Carbohydrate-binding protein 35. Isoelectric points of the polypeptide and a phosphorylated derivative

• Published in: The Journal of biological chemistry 1990-10, Vol.265 (29), p.17706-17712
• Main Authors: Cowles, E A, Agrwal, N, Anderson, R L, Wang, J L
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Antigens, Differentiation - genetics; Antigens, Differentiation - isolation & purification; Antigens, Differentiation - metabolism; Base Sequence; Binding and carrier proteins; Biological and medical sciences; Carrier Proteins - genetics; Carrier Proteins - isolation & purification; Carrier Proteins - metabolism; Cell Line; Chromatography, Affinity; Cloning, Molecular; Electrophoresis, Gel, Two-Dimensional; Escherichia coli - genetics; fibroblasts; Fundamental and applied biological sciences. Psychology; Galectin 3; Hemagglutinins - genetics; Hemagglutinins - isolation & purification; Hemagglutinins - metabolism; Isoelectric Focusing; Kinetics; Mice; Molecular Sequence Data; Phosphorylation; Proteins; Receptors, Cell Surface - isolation & purification; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)38221-8

Purified carbohydrate-binding protein 35 (CBP35) and extracts of mouse cells containing CBP35 were analyzed by two-dimensional gel electrophoresis. Such an analysis on recombinant CBP35, obtained by expression of a cDNA clone in Escherichia coli, yielded a pI value of 8.7. When extracts of mouse 3T3 cells were subjected to two-dimensional gel electrophoresis and immunoblotting, two spots were observed, corresponding to pI values of 8.7 and 8.2. The pI 8.2 species represents post-translational modification of the CBP35 polypeptide (pI 8.7) by the addition of a single phosphate group. This conclusion is based on the finding that purified CBP35 contained a pI 8.2 species that was labeled with 32PO4 and could be converted to the unlabeled pI 8.7 species by alkaline phosphatase treatment. The phosphorylated (pI 8.2) form of CBP35 is found in both the cytosolic and nuclear fractions, whereas the unphosphorylated (pI 8.7) species is found exclusively in the nuclei. Quiescent populations of 3T3 fibroblasts (confluent monolayers or serum-starved sparse cultures) are characterized by the predominance of phosphorylated CBP35. Stimulation of the same cells into the proliferative state resulted in an increase in the amount of the phosphorylated species; more dramatic, however, is the elevation of the level of the unphosphorylated form, which is barely detectable in quiescent cells.