Cathodal proteins from primitive (embryonic) red cells of amphibia. Isolation and characterization.

Ec Theil

doi:10.1016/s0021-9258(19)41470-1

Abstract

A group of abundant (15% of the soluble protein) nonhemoglobin proteins was isolated from the primitive (embryonic) red cells found in tadpoles, using the cationic properties of the proteins at pH 8.6 to separate them from hemoglobin and other red cell proteins. The cathodal proteins (CP) were resolved into five components, and the two most predominant proteins were separated and characterized. Purified CP-1b and CP-2 had an amino acid composition similar to that of unfractionated cathodal proteins and to each other, except for small variations in the lysine and half-cystine content. The molecular weight of the purified CP-1b and CP-2 was 13 to 14,000, determined by gel filtration chromatography and electrophoresis in the presence of sodium dodecyl sulfate. Cathodal proteins were immunologically related although there were quantitative differences in reactivity. The concentration of cathodal proteins in primitive (embryonic) red cells was 100 times that in definitive (adult) red cells coincided with the replacement of primitive red cells. The synthesis of the cathodal proteins appeared to continue throughout the life of the primitive red cells; when hemoglobin synthesis declined in primitive red cells, approximately half of the protein synthesized by the cells was cathodal protein. Although the function of the cathodal proteins is as yet unknown, the data suggest that the cathodal proteins are a unique characteristic of erythroid differentiation in early development.

Highlights

Separation of the cathodal proteins from hemoglobin and other proteins in the supernatant solution was achieved by chromatography on DEAE-cellulose in 0.05 M Tris-HCI, pH 8.6; the cathodal proteins were recovered in the first wash of the column, free of any other proteins
That the abundance of cathodal proteins is a property of the primitive red cells is shown by the coincidence of the decline of primitive cells in the circulation and the decrease in the red cell concentration of cathodal proteins (Fig. 4)
Whether the small amount of cathodal protein detected in definitive cells

Summary

Methods

Collection of Blood, Labeling of Red Cells, and Preparataon of LysatesBlood was collected ized capillary pipettes from anesthesized animals using heparin-(7). Collection of Blood, Labeling of Red Cells, and Preparataon of Lysates. Blood was collected ized capillary pipettes from anesthesized animals using heparin-. Red cells were suspended in a buffered medium containing glucose and amino acids and were labeled with [“Clleucine for 2 hours at 28” [14]. Lysates were prepared by shaking the cells with an equal volume of water and 3s volume of chloroform [10]. When ldC-labeled proteins were purified, the lysates were mixed with carrier lysate prepared from red cells of the same animals. Frozen, labeled cells were separated into solubIe and insoluble fractions by adding 50 volumes of 5% trichloroacetic acid [14]

Results

Discussion

Conclusion