The Isoelectric Focusing of Creatine Kinase Variants: I. The Heterogeneity of Creatine Kinase in Human Heart Cytosol and Mitochondria

Abstract

|Summary: Creatine kinase isoenzymes in cytosolic and mitochondrial fractions from human cardiac tissues i· were studied by analytical and preparative isoelectric focusing (IEF), electrophoresis and immunoinhibition. ;Analytical IEF on agärose gels revealed many creatine kinase variants in human cardiac cytosol prepared ! by extraction with a hypotonic medium. The bands located at approximately pH 5.5 were shown to contain creatine kinase-MB and minute creatine kinase-BB bands by electrophoresis. Two bands which focused closely together in IEF (pH 6.85-7.0) showed an electrophoretic migration pattern similar to creatine kinase-MM. One of them (IP 6.85) showed a complete Inhibition by anti-creatine kinase-M antibodies, whereas the other showed only 50% Inhibition.Increasing the salt concentration of tris-HCl (0.1 mol/1) in the extraction medium resulted in additional creatine kinase variants, They were characterized by high alkaline isoelectric points and were not inhibited by anti-creatine kinase-M antibodies. These variants corresponded to two cathodic bands in electrophoresis. iThe treatment of washed mitochondria with phosphate buffer resulted in a release of mitochondrial variants with differestt isoelectric points, äs shown by analytical IEF in agärose gels. l The saine pattern was pbtained by using preparative IEF. Variants with high alkaline isoelectric points gave rise,to two cathodic bands üpon electrophoresis. These two bands resembled those present in cytosol after extraction with high salt concentration. No complete Inhibition with anti-creatine kinase-M was observed in l any of the eluates. ! lThe mitochondrial variants exhibited different affinities towards creatine phosphate and ADR Variants with ,;j higher alkaline isoelectric points showed lower Äm-values for these Substrates than those with less alkaline isoelectric points.

The Isoelectric Focusing of Creatine Kinase Variants: II. The Heterogeneity of Creatine Kinase in Human Serum with Normal and Elevated Catalytic Concentrations

“…In contrast to several authors (32-33), our fmdings in tissue studies confirm that creatine kinase exists in many forms in heart muscle cells (20). We postulate that, depending on the severity of the cell damage, variable amounts of these different forms are released into the serum.…”

“…In a previous publication (20) we described our findings on the heterogeneity of creatine kinase in human heart cytosol and mitochondria. The studies indicated considerable differences among these fractions in their isoelectric points, electrophoretic behaviour and response to immunoinhibition.…”

“…By analysing this serum by IEF on Isogel using pH 3.5 -9.5, we noticed that the creatine kinase-BB focused almost in the same position äs creatine kinase-MB (20). Although the frequency of creatine kinase-BB interference is in the Order of 1% (29), its presence with creatine kinase-MM may reduce the specificity of the IEF method for the diagnosis of myocardial infarction.…”

Cellulose Acetate and Electroendosmosis-Low Agarose Electrophoresis: Advanced Methods for the Separation and Quantitative Determination of Serum Creatine Kinase Isoenzyme Levels

“…Additionally, the immunoinhibition test lacks reproducibility at lower creatine kinase-MB levels (bel ow 10 U/l). Therefore, the application of sensitive and specific electrophoresis methods (cellulose acetate, agarose) is meaningfül in cases where either creatine kinase^BB and or macro creatine kinase prevails or higher sensitivity is required (22,23). The same is true for some patients with various types of carcinoma (23 -25).…”