The Amino‐Acid Sequence of the Most Acidic Major Parvalbumin from Frog Muscle

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

doi:10.1111/j.1432-1033.1975.tb02224.x

Cited by 36 publications

(16 citation statements)

References 20 publications

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“…The rabbit parvalbumin sequence determined by Enfield et al [29] on the basis of a sowewhat different strategy than that used here, and which was made available after the present work was completed and had been published in part [5,6], is in full agreement with the data presented above. Even the abnormal situations which were encountered here (e.g.…”

Section: Discussionsupporting

confidence: 81%

Parvalbumin from Rabbit Muscle. Isolation and Primary Structure

Capony¹,

Pina²,

Péchère³

1976

Eur J Biochem

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A parvalbumin, with its characteristic low molecular weight (∼ 12000), acidic isoelectric point (∼ 5.5), ultraviolet spectrum (maxm 259 nm) and Ca2+‐binding capacity (2 mol/mol protein) has been isolated from rabbit (Oryctolagus cuniculus) muscle. Its primary structure has been determined from a study of its tryptic peptides and of overlapping peptides generated by limited tryptic digestion and by chymotryptic and thermolytic digestions of the protein. The amino acid sequence so obtained is considered in comparison with those known for other parvalbumins and for rabbit troponin C.

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Section: Discussionsupporting

confidence: 81%

Parvalbumin from Rabbit Muscle. Isolation and Primary Structure

Capony¹,

Pina²,

Péchère³

1976

Eur J Biochem

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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%

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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“…Only 13 residues of glutama,te/glutamine were found in the sequence, while 14 residues appeared after hydrolysis. This discrepancy, not yet explained, was previously found in other parvalbumins [9,11,24]. The analysis showed also the presence of three residues as cysteic acid or carboxymethylcysteine, while two residues only are present in the sequence.…”

Section: Discussionmentioning

confidence: 46%

“…Tryptic, chymotryptic and thermolytic digestions and the separation of the tryptic peptides were performed as previously described [9,11,12].…”

Section: Tryptic Chymo Tryp T Ic and Thermolytic Digest Ionmentioning

confidence: 99%

“…Frog skeletal muscle is especiadly interesting in that the same cell contains the two major parvalbumins [8], one of which, the PI = 4.50 component [9], exhibits the typical amino-acid sequence of a /?-parValbumin, while the other component, PI = 4.88, appeared rather to be an a-parvalbumin. The Ca2+-binding properties of both frog parvalbumins were studied in detail [6].…”

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confidence: 99%

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Amino‐Acid Sequence of an α‐Parvalbumin, PI = 4.88, from Frog Skeletal Muscle

Jáuregui-Adell¹,

Pechere²,

Briand³

et al. 1982

European Journal of Biochemistry

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The primary structure of the most basic (PI = 4.88) of the two major parvalbumins from frog skeletal muscle (Rana esculenta:) has been determined by partial automatic sequencing of the protein which exhibits a free N terminus, and a study of overlapping peptides obtained by cyanogen bromide cleavage and digestion with trypsin, thermolysin and Armillaria mellea protease.This protein shows the typical structure of an a-parvalbumin. Comparison of the primary structure of ionbinding loops of a anld @-parvalbumins does not provide a clear-cut explanation of their differences in ionbinding properties [Haiech, J., Derancourt, J., Pechere, J. F., and Demaille, J. G. ( ) Biochemistry, 18, 2752 Calcium ions are now widely recognized as an intracellular second messenger, which, upon increase of their cytosolic concentration from pCa7 to pCa 5, trigger a number of metabolic events, e.g. contraction and secretion [I]. Ca2+ only acts through binding to intracellular Ca2+-binding proteins, all derived from a common 40-residue-long ancestral peptide which doubled and then quisdrupled in length to yield an ancestral four-domain Ca2 +-binding protein. The latter evolved either to calmodulin and troponin C, which are involved in the triggering of Ca2 +-de:pendent effects, or toward 'relaxing' proteins, such as parvalb'umins [2 -41.Parvalbumins are low-molecular-weight acidic proteins found in the cytosol o f fast twitdh skeletal muscles and in nervous tissue IS], which exhibit only two high-affinity Ca2+/ Mg2+-binding sites in domains 111 and IV [6]. Considerable work in this and other laboratories has been devoted to the study of their primary structure and their evolution, leading to the recognition of two major lineages, a and fl [2]. While a number of /?-parvalbumins were exhaustively studied, up to the three-dimensional structure of one of them [7], the tertiary structure of a-parvalbumins is still unknown and only three amino-acid sequences were determined, namely the rabbit PI = 5.5, the pike PI = 5.0 and the coelacanth PI = 5.0 components (see [2] and [3] for review and references).Frog skeletal muscle is especiadly interesting in that the same cell contains the two major parvalbumins [8], one of which, the PI = 4.50 component [9], exhibits the typical amino-acid sequence of a /?-parValbumin, while the other component, PI = 4.88, appeared rather to be an a-parvalbumin. The Ca2+-binding properties of both frog parvalbumins were studied in detail [6].The present report confirms that frog parvalbumin PI = 4.88 belongs to the a-lineage. Direct inspection o f the sequence of Ca'+-binding sites does not provide any clue as to the differences in ion-binding properties of CI and /?-parvalbumins, which therefore probably stems from differences in their tertiary structure. MATERIALS AND METHODS Protein PreparationFrogs (Rana esculenta) were purchased from the Elevages Couetard (St Hilaire de Riez, France). The two major parvalbumins were prepared as described by Haiech et al. [10]. The parvalbumins appeared to be homogenous upon ...

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The Amino‐Acid Sequence of the Most Acidic Major Parvalbumin from Frog Muscle

Cited by 36 publications

References 20 publications

Parvalbumin from Rabbit Muscle. Isolation and Primary Structure

Parvalbumin from Rabbit Muscle. Isolation and Primary Structure

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

Amino‐Acid Sequence of an α‐Parvalbumin, PI = 4.88, from Frog Skeletal Muscle

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