Distinct response to heparin by two chicken brain type creatine kinase subunits.
In the chicken, two creatine kinase-type B (B-CK) isoproteins, Ba- and Bb-CK, both of which are derived from a single copy gene by alternative splicing, dimerize in neural tissues. The two isoproteins contain distinct N-terminal portions, but their functional difference remains unknown. We investigated the binding affinities of Ba- and Bb-CK to heparin, hyaluronan and chondroitin sulfates, and examined the influence of these glycosaminoglycans on enzyme activity. Chicken retinal samples analyzed by Western blotting and amino acid sequence study after two-dimensional gel electrophoresis showed that heparin binds Bb-CK, but not Ba-CK, while hyaluronan and chondroitin sulfates showed no interaction with either isoprotein. Using fusion proteins covering the distinct N-terminal portions, we also showed that heparin did not react with the N-terminus of Ba-CK, but did react with that of Bb-CK. Site-directed mutagenesis of basic amino acids found in the N-terminal portion of Bb-CK identified three basic amino acids critical for this binding. Furthermore, heparin dose-dependently inhibited the enzymatic activities of Ba-CK; Bb-CK activities were less intensely inhibited. Hyaluronan and chondroitin sulfates had no effects on the activities of these enzymes. Thus, the N-terminal portion of B-CK is critical to mediate its affinity to heparin and control enzyme activity, which may be important for regulating energy metabolism in neural tissues such as brain and retina, unique organs abundant in heparan sulfates.
Kataoka
Takuya
T
Department of Ophthalmology, Aichi Medical University, Nagakute, Japan.
Yoneda
Masahiko
M
Takeyama
Masayuki
M
Ohno-Jinno
Akiko
A
Sugita
Iichiro
I
Li
Huili
H
Isogai
Zenzo
Z
Iwaki
Masayoshi
M
Zako
Masahiro
M
D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000378:metabolism / Q000494:pharmacology*; D001923:Brain Chemistry / Q000187:drug effects*; D002645:Chickens / Q000378:metabolism*; D003402:Creatine Kinase / Q000378:metabolism*; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D006025:Glycosaminoglycans / Q000494:pharmacology; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology*; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D012160:Retina / Q000187:drug effects / Q000254:growth & development
D000925:Anticoagulants; D018076:DNA, Complementary; D006025:Glycosaminoglycans; D007527:Isoenzymes; D012313:RNA; D006493:Heparin; D003402:Creatine Kinase