Creatine kinase in human erythrocytes: A genetic anomaly reveals presence of soluble brain-type isoform
Introduction
Isoforms of creatine kinase (CK) are key players in cellular energy homeostasis by providing together with creatine and phosphocreatine an efficient energy buffering and transfer system [1], [2], [3], [4]. High CK levels are therefore present in tissues and organs with high and/or fluctuating energy demands such as muscle, brain and epithelia, while this enzyme is much less abundant or even undetectable in some other cell types including hepatocytes and human blood cells. However, in erythrocytes of three probands from a family of Italian origin, unusually high CK activity in peripheral blood cells was described by Arnold et al. (1978) [5]. The CK isoform responsible for this activity was identified to be the brain-type CK (BCK) [6]. This ectopic expression of BCK (CKBE, MIM ID 123270) in blood cells is determined by a positive CK activity test in blood cells and normal CK activity in serum. It represents an autosomal dominant inherited anomaly with a frequency of about 1 out of 5000 persons. Most of the probands are in good health and do not show any hematologic disorder [6]. The mutation underlying the CKBE phenotype has been mapped to chromosome 14q32 where the BCK gene is located, but the molecular basis of this anomaly remains unknown [6]. CK in platelets and leukocytes has been analyzed in more detail in a recent study comparing CKBE probands of eight unrelated families or normal control individuals [6]. CK activity in probands was 50- and 7-fold higher in platelets and leukocytes, respectively, as compared to control individuals. Probands also showed significantly higher BCK mRNA levels that correlated with CK protein abundance.
Much less is known about CK in erythrocytes. Earlier data indicated that in contrast to other peripheral blood cells, human erythrocytes contain muscle-type CK (MCK) that is present in the membrane fraction [7], and this has been confirmed by first proteomic approaches [8]. Appreciable CK activities of 6.0 U/mg hemoglobin (Hb) and up to 37.3 U/mg Hb were also reported for rainbow trout and domestic chicken, respectively [9], [10], and in the latter case again MCK was suggested as the principal erythrocyte CK. However, more recent proteomic studies do not identify any CK isoform in erythrocytes [11], [12], [13], [14] and CK mRNA has neither been detected [15]. Thus, the questions remain whether red blood cells still express any CK activity, and if this is the case, which isoform and which putative function this would represent, and in particular how this activity would be affected in CKBE individuals.
In this study, we re-investigated the issue of erythrocyte CK by comparing CKBE and control probands [6]. We detected varying degrees of erythrocyte CK activity, but significantly higher in CKBE probands as compared to control individuals, and could identify this CK as a soluble, cytosolic form of BCK [4].
Section snippets
Subjects
The subjects included in this study are identical to those described before [6]: 22 probands from eight unrelated families previously diagnosed with high levels of CK activity in peripheral blood cells and ten normal subjects (control group) composed of three normal individuals from these families and seven unrelated controls (healthy volunteers). Serum levels of CK activity were normal and comparable in CKBE probands and controls [6]. For blood collection, standard systems (S-Monovette®;
Creatine kinase activity and creatine
CK activity in erythrocytes was measured by a carefully controlled spectrophotometric assay in 10 control individuals (7 unrelated individuals and 3 normal individuals from three proband families) and 22 CKBE probands, the same previously analyzed for platelet and leukocyte CK [6]. The individual values are summarized in Fig. 1. CK activity in all controls was very low, with absolute values not exceeding 0.70 U/g Hb. Most probands showed a slightly higher CK activity, but not exceeding 1.20 U/g
Discussion
Creatine kinase is well known for its functions in maintaining cellular energy homeostasis by providing energy buffer and energy transfer functions, but this knowledge is restricted to few cell types. Peripheral blood cells have been assumed to possess very little or no CK, and in particular for erythrocytes, conflicting data have been published. Here we evidence low but detectable activity of CK that increases in probands carrying the CKBE anomaly, and identify this activity as a soluble BCK
Acknowledgements
We thank Hubert Roth (LBFA and CHU Grenoble, France) for statistical analysis and Be Wieringa (Univ. Nijmegen, The Netherlands) for the monoclonal BCK antibody.
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