Phosphorylation of chicken brain‐type creatine kinase affects a physiologically important kinetic parameter and gives rise to protein microheterogeneity in vivo

[摘要]

In addition to the two monomer subunits of chicken brain-type creatine kinase (B-CK, EC, 2.7.3.2), termed Bb (basic) and Ba (acidic), another subspecies called Bb^∗ was identified by chromatofocussing in the presence of 8 M urea (Quest et al., [20]). The latter low abundance protein species, isolated from tissue extracts, comigrated on 2D-gels with three minor species (Bbl-3), initially identified in immunoprecipitated, [³⁵S]methionine labeled in vitro translation products of cDNA coding for the basic monomer Bb. During in vitro translation experiments in the presence of [³²P]-γ -ATP, Bbl-3 were labeled while phosphatase treatment eliminated these minor species. It is concluded that Bb^* is identical to Bbl-3 and represents phosphorylated derivatives of Bb. B-CK dimer populations from different tissues were separated by ion-exchange chromatography and the K_m values of the resulting fractions were determined under phospho-creatine (CP)-limiting conditions. In fractions containing only Bb and Bb^∗ two kinetically different enzyme species were detected (K_m values for CP = 1.6 mM and 0.8 mM), while fractions containing B-CK dimers composed of the major Ba and Bb monomers, but no Bb^∗, were homogeneous in this respect (K_m for CP = 1.6 mM). Phosphorylation of Bb to yield Bb^∗ is concluded to reduce the K_m of B-CK dimers for CP by about 50%. This K_m shift is within the range of CP concentrations found in tissues expressing the B-CK isoform and may therefore be of physiological relevance.

[学科分类] 生物化学/生物物理

[关键词] Phosphorylation of creatine kinase;Regulation of creatine kinase activity;Intracellular ATP level;Energetics;The term B-CK stands for the dimeric brain-type creatine kinase isoenzyme (enzymatically active form). Bb;Ba;Bb∗ and Bbl-3 represent different B-CK monomers