Chemical Determination of Tryptophan

Spies, Joseph R.; Chambers, Dorris C.

doi:10.1021/ac60013a006

Cited by 394 publications

(104 citation statements)

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“…The number of sulfhydryl groups was estimated by Ellman's reagent [30]. Tryptophan was determined according to Spies and Chambers [31]. For the determination of the total amide content Nesslers reagent was used according to Steegeman [32].…”

Section: Amino Acid Analysis and Peptide Mappingmentioning

confidence: 99%

The Malate Dehydrogenase Isoenzymes of Saccharomyces cerevisiae. Purification, Characterisation and Studies on Their Regulation

1978

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1. One mitochondrial and one cytoplasmic malate dehydrogenase isoenzyme could be purified from acetate grown cells of the yeast Saccharomyces cerevisiae. 2. The purification procedure uses chromatography on dextran blue columns as an essential step for enrichment, and reverse ammonium sulfate chromatography on celite for isoenzyme separation. 3. The homogeneity of the preparations was established by gel electrophoreses in the presence of sodium dodecylsulfate and by a sedimentation run in the analytical ultracentrifuge. 4. Both enzymes are dimers with a molecular weight of 75 000 for the cytoplasmic and of 68 000 for the mitochondrial enzyme. 5. Amino acid analysis and peptide mapping showed that both enzymes are closely related, but genetically different (true isoenzymes). 6. The cytoplasmic enzyme shows electrophoretic splitting. This is most likely due to post‐translational deamination in vivo. 7. Antibodies to both isoenzymes could be obtained in rabbits. The antisera to cytoplasmic malate dehydrogenase were specific for this enzyme. Antisera to mitochondrial malate dehydrogenase react with both isoenzymes. Neither type of antisera precipitated an inactive protein after the glucose‐dependent inactivation of cytoplasmic malate dehydrogenase in vivo.

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Section: Amino Acid Analysis and Peptide Mappingmentioning

confidence: 99%

The Malate Dehydrogenase Isoenzymes of Saccharomyces cerevisiae. Purification, Characterisation and Studies on Their Regulation

1978

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“…Tryptophan was also determined chemically using method C of Spies and Chambers [25]. Cysteine was determined using Nbs, as described by Ellman [26].…”

Section: Methodsmentioning

confidence: 99%

Phosphoglycerate Kinase B from Ram Testis

Stewart

Scopes

1978

European Journal of Biochemistry

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The testis‐specific isoenzyme of phosphoglycerate kinase (phosphoglycerate kinase B) has been isolated from ram testes using a procedure which separates it from ‘normal’ phosphoglycerate kinase which is also present in testis tissue. The purification procedure is described. The best preparations had no detectable impurity on electrophoresis, and had specific activities comparable with the same enzyme from other sources. Kinetic studies indicated that the two isoenzymes have identical properties, within experimental error, for substrate affinity (for MgATP, 3‐phosphoglycerate and MgADP), energy of activation and thermal denaturation. The molecular weights of both isoenzymes were not distinguishably different from those previously reported, as measured by polyacrylamide/dodecylsulphate electrophoresis. The amino acid compositions showed only slight differences, and tryptic peptide maps showed that there was close homology of sequence. Starch gel electrophoresis at pH 6.5 indicates that the B isoenzyme has 1–2 more positive charges than the A. Phosphoglycerate kinase A isolated from sheep muscle was shown, within experimental error, to be identical to the phosphoglycerate kinase A isolated from testis. The results further substantiate the suggestion that the B isoenzyme is coded by a gene which was duplicated from the phosphoglycerate kinase A gene.

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“…Tryptophan was estimated according to the method of Spies and Chambers [18], as modified by Harrison and Hofmann [19].…”

Section: Amino Acid Analysismentioning

confidence: 99%

“…Hydrolysis with trypsin was used in three separate sets of experiments, involving the whole light chain, [18,19].…”

Section: Tr-vp T Ic Pep T Idesmentioning

confidence: 99%

Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin

Schiff¹,

Fougereau²

1975

European Journal of Biochemistry

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The amino acid sequence of the light chain of the mouse monoclonal MOPC 173 immunoglobulin molecule (IgG2a,x) is presented. This kappa chain contains 214 residues. Comparisons of this sequence with murine kappa chains already published by other workers bring a confirmation of the large size of the murine Vx chain pool. A complete identity was found with the constant region of the light chain of MOPC 21 from residue 97 to residue 214.We have undertaken the determination of the complete amino acid sequence of the murine MOPC 173 immunoglobulin molecule, which is a monoclonal protein of the IgG2a (x) class [I]. A topological model of the molecule has been proposed, on the grounds of results derived from the isolation and characterization of the CNBr fragments of the entire molecule [2], and from the identification of the interchain and intrachain disulfide bridges [3,4]. The sequence of the CNBr fragments H1, H2, H3 (covering most of the V, region), and that of fragments H5 to H10, accounting for the hinge region and the Fc fragment have already been published [5 -71.We report in the present paper the amino acid sequence of the light chain of this molecule. MATERIALS AND METHODS Isolation of the MOPC 173 ProteinPlasmacytomas which originated from Dr Potter's laboratory, were serially transplanted in Balb/c mice, in solid form, by subcutaneous injection of small tumor fragments. On the average, mice were bled three weeks after the transplantation. The immunoglobulin fraction of pooled sera was precipitated with ammonium sulfate (33 % saturation, final concentration), and was further purified by chromatography on DEAE cellulose (DE 52, Whatman) equilibrated with 0.017 M phosphate buffer (pH 7.3). Purity was checked by immunoelectrophoresis [8], using a rabbit antiserum against whole Balb/c mouse serum. Isolation of the Light ChainsThe MOPC 173 immunoglobulin molecule (500mg) was mildly reduced in 0.1 M Tris buffer, 0.15 M NaC1, 0.002 M EDTA, pH 8.2, with dithiothreitol (Calbiochem), according to Frangione et al. [9] (except that the molarity of the reagent was 8 mM) and alkylated with a 2.5 molar excess of iodoacetamide. Chains were then separated by gel-filtration on a Sephadex G-100 superfine column ( 5 x 100 cm) equilibrated in 1.0 M propionic acid. Purity of the light chains was tested by polyacrylamide (7.5 %) gel electrophoresis according to Shapiro and Maize1 [lo].Complete reduction and alkylation of the isolated light chains was made according to Waxdal et al.[ 1 1 1.except that 8 M urea was used instead of guanidine. Whenever labelled peptides were required, alkylation was performed, according to Frangione et al. [9], by making the reduced solution 0.02 M with iodo['"C]acetic acid (Amersham), using a 0.1 M stock solution (specific activity 83 pCi/ml). Radioactivity was counted in a Packard spectrometer 3380. Isolation of the CNBr FragmentsCNBr fragments were prepared by direct attack of the whole MOPC 173 molecule. Fragments L1 and L3 were isolated by successive steps of gel filtration, as previously descri...

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Chemical Determination of Tryptophan

Cited by 394 publications

References 13 publications

The Malate Dehydrogenase Isoenzymes of Saccharomyces cerevisiae. Purification, Characterisation and Studies on Their Regulation

The Malate Dehydrogenase Isoenzymes of Saccharomyces cerevisiae. Purification, Characterisation and Studies on Their Regulation

Phosphoglycerate Kinase B from Ram Testis

Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin

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