Abstract
The subcellular location of hexokinase was investigated in rat kidney. Both soluble and particulate locations are indicated by differential centrifugation. The particulate form is predominant, representing about 80% of the total activity. None of the activity is latent. Density gradient centrifugation followed by marker enzyme analysis reveals the presence of two populations of mitochondria with distinct densities. Hexokinase is associated primarily with the mitochondrial population having the lower density. Association of hexokinase with brush border, plasma membrane, lysosomes, and endoplasmic reticulum is considered unlikely on the basis of density gradient centrifugation and enzyme analysis. About 95% of the hexokinase activity associated with the mitochondrial fraction can be released in soluble form by repeated incubations with glucose 6-phosphate. An incubation time of about 4 min at 30 degrees C is required to achieve a maximal solubilizing effect. Release is accomplished without disrupting the mitochondrial compartments. Hexokinase is released also by treatment of the mitochondrial fraction with increasing concentrations of digitonin. This technique disrupts and differentially releases the mitochondrial compartments. As observed with liver, but in contrast to that observed with tumor (Parry, D. M., and Pedersen, P. L. (1983) J. Biol. Chem. 258, 10904-10912), the release of hexokinase from the mitochondrial fraction of kidney does not correlate with the release of enzymes known to mark the mitochondrial membranes or compartments. These studies provide the first critical evidence about the subcellular location of hexokinase in kidney. They show that in this tissue hexokinase is associated primarily with low density mitochondria, a finding that adds credibility to the existence of this discrete population of mitochondria in vivo. Significantly, this association of hexokinase with kidney mitochondria appears unique in that its release on submitochondrial fractionation does not correlate with the release of known mitochondrial marker enzymes. These results are directly relevant to those cells in the kidney which utilize glucose as an energy source. It is suggested that the enhanced glycolytic capacity of these cells may be due, at least in part, to an association of hexokinase with low density mitochondria.
Highlights
From the Laboratory for Molecular and Cellular Bioenergetics,Department of Physiological Chemistry,Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205
This assumption is based, uncritically, on its association with crude mitochondrial fractions obtained from homogenized renal tissue by differential centrifugation [1, 3] and on the fact that mitochondria isolated from many other tissues have been reported to bind or contain hexokinase [12,13,14,15,16]
Treatment of Washed Mitochondrial Fraction with Digitonin and Glucose 6-Phosphate-The mitochondrial fraction was treated with increasing concentrations of digitonin under isotonic conditions to assess the latency and therelease of hexokinase and marker enzymes as previously described [16].Treatment of the mitochondrial fraction with 2 mM glucose 6-phosphate was carried out according to Ref. 16
Summary
From the Laboratory for Molecular and Cellular Bioenergetics,Department of Physiological Chemistry,Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205. M., and Pedhas been undertaken previously to localize renal hexokinase, it is generally held that its particulate location in kidney is mitochondrial [1, 10, 11] This assumption is based, uncritically, on its association with crude mitochondrial fractions obtained from homogenized renal tissue by differential centrifugation [1, 3] and on the fact that mitochondria isolated from many other tissues have been reported to bind or contain hexokinase [12,13,14,15,16]. Tion of kidney does not correlate with the release of the crude mitochondrial fractions with which renal hexokienzymesknown to mark themitochondrial membranes nase has been shown to be associated were contaminated to or compartments These studies provide thefirstcritical evidence about the subcellular location of hexokinase in kidney.
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