European Journal of Clinical Investigation (1979) 9,349-354

The subcellular localization of peptidase activity in the human jejunum J. A. NICHOLSON & T. J. PETERS, Department of Medicine, Royal Postgraduate Medical School, London Received 5 February 1979 and in revised form 28-May 1979

Abstract. The subcellular localization of peptidase activity in the normal human jejunum has been investigated. Subcellular organelles were fractionated by density gradient centrifugation. The localization of peptidases was determined by comparing the distributions of peptidase activities with those of organelle ‘marker’enzymes. The organellesand their markers were: cytosol-lactate dehydrogenase; brush border-neutral a-glucosidase, y-glutamyl transferase and leucyl-2 naphthylamidase; plasma membrane-5’-nucleotidase; lysosomes-N-acetyl-0-glucosaminidase; mitochrondria-malate dehydrogenase; endoplasmic reticulum -alkaline a-glucosidase; peroxisomes-catalase. Thirteen dipeptides, seven tripeptides, two tetrapeptides, two pentapeptides and a hexapeptide were used as substrates. The distribution of dipeptidyl peptidase TV was also determined. Irrespective of whether the NH2-terminal or CQOH-terminal amino acid was neutral, basic or acidic, the major or exclusive locus of dipeptidase activity was cytosolic. All of the activity against a dipeptide with the amino acid proline at the NH2-terminus was in the cytosol. The distribution of tripeptidase activity was quite different. Although the cytosol hydrolysed all tripeptides, as much as 50% of tripeptidase activity was particulate. For both tetrapeptides, one of the pentapeptides and the hexapeptide, the major or exclusive locus of activity was the brush border membrane. Pentaphenylalanine, however, was hydrolysed by both the cytosol and the brush border. Dipeptidyl peptidase IV was localized in the brush border.

These enzymes are believed to play an important role in the digestion and absorption of peptides derived from the gastric and pancreatic digestion of protein [ 1, 21. Studies of the subcellular distribution of peptidases which included analysis of various organelle ‘marker’ enzymes have been performed on the small intestine of the guinea-pig [3] and the rat [4, 51 and the main intracellular locations identified. The study of the subcellular distribution of enzymes in the human intestine, however, has posed special problems. The results from investigations of postmortem material are ambiguous and it is rare to obtain sufficient non-autolysed tissue to permit rigorous analysis of subcellular fractions isolated by conventional fractionation methods. Recently, however, a microanalytical technique has been developed [6] which allows the subcellular distribution of enzymes to be determined using only milligram quantities of human tissue obtained from living subjects. In addition it has also been necessary to develop sufficiently sensitive assays using fluorescence procedures to analyse the density gradient fractions when these very small amounts of tissue are processed [7, 81. This paper presents the results of an investigation of the subcellular distribution of peptidase activities in the normal human jejunum using a variety of peptide substrates composed of from two to six amino acid residues. A preliminary report of part of this work has been published [9].

Key words. Peptide hydrolase, peptidase, jejunum, subcellular localization, peptides.

Chemicals. Chromatographically-pure amino acids and peptides used in the assays of peptidase activity were obtained from Sigma (London) Chemical Co., Kingston-upon-Thames, Surrey, U.K. Stock solutions of amino acids were stored at - 20°C for up to 1 month but peptide solutions were prepared on the same day as the peptidase assays were performed. Glycyl-L-proline-2-naphthylamide, used as substrate for dipeptidyl peptidase IV [lo], was obtained from Bachem Feinchemikalein AG, Leistal, Switzerland. Reagents for the assays of peptidase activity; L-amino acid oxidase, horse radish peroxidase, chloramine-T and acetyl acetone were all purchased from Sigma (London).

Introduction

The mucosa of the mammalian small intestine contains several enzymes with peptidase activity (EC 3.4.--). Correspondence: Dr J. A. Nicholson, Division of Clinical Cell Biology, Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ. 0014-2972/79/1000-0349$02.00 01979BiackwellsScientificPublications

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Humanjejunal tissue. Jejunal biopsies were obtained with a Crosby capsule from adult subjects undergoing investigations for possible gastrointestinal disease. The capsule was positioned under fluoroscopic control and all biopsies were taken from a site just distal to the ligament of Treitz. Approximately one quarter of the biopsy was removed for histological examination and the remainder (about 15 mg wet weight) was placed in ice-cold homogenizing medium. None of the patients for whom data are presented were subsequently found to have organic intestinal disease as judged by histological, functional or radiological criteria. The studies were approved by the local ethical committee. Subcellularfractionation. The conditions for analytical fractionation of human jejunal biopsies by sucrose density centrifugation employing a Beaufay automatic zonal rotor are described in detail in Ref. 6. The protocol followed in the present study differed only in so far as ethylenediamine tetra-acetic acid (EDTA) and ethanol were omitted from the homogenizing medium and from the density gradient. EDTA was found to inhibit hydrolysis of several peptides. Peroxisomal catalase [6] could be identified in the absence of ethanol. Briefly, each biopsy was homogenized in 3 ml of ice-cold sucrose solution (0.3 mol/l) containing 3 mmol/l imidazole-HC1, pH 7.2. Homogenization was performed in a small Dounce homogenizer with ten strokes of a loose-fitting (type A) pestle. The homogenate was centrifuged at 800 g for 10 min and the supenatant decanted and saved. The pellet was resuspended in a further 2 ml of medium with three strokes of the pestle and the suspension was again centrifuged. The resulting supernatant was combined with that from the first centrifugation to form a ‘post-nuclear fraction’ (PNS). The low-speed pellet was supended in 2 ml of medium with ten strokes of a tight-fitting (type B) pestle. This pellet, consisting of cell nuclei, large membrane fragments and unruptured cells, is termed the ‘nuclear fraction’ (N fraction). In some experiments homogenization was performed in the standard way but with either 0.12 mmol/l digitonin or 0.8 mol/l Tris base in the homogenizing medium. Density gradient centrifugation of the PNS fraction was precisely as described previously [6] with the exception that the sucrose gradient solutions contained 3 mmol/l imidazole-HC1, pH 7.2, and the EDTA and ethanol were omitted. After centrifugation the fractions were collected into tared tubes which were reweighed and each fraction was mixed and its density determined by refractometry. Assay of marker enzymes. The locations of subcellular organelles in the density gradients were determined by assaying marker enzymes. Details of each assay are given in Ref. 6. The marker enzymes used in this study were: brush border-a-glucosidase (EC 3.2.1.20) measured at pH 6.0 in the presence of 2.8 nimol/l Zn2+,y-glutamyl transferase (EC 2.3.2.2) and leucyl-2-

naphthylamidase (EC 3.4.1 1.1);endoplasmicreticulum -a-glucosidase measured at pH 6.0 in the presence of 25 mmol/l Tris; lateral and basal plasma membrane5’-nucleotidase (EC 3.1.3.5); mitochondria-malate dehydrogenase (EC 1.1.1.37); 1ysosomes-N-acetyl-Pglucosaminidase (EC 3.2.1.30); peroxisomes-catalase (EC 1.1I . 1.6); cytosol-lactate dehydrogenase (EC 1.1.1.27). Peptide assays. Hydrolysis of peptides was studied at pH 8.0 and at a substrate concentration of 5 mmol/l except in the case of phenylalanine homopeptides. These peptides, because of their limited solubilities, were studied at pH 8.5 and at substrate concentrations of 1 mmol/l in the case of (Phe)2.4and 0.5 mmol/l in the case of (Phe)s. All of the assays were performed at 37°C. In all assays free amino acids released by hydrolysis represented less than 2% of initial substrate concentration. The amount of amino acid released was directly proportional to the assay time and to the concentration of enzyme. A fluorimetric L-amino acid oxidase assay 171 was used to assay peptidase activity against dipeptides containing leucine, methionine or phenylalanine and tri-, tetra- and penta-peptides containing these amino acids at the NH2-terminus. When glycine homopeptides, glycyl-L-alanine or glycyl-glycyl-L-alaninewere used as substrates, a novel method for peptidase assay was developed [8]. Dipeptidyl peptidase IV was assayed with glycyl-~-prolyl-2-naphthylamide as substrate as described by Kenny et al. [lo] except that a final substrate concentration of 0-15 mmol/l in a volume of 0.35 ml was used. The liberated P-naphthylamide was assayed fluorimetically [I I]. For all enzymes linear kinetics with respect to time and amount of enzyme were established.

Results Recovery of enzymes after homogenization. The percentages of marker enzymes and peptide hydrolases recovered in the post-nuclear supernatant following tissue homogenization and low-speed centrifugation was between 65% and 15%, indicating that between two-thirds and three-quarters of the organelles in the tissue biopsy are released into the PNS fraction. Density gradient experiments. The distributions of enzyme activities in the density gradients following isopycnic centrifugation are represented in the frequencydensity histograms. Fig. 1 shows the distribution of the various marker enzymes in the density gradients. The results of several separate experiments have been pooled and averaged as described by Leighton et al. [12]. Inevitably, some loss of resolution has occurred; however, even on individual runs certain organelles are not completely separated. Thus the distribution and modal density of the brush border markers, leucyl-2-naphthylamidase, y-glutamyl transferase and Zn2+-resistant a-

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Figure 1. Isopycnic centrifugation of 8000 g-min supernatant from jejunal biopsy homogenates. Graphs show frequency-density histograms for marker enzymes. Frequency (mean k SD) is defined as the fraction of total recovered activity present in each gradient fraction divided by the density span covered. The percentages ( & SD) of recovered activity with numbers of experiments in parentheses are: malate dehydrogenase, 108 f 17 (10); N-acetyl-P-glucosaminidase, 87 f 19 (8); leucyl-2-naphthylamidase. 82 f 10 (3); lactate dehydrogenase, 104+ 17 (8); catalase, 94& 12 (8); Tris-resistant a-glucosidase, 105 18 (4); y-glutamyl transferase, 87 5 (3); Zn*+-resistant a-glucosidase, 80 f8 (7).

Figure 2. Effect of homogenization in the presence of digitonin on enzyme distributions. The biopsies were homogenized either in the presence of 0.12 mmol/l digitonin in the homogenizing medium (-) or in its absence (----). Details as for Fig. I . Peptidase substrates were L-leucyl-L-leucyl-L-leucine(leu-leu-leu) and Lmethionylglycylglycine(met-gly-gly). The percentages of recovered activity, with numbers of experiments in parentheses, are: malate dehydrogenase, 85 (2); N-acetyl-fi-glucosaminidase, 79 (2); lactate dehydrogenase, 106 (2); catalase, 107(2); Tris-resistant a-glucosidase, 103 (2); Zn2+-resistant a-glucosidase, 77 (2).

glucosidase are quite similar to those of the lysosomal N-acetyl-P-glucosaminidase. Fig. 2 compares the sucrose gradient distributions of biopsy homogenates prepared in isotonic sucrose with or without perturbant concentrations of digitonin. In the control experiments it is only possible to localize the tripeptidase activities to either the lysosomes (N-acetyl-P-glucosaminidase) or the brush border (Zn2+-resistant u-glucosidase). However, in the presence of 0.12 mmol/l digitonin disruption of the lysosomes occurred with almost complete recovery of their enzymic activity in the soluble fractions. The brush borders showed an increase in median density and it is clear that the particulate component of the peptidases is localized to this organelle. Figs. 3-5 display the distributions of activities against the peptides studied. It is evident that there is a

striking difference in the distribution of activities depending on the peptide used as substrate. For all dipeptides, hydrolase activity is largely or almost entirely soluble. For tripeptidases there is a bimodal distribution with a variable but always substantial proportion of the activity associated with a particulate component that has a distribution and modal density identical to that of the brush border markers. As exemplified by the series of glycine peptides (Gly)2-(GIy)s(Fig. 4) and (Gly)6 (Fig. 5), the proportion of activity associated with the brush border increases with the chain length of the peptide. A similar phenomenon is seen for the peptide series (Phe)2.4 (Fig. 4). For (Phe)s, however, in contrast to (Gly)S there is a substantial proportion of activity associated with the soluble fraction. Fig. 5 also shows the distribution of the enzyme dipeptidyl

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Density Figure 3. Distribution of peptidase activities following isopycnic centrifugation of post-nuclear supernatant. Details are as for Fig. 1. Substrates and the percentage of recovered enzymic activity, with number of experiments in parentheses, are: glycyl-L-methione (glymet), 83 (1); L-methionylglycine (met-gly), 80 (2); L-leucylglycine (leu-gly), 75 (3); glycyl-L-leucine (gly-leu). 87 (2); L-leucylglycylglycine (leu-gly-gly), 88 (I); L-methionylglycyl-glycine(met-gly-gly), 87 (I); trileucine (leu-leu-leu), 86 (3); glycylglycyl-L-alanine (gly-glyala), 94 (1).

peptidase IV. This enzymic activity is localized in the brush border. For some dipeptides a small proportion of activity appeared to be particulate. This was particularly evident in the case of L-phenylalanyl-glycine peptidase activity (Fig. 6 ) . It was not certain whether this particulate activity was merely due to adsorption of the cytosolic component or whether there was intrinsic membrane-bound activity. Studies with the sulphydrylbinding reagent p-hydroxymercuribenzoate, however, suggested that the small proportion of membraneassociated activity was intrinsic. This reagent inhibited the soluble peptidase activity but not the membraneassociated activity. Hence, the frequency-density histograms of peptidase activity in the gradient fractions assayed in the presence of this inhibitor are significantly altered (Fig. 6 ) . The particulate component is now predominant for L-phenylalanyl-glycine and L-phenylalanyl-glycyl-glycine and prominent for L-leucyl-L-leucinepeptidase activities. The distribution of this particulate component is characteristic of brush border enzymes. Homogenization in the presence of 0.8 mmol/’lTrisbase [13] did not alter the distribution of peptidase activity in the gradients (not shown). In particular, the

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Density Figure 4. Distribution of peptidase activies following isopycnic centrifugation of post-nuclear supernatant. Details are as for Fig. 1. Substrates and the percentage of recovered enzymic activity, with number of experiments in parentheses, are: diglycine (gly-gly), 86 (2); triglycine, 76 (2); tetraglycine, 75 (2); pentaglycine, 82 (2); diphenylalanine (phe-phe), 84 (2); triphenylalanine, 75 (2); tetraphenylalanine, 77 (2); pentaphenylalanine, 79 (2).

peptidase activities associated with the brush border membrane was unaffected by these conditions, indicating an intrinsic association of these enzymes with the membrane. Discussion It has long been known that peptidases are widely distributed in mammalian tissues [ 141. Some understanding of their biological role in the intestine has been gained by studies of purified enzymes [ 15-19] but relatively few studies of their subcellular distribution have been performed. Methods which employ differential centrifugation have been used to investigate the subcellular localization of activity against dipeptides and tripeptides in the intestine of the rat [4, 51 and the guinea-pig [3]. In both species most of the dipeptidase activity (75-90%) was associated with the soluble cytoplasmic fraction (cytosol) of mucosal homgenates and approximately 10% of activity was bound to the brush border membrane of the intestinal epithelial cells. Species differences were found, however, when tripeptides were used as substrates. In the guinea-pig intestine [3] 50% or more of the tripeptidase activity against three different tripeptides was associated with the brush border fraction. In contrast, in the rat [5]

LOCALIZATION OF PEPTIDASES IN HUMAN JEJUNUM

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Density Figure 5. Distribution of peptidase activies following isopycnic centrifugation of post-nuclear supernatant. Details are as for Fig. 1 . Substrates and percentage of recovered enzymic activity, with (argnumber of experiments in parentheses, are: ~-arginyl-~-leucine leu), 79 (2); ~-lysyl-~-phenylalanine (lys-phe), 76 (2); L-prolyl-L-leucine (pro-leu), 84 (2); L-methionyl-L-glutamicacid (met-glu), 74 (2); glycyl-L-phenylalanine, 94 (3); glycyl-L-alanine (gly-ala), 89 (I); hexaglycine, 75 (2). Recovery of dipeptydyl peptidase IV was 79% (2).

the distribution of enzymic activity hydrolysing a tripeptidewassimilar to thedistributionofdipeptidases, with the greater proportion of activity (95%) associated with the cytosol. The present study indicates that in the human jejunum the major subcellular locus of intestinal dipeptidase activity is the soluble cytoplasmic fraction. This distribution of enzymic activity correlates well with evidence which suggests that dipeptides presented to lumen of the human intestine are absorbed into the cell in an intact form and are not hydrolysed to any extent at the luminal surface of the cell membrane [20]. A significant percentage of tripeptidase activity measured at pH 8.0 was membrane-bound. Under physiological conditions, however, jejunal intraluminal pH is usually less than 6.0 which is below the pH optimum of the brush border peptidases. It is possible, therefore, that our in uitro studies may overestimate the physiological role of brush border tripeptide hydrolysis. From a consideration of the distribution of peptidase activities when substrates contain more than three amino acid residues, it seems likely that membrane hydrolysis plays a functionally significant role during their absorption. The most obvious function would be the degradation of larger peptides to amino acids,

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Density Figure 6. Effect of p-hydroxymercuribenzoate on the distribution of peptidase activities following isopycnic centrifugation of postnuclear supernatant (PNS). Details are as for Fig. 1 . PNS and gradient fractions were measured in the presence of 0.5 mmol/l p-hydroxymercuribenzoate (+PHMB) or in the absence of this reagent. Substrates and the percentage ofrecovered enzymic activity, with number of experiments in parentheses, are: ~-leucyl-~-leucine (leu-leu), 107 (2); leu-leu (+PHMB), 109 (I); L-phenylalanylglycine (phe-gly), 94 (2); phe-gly (+PHMB), 94 (I); L-phenylalanylglycylglycine (phe-gly-gly), 97 (2); phe-gly-gly (+PHMB), 102 (I).

dipeptides and tripeptides. These smaller peptides and amino acids could then be absorbed by the carrier mechanisms which have been postulated on the basis of transport kinetics performed on the intestine of laboratory animals and man [20]. As shown in the present studies the major locus with hydrolase activity for the larger peptides is the brush border with only trivial or no cytosolic peptidase activity. Similar conclusions were drawn by Kim et al. [21] who studied the substrate specificities of peptidases partially purified from either the cytosol or the brush border of human jejunum. Two tetrapeptides, a pentapeptide and a hexapeptide could not be hydrolysed by cytosolic enzymes but all were hydrolysed by the brush border membrane. The present results, in general, confirm these findings. However, it is noteworthy that pentaphenylalanine was hydrolysed by thecytosol. Thus it may well be that the cytosolic enzymes can hydrolyse certain large peptides, and no definite statement about the specificity of cytosolic peptidase can be made until more substrates have been investigated. Studies with glycyl-~-prolyl-2-naphthylamide, a

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substrate for dipeptidyl peptidase IV, indicate that this enzyme is also located in the brush border membrane of the human intestine. This enzyme releases dipeptides from peptides containing proline or alanine as the penultimate NHz-terminal amino acid. Its localization in the brush border membrane of pig kidney tubule has previously been demonstrated by Kenny et a1.[10].It is of interest that the brush border peptidase purified from rat intestine did not hydrolyse substrates containing proline as the penultimate NH2-terminal amino acid [19]. If this restriction in the range of activities is true for human intestinal brush border peptidases, the brush border dipeptidyl peptidase IV may play a signficant role in the degradation of peptidase derived from wheat and other cereal gliadins, proteins that are rich in L-proline residues.

Acknowledgments

The expert technical assistance of P. J. White is gratefully acknowledged. Jean de Luca kindly typed the manuscript. This work is supported by the Medical Research Council.

References I Kim Y.S., Nicholson J.A. &Curtis K.J. (1974) Intestinal peptide hydrolases: peptide and amino acid absorption. Med CIin North Am 58, 139771412, 2 Peters T.J. (1970) Intestinal peptidases. Gur 11,720-72s. 3 Peters T.J. (1970) The subcellular localization of di- and tripeptide hydrolase activity in guinea-pig small intestine. Biochem J 120,195-203. 4 Robinson G.B. (1963) The distribution of peptidases in subcellular fractions from the mucosa of the small intestine of the rat. Biochem J 88, 162-168. 5 KimY.S., Birtwhistle W.&KimY.W.(1972)Peptidehydrolases in the brush border and soluble fractions of small intestinal mucosa of rat and man. J Clin Invest 51, 1419-1430.

6 Peters T.J. (1976) Analytical subcellular fractionation of jejunal biopsy specimens: methodology and characterization of the organelles in normal tissue. Clin Sci Mol Med 51,557-574. 7 Nicholson J.A. & Peters T.J. (1978) Fluorometric assay for intestinal peptidases. Anal Biochem 87,418424. 8 Nicholson J.A. & Peters T.J. (1979) Fluorimetric assay for human intestinal glycinepeptidases. Clin Chim Acra 91,153-158. 9 Nicholson J.A. & Peters T.J. (1978) Subcellular distribution of hydrolase activities for glycine and leucine homopeptides in human jejunum. Clin Sci Mol Med 54,205-207. 10 Kenny A. J., Booth A.G., George S.G.,Ingram J., Kershaw D., Wood E.J. & Young A.R. (1976) Dipeptidyl peptidase. 1V. A kidney brush border serine peptidase. Biochem J 155, 169-182. 11 Seymour C.A. & PetersT.J. (1977) Enzyme levels in human liver biopsies. Assay methods and activities of some lysosomal and membrane-bound enzymes in control tissue and serum. Clin Sci Mol Med 52,229-239. 12 Leighton F., Poole B., Beaufay H., Baudhuin P., Coffey J.W., Fowler S. & de Duve C. (1968) The large-scale separation of peroxisomes, mitochondria and lysosomes from livers of rats injected with Triton WR 1339. J Cell Biol37,482-513. 13 Schmitz J., Preiser H., Maestracci D., Ghosh B.K., Cerda J . & Crane R.K. (1973) Purification of the human intestinal brush border membrane. Biochim Biophys Acta 323,98-112. 14 Smith E.L. (1951) The specificity of certain peptidases. Ado Enzymoll2, 191-257. 15 Das M. & Radhakrishnan A.N. (1973) Glycyl-L-leucine hydrolase, a versatile ‘master’ dipeptidase from monkey small intestine. Biochem J 135,609415. 16 Donlon J. & Fottrell P.F. (1973) Purification and characterization of one of the forms of peptide hydrolases from guinea-pig small intestinal mucosa. Biochim Biophys Acfa 327,425-436. 17 Noren 0.. Sjostrom H. & Josefsson L. (1973) Studies on soluble dipeptidase from pig intestinal mucosa. I. Purification and specificity. Biochim Biophys Acla 327,446456. 18 Maroux S., Louvard D. & Baratti J . (1973) The aminopeptidase from hog intestinal brush border. Eiochim Siophys Acta 321, 282-295. 19 Yim Y.S., Brophy E.J. & Nicholson J.A. (1976) Rat intestinal brush border membrane peptidases. 11. Enzymatic properties, immunochemistry, and interactions with lectins of two different forms of the enzyme. J Biol Chem 251,3206-32 12. 20 Matthews D.M. (1975) Intestinal absorption of peptides. Physiol Rev 55,537-608. 21 Kim Y.S., Kim Y.W. & Sleisenger M.H. (1974) Specificities of peptide hydrolases in brush border and cytosol fractions of rat small intestine. Biochim Biophys Acra 370,283-296.

The subcellular localization of peptidase activity in the human jejunum.

European Journal of Clinical Investigation (1979) 9,349-354 The subcellular localization of peptidase activity in the human jejunum J. A. NICHOLSON &...
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