Isolation and Characterization of Parvalbumins from the Skeletal Muscle of Higher Vertebrates*

existed in mammalian systems, there was a strong suspi-cion that classical parvalbumins might also be present. This manuscript describes the isolation of parvalbumins from turtle, chicken, rabbit, and human skeletal muscle. The finding that these low molecular weight, calcium-binding proteins are not confined to aquatic animals strongly suggests that they must possess a definite physiological function possibly related to the contractile process.

exclusively from fish and amphibians, have been obtained in sizable amounts from the skeletal muscle of turtle, chicken, rabbit, and man. The finding that these proteins are not confined to lower vertebrates but have been conserved throughout evolution strongly suggests that they must possess a definite physiological function possibly related to the contractile process.
Parvalbumins are low molecular weight, soluble, calcium-binding proteins found hitherto exclusively in the muscle of lower species (fish and amphibians).
They display an unusual ultraviolet spectrum due to a high phenylalanine to tyrosine and tryptophan ratio. Their amino acid sequence has heen determined (l-3), as well as their tertiary structure by x-ray crystallography (4)(5)(6)(7). However, no physiological function could be ascribed to them as yet.
Recently, a protein related to parvalbumin was isolated from dogfish skeletal muscle with the main difference that it could be readily phosphorylated by an endogenous protein kinase (8 Upon further purification, they could also be characterized by their unusually high (greater than 1) 260:2&?0 nm absorbance ratio, and by their characteristic ultraviolet spectra.

RESULTS
Since the purification and properties of the parvalbumins from turtle, chicken, rabbit, and human skeletal muscle are very similar, only the characterization of the rabbit protein will be described in some detail.
Purification of Rabbit Muscle Parvalbumin-All steps were carried out at 4". Fresh rabbit skeletal muscle (1 kg) was ground in a meat grinder, homogenized for 1 mm in a Waring Blendor at full speed in 2.5 volumes of distilled water, pH 7.0, and centrifuged for 30 mm at 14,000 x g; the supernatant was lyophilized.
The residue was dissolved in 100 ml of 10 mM sodium acetate, pH 5.7, and dialyzed against the same solution. The dialyzed material (after clarification by centrifugation) was applied to a column (65 x 5 cm) of DE52-cellulose equilibrated in the same solution; elution was carried out with a linear gradient of 10 to 500 mu sodium acetat.e, pH 5.7. The fractions containing the low molecular weight protein (see "Materials and Methods") were pooled and the solution was lyophilized. The residue was dissolved in 10 mM imidazole HCl, pH 6.8, and passed through a column (2.5 x 100 cm) of Sephadex G-75 equilibrated in the same solution.
The pure fractions as judged by gel electrophoresis in the presence and absence of sodium dodecyl sulfate (and their characteristic ultraviolet spectra) were pooled; following dialysis against 5 mu ammonium acetate, pH 8.0, and lyophilization, a salt-less powder was obtained. From 1 kg of fresh rabbit muscle, 100 to 300 mg of pure parvalbumin can be isolated.

Test of Purity and Molecular
Weight-Parvalbumins from the turtle, chicken, rabbit, and man were pure as judged by the criteria of polyacrylamide gel electrophoresis (Fig. l), sedimentation velocity, and equilibrium experiments in the ultracentrifuge (not illustrated). The faster migration observed on normal gels (Fig. 1B) for turtle and human (versus chicken and   Absorption Spectrum--The absorption spectrum of rabbit parvalbumin is illustrated in Fig. 2; similar spectra, characteristic of a high phenylalanine to tyrosine and tryptophan ratio, were displayed by material obtained from the other species except that turtle and chicken parvalbumins which contain 1 tyrosyl residue show an additional shoulder or peak at 278 nm. All four parvalbumins have a low absorbance index Aifr of about 1.2 (versus biuret) and 2.0 (versus dry weight, synthetic boundary, etc.).
Ami?zo Acid Composition-The amino acid composition of rabbit parvalbumin is listed in Table I; almost identical  values  were obtained for the parvalbumins from the other species investigated here, except that the proteins from turtle and from chicken had 1 tyrosyl residue per molecule; these values, in turn, were very similar to those obtained from lower vertebrates including the hake (1, 2), the carp (3), the pike (18), the ray (lQ), the frog (18), and the dogfish.' All four proteins have a blocked NH2 terminus as revealed by analysis on the sequencer. Metal An&siz+The four parvalbumins described here were isolated as calcium metalloproteins even though the purification was carried out in the absence of added Ca* ions or even in the presence of 0.1 mu EDTA (for the turtle and chicken proteins). The purified materials were free of magnesium, manganese, iron, cobalt, copper, and zinc as determined by atomic absorption. Calcium-binding measurements by equilibrium dialysis and the Chelex partition procedure (15) indicated that all four parvalbumins display two strong binding sites for calcium with a Kdim of 0.2 PM or below, an affinity similar to that previously reported for hake parvalbumin (20).

DISCUSSION
The finding that parvalbumins are present in sizable amounts in the muscle of higher vertebrates including man raises more than ever the question of their physiological function. While 100 to 300 mg of the low molecular weight calcium-binding proteins could be isolated per kg of fresh rabbit muscle, more than 1 g per kg was obtained from the turtle.
Assuming a conservative yield of 50 y0 during purification, the concentration of parvalbumins would be approximately xe to g that of TN-C* (21). Evidence for a relationship between these two proteins was recently presented (l&22), based on a degree of homology between their amino acid sequences.
On the other hand, parvalbumins cannot originate as mere breakdown products of the larger TN-C. First, while the greater similarity was observed between the COOH-terminal portion of TN-C and parvalbumins (22), the latter proteins could only come from the NH2 end of TN-C since all but one (18) have a blocked NH2 terminus. Second, the rabbit parvalbumin described here contains 16 lysyl residues per molecule while only 9 are found in rabbit TN-C (22).
for carrying out the amino acid analyses, to K. A. Peters for the ultracentrifuge runs, and to L. H. Ericsson and Dr. K. A. Walsh for analyzing the NH2 terminus of the parvalbumins.