Two Subunits of Brain Type Creatine Kinase Have Different Heparin Binding Activity in the Chick Retina

Abstract

Abstract: : Purpose:Molecular cloning and characterization of heparin binding proteins which vary the amount of expression during chick retinal development. Methods:PBS–soluble samples from retinas at embryonic day from 12 (E12) to 20 (E20) were separated by two–dimensional electrophoresis. After the Western–blotting, the membranes were stained with biotin–labeled heparin. Amino acid sequencing was carried out on the most varied band. Recombinant proteins made on the basis of the identified sequence were used to examine the heparin binding activity. Results: An expression of 42 kDa heparin binding band significantly decreased during retinal development. Amino acid sequencing showed the identified molecule is chicken brain type creatine kinase subunit b (CK–Bb). Another band quite similar to CK–Bb without heparin binding activity was detected by Coomassie brilliant blue staining. Amino acid sequencing indicated the molecule is brain type creatine kinase subunit a (CK–Ba). Amino acid sequence analysis showed a striking difference between them in the amino–terminal. The genes for each amino–terminal (termed SB and SA, respectively) cloned from the chick retinal cDNA library were recombined to pGEX6P–1 vector, and then transformed to E.coli SB also showed more significant heparin binding activity than SA by the fusion protein analysis. Conclusion: Two subunits of CK–B having different heparin binding activity may be important for the retinal development. The difference in heparin binding activity between them may be derived from the different structure in amino–terminal of each molecule.

Keywords: cytology • retina: distal (photoreceptors, horizontal cells, bipolar cells) • extracellular matrix