to address the question of how many heparanases are responsible for this process by analyzing short heparan sulfate chains produced in vivo by Chinese hamster ovary (CHO) cell heparanases. Short heparan sulfate chains were purified from CHO cells and labeled at the reducing end with [ 3 H]NaBH 4 . Hydrolysis of the chains to monosaccharides and analysis of the 3 H-sugar alcohols indicate that heparanase activities in CHO cells are endo-␤-glucuronidases. The modification state of the heparanase-derived glycosaminoglycans was examined by treating the [ 3 H]heparan sulfate chains with nitrous acid or bacterial heparin lyases, which cut the chain at specific sequences, and analyzing the products by P2 gel filtration chromatography. Two populations of short chains were identified that differ in the extent of modification on the nonreducing side of the heparanase cleavage site. One class of chains is unmodified for at least 9 residues from the reducing end, while the other group has a modified domain within 3-7 residues from the heparanase cleavage site. Our results suggest a model of heparanase action where the enzymes recognize differences in sulfate content between modified and unmodified regions and bind to sites that encompass both domains. The enzymes then cleave the glycosaminoglycan at junctions between the modified and unmodified sequences to produce the different populations of short heparan sulfate chains.

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Rapid Paper Chromatography or Carbohydrates and Related Compounds

Cited by 388 publications

References 11 publications

Kinetic Properties of Phenylalanyl‐tRNA and Seryl‐tRNA Synthetases for Normal Substrates and Fluorescent Analogs

Kinetic Properties of Phenylalanyl‐tRNA and Seryl‐tRNA Synthetases for Normal Substrates and Fluorescent Analogs

A Novel Epimerase That Converts GlcNAc-P-P-undecaprenol to GalNAc-P-P-undecaprenol in Escherichia coli O157

Heparanases Produce Distinct Populations of Heparan Sulfate Glycosaminoglycans in Chinese Hamster Ovary Cells

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