Use of parvalbumin as a protecting protein in the sequenator: An easy and efficient way for sequencing small amounts of peptides

Rochat, Hervé; Bechis, Guy; Kopeyan, Charles; Gregoire, J; Rietschoten, J. Van

doi:10.1016/0014-5793(76)80337-7

Cited by 34 publications

(15 citation statements)

References 19 publications

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“…These results, together with others [19] strongly support the usefulness of the dimethylbenzylamine-parvalbumin program that was worked out in our laboratory for sequencing small amounts of peptides [4].…”

Section: Discussionsupporting

confidence: 87%

“…Trypsin treated with ~-tosylamido-2-phenylethyl chloromethyl ketone (batch 256 U/90) and carboxypeptidase A treated with diisopropylfluorophosphate were obtained from Worthington (Freehold, N.J., U.S.A.). Imidodipeptidase was from Miles (Lausanne, Switzerland) and both aminopeptidase M and papain from Sigma from the white muscles of the hake Merluccius merluccius [4]. Sephadex G-50, G-15 and G-25 (beads, fine grade) were from Pharmacia (Uppsala, Sweden), Biogel P, and P4 (200-400 mesh) from Biorad Laboratories (Richmond, Cal., U.S.A.) and diethylaminoethylcellulose DE-52 from Whatman (Maidstone, England).…”

Section: Methodsmentioning

confidence: 99%

“…For the reduced and methylated neurotoxins I and 111, the quadrol protein programm was used following closely the procedure of Edman and Begg [15]. For peptides, a new technique, which has been described in detail in another communication, was used [4]. The degradation was made in the presence of a naturally a-NH,-blocked protein, the hake major parvalbumin.…”

Section: Determination Of N-terminal Sequencesmentioning

confidence: 99%

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Amino Acid Sequences of Neurotoxins I and III of the Elapidae Snake Naja mossambica mossambica

Gregoire

Rochat

1977

European Journal of Biochemistry

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The amino acid sequences of two neurotoxins of the African cobra Naja mossambica mossambica have been determined using almost uniquely phenylisothiocyanate degradation in a liquid protein sequencer programmed alternatively with ‘protein’ and ‘peptide’ programs. When compared to known sequences of so‐called ‘short’ neurotoxins belonging to other Elapidae snakes, neurotoxins I and III of Naja mossambica mossambica are very similar to the cobrotoxin, a neurotoxin isolated from the formosan cobra Naja atra atra.

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Section: Discussionsupporting

confidence: 87%

Section: Methodsmentioning

confidence: 99%

Section: Determination Of N-terminal Sequencesmentioning

confidence: 99%

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Amino Acid Sequences of Neurotoxins I and III of the Elapidae Snake Naja mossambica mossambica

Gregoire

Rochat

1977

European Journal of Biochemistry

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“…Some peptides were automatically degraded in the presence of 1 mg polybrene [I 51 and 1.5 mg cod parvalbumin [16] as carriers, with 0.1 M quadrol as buffer and a single-cleavage program adapted from Brauer et al [17].…”

Section: Sequence Determinationmentioning

confidence: 99%

p‐Hyroxybenzoate Hydroxylase from Pseudomonas fluorescens

Hofsteenge

Weijer

Jekel

et al. 1983

European Journal of Biochemistry

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As a final step in the elucidation of the primary structure ofp-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, the amino acid sequences of a CNBr peptide (CBI,, that accounts for the middle part of the sequence, and the C-terminal CNBr peptide (CB2, positions 277-394) from the enzyme were determined. Important sequence information was obtained from two subfragments that were formed by the cleavage with CNBr of the Met-Thr sequence (positions 346 -347) in peptide CB2.The alignment of the two subfragments from peptide CB2 and three one-residue overlaps between peptides from one of these subfragments were confirmed by investigation of well-resolved parts of a 0.25-nm electron-density map. The sequence of residues 343 -346 could not be determined with chemical methods and was assigned from the size and shape of the amino acids in the electron-density map.An important tool in the analysis of the amino acid sequence of peptide CBI was the proteinase Lys-C from Lysobacter enzymogenes, which preferentially cleaves at lysine residues.The flavoprotein p-hydroxybenzoate hydroxylase is an FAD-containing monooxygenase. It catalyses the incorporation of one atom 0 from molecular oxygen into the substrate p-hydroxybenzoate with NADPH as electron donor. Both the primary and the three-dimensional structure of the enzyme from Pseudomonasfluorescens are under investigation [ 1 -31.The enzyme occurs as a dimer, composed of identical monomers, both in the crystalline state and in solution [2, 4, 51.Five fragments were isolated after CNBr cleavage of this enzyme, which were aligned on the basis of the 0.25-nm electron-density map [3]. The amino acid sequence of three of these peptides was reported previously [6]. This sequence contained many residues involved in the binding of FAD.Tn this paper we present the amino acid sequence determination of the other two CNBr peptides: CB1, the fragment that accounts for the middle part of the primary structure of the protein, and CB2, the C-terminal fragment. The sequence of CB2 was derived not only from analysis of this peptide itself, but also from two additional CNBr subfragments (CB2a and CB2b) which could be isolated from the CNBr digest in low yield and which were formed by partial cleavage of a Met-Thr bond (positions 346 -347) in peptide CB2. The presence of these two subfragments in the CNBr digest was not demonstrated before [3]. ~~The studies on peptide CB1 will form part of the Ph. D. Thesis of W. J. Weijer to be presented to the Rijksuniversiteit Groningen; the studies on peptide CB2 were part of the Ph. D. Thesis of J. Hofsteenge presented in 1981 to the same university.Abbreviations. dansyl, 5-dimethylaminonaphthalene-1 -sulfonyl ; DEAE, diethylaminoethyl; TosPheCH,CI, L-1-tosylamido-2-phenylethyl chloromethyl ketone; BNPS-skatol, 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine. In the miniprint supplement: TLC, thin-layer chromatography; HVPE, high-voltage paper electrophoresis: BAWP, butanol/acetic acid/water/pyridine, 75 :I 5 :60:50, vjv.

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“…The degradation of the peptides was performed with dimethylbenzylamine buffer prescribed by Hermodson et al [21] in the presence of hake parvalbumin as protein carrier [12] to avoid losses of material.…”

Section: Automatic Sequential Degradationsmentioning

confidence: 99%

Primary structure of toxin IV of Leiurus quinquestriatus quinquestriatus

1985

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We report the sequence of toxin IV of Leiurus quinquestriatus quinquestriatus, one of the live toxins lethal for the mouse purified from the venom of this scorpion. Automatic sequencing of the S-carboxymethylated protein and derived peptides obtained by action of the Staphylococcus aureus protease allowed us to work out the complete sequence of 65 residues. The toxin which was found to be blocked at the C-terminal end has an extra residue at the N-terminus as compared with other toxins from Asian and African scorpions. Moreover, toxin IV must be classified as an a-scorpion toxin since it was shown to displace competitively rz51-toxin II of Androctonus australis Hector ( lzsI-AaH II) from its binding site on rat brain synaptosomes. Nevertheless, it cannot be classified in one of the groups of scorpion toxins previously defined on the level of their antigenic properties. Again the classifications of scorpion toxins based on sequence homologies or antigenic properties support one another. Scorpion toxin Amino acid sequence Automatic sequencing Scorpion toxin classiJication

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Use of parvalbumin as a protecting protein in the sequenator: An easy and efficient way for sequencing small amounts of peptides

Cited by 34 publications

References 19 publications

Amino Acid Sequences of Neurotoxins I and III of the Elapidae Snake Naja mossambica mossambica

Amino Acid Sequences of Neurotoxins I and III of the Elapidae Snake Naja mossambica mossambica

p‐Hyroxybenzoate Hydroxylase from Pseudomonas fluorescens

Primary structure of toxin IV of Leiurus quinquestriatus quinquestriatus

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