Analytical cell fractionation of isolated rabbit renal proximal tubules

Abstract

Proximal tubules were isolated in highly pure form from rabbit cortices by a mechanical procedure that is known to preserve the structural and metabolic aspects of the tubular cells. Postnuclear supernates prepared from the isolated tubules were subjected to isopycnic centrifugation in linear sucrose gradients. The enzyme activities associated with the plasma membrane (γ-glutamyl transpeptidase, amino-peptidase M, alkaline phosphatase, Na-K-ATPase, and phosphodiesterase I) exhibited sharp unimodal frequency-density profiles with a median density near 1.16 g/ml, which shifted to a heavier density when treated with digitonin. The lysosomal enzymes, N-acetyl-β-glucosaminidase, α-mannosidase, and cathepsin B, and the peroxisomal enzyme catalase exhibited particle-associated activity near a density of 1.22 g/ml. Disruption of these particles by freezing and thawing resulted in these activities appearing in the ρ = 1.10 g/ml region of the gradient where the soluble cytosolic enzyme, phosphoglucomutase, exhibited activity. Cytochrome oxidase activity typical of mitochondria gave a sharp unimodal profile at ρ = 1.18 g/ml. Microsomal glucose-6-phosphatase and NADPH:cytochrome c reductase activities gave median densities near 1.16 g/ml, which did not change after incubation with digitonin. Galactosyl transferase activity gave a skewed profile at ρ = 1.16 g/ml and showed a slight shift to heavier density after digitonin. This study of the enzyme activities and density gradient distribution of the components of the proximal tubule cells provides the methodology for the further study of the cellular processing of endogenous and exogenous substances by this vital cell type.