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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 distinct 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 jejunum.

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 certain 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) containing 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 automatic 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 collected 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 modification (Peters et al., 1976) of the technique of Dahlqvist (1 964).

Inhibitor studies

For certain enzymes, analysis of the gradient fractions 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 selective 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 histograms. The results in the lightest fractions, up to

TA

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Subcellular fractionation of human jejunum 561

TABLE 2. Specific activities and percentages of released enyzmes in homogenized jejunal biopsies Specific activity is expressed as mean value ~ S E (munits of activitylmg of protein). Number of specimens assayed is shown in parentheses. Protein is expressed as mg+ SE. p-CMB, p-chloro-

mercuribenzoate.

Specific Activity in Enzyme EC no. activity PNS fraction

(%)

Alkaline phosphatase Leucyl 8-naphthylamidase y-Glutamyl transpeptidase Total a-glucosidase (pH 6.0) Zn2+-resistant a-glucosidase (pH 6.0) Total 8-galactosidase (pH 6.0) p-CMB-resistant 8-galactosidase (pH 6.0) Catalase N- Acetyl-8-glucosaminidase N-Acetyl-8-galactosidaminidase Acid phosphatase Cathepsin C 8-Galactosidase (pH 3.5) Acid diesterase 8-Glucuronidase /l-Glucosidase Malate dehydrogenase Glutamate dehydrogenase Monoamine oxidase Lactate dehydrogenase a-Glucosidase (pH 8.0) Zn2+-sensitive a-glucosidase (pH 6.0) 5’-Nucleotidase Total protein

3.1.3.1 3.4.1 1 . I 2.3.2.2 3.2.1.20 3.2.1.20 3.2.1.23 3.2.1.23 1.11.1.6 3.2.1.30 3.2.1.53 3.1.3.2 3.4.14.1 3.2.1.23 3.6.1 . I 1 3.2.1.31 3.2.1.21 1.1.1.37 1.4.1.2 1.4.3.4 1.1.1.27 3.2.1.20 3.2.1.20 3.1.3.5 -

64.7k6.3 (IS) 4.252 0.78 (9) 12.1k1.4 (11) 9.542 1.0 (13) 7.872 1.0 (3) 1.11+0.18 ( 5 )

0.3 13 & 0.056 (5) 79.9+5.5 (15)

6.08 f 0.47 (14) 2.13f0.23 (4) 39.1f3.8 (7) 2.68f0.5 ( 5 ) 1.79f0.93 (4) 1.15f0.19 (5) 3.05f0.20 (7) 1.46f0.19 (3) 3720f 305 (15) 7.08f0.64 (4) 41.8k6.7 (7) 387240 (5) 1.13+0.10 (14) 1.60f0.3 (3) 41.1f4.1 (8) 1.05+0.14 (25)

36.42 2.2 53.7f 5.1 39.6f 4.0 39%+ 2. I 35.12 2.9 75.8f 3.5 32.42 6.4 82*6* 2.0 55.7f 3.1 62.0f 8.1 61.1f6.4 73.4f 3.9 71.5f4.8 64.Ok 7.0 66,9f 6.0 73.6f 5-4 81-7f 1.40 64.3+ 3.7 75.6f 3.0 73.8 2 4.2 55.7+ 3.1 -

50.1 If: 2.4 75 f2

a density of 1.10, which relate mostly to soluble activity remaining in the starting layer, were pooled and plotted over the density interval 1.05-1.10. The corresponding block is shaded in the histogram to indicate that it is not part of the density distribution proper. All calculations and plots were performed by computer (Beaufay, Jacques, Baudhuin, Sellinger, Berthet & de Duve, 1964; Leighton et af., 1968). All histograms were normalized and the percentage recoveries (sum of gradient fractions against tissue extract injected into rotor) are given. Pooling and averaging of several distributions were performed by computer (Leighton et al., 1968). The method re- quires conversion of all the histograms into the same pre-set density intervals with some resultant loss of resolution.

Results

Tissue enzynzes and released actiuity

The specific activities of the various enzymes estimated in the homogenates of the jejunal biopsies (Table 2) form the control values against which the results of measurements in diseased tissue will subsequently be compared. Sufficiently sensitive assays have been established so that the activities of these enzymes and of protein can be determined in the density gradient fractions. The percentageactivity in the PNS fraction is also listed (Table 2), reflecting the proportion of cells disrupted and the amount of enzyme activity not sedimented at 800 g for 10 min. Enzymes predominantly localized to the brush border (e.g. alkaline phosphatase) are found in the

562 T. J . Peters

Alkaline ptosphatase

Dfnsi!!

FIG. 1. Isopycnic centrifugation of 8000 g-min supernatant from jejunal biopsy homogenate. Graphs show frequency-density histograms for marker enzymes. Frequency (mean+ SD) is defined as the fraction of total recovered activity present in the subcellular fraction divided by the density span covered. The cross-hatched area represents, over an arbitrary abscissa interval, the enzyme remaining in the sample layer. The percentages (+ SD) of recovered activity, with numbers of experiments in parentheses, are: alkaline phosphatase, 81 + 18 (15); glutamate dehydrogenase, 84+ 14 ( 5 ) ; 5’-nucleotidase, 98+ 10 ( 5 ) ; N-acetyl-Sglucosaminidase, 84+ 17 (14); catalase, 101k I 1 (7); lactate dehydrogenase, 101+21 (4); a-glucosidase @H 8.0), 109223 (16); protein, 8 8 k IS (17).

supernatant only to an extent of 35%. Similar values are found for y-glutamyl transpeptidase, Zn2 + - resistant a-glucosidase and p-chloromercuribenzoate- resistant 8-galactosidase. Leucyl-8-naphthylamidase has a higher released activity than the other brush- border enzymes. Other enzymes are released into the extract to between 50 and 75 %. Enzymes having a major cytosol component, e.g. catalase, malate dehydrogenase and lactate dehydrogenase, are released

to a greater extent than the predominantly organelle- bound enzymes, e.g. acid hydrolases. Allowing for the fact that certain enzymes, e.g. lactate dehydrc- genase (de Duve & Berthet, 1954) and N-acetyl-8- glucosaminidase (Baccino, Rita & Zuretti, 1971), tend to become adsorbed to other organelles, approximately three-quarters of the cells in the biopsies have been disrupted. As mentioned previously (Peters et al., 1975) the gentle homogeniza-


tion procedures employed tend to leave the nonepithelial cells of the biopsy intact, and these are therefore sedimented in the low-speed pellet, and so not fractionated on the sucrose gradients.

Density gradient experiments

Organelle distributions: averaged data. The distribution of the principal marker enzymes in the sucrose density gradients include the averaged data from several experiments (Fig. 1 and Fig. 2). Alkaline phosphatase shows a peak with a modal density of 1.21, with skewing of activity to lower densities (Fig. 1). 5'-Nucleotidase shows a peak with a modal

15r a- Glucosidase

0 1 I Leucyl-p-naphthybmidase r

h 1 y-Glutamyl tmnspeptidase

L M a y e dehydmqenase I0

5

0 1.05 1-10 145 1.20 125

density of 1.12 and a shoulder at density of 1.21, the latter corresponding to that of the alkaline phosphatase. The distribution of 5'-nucleotidasein the sucrose gradients was also determined with selective inhibitors of alkaline phosphatase and of 5'-nucleotidase, with no significant difference in the enzyme distribution. In particular, the relative amounts of activity at modal densities 1-11 and 1.21 were identical although the absolute enzyme activities were, of course, reduced by the inhibitors.

Catalase shows a major soluble component with a particulate component at a modal density of 1.18. D-Amino acid oxidase assayed in two experiments was found to be associated with particulate catalase.

Acid p-qalactasidase

T

Acid phosphatase

Acid diesterase

Densily

FIG. 2. Isopycnic centrifugation of 8000 g-min supernatant from jejunal biopsy homogenate. Details are as given for Fig. 1. The percentages ( ~ s D ) of recovered activity. with numbers of experiments in parentheses, are: a- glucosidase (PH 6.0), 87+ 16 (7); acid j'-galactosidase, 90+ 13 (4); leucyl-&naphthylamidase, 119+ 16 (7); acid phosphatase, El+ 19 (7); y-glutamyl transpeptidase. 98+ 15 (10); acid diesterase, 81+ 12 (5); malate dehydrogenase, 101k 19 (15); monoamine oxidase, 87k 1 1 (7).

D

564 T. J .

or-Glucosidase assayed at pH 8.0 shows a peak with a modal density of 1.16. Glutamate dehydrogenase has a small soluble component with a sharp peak in the density region of 1.16. A similar distribution (not shown) was found for succinic dehydrogenase. Monoamine oxidase is almost completely particulate with a modal density also at 1.16. N-Acetyl-8- glucosaminidase shows a soluble component with a symmetrical peak at a modal density of 1.21 but with an estimated median density slightly less than that of alkaline phosphatase. Lactate dehydrogenase is mainly localized in the soluble fractions but significant activity is found in the gradient, with

Peters

1 Leucyi-B- nophthylomdose

p y - Glutornyl tronsferose

a shoulder at density 1.16, corresponding to that of or-glucosidase (pH 8.0). Protein is also mainly recovered in the soluble components with a broad peak in the region 1.15-1.18 corresponding principally to the distributions of glutamate dehydrogenase, malate dehydrogenase and monoamine oxidase.

a-Glucosidase assayed at pH 6.0 has a small soluble component with the particle-bound activity showing a bimodal peak at densities of 1.21 and 1.16, corresponding to alkaline phosphatase and neutral a-glucosidase (pH 8.0) respectively (Fig. 2). Leucyl- /3-naphthylamidase and y-glutamyl transpeptidase show similar distributions with a bimodal distribu-

a-Glucosidase IpH 6.0)

a-Glucosidase (pH 8.01

I

Alkaline phosphatase

5'-Nucleotidose

Density

FIG. 3. Isopycnic centrifugation of 8OOO g-min supernatant from homogenate of jejunal biopsy. Homogenate was prepared and gradient formed from solutions of purified sorbitol. Details are as given for Fig. I . The percentages of recovered activity are: maltase, 68; a-glucosidase (PH 6.0). 78; leucyl-/?-naphthylamidase, 83 ; a-glucosidasc (pH 8.0). 73 ; y-glutamyl transferase, 73 ; alkaline phosphatase, 87 ; malate dehydrogenase, 103 ; 5'-nuclcotidase, 79.


tion at densities 1-21 and 1.12 corresponding to alkaline phosphatase and 5’-nucleotidase respectively. Malate dehydrogenase has a major soluble component with particulate activity at a density of 1.16. Acid Bgalactosidase has a broad peak of activity at a density of 1-21 and is similar to that of N-acetyl-Bglucosaminidase. Acid phosphatase and acid diesterase also have broad particulate components but with a significantly lower modal density of 1.18.

Fractionation on sorbitol density gradknts. Fig. 3 shows the distribution of the various marker enzymes after collection and disruption of the biopsy in sorbitol and density gradient centrifugation of the tissue extract on a sorbitol gradient. The organelles are separated in a similar manner to that achieved on sucrose gradients although they have higher equilibrium densities. Thus 1eucyLBnaphthyIamidase has a modal density of 1.24, compared with a value of 1.21 in sucrose. Maltase and sucrase (not shown) have distinct peaks also with modal densities of 1.24.

Clear separation of the a-glucosidases when assayed at pH 6.0 and pH 8-0 is achieved.

Effect of inhibitors on hyakolase distributions. Fig. 4 shows the distribution of certain acid and neutral hydrolases in the sucrose density gradients. BGalactosidase assayed at pH 3.5 shows a symmetrical peak at modal density of 1.21 with a significant soluble component. Total Bgalactosidase assayed at pH 6.0 (not shown) has a complex distribution with soluble component and broad peak in the region 1.18-1-22. When activity is assayed in the presence of pchloromercuribenzoate (0.18 mmolfl) (resistant enzyme activity) a sharply de5ed peak at a modal density of 1.22 is obtained. The pchloromercuribenzoate-sensitive Bgalactosidase activity, obtained by plotting the difference in activity of the enzyme when assayed with and without the inhibitor, shows a similar distribution to that of the acidic Bgalactosidase, although there is a larger soluble component.

/?-Glucosidase (Fig. 4) shows a large soluble

Density

FIO. 4. hpycnic centrifugation of 8OOO g-min supernatant from jejunal biopsy homopenate. Details ae as given for Fig. 1. The parcentages of m v m d activity are: &galactosida8e (pH 3.5),79; figlucosidase, 92; figdactoei- d w (PH 60, p c h l o r o m e r m r i b c o z o a ~ M B ~ ~ ~ i t i v e J , 71; a-glucaidasc (pH 60). 73; ~gahtosidasc [PH 6.0, pchioromercuribanzoate(pCMB)-resiltant], 89; a-glucosldasc (pH 8.0). 103.

566 T. J. Peters

component with activity throughout the gradient and a distinct component at density 1.22. The distribution of Bglucosidase assayed at pH 6.0 shows a bimodal distribution with peaks of activity at 1.22 and 1.16. BGlucosidase, assayed at pH 8.0, shows a distinct peak at density 1.16 corresponding to the low-density peak found when the enzyme is assayed at pH 6.0. In addition there is a shoulder of activity at density 1.22.

The distribution of a-glucosidase assayed with and without inhibitors shows that when assayed at pH 6.0 there is a distinct peak at density 1.22 with a shoulder at 1.15 (Fig. 5) , corresponding to the peak when a-glucosidase is assayed at pH 8 0 . Discrimina- tion between the high- and low-density a-glucosidase activities can be clearly achieved by assaying the activity at pH 6.0 with and without Zn2 + (2-8 m o l l

1). The activity in the soluble fractions and in the density region 1.141.18 is strongly inhibited by this ion. Activity deeper in the gradient is relatively unaffected. A partial discrimination between the high- and low-density a-glucosidase activities can be achieved by assaying the activity in the presence and absence of mturanose (32 mmol/l). The activity at density 1.22 is more sensitive to this inhibitor than the activity at density 1.15.

Fractionation of brush-border components. The distribution of certain hydrolases in the sucrose gradients when Trisdisrupted brush borders are fractionated on a density gradient (Fig. 6) shows that, with the exception of Bglucuronidase, for all there is a peak of activity at density 1.23, which corresponds to a peak in the proteindistribution. BGlucuronidase shows a broad peak in the density region 1.13-1-17,

15r a-Glucosidose ( p ~ 6 0 )

y 5 :t 0

a-Glucosidase (pH 601

15t ;-$;?dose

Turanosa-resistant

1.05 k 1 0 1-15 1-20 1.25 I

a-Glucosidose (DH 801

a-Glucosidase (DH 6.0)

Density

FIG. 5. Isopycnic centrifugation of 8OOO g m i n supernatant from jejunal biopsy homogenate. Details arc as given for Fig. 1. The percentages of recovered activity are: a-glucosidase @H 6.0). 71 ; a-glucosidasc (pH 6.0, ZnZ+- sensitive), 107; a-glucosidase (PH 6.0, Zn*+-resistant), 65; a-glucosidase @H 8-0), 106; a-glucosidase @H 6.0, turanose-resistant), 66; a-glucosidase (pH 6.0, turanose-sensitive). 66.

Subcellular fractwnution of human jejunum 567

I Protein I 8-Glucuronidase

Density

FIG. 6. Isopycnic centrifugation of 8000 g-min supernatant from homogenate of isolated jejunal brush borders. Details are as given for Fig. 1. The percentages of recovered activity are: leucyl-8-naphthylamidase, 106; y- glutamyl transpeptidase, 81 ; a-glucosidase (PH 6.0), 96; S’-nucleotidase, 88; 8-glucosidase. 74; alkaline phosphatase. 64; protein, 66; ~-glucuronidase, 66.

some soluble activity, but no signiscant activity in the density 1.23 region. 5’-Nucleotidase shows a similar distribution to that of alkaline phosphatase, although there is a small peak of activity in the region of density 1-12. Protein is distributed throughout the gradient with major components in the soluble and low-density region of the gradient.

Distribution of acid hydrohes. Fig. 7 shows the distribution of six acid hydrolases assayed in the gradient fractions from one experiment. The distributions of the individual enzymes differ sign& cantly from each other, soluble activity varying from Bglucuronidase, showing the least, to acid diesterase, showing the most. The density distributions of the particulate components vary with /?-glucuronidase

having a modal density of 1.16 and acid /?-galactosidase a density of 1.21. More detailed analysis indi- cates the existence of at least three distribution patterns with the following modal densities: 1.20-1.21 (N-acetyl-Bglucosaminidase, N-acetyl-/%galactosaminidase, Bgalactosidase); 1.17-1.18 (cathepsin C, acid diesterase); 1.16-1-20 (acid phosphatase, B glucuronidase).

Eflects of digitonin on enzyme distributions. The densitydistribution profiles of the various markex enzymes after homogenization of the biopsy in SVE d m containing digitonin (0.12 mmol/l) shows striking differences when compared with the un- treated tissue (Fig. 8). Zinc-sensitive a-glucosidase assayed at pH 6 9 shows a modal density of 1.12,

568 T. J. Peters

, L N - Ace1 y I -p - G l u c m n i d o s e

I0 -

C

Acid phospholose

I0 c

Cuthepsin C

Acid diesterase

11.05 1.10 1.15 1.20 1.25 1.30 Oensily

FIG. 7. Isopycnic centrifugation of 8000 g-min supernatant from jejunal biopsy homogenate. Details are as given for Fig. 1. The percentages of recovered activity are: N-acctyl-8-glucosaminidase, 72; 8-glucuronidase, 94; acid 8-galactosidase, 84; cathepsin C, 65; acid phosphatase, 98; acid diesterase, 109.

significantly less than the density of 1.16 found in the control experiments. Zinc-resistant a-glucosidase @H 6.0), alkaline phosphatase, leucyl-pnaphthyla- midase (not shown) and y-glutamyl transpeptidase (not shown) all show a smaller but consistently increased modal density of 1.22-1.23 compared with 1.21-1.22 in the control experiments.

The distribution of N-acetyl-pglucosaminidase is strikingly different, with complete loss of the particulate component with nearly all the enzyme activity being recovered in the soluble fraction. In agreement with this, measurements of latent N-acetyl-8 glucosaminidase (Peters et al., 1975) consistently give values of less than 3%, compared with the value of approximately 60% in control tissue (Peters et af., 1975). Other acid hydrolases showing similar distributions to that of N-acetyl-Bglucosaminidase after digitonin treatment include: Bgalactosidase, acid diesterase, N-acetyl-@-galactosaminidase and cathepsin C. S’-Nucleotidase shows a significant increase in modal density compared with the control experiments, with a shift from a density of 1.12 to 1.16. Acid phosphatase shows a greater amount of

activity remaining in the sample layer but much particulate activity remains. 8-Glucuronidase (not shown) behaves similarly to acid phosphatase except that there is a disproportionately greater loss of particulate activity in the higher density region. The distribution of catalase and malate dehydrogenase in the sucrose gradients is similar in both the control and digitonin-treated experiments, apart from a slightly higher soluble catalase activity.

Subcellular fractionation experiments were also performed, in which digitonin (0.06 mmol/l) was included in the homogenization medium. Only partial solubilization of the N-acetyl-/?-glucosaminidase (latent activity approximately 20%) was achieved and the density shifts of the membrane bound enzymes were less than those achieved with digitonin (0.12 mmol/l).

Possible variables in the fractionation procedures were also studied. The tissue-homogenization procedure has been optimized to provide highest lysosomal integrity, as assessed by latent N-acetyl-8 glucosaminidase (Peters et al., 1975). Tissue extracts prepared by more vigorous homogenization than the

Subcellular fiactwnatwn of human jejunum 569

I Zn*+-sensitive 5- Nucleotidase a-glucosidase

Density

FIG. 8. Isopycnic centrifugation of 8000 g-mh supernatant from jejunal biopsy homogenized in isotonic sucrose (SVE medium) containing digitonin (016 mmol/l). Details are as given for Fig. 1. The percentages of recovered activity are: a-glucosidase @H 60. Zn'+-scnsitive), 106; S'-nucleotidase, 97; a-glucosidase (PH 6.0. a'+- resistant), 82; acid phosphatase, 102; alkaline phosphatasc, 103; catalast. 93; N-acetyl-8-glucosaminidase, 93 ; malate dehydrogenase. 8 1.

standard procedure were also subjected to centrifugation on sucrose density gradients. Essentially similar enzyme distributions to those from the standard procedure were found except that more acid hydrolase and catalase activities were found in the soluble fractions. However, the more gentle procedure was used to preserve organelle integrity, particularly in diseased tissues. A gentle homogenization procedure also minimized the contribution of the nonepithelial cell organelles to the tissue extract. Phasecontrast microscopy of the low-speed pellet (N fraction) revealed many intact interstitial cells.

Tissue extracts, prepared by the standard procedure were also subjected to centrifugation for longer periods. After centrifugation for five times the standard time the organelles had similar modal densities to those after 35 min centrifugation. How- ever, the particulate enzyme activities tended to have more distinct and symmetrical peaks. Thus alkaline phosphatase had a sharp peak at density 1.21-1-22 with significantly less activity in the low-density region of the gradient. However, the standard fractionation conditions were used to keep centrifugation as short as possible, consistent with adequate

570 T. J . Peters

resolution of the organelles. More recently a titanium version of the Beaufay rotor has achieved complete resolution of the organelles from jejunal biopsies by centrifugation at 50000 rev./min for 15 min. Pro- longed centrifugation leads to organelle damage and disruption, particularly with pathological tissue, owing to the hypertonic sucrose solutions of the gradient. Sedimentation of soluble protein into the body of the gradient may also obscure the low- density organelles.

Discussion These studies form the first descriptions of analytical subcellular fractionation of jejunal biopsy specimens in which all the principal organelles have been characterized. In establishing the fractionation procedure techniques had to be devised for separating the organelles from milligram quantities of tissue and for assaying the very small amounts of enzyme activity associated with these organelles. With suitable modification the procedures developed for study of lysosomes of normal and diseased smooth muscle cells were used (Peters et al., 1972; Peters & de Duve, 1974). In essence, the small-volume automatic zonal ultracentrifuge rotor of Beaufay (1966) was used for separating the organelles and highly sensitive enzyme assay techniques employing fluori- genic or radiolabelled substrates were used to locate the organelles in the sucrose gradients by means of their marker enzymes.

It is stressed that these studies employ an analytical rather than preparative approach. Crane and his colleagues have recently applied a preparative subcellular fractionation procedure to frozen specimens of human jejunum mainly obtained by open surgical biopsy (Welsh, Preiser, Woodley & Crane, 1972; Schmitz, P rek r , Maestracci, Ghosh, Cerda & Crane, 1973). They obtained an enriched fraction of brush- border membranes by a complex multi-step subcellular fractionation procedure. The aim of a preparative procedure is to obtain a sample of the particular organelle in a high state of purity but often with little concern for yield. In humans the preparative approach has given valuable information on the properties of the brush border (Maestracci, Schmitz, Preiser & Crane, 1973; Preiser, Mennard, Crane & Cerda, 1974; Schmitz, Preiser, Maestracci, Crane, Troesch & Hadorn, 1974) but not on the other organelles of the enterocyte. In addition the preparative approach to subcellular fractionation of human jejunum is not readily applicable to pathological tissue for the centrifugal properties of diseased organelles may differ significantly from similar organelles in normal tissue. In the present study a simple single-step procedure is used to resolve the principal organelles from the jejunal biopsy. The activities and certain properties of all the organelles can be determined in a single biopsy and the technique is readily applicable to diseased tissue.

The centrifugation properties of the individual organelles and their enzyme contents as deduced

TABLE 3 . Principal organelles, equilibrium density in sucrose and their associated enzyme acrivities

Organelle Equilibrium density

Associated enzyme activities

Basal-lateral membranes 1.12

Endoplasmic reticulum 1.16

Mitochondria 1.16

Peroxisome Lysosomes

1.18 1.16-1.18

1.18 1.20

Brush border 1.21

5’-Nucleotidase, leucyl-B-naphthylamidase (part), y-glutamyl transpeptidase (part), alkaline phosphatase (part) Zn2+ -sensitive a-glucosidase, alkaline a-glucosidase, acid phosphatase (part), j7-glucuronidase (part), B-glucosidase (?) (part) Succinic dehydrogenase, glutamate dehydrogenase, monoamine oxidase, malate dehydrogenase (part) Catalase, D-amino acid oxidase Acid phosphatase (part), B-glucuronidase (part) Cathepsin C, acid diesterase N-Acetyl-8-glucosaminidase, acid 8-galactosidase, acid phosphatase (part), 8-glucuronidase (part) Maltase, alkaline phosphatase, leucyl-8-naphthylamidase, y-glutamyl transpeptidase, Zn’+-resistant a-glucosidase, p-chloromercuribenzoate- resistant 8-galactosidase, 8-glucosidase (part)


from the fractionation techniques are summarized in Table 3.

Plasma membrane (basal-lateral membranes) The distribution of 5’-nucleotidase suggests that

it is associated with the plasma membrane. Similar conclusions have been reached with isolated enterocytes from both rat (Douglas, Kerley & Isselbacher, 1972; Peters & Shio, 1976) and guinea-pig (Lewis, Elkin, Michell & Coleman, 1975a). This enzyme is also localized predominantly to the plasma membrane in a wide variety of cell types (see Solyom & Trams, 1972). There is a second component of 5’- nucleotidase, deeper in the gradient, which is associated with the brush-border elements. Non- specific brush-border alkaline phosphatase may be hydrolysing the 5’-nucleotidase substrate, but this cannot account for all the brush-border 5’-nucleotidase activity. The assay used, based on that of Douglas et al. (1972), has a high selectivity for this enzyme. In addition, if gradient fractions are assayed with alkaline phosphatase or 5’-nucleotidase inhibitors, the relative distribution of 5’-nucleotidase in the gradient is unchanged, indicating that the brush- border activity is not due to non-specific alkaline phosphatase. It thus appears that 5‘-nucleotidase is an intrinsic brush-border enzyme. Although the brush border of the enterocyte is a highly specialized plasma membrane, it is reasonable to suggest that despite its specific functional adaptations it has pre- served, to some extent, attributes of the normal plasma membrane.

The increase in density of the plasma membrane after digitonin treatment is similar to that found in rat liver (Thinks-Sempoux, Amar-Costesec Beaufay, & Berthet 1969; Tilkray & Peters, 1976) and in rat embryo fibroblasts (Tulkens, Beaufay 8c Trouet, 1974), and reikcts its high concentration of digitonin- complexable cholesterol (Amar-Costesec, Wibo, Thinks-Sempoux, Beaufay & Berthet, 1974).

As judged by their distribution profiles a small proportion of leucyl-/?-naphthylaxnidase and y- glutamyl transpeptidase activities is also associated with the plasma membranes. A portion of the aklaline phosphatase may be similarly located, as confirmed by finding digitonin txeatment of the homogenate to caw a redistribution of the plasma membrane component to higher densities (Fig. 8).

Endophmic reticulum This is the principal component of the ‘microsomal

fraction’ of differential centrifugation experiments. Recent studies have cordinned the heterogeneous nature of this fraction, which contains elements derived from the plasma membrane, Golgi complex, outer mitochondria1 membranes as well as endoplasmic reticulum granulated with ribosomes to various degrees (Amar-Costesec et al., 1974; Tilleray & Peters, 1976). Owing to the relatively small amounts of endoplasmic reticulum in the enterocyte it has not been possible to assay a range of enzymes localized to different subcomponents of the microsomal fraction in the human jejunal biopsies. a- Glucosidase, assayed at alkaline pH in order to dis- criminate against the very high activity of the brush- border a-glucosidase, has been used as a marker for this organelle. The endoplasmic and cytoplasmic a-glucosidase activities are strongly inhibited by Zn2+ and thus by plotting the Zn2+-inhibitable a-glucosidase activity the distribution profile of the endoplasmic reticulum in the density gradients can be accurately determined. The use of neutral a-glucosidase as a marker for the endoplasmic reticulum has been clearly demonstrated for rat liver (Lejeune et al., 1963; Tilleray & Peters, 1976), smooth muscle cells (Peters et al., 1972; Peters & de Duve, 1974), skeletal muscle (Angelini & Engel, 1973), cardiac muscle (Bloomfield & Peters, 1974) and human liver (Brown & Brown, 1965; Gamklou & Schersten, 1972; Seymour, Neale & Peters, 1974). The distribution of alkaline a-glucosidase in the

sucrose gradients is affected by digitonin treatment of the biopsy homogenate. Unlike the plasma membrane and brush border, which show increased densitits, the endoplasmic reticulum (reflected by the distribution of a-glucosidase), shows a significant reduction in density. The mechanism of this response is uncertain but is presumably related to the deter- gent action of the digitonin and also occurs with rat liver microsomes (Tilleray & Peters, 1976). There is also evidence for a partial localization of acid phosphatase, Bglucuronidase and perhaps Bglucosidase to endoplasmic reticulum of the human intestine, these enzymes having significant particulate activities in the same density region as a-glucosidase, at 1.16. BGlucuronidase and Bglucosidase show similar decreases to a-glucosidase after digitonin treatment. Acid phosphatase does not show this effect but similar differential responses of endoplasmic reticulum enzymes, apparently in the same microsomal subfraction, OCCUT in rat liver microsomes (Tilleray & Peters, 1976). Studies in rat liver also show signscant

572 T. J. Peters

amounts of microsomal acid phosphatase (Neil & Horner, 1964; Beaufay, 1972) and Bglucuronidase (de Duve, Pressman, Gianetto, Wattiaux 8c Appel- mans, 1955; Paigen, 1961; Fishman, Goldman & Delellis, 1967) in this subcellular fraction in addition to the lysosomal components.

Mitochondria

These have a modal density of 1.1 6 but, compared with theendoplasmicreticulum, the density spread is considerably less. Enzymes for certain of the mitochondrial subcomponents have been assayed. Thus succinic dehydrogenase (inner membrane), malate and glutamate dehydrogenase (matrix) and monoamine oxidase (outer membrane) marker enzymes (Ernster & Kuylenstierna, 1969) all had identical distributions with a sharply deiined modal density of 1-16. This is significantly lower than the modal density of 1.19 reported for rat (Beaufay et al., 1964; Leighton et al., 1968; Peters & Shio, 1976) and human liver (Seymour et al., 1974). However, a similar figure of 1.16-1.17 was found for the equilibrium density of rat enterocytes when fractionated under similar conditions (Peters & Shio, 1976). The outer and inner membrane marker enzymes have similar distributions, indicating that there is no significant disruption of the mitochondria during the fractionation. Any dissociated outer mitochondria1 membrane would be expected to have a significantly lower equilibrium density (Parsons, Williams & Chance, 1966; Peters-Joris, Vandevoorde & Baudhuin, 1975), and if the inner membrane is damaged and rendered permeable to sucrose the equilibrium density would be approximately 1.22 (Wattiaux, 1974).

Peroxisomes

The demonstration of latent and sedimentable catalase in the tissue extracts and the co-sedimentation of catalase and D-amino acid oxidase on the density gradients satisfies the biochemical criteria for peroxisomes (Baudhuin, Beaufay, Rahman-Li, Sel- linger, Wattiaux, Jacques & de Duve, 1964) and thus this is the first report of their occurrence in human jejunum, although they have been conclusively demonstrated in rat (Peters & Shio, 1976) and guinea- pig (Connock, Kirk & Sturdee, 1974) enterocytes. Their significantly lower density than rat liver peroxisomes (Baudhuin ef al., 1964; Leighton et al.,

1968; Peters & Shio, 1976) is consistent with the absence of uricase from homogenates of human jejunum (T. J. Peters, unpublished result). In rat liver the cores of the peroxisomes contain most of the uricase and contribute to their high density (de Duve & Baudhuin, 1966; Leighton et al., 1968).

Lysosoms Lysosomes have been shown previously to occur

in human jejunum (Peters et al., 1975) according to the accepted biochemical criteria (de Duve & Wat- tiaux, 1966). Evidence is now presented for the heterogeneity of these lysosomes. The principal lysosomal component has an equilibrium density of 1-20-1.21 and is completely disrupted by digitonin treatment. A second population, identi6ed by its unique content of cathepsin C and acid diesterase, has a lower equilibrium and is solubilized by digitonin treatment. BGlucuronidase and acid phosphatase, although partially localized to the high-density population of Iysosomes, probably contribute mainly to a third low-density population of lysosomes which appear to be relatively resistant to disruption by digitonin. On the present evidence it is difficult to distinguish definitively this third population of lysosomes from elements of the endoplasmic reticulum containing these enzymes. The functional significance of these distinct populations of lysosomes remains to be determined.

Brush borders Brush borders have the highest equilibrium den-

sity (1.21) of the organelles studied, being characterized by disaccharidases and other hydrolases (alkaline phosphatase, p-chloromercuribenzoate- resistant bgalactosidase, leucyl-Bnaphthylamidase, y-glutamyl transpeptidase and Zn’ +-resistant a- glucosidase), many of which have been localized to this organelle both histochemically and by analysis of highly purified brush-border membranes isolated by preparative fractionation procedures (see Schmitz et al., 1973). This study confirms these findings, but demonstrates that certain of the enzymes have significant extra-brush-border components in other organelles. The brush borders increase in equilibrium density after digitonin treatment, although the increase is less than that for the basal-lateral membrane. This presumably reflects the different lipid composition of the brush-border membranes (Doug-

Subcellular fructwtuatwn of human jejunum 573

las et al., 1972; Kwai, Fujita & Nakao, 1974; Lewis, Gray, Coleman & Michell, 1975b). Similar differences in response to digitonin have been noted for basal-lateral and brush-border membranes from guinea-pig enterocytes (Lewis et al., 1975a).

The subcellular fractionation and separation of the organelles in human jejunal biopsies proved easier than in isolated rat enterocyte8, where all the organelles, apart from the basal-lateral membrane, had very similar equilibrium densities and complex gradients were neccsp~ly to achieve separations (Peters & Shio, 1976). Having established the distribution of the various organelles in the gradients, the localization of hitherto unassigned enzymes and other components can be determined. The technique will be used to investigate the subcellular pathology of small-intestinal diseases including the coeliac syndrome (Peters, Heath, Jones & Peacham, 1975). Although this new technique has been applied in the 6rst instance to human jejunum it is applicable to the study of the physiology or pathology of any organ amenable to biopsy, and thus has potentially a very wide application in the study of cell pathology.

Acknowlerllpnents

I am particularly grateful to Ms Janet Heath, Mr P. Hope and Mr P. White for their expert technical assistance, to Ms Margaret Chandler for performing the computations and Ms Jean de Luca for typing the manuscript. The constructive criticism of colleagues, particularly Mr R. Batt, and the enthusiastic support of Professor C. C. Booth, are gratefully acknowledged. This work is supported by the Medical Research Council and The Wellcome Trust.

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