Analytical Subcellular Fractionation of Jejunal Biopsy ... Analytical subcellular fractionation of jejunal

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  • Clinical Science and Molecular Medicine (1976) 51, 557-574.

    Analytical subcellular fractionation of jejunal biopsy specimens : methodology and characterization of the organelles in normal tissue

    T. J. PETERS Department of Medicine, Royal Postgraduate Medical School, London

    (Receiued 14 May 1976)

    Summary 1. Portions of closed jejunal biopsies were gently

    homogenized in isotonic sucrose or sorbitol and centrifuged at 800 g for 10 min to prepare a cell extract.

    2. The extract was fractionated in a single-step procedure by isopycnic centrifugation on a con- tinuous sucrose or sorbitol density gradient with the Beaufay automatic zonal rotor.

    3. The subcellular organelles were located in the density gradient by assay of marker enzymes and previously unassigned enzymes were localized to particular organelles.

    4. The following organelles were characterized by their modal equilibrium densities in sucrose density gradients: brush borders (1~21)~ peroxisomes (1.18), mitochondria (1.16), endoplasmic reticulum (1016)~ basal-lateral membranes (1.12). At least three dis- tinct populations of lysosomes with different modal densities and enzyme content were demonstrated.

    5. This analytical fraction technique can be used to study the subcellular pathology of human jeju- num.

    Key words: brush borders, closed biopsy, cytosol, disaccharidases, endoplasmic reticulum, lysosomes, microsomes, mitochondria, plasma membrane, subcellular fractionation, jejunum.

    Introduction The absorptive epithelial cells of the small intestine

    Correspondence: Dr T. J. Peters, Department of Medicine, Royal Postgraduate Medical School, Du Cane Road, London W12 OHS.

    (enterocytes) are known to have a complex structure. Enterocytes are implicated in the digestion, absorp- tion and metabolism of dietary components and their properties and functions are disturbed to various degrees in most small-intestinal diseases. Afthough there are considerable data on the properties of cer- tain organelles of the enterocyte of animal intestine, little is known quantitatively about these organelles in human intestine. Some information can be derived from morphological and histochemical studies but analytical subcellular fractionation techniques per- mit quantitative assessment of the properties of the individual organelles in health and disease. The application of fractionation procedures to human tissue has hitherto been limited by the technical difficulty of processing the milligram quantities of tissue obtained by closed jejunal biopsy. The development of a suitable single-step fractionation technique coupled with highly sensitive marker- enzyme assay procedures has permitted analytical fractionation techniques to be applied to human biopsies for the first time.

    Methods

    Patients Jejunal biopsies were obtained with either a Crosby

    capsule or the Debri multiple biopsy capsule. The biopsies were collected from adult subjects of both sexes undergoing investigation for possible gastro- intestinal disease. They were shown under dis- secting-microscope and histological examination to be completely normal. The disaccharidase activities of the tissue were within normal limits (Peters, Batt,

    557

  • 558 T. J. Peters

    Heath & Tilleray, 1976). The studies reported in this paper have been approved by the local ethical committee.

    Tissue homogenization A portion of the biopsy, approximately 10 mg wet

    weight, was fixed in formalin-saline and processed for routine histological examination; another, approximately 10 mg wet weight, was collected in 2 ml of ice-cold sucrose solution (0.3 mol/l) con- taining disodium EDTA (1 mmol/l), pH 7.4, and ethanol (22 mmol/l) (SVE medium). The tissue was disrupted with ten strokes of a loose-fitting (type A) pestle in a small Dounce homogenizer (Kontes Glass Co., Vineland, N.J., U.S.A.) and centrifuged at 800g for 10 min in an MSE 4L centrifuge (Measuring and Scientific Equipment, Crawley, Sussex, U.K.). The pellet was resuspended in a further 2 ml of SVE medium with three strokes of the type A pestle and centrifuged again. The supernatants were combined (PNS fraction). The low-speed pellet, consisting of nuclei, large brush-border fragments and interstitial cells (N fraction), was resuspended in 2 ml of SVE medium with a tight-fitting (type B) pestle.

    Density gradient centrifugation Approximately 3.5 ml of PNS fraction was layered

    on to 30 ml of density gradient extending linearly with respect to volume, from a density of 1-05 to one of 1.28 and resting on a 4 ml cushion of density 1-32, in the Beaufay (1966) automatic zonal rotor. All solutions contained disodium EDTA (1 mmol/l), pH 7-4, and ethanol (22 mmol/l). The rotor was accelerated to 35 000 rev./min and run for 35 min with an integrated force of 3.3 x 10'O rad2 s-'. The rotor was then slowed to 8000 rev./min for auto- matic unloading and collection of the gradient fractions (Leighton, Poole, Beaufay, Baudhuin, Coffey, Fowler & de Duve, 1968). Some sixteen fractions were collected into tared tubes, thoroughly mixed, weighed, and then density was determined with an Abbe refractometer (de Duve, Berthet & Beaufay, 1959). Certain enzymes, including alkaline phosphatase, the dehydrogenases, D-amino acid oxidase and neutral 8-galactosidases, were assayed on the same day as the fractionation: the samples were then stored at -20°C. Sucrose was always used as the density gradient medium but purified sorbitol (Messer & Dahlqvist, 1966) was also used both for

    homogenization and the density gradient media. Brush borders were isolated from jejunal mucosa

    by homogenization in a small Dounce homogenizer in 3 ml of TrisIEDTA (5 mmol/l),pH7.4(Millington7 Critchley & Tovell, 1966) with five strokes of a loose- fitting (type A) pestle. The brush borders were col- lected by centrifugation at 8000 g and were twice resuspended and centrifuged in EDTA/Tris buffer. The final pellet was suspended in 10 ml of Tris/HCI buffer (1 mol/l), pH 7.4, and stirred gently for 1 h at 4°C. A portion (3-5 ml) of the Tris-disrupted brush- border suspension was then subjected to analytical isopycnic centrifugation on a sorbitol density gradient as described above.

    Enzymic analyses

    Table 1 shows the assay conditions used for analysis of the density gradient fractions (Peters, Heath, Wansbrough-Jones & Doe, 1975). Protein was assayed fluorimetrically as described previously (Peters, Miiller & de Duve, 1972). Maltase, sucrase and lactase were assayed by the fluorimetric modifi- cation (Peters et al., 1976) of the technique of Dahlqvist (1 964).

    Inhibitor studies

    For certain enzymes, analysis of the gradient frac- tions was carried out with selective inhibitors. Parallel incubations were carried out under identical conditions with and without the inhibitor. 5'-Nucleo- tidase was assayed in the presence of nickel chloride (5 mmol/l), a selective inhibitor of nucleotidase activity (Ahmed & Reiss, 1958; Campbell, 1962) or in the presence of glycine buffer (0.1 mol/l), a selec- tive inhibitor of alkaline phosphatase (Bodansky, 1946). a-Glucosidase was assayed in the presence of D-turanose (32 mmol/I), an inhibitor of lysosomal glucosidase activity (Lejeune, Thinks-Sempoux & Hers, 1963) or with zinc chloride (2.8 mmol/l), an inhibitor of neutral microsomal activity (Suzuki & Kushida, 1973). 8-Galactosidase was assayed with and without p-chloromercuribenzoate (0.1 8 mmol/l), an inhibitor of non-brush-border-enzyme activity (Asp & Dahlvqist, 1972).

    Presentation of enzyme distribution results Results are expressed as frequency-density histo-

    grams. The results in the lightest fractions, up to

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