Abstract
Differential extraction of creatine kinase activity (CK, EC 2.7.3.2) from rat brain mitochondria by graded concentrations of digitonin all yielded supernates varying in CK activity. As analyzed by isozyme electrophoresis and gel permeation chromatography the extracts contained different species of creatine kinase: (i) one third of the total CK activity consisting of contaminating cytosolic brain-type CK (B-CK) was liberated by 100 μg digitonin/mg of mitochondrial protein, (ii) approx. 20% more CK activity consisting of B-CK, as above, plus dimeric and octameric mitochondrial CK (Mi-CK), was extracted by 300 μg/mg digitonin, wheras (iii) all CK activity, consisting of B-CK and mainly octameric Mi-CK, were liberated by 700 μg/mg digitonin. In contrast to Mi-CK, B-CK associated with contaminating synaptic vesicles was readily extracted even by low concentrations of digitonin, but on the other hand octameric Mi-CK was significantly more resistant to digitonin extraction than the dimeric enzyme species. It appeared that the Mi-CK resistant to treatment with 300 μg/mg digitonin consisted to a large percentage of octamers and was organized as a complex between the two envelope membranes, for its activity was latent and still remained regulated by the outer membrane pore, that is: (i) the Mi-CK activity in such mitoplasts could be inhibited reversibly by cessation of the adenine nucleotide transport through the outer membrane pore with a polyanion, (ii) the ADP produced by Mi-CK in mitoplasts was not available to external pyruvate kinase, (iii) approx. 50% of total CK activity was not susceptible to inhibition by iodo acetate and phosphocreatine. In agreement with these findings a preferential association of octameric Mi-CK was also found in isolated contact site fractions indicating a physiological role of Mi-CK in energy transfer and a structure-function relationship of Mi-CK octamers at these sites [1]. In addition some evidence for an interaction of Mi-CK with the adenylate translocator is presented.
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More From: Biochimica et Biophysica Acta (BBA) - Biomembranes
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