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Biochim Biophys Acta. 1993 May 6;1142(3):228-39.

Effect of macromolecules on the structure of the mitochondrial inter-membrane space and the regulation of hexokinase.

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  • 1Fakultät für Biologie, Universität Konstanz, Germany.

Abstract

Macromolecules as components of the physiological mitochondrial environment were substituted by addition of 10% dextran 70. This led to a significant reduction of the space between the two envelope and the crista membranes and to an increase of contact sites as observed by freeze-fracture analysis. The preferential binding of hexokinase in these sites was employed to further analyze the dextran effect: (i) desorption of the enzyme by digitonin treatment was found to be significantly reduced in the presence of dextran although liberation of adenylate kinase and monoamine oxidase were not affected, (ii) the affinity of isolated hexokinase isozyme I to liver mitochondria was increased by dextran. Generally the binding of hexokinase to intact mitochondria (also control mitochondria) followed a co-operative mechanism and led to an activation. Cooperativity and activation were not observed when the contact formation was suppressed by dinitrophenol or glycerol. The binding of hexokinase to the isolated outer membrane resembled that of mitochondria in the absence of contacts (i.e., no cooperativity and activation). Conversely to the observation in intact mitochondria, dextran rather reduced the affinity of hexokinase to the isolated outer membrane. Kinetic analyses of the dextran effect served to explain the function of contact site specific hexokinase binding. We observed that dextran improved the hexokinase dependent stimulation of the oxidative phosphorylation (state 3 respiration), while the activity of the enzyme with internal or external ATP remained unaffected. The results suggest three things: (i) that contact sites are probably more frequent in the intact cell than in vitro in the absence of macromolecules, (ii) that the contact preference of hexokinase serves rather the ADP supply of the translocator than the ATP transfer to the enzyme and (iii) that the total cellular hexokinase activity may be regulated by specific binding of the enzyme to the contact sites, either because of a different pore structure or because of additional components exclusively exposed in these sites.

PMID:
7683206
[PubMed - indexed for MEDLINE]
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