Phosphorylation of beta-crystallin B2 (beta Bp) in the bovine lens.
Three major 32P-labeled polypeptides were found in the soluble fraction of bovine lenses cultured in a medium containing [32P]orthophosphate. Two of the polypeptides corresponded to the phosphorylated A and B chains of alpha-crystallin. In this communication, the third polypeptide is now identified. This polypeptide is characterized by a molecular weight of 27,000 and a pI of 6.6, eluted exclusively in the beta Low fraction of a CL-6B gel filtration separation of lens soluble material, and could be further purified by DE52 anion exchange chromatography. The only 32P-labeled amino acid detected was phosphoserine. A single 32P-labeled peptide was observed after tryptic digestion and two-dimensional mapping. The amino acid sequence of the purified peptide is Gly-Ala-Phe-His-Pro-Ser-Ser. This sequence exactly matches the expected C-terminal tryptic fragment, residues 198-204, of the bovine beta-crystallin B2. The results of carboxypeptidase A digestion of the 32P-labeled peptide suggest that only Ser203 is phosphorylated. By using the catalytic subunit of the cAMP-dependent protein kinase, purified beta B2 was phosphorylated in vitro, generating a single 32P-labeled polypeptide with the identical pI as the phosphorylated polypeptide obtained from lens culture. On the basis of these data, the Mr 27,000 32P-labeled polypeptide is identified as the phosphorylated form of the beta-crystallin B2.
Kleiman
N J
NJ
Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Chiesa
R
R
Kolks
M A
MA
Spector
A
A
D000818:Animals; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D002417:Cattle; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003459:Crystallins / Q000302:isolation & purification / Q000378:metabolism*; D007525:Isoelectric Focusing; D007908:Lens, Crystalline / Q000378:metabolism*; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation
D003459:Crystallins; D010710:Phosphates; D010748:Phosphopeptides; D002268:Carboxypeptidases; D043422:Carboxypeptidases A