Carp Myogens of White and Red Muscles. Properties and Amino Acid Composition of the Main Low-Molecular-Weight Components of White Muscle

BiochemistryUnfolding and refolding occur much faster for a proline-free protein than for most proline-containing proteins ( Communicated by Walter Kauzmann, June 27,1977 ABSTRACT The kinetics for unfolding and refolding of a parvalbumin (band 5) have been examined as a function of pH near the transition region, using stopped-flow techniques. This protein is rather unusual in that it has no proline residues, and therefore serves as a good example to test the hypothesis that the rate-limiting step seen in denaturation reactions is due to the cis-trans isomerization of proline peptide bonds in the denatured state.The kinetics for parvalbumin unfolding and refolding are complex, with the data being resolvable into two fast phases at

Section: Methods and Resultsmentioning

confidence: 99%

Unfolding and refolding occur much faster for a proline-free proteins than for most proline-containing proteins.

Brandts¹,

Brennan²,

Lin³

1977

“…In view of this property, this protein has been designated carp-muscle calcium-binding protein. These proteins apparently are not involved in glycolysis or osmotic regulation, nor do they serve any known nutritional role (2,3). However, several features suggest that they are related to mammalian troponin A (4).…”

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confidence: 99%

“…They are defined by the following characteristics: (a) high solubility in water, (b) precipitation between 70-and 95%-saturated ammonium sulfate, (c) unusual amino-acid compositions, with 10% phenylalanine, 20% alanine, and only one or no tryptophan, tyrosine, arginine, histidine, cysteine, proline, or methionine residues, (d) isoelectric points between pH 4.0 and 4.5, and (e) molecular weights about 12,000 (1)(2)(3). Pechere (4) has shown that one of the hake parvalbumins binds calcium, as does the carp protein described here.…”

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confidence: 99%

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Structure of a Calcium-Binding Carp Myogen

Nockolds

Kretsinger

Coffee

et al. 1972

The amino-acid sequence and three- alanine, and only one or no tryptophan, tyrosine, arginine, histidine, cysteine, proline, or methionine residues, (d) isoelectric points between pH 4.0 and 4.5, and (e) molecular weights about 12,000 (1-3). Pechere (4) has shown that one of the hake parvalbumins binds calcium, as does the carp protein described here. In view of this property, this protein has been designated carp-muscle calcium-binding protein. These proteins apparently are not involved in glycolysis or osmotic regulation, nor do they serve any known nutritional role (2, 3). However, several features suggest that they are related to mammalian troponin A (4). This relation will be described below.Preparation. The myogens were extracted from ground carp muscle with water and dialyzed against water, which precipitated all proteins except the albumins. Most of these albumins are precipitated when ammonium sulfate is added to 70% saturation, and the remaining proteins in solution were easily divided into a high-and a low-molecular-weight fraction by chromatography on Sephadex G-75. The low molecular weight myogens or parvalbumins were separated by DEAE-cellulose ion-exchange chromatography (5) into three fractions-A, B, and C-corresponding to components 5, 3, and 2 obtained by * Present address: Electron Microscopy Unit, University of Sydney, Sydney, Australia. 581Konosu et al. (2) by moving boundary electrophoresis. The B component was used in the structural studies because of its preferable crystallographic properties, although preliminary sequence analysis of the A and C fractions indicates they are very similar to the B proteins (unpublished results). All preparative work was done in a cold room at 40C.Amino-Acid Sequence Determination. The general approach was to purify the 15 tryptic peptides by ion-exchange chromatography. The sequence of each peptide was determined by a combination of Edman degradation, aminopeptidase M digestion, and carboxypeptidase A and/or B digestion. The amino-terminal peptide, amino acids 1-19, was hydrolyzed into four peptides by thermolysin digestion, and subsequently sequenced as were the tryptic peptides. The amino-terminal N-acetylalanine was identified with a mass spectrometer. Similar results for hake parvalbumin have been obtained by nuclear magnetic resonance (NMR) analyses (Dr. J. F. Pechere, personal communication).Ordering of Peptides. These peptides, without any overlap data, were sequenced at the same time as the first 2.0-resolution electron density map was calculated. With just these results, six of the sequenced peptides, containing 53 residues, could be placed with certainty. Five additional peptides, with 32 residues, were positioned with less confidence. Even though the remaining four peptides, containing 23 amino acids, could not be assigned unambiguously, it is clear that close coordination of crystallographic and sequence studies certainly facilitates the determination of protein structure.After these analyses, Dr. Pechbre kindly sent us the sequence for...

“…The parvalbumins are low molecular weight, acidic, watersoluble, calcium-binding proteins long believed to occur exclusively in the white muscle of lower vertebrates and for which no physiological function has been determined as yet (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Amino-acid sequences indicate that they are homologous (10,12,13) and that this homology extends to other muscle calcium-binding proteins, such as troponin C and one of the light chains of myosin (14).…”

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confidence: 99%

Amino-acid sequence of parvalbumin from rabbit skeletal muscle.

Enfield¹,

Ericsson²,

Blum³

et al. 1975

Determination of the complete aminoacid sequence of rabbit skeletal muscle parvalbumin is described. The sequence of 86 of the 109 total residues was determined automatically by sequenator analyses of peptides obtained after cleavage with CNBr or with trypsin. The positions of the remaining 23 residues were determined by subtractive Edman degradation of tryptic and chymotryptic peptides. The protein has an acetylated amino terminus. Comparison of the rabbit parvalbumin with those from carp, hake, and pike and with the calcium-binding subunit of rabbit muscle troponin indicates that these proteins are homologous. Among the parvalbumins a high degree of identity is observed, especially of residues involved in the binding of calcium or in the formation of the hydrophobic core.