Parvalbumin from Rabbit Muscle. Isolation and Primary Structure

Capony, Jean‐Paul; Pina, Concepción; Péchère, Jean-Claude

doi:10.1111/j.1432-1033.1976.tb10963.x

Cited by 44 publications

(19 citation statements)

References 21 publications

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“…The parvalbumin isolated from chicken leg-muscle is very similar to the rabbit protein in a number of properties such as molecular weight, amino acid composition, unusual ultraviolet absorption spectrum and Ca2+-content [2,7,8]. The much lower tissue concentration of only 1 O-20% of the amount found in rabbit muscle and the presence of 1 tyrosine residue/molecule are, however, distinct features to the rabbit counterpart.…”

Section: Discussionmentioning

confidence: 99%

“…Approximately lo-20 mg parvalbumin was obtained from 1 kg muscle. This corresponds to approximately 20% of the amount of parvalbumin isolated from rabbit muscle [8]. Step 6, the absorption coefficientd~s~mn of 1.85 calculated for the purified parvalbumin was used.…”

Section: Purification Of Chicken Parvalbuminmentioning

confidence: 99%

“…the only other parvalbumin from vertebrate muscle fully characterized up to now [7,8] and to PVlP [9], the latter having been extracted under identical conditions as parvalbumin and copurified through several steps of purification.…”

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Isolation and characterization of parvalbumin from chicken leg‐muscle

1977

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Section: Discussionmentioning

confidence: 99%

Section: Purification Of Chicken Parvalbuminmentioning

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Isolation and characterization of parvalbumin from chicken leg‐muscle

1977

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“…The standard sequencing methods used throughout this work, including the nomenclature of peptides, have been fully documented earlier [5,11,12] ' . Carboxypeptidase A digestion of peptides was carried out by the method of Ambler [26] and free amino ' In order to facilitate the reading of the reciprocal connection existing between the experimental data (Tables) and the full sequence (Fig.…”

Section: Other Methodsmentioning

confidence: 99%

“…Genetic duplication and genetic fusion seem to have played a remarkable role in the diversification of all these proteins from a single ancestral gene [2-41. Previous work on the primary structure of muscular parvalbumins has been concerned with components isolated from different species of teleostean fishes, [l, 5 -103, from an amphibian [l 1 ] and from a mammal [12,13]. This exploration of the molecular evolution of vertebrate parvalbumins has now been extended by examining the amino acid sequence of the protein from a species considered morphologically primitive.…”

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The Amino-Acid Sequence of the Major Parvalbumin from Thornback-Ray Muscle

Thatcher

Péchère

1977

Eur J Biochem

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ParvalbuminRecherche Scientifique, Montpellier (PI = 4.45) from the cartilagenous fish Raja The primary structure of the major parvalbumin clavata has been determined. The amino acid sequence was dehuced by the analysis of peptides derived from tryptic digestion of the oxidized protein. These peptides were aligned by comparison with (a) overlapping peptides produced by limited tryptic and chymotryptic digestion, and (b) by comparison with the known structures of other fish parvalbumins. The molecular evolution of thornback ray parvalbumin is briefly discussed.The investigation of the molecular evolution of muscular parvalbumins [l] is interesting both in itself and because the close relationship existing between this family of proteins and other proteins, in particular troponin-C and the light chains of myosin. Genetic duplication and genetic fusion seem to have played a remarkable role in the diversification of all these proteins from a single ancestral gene [2-41.Previous work on the primary structure of muscular parvalbumins has been concerned with components isolated from different species of teleostean fishes, [l, 5 -103, from an amphibian [l 1 ] and from a mammal [12,13]. This exploration of the molecular evolution of vertebrate parvalbumins has now been extended by examining the amino acid sequence of the protein from a species considered morphologically primitive. Thornback ray, a cartilagenous fish, was chosen, as previous studies [14,15] had shown that a major parvalbumin could be readily isolated from the muscles of this animal. The present report will describe the establishment of its primary structure as well as those characteristics which show it to be closely related to the molecular ancestor of all presently known parvalbumins. MATERIALS AND METHODS Preparation of the ProteinThornback rays (Raja clavata) were kindly provided by the Institut des Ptches Maritimes, Sete. The Pro teinase Digest ionThe parvalbumin (50 mg) was first oxidized by the method of Hirs [18], desalted into 5 % formic acid and freeze-dried. A solution of 20 mg of oxidized parvalbumin per ml was then made up in 0.05 M ammonium acetate buffer, pH 8.5, 6 M with respect to guanidinium chloride. The protein solution was heated at 50 "C for 4 h, before desalting into 0.05 M ammonium acetate, pH 8.5 on Sephadex G-25. The parvalbumin fractions were pooled and then boiled for 15 min. A thick white precipitate formed on cooling. Trypsin or chymotrypsin were added to the parvalbumin suspension to give a final concentration (w,/w) of 1 : 40 for the proteinase: substrate ratio. The precipitate cleared within 5 min and digestion was allowed to continue for a further hour at 37 "C. The digest was then freeze-dried. Pep tide Fructionut ionBoth the ion-exchange methods developed by Jones [19] and paper methods [20] were used to isolate peptides. Initially tryptic peptides were separated by cation-exchange chromatography, using either a Chromobeads P column or a column of DAI-X2 resin as described previously [ 121. Peptide fractions

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Peptide‐mass fingerprinting and the ideal covering set for protein characterisation

1997

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The rules that govern the dynamics of protein characterisation by peptide-mass fingerprinting (PMF) were investigated through multiple interrogations of a nonredundant protein database. This was achieved by analysing the efficiency of identifying each entry in the entire database via perfect in silico digestion with a series of 20 pseudo-endoproteinases cutting at the carboxy terminal of each amino acid residue, and the multiple cutters: trypsin, chymotrypsin and Glu-C. The distribution of peptide fragment masses generated by endoproteinase digestion was examined with a view to designing better approaches to protein characterisation by PMF. On average, and for both common and rare cutters, the combination of approximately two fragments was sufficient to identify most database entries. However, the rare cutters left more entries unidentified in the database. Total coverage of the entire database could not be achieved with one enzymatic cutter alone, nor when all 23 cutters were used together. Peptide fragments of > 5000 Da had little effect on the outcome of PMF to correctly characterise database entries, while those with low mass (near to 350 Da in the case of trypsin) were found to be of most utility. The most frequently occurring fragments were also found in this lower mass region. The maximum size of uncut database entries (those not containing a specific amino acid residue) ranged from 52,908 Da to 258,314 Da, while the failure rate for a single cutter in identifying database entries varied from 10,865 (8.4%) to 23,290 (18.1%). PMF is likely to be a mainstay of any high-throughput protein screening strategy for large-scale proteome analysis. A better understanding of the merits and limitations of this technique will allow researchers to optimise their protein characterisation procedures.

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Parvalbumin from Rabbit Muscle. Isolation and Primary Structure

Cited by 44 publications

References 21 publications

Isolation and characterization of parvalbumin from chicken leg‐muscle

Isolation and characterization of parvalbumin from chicken leg‐muscle

The Amino-Acid Sequence of the Major Parvalbumin from Thornback-Ray Muscle

Peptide‐mass fingerprinting and the ideal covering set for protein characterisation

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