A new technique is described for determining the distribution of metabolites between the cytosol and mitochondria. Rapid lysis of the cell plasma membrane is obtained by forcing isolated liver cells under high pressure through a small diameter needle. The cells, after disruption by the shearing forces generated during the turbulent flow through the needle, are exposed to mitochondrial anion transport inhibitors to prevent efflux of mitochondrial metabolites. Maximal release of cytosolic metabolites was obtained when release of lactate dehydrogenase was greater than 70%, which corresponded with minimal release of mitochondrial enzymes (5-9%). A measured Reynolds number between 7600 and 8000 was indicative of optimal disruption. Mitochondria in the disrupted cells were still functional, as shown by the ability of ADP to stimulate respiration when glutamate plus malate were provided as substrates. Measurement of the subcellular volumes yielded values of 2.0 and 0.2 ml/g dry wt, respectively, for the cytosol and mitochondria. Calculation of the mitochondrial ΔpH (pHin-pHout) in the isolated liver cell based on 22 individual measurements of mitochondria/cytosol gradients for citrate, isocitrate, α-ketoglutarate, malate, glutamate, and pyruvate yielded a value of 0.41 ±0.03. The excellent relationship of these gradients to a common ΔpH lends credence to the technique. Cytosolic and mitochondrial ATP ADP ratios were similar in liver cells isolated from starved and fed rats. Fed rat liver cells, however, had a higher cytosolic adenine nucleotide content (16.8 μmol/g dry wt) than those from starved rats (14.5 μmol/ g dry wt) whereas the mitochondrial content was the same (16 nmol/mg of mitochondrial protein). Data obtained by the disruption technique are compared with other previously published data obtained using either digitonin treatment of isolated hepatocytes or nonaqueous solvent extraction of lyophilized freeze-clamped perfused livers.
ASJC Scopus subject areas
- Molecular Biology