An examination of conditions for the cleavage of polypeptide chains with cyanogen bromide: Application to catalase

Schroeder, Werner; Shelton, J. C.; Shelton, J. R.

doi:10.1016/0003-9861(69)90069-1

Cited by 284 publications

(82 citation statements)

References 11 publications

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“…When the enzyme was cleaved with cyanogen bromide and the mixture of fragments was sequenced, the N-termini of CBI, CB2, and CB3 were found to be present in yields of 100%, 82%, and 70%, respectively. This improvement, compared with the previous results (44), is explained by the trifluoroacetic acid effecting better cleavage than the weaker formic acid of the Met-Set bond (39). The 2-pyridylethylated CB fragments were separated by ion exchange chromatography on DEAESephacel (Figure 1).…”

Section: Cyanogen Bromide Fragmentsmentioning

confidence: 70%

The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger

Svensson

Larsen

Svendsen

et al. 1983

Carlsberg Res. Commun.

189

158

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The primary structure ofglucoamylase G1 (EC 3.2.1.3) from Aspergillus niger has been determined. Fragments of GI were obtained by cleavage with either cyanogen bromide, hydroxylamine, or S. aureus V8 protease. The resulting peptides were separated using ion exchange chromatography on DEAE-Sephacel, gel filtration, and affinity chromatography on Con A-Sepharose. Secondary fragments were generated by cleavage with either o-iodosobenzoic acid or BNPS-skatole as well as by digestion with S. aureus V8 protease, trypsin, and endoproteinase Lys-C. These peptides were purified by the procedures mentioned above and by reverse phase HPLC. The present fragments were amino acid sequenced and this permitted, in combination with the tryptic peptides (Carlsberg Res. Commun. 48, 517-527 (1983)), identification of 574 of the 614 amino acid residues in Gl. Sequencing of glucoamylase Gl cDNA, constructed from A. niger total mRNA, enabled deduction of the sequence of the remaining 40 amino acid residues localized to 6 short stretches. From the alignment of the fragments the complete primary structure of the enzyme was established. The amino acid sequence corresponds to a molecular weight of the polypeptide moiety of 65,424. Including both hexosamine and neutral carbohydrate contents the molecular weight of the present sample of G1 was calculated to be about 82,000.The majority of the carbohydrate of Gl is found in a highly glycosylated region of 70 amino acid residues which comprises about 35 O-glycosyl serine and threonine residues. This region ends approximately 100 residues from the C-terminus of the enzyme. Two N-glycosylated positions were found in the central part of the polypeptide chain. The molecule contains 9 half-cystine residues. No homology is apparent between the sequence of glycoamylase and various at-amylases.Abbreviations: BNPS-skatole = 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine; ca = citraconyl; Con A = concanavalin A; DFP = diisopropylfluorophosphate; DPCC = diphenylcarbamyl chloride; EDTA = ethylenediaminetetraacetic acid, disodium salt; Hepes = N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid; Nemac = N-ethylmorpholine acetate; HPLC = high pressure liquid chromatography; PTH = phenylthiohydantoin; SDS-PAGE = polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; 2-pe = 2-pyridylethyl; G I designates the larger and G2 the smaller of the forms ofglucoamylase from A. niger (44).

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Section: Cyanogen Bromide Fragmentsmentioning

confidence: 70%

The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger

Svensson

Larsen

Svendsen

et al. 1983

Carlsberg Res. Commun.

189

158

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“…Cleavage of Met-Thr bonds by CNBr has indeed been reported to occur in low yields [21] under these conditions (70o70 formic acid). We thus tested various solvents in order to achieve cleavage of the c~-Met-243-Thr-244 bond.…”

Section: Analysis Of Cnbr Sub Fragments Of the Tryptic Digestmentioning

confidence: 93%

“…[3H]CPZ (20)(21)(22)(23)(24)(25) Ci/mmol; 1 Ci = 37 GBq) was purchased from New England Nuclear; carbamoylcholine chloride and unlabeled CPZ from Sigma; dimethyl sulfide from Fluka; L-l-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin from Merck (Darmstadt); and cyanogen bromide from Eastman-Kodak.…”

Section: Methodsmentioning

confidence: 99%

The noncompetitive blocker [³H]chlorpromazine labels segment M2 but not segment M 1 of the nicotinic acetylcholine receptor α‐subunit

et al. 1989

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The membrane bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [~H]chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The radioactivity incorporated into the AChR subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The ~t-subunit was purified and digested with trypsin and/or CNBr and the resulting fragments fractionated by HPLC. Sequence analysis resulted in the identification of Ser-248 as a major residue labeled by pH]chlorpromazine in a phencyclidine-sensitive manner. This residue is located in the hydrophobic and putative transmembrane segment M2 of the ~t-subunit, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the Nchain [1] and the Ser-254 and Leu-257 in the//-chain [2]. Extended sequence analysis of the hydrophobic segment M 1 further showed that no labeling.occurred in this region.

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“…The labeled enzyme was cleaved by incubation with CNBr (3/stool) in 40,ul of 70% TFA [11]. After 24 h at room temperature m the dark, the reaction mixture wa~ dried.…”

Section: Active Site Labehagmentioning

confidence: 99%

“…They correspond most probably to partial cleavages of the protein by CNBr. Indeed, 6 Met-Thr bonds which are known to be poorly cleaved by CNBr [11], are present in PepX out of 12 Met-X bonds. Two of these bonds preceeds the active site and their incomplete cleavage could lead to radioactive cleavage products of various sizes.…”

Section: Sequence Determination Of the Active Site Peptidementioning

confidence: 99%

Identification of the active site serine of the X‐prolyl dipeptidyl aminopeptidase from Lactococcus lactis

et al. 1992

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The active site ser, ne of the X-prolyl dipept~dyl ammopcptidase from Lactococcus lactzs (PepX) was identified. The enzyme was labeled by [~H]DFP, treated by CNBr and the resulting pepUdes were separated by reverse-phase-HPLC. The mum rad~olabeled peptide was sequenced. Ser-348, in the following sequence, Gly-L~s-Ser-Tyr-Leu-Gly, was identified as the active site serme. A sequen¢~ comparison between the active site of l"epX and other serme proteases was made, ~howing only limited ~equence homologies m this area. The consensus sequence surrounding the active site ~grine in the three known X-prolyl dipeptidyl ammopeptidases (mammalian DPPIV, :)'east DPAB and PcpX) is G-X-S-Y-X-G, where X is a non-conserved amino acid.

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An examination of conditions for the cleavage of polypeptide chains with cyanogen bromide: Application to catalase

Cited by 284 publications

References 11 publications

The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger

The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger

The noncompetitive blocker [³H]chlorpromazine labels segment M2 but not segment M 1 of the nicotinic acetylcholine receptor α‐subunit

Identification of the active site serine of the X‐prolyl dipeptidyl aminopeptidase from Lactococcus lactis

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An examination of conditions for the cleavage of polypeptide chains with cyanogen bromide: Application to catalase

Cited by 284 publications

References 11 publications

The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger

The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger

The noncompetitive blocker [3H]chlorpromazine labels segment M2 but not segment M 1 of the nicotinic acetylcholine receptor α‐subunit

Identification of the active site serine of the X‐prolyl dipeptidyl aminopeptidase from Lactococcus lactis

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The noncompetitive blocker [³H]chlorpromazine labels segment M2 but not segment M 1 of the nicotinic acetylcholine receptor α‐subunit