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

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Abstract
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 Km 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 (Km 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 (Km for CP = 1.6 mM). Phosphorylation of Bb to yield Bb∗ is concluded to reduce the Km of B‐CK dimers for CP by about 50%. This Km shift is within the range of CP concentrations found in tissues expressing the B‐CK isoform and may therefore be of physiological relevance.