A simple solid-phase amino acid sequencer employing a thioacetylation stepwise degradation procedure

Doolittle, L.; Mross, George A.; Fothergill, Linda A.; Doolittle, Russell F.

doi:10.1016/0003-2697(77)90109-9

Cited by 28 publications

(18 citation statements)

References 9 publications

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“…Cyanogen bromide was supplied by Eastman Kodak Company. Reagents and solvents for automated sequencing were from Rathburn Chemicals, except that Quadrol-trifluoroacetie acid buffer (0.25 m stabilized with aminoethylcellulose was from Fluka AG, and thioacetylthioglycollic acid was prepared according to Doolittle et al (1977). All other chemicals were from BDH Chemicals, and were Analar or Aristar grade.…”

Section: Methodsmentioning

confidence: 99%

The amino acid sequence of yeast phosphoglycerate mutase

Fothergill

Harkins

1982

Proc. R. Soc. Lond. B.

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The complete amino acid sequence of yeast phosphoglycerate mutase comprising 241 residues has been determined. The sequence was deduced from the two cyanogen bromide fragments, and from the peptides derived from these fragments after digestion by a number of proteolytic enzymes. Determination of this sequence now allows a detailed interpretation of the existing high-resolution X-ray crystallographic structure. A comparison of the sequence reported here with the sequences of peptides from phosphoglycerate mutases from other species, and with the sequence of erythrocyte diphosphoglycerate mutase, indicates that these enzymes have a high degree of structural homology. Autolysis of phosphoglycerate mutase by yeast extracts leads to the complete loss of mutase activity, and the formation of electrophoretically distinguishable forms (R. Sasaki, E. Sugimoto & H. Chiba, Archs Biochem. Biophys. 115, 53-61 (1966)). It is apparent from the amino acid sequence that these changes are due to the loss of an 8─12 residue peptide from the C-terminus.

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Section: Methodsmentioning

confidence: 99%

The amino acid sequence of yeast phosphoglycerate mutase

Fothergill

Harkins

1982

Proc. R. Soc. Lond. B.

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“…After drying, 2 l of a saturated solution of ␣-cyanohydroxycinnamic acid in 67% (v/v) acetonitrile plus 0.1% (v/v) trifluoroacetic acid was added, and the peptide ladder was determined by MALDI-TOF-MS. The method described is based on that by Gu and Prestwich (8) using the reagents introduced by Doolittle et al (9). where a ϭ 1 Ϫ b, b represents the methyl-D 3 -labeled fraction of the total methyl content of the sample, and n is the number of possible labeling sites.…”

Section: Matrix-assisted Laser Desorption Ionization-time Of Flight Mmentioning

confidence: 99%

The Biosynthesis of Methylated Amino Acids in the Active Site Region of Methyl-coenzyme M Reductase

Selmer¹,

Kahnt²,

Goubeaud³

et al. 2000

Journal of Biological Chemistry

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The global production of the greenhouse gas methane by methanogenic archaea reaches 1 billion tons per annum. The final reaction releasing methane is catalyzed by the enzyme methyl-coenzyme M reductase. The crystal structure of methyl-coenzyme M reductase from Methanobacterium thermoautotrophicum revealed the presence of five modified amino acids within the ␣-subunit and near the active site region. Four of these modifications were C-, N-, and S-methylations, two of which, 2-(S)-methylglutamine and 5-(S)-methylarginine, have never been encountered before. We have now confirmed these modifications by mass spectrometry of chymotryptic peptides. With methyl-coenzyme M reductase purified from cells grown in the presence of L-[methyl-D 3 ]methionine, it was shown that the methyl groups of the modified amino acids are derived from the methyl group of methionine rather than from methyl-coenzyme M, an intermediate in methane formation. The D 3 labeling pattern was found to be qualitatively and quantitatively the same as in the two methyl groups of the methanogenic coenzyme F 430 , which are known to be introduced via S-adenosylmethionine. From the results, it is concluded that the methyl groups of the modified amino acids in methyl-coenzyme M reductase are biosynthetically introduced by an S-adenosylmethioninedependent post-translational modification. A mechanism for the methylation of glutamine at C-2 and of arginine at C-5 is discussed.Methyl-coenzyme M reductase catalyzes the final reaction step in the formation of methane by methanogenic archaea. It is a 300-kDa protein composed of three different subunits in an ␣ 2 ␤ 2 ␥ 2 arrangement and contains per mol 2 mol of the nickel porphinoid coenzyme F 430 as prosthetic group (1, 2). The crystal structure of the enzyme from Methanobacterium thermoautotrophicum has recently been solved at 1.45-Å resolution (3). The electron density map suggested the presence of five modified amino acids in the ␣ subunits either at or very near the active site region: 1-N-methylhistidine ␣257 (3-methylhistidine according to IUPAC nomenclature), 5-(S)-methylarginine ␣271, 2-(S)-methylglutamine ␣400, S-methylcysteine ␣452, and thioglycine ␣445, forming a thiopeptide bond (Fig. 1). Thioglycine has been proposed to function as a one-electron relay in the catalytic mechanism (2). Methylation of histidine ␣257, which is involved in substrate binding, probably influences the substrate affinity of the enzyme (3). For the methylation of the three other amino acids, an accidental methylation via methylcoenzyme M-derived methyl radicals has been envisaged. We have now ruled out this possibility by showing that the methyl group of all four methylated amino acids is derived from the methyl group of methionine, most likely via S-adenosylmethionine (SAM).1 Our experiments are based on the previous finding that methionine is taken up from the medium by growing M. thermoautotrophicum and that the methionine taken up is used for protein synthesis and in SAM-dependent methylation reactions rather than in met...

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“…Cyanogen bromide digestions were carried out by dissolving the protein or peptide (5-7 mg/ml) in 70% formic acid followed by the addition of cyanogen bromide at a concentration of 10 mg/ml. Digestions were for [16][17][18][19][20][21][22][23][24] hr at room temperature, after which the mixtures were diluted with cold distilled water and lyophilized.…”

Section: Methodsmentioning

confidence: 99%

“…Amino acid analyses were performed on either a Spinco model 121 or a Spinco 119 Amino Acid Analyzer. Sequences were determined by a variety of methods, but especially by a thioaoetylation procedure (15) either manually or automatically on a solid phase sequencer (16). The dansyl chloride/phenylisothiocyanate procedure (17) was also used in many instances and particularly for the confirmation and/or identification of serine and threonine residues.…”

Section: Methodsmentioning

confidence: 99%

Amino acid sequence of the beta chain of human fibrinogen: homology with the gamma chain.

Watt¹,

Takagi²,

Doolittle³

1978

Proc. Natl. Acad. Sci. U.S.A.

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The ft chain of human fibrinogen is composed of 452 ± 5 amino acid residues, 14 of which are methionines. Consistent with these findings we MATERIALS AND METHODSMost of the materials and procedures used in this study have been fully described in previous publications from this laboratory (11,12). In essence, fibrinogen was prepared from blood bank plasma, reduced with dithiothreitol in 6 M guanidine and carboxymethylated with iodo['4C]acetic acid; the chains were separated by chromatography on carboxymethylcellulose.Amino acid analyses were conducted after 24, 48, and 72 hr of acid hydrolysis; tryptophan was determined colorimetrically (13). Fragments D and E were isolated by DEAE-cellulose chromatography of an exhaustive plasmic digest of human fibrinogen (14). Cyanogen bromide digestions were carried out by dissolving the protein or peptide (5-7 mg/ml) in 70% formic acid followed by the addition of cyanogen bromide at a concentration of 10 mg/ml. Digestions were for 16-24 hr at room temperature, after which the mixtures were diluted with cold distilled water and lyophilized.Peptides were isolated by gel filtration on Sephadex G-50 followed by paper electrophoresis at pH 6.5 and/or pH 2.0. Amino acid analyses were performed on either a Spinco model 121 or a Spinco 119 Amino Acid Analyzer. Sequences were determined by a variety of methods, but especially by a thioaoetylation procedure (15) either manually or automatically on a solid phase sequencer (16). The dansyl chloride/phenylisothiocyanate procedure (17) was also used in many instances and particularly for the confirmation and/or identification of serine and threonine residues. Enzymes used to degrade peptides included trypsin, chymotrypsin, thermolysin, and the staphylococcal protease V-8 (18). RESULTS Amino Acid Composition. The amino acid composition of 3 chains isolated from human fibrinogen is tabulated in Table 1. Our results are compared with previous reports, as well as with the theoretical composition obtained by summing the residues found by determining the sequence of the 15 cyanogen bromide-fragments. In general, the agreement is quite good.Cyanogen Bromide Peptides. The cyanogen bromide digest was fractionated by gel filtration on Sephadex G-50 (Fig. 1); seven pools were routinely prepared.-Each run was made with approximately 60 mg of purified carboxymethylated chain (about 1 ,umol). Altogether more than 500 mg of ,B chains were treated in this fashion. Pools III-VII were purified further by paper electrophoresis at pH 6.5 and/or pH 2.0. Pools I-III were purified further by rechromatography on Sephadex G-50. Al-* Present address: Biological Institute, Tohoku University, Sendai, Japan.

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A simple solid-phase amino acid sequencer employing a thioacetylation stepwise degradation procedure

Cited by 28 publications

References 9 publications

The amino acid sequence of yeast phosphoglycerate mutase

The amino acid sequence of yeast phosphoglycerate mutase

The Biosynthesis of Methylated Amino Acids in the Active Site Region of Methyl-coenzyme M Reductase

Amino acid sequence of the beta chain of human fibrinogen: homology with the gamma chain.

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