The isolation and purification of a proteinase with chymotrypsin-like properties from ovine mucosal mast cells

lnt. J. Biochem. Vol. 18, No. 8, pp. 673-682, 1986 Printed in Great Britain

0020-711X/86 $3.00+0.00 Pergamon Journals Ltd

THE ISOLATION A N D PURIFICATION OF A PROTEINASE WITH CHYMOTRYPSIN-LIKE PROPERTIES FROM OVINE MUCOSAL MAST CELLS J. F. HUNTLEYl*, S. GIBSON2, D. KNOX l and H. R. P. MILLER I IMoredun Research Institute, 408 Gilmerton Road, Edinburgh EHI7 7JH, U.K. [Tel. (031) 664-3262]. 2Gastro-Intestinal Unit, Western General Hospital, Crewe Road, Edinburgh, U.K. (Received 16 December 1985)

Akstraet--l. A mast cell granule proteinase was purified from isolated ovine mucosal mast cells by cation exchange chromatography, which defined the conditions for enzyme purification from sheep gastric mucosae. 2. Antibodies raised against the proteinase were used in subsequent purification procedures which yielded 78 #g of enzyme per 5 g wet wt of abomasal tissue. 3. Immuno-histochemistry confirmed that mucosal mast cells were the source of the enzyme. 4. The proteinase had chymotrypsin-like esterase activity, with a molecular weight between 19,000 and 25,000.

INTRODUCTION

In a previous histochemical study the presence of a chymotrypsin-like serine esterase within ovine rnucosal mast cells ( M M C ) was reported (Huntley et al., 1985a). The esterase(s) was considered to be similar to rat mast cell protease II ( R M C P I I ) , a granulespecific neutral proteinase (Woodbury and Neurath, 1980) which is released systemically from M M C during infection with nematode (Miller et al., 1983a; W o o d b u r y et al., 1984) or protozoan (Huntley et al., 1985b) parasites. Since M M C may play an important role in immunity to nematode infections of sheep (reviewed in Miller, 1984), a sheep mast cell proteinase (SMCP) from nematode-infected gastric rnucosae of sheep has been isolated and partially characterised. Repeated infection with these nematodes causes a substantial increase in the number of M M C in the mucosa and permits the recovery of highly enriched populations of viable M M C (Huntley et al., 1984). Initial recovery of S M C P was, therefore, from isolated cells enriched for M M C . This technique facilitated subsequent attempts to purify S M C P from tissue homogenates. MATERIALS AND METHODS

Animals Eight month old Suffolk cross lambs (out of Greyface ewes) were used. Infection with nematode larvae Sheep were dosed orally with either 10,000 Ostertagia circumcincta or I0,000 Haemonchus contortus larvae per day for 8-10 weeks before being killed. Preparation o f tissues Excised abomasal (gastric) folds were washed briefly in tap water, and either processed immediately for enzyme extraction, or were stored at -80°C. Abomasal tissue for

*Author to whom reprint requests should be addressed, B.C. 18/8--A

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immuno-histochemistry was fixed in Bouin's fluid for 6 hr (Newlands et al., 1984) and was trimmed and prepared as previously described (Miller et al., 1983b). Histochemical staining o f M M C Sections were stained with toluidine blue pH 0.5 (Enerback, 1966). Isolation o f abomasal mast cells Viable mucosal mast cells were isolated from the abomasum and enriched for MMC as previously described (Huntley et al., 1984). Cell smears were prepared in a Shandon cytocentrifuge and fixed in Bouin's fluid for 15 min at 4°C (Huntley et al., 1984). Smears were then transferred into 70% alcohol, left overnight at 4°C, and rehydrated and stained the following day. Protein determination Unless stated otherwise, protein concentrations were measured by the method of Lowry et al. (1951) with purified chymotrypsin (Sigma) as standard. Enzyme activity The standard assay developed for monitoring enzyme activity using the esterase substrate, carbobenzoxyL-tyrosine-4-nitrophenyl ester (CBZ-L-Tyr-4-NPE), was as follows: to 150#1 of H20, were added 50pl of 0.25M Tris-HCl buffer, pH 7.5, 50/~1 of sample solution and 10/~1 of 0.01 M CBZ-L-Tyr-4-NPE dissolved in dimethyl sulphoxide. Esterase activity was terminated after 5 min at 20:'C by the addition of 1 ml of 70o methanol and the reaction product at E405 was measured. The free 4-nitrophenol liberated by hydrolysis of the substrate was read from a standard curve prepared with varying concentrations of 4-nitrophenol, and expressed as /~M of 4-nitrophenol released. At each purification step, enzyme recovery was estimated with the aid of an I.L Multistat III F/LS microcentrifugal analyser (Allied Instrumentation Laboratory, Warrington) as described by Knox et al. (1985). Column chromatography Ion-exchange, gel-filtration and affinity chromatography were carried out with the aid of fast protein liquid chromatography equipment (FPLC, Pharmacia).

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Recovery of SMCP .from isolated MMC Isolated MMC (2 x 108) in 1 ml of 0.02 M Tris-HCl pH 7.5 (Tris buffer) were disrupted by 6 rapid freeze/thaw cycles. After centrifugation at 30,000g for 5 min, the supernatant was stored at -80°C. The pellet was dissolved in 1 ml of Tris buffer containing 2 M KCI and dialysed against Tris buffer; a precipitate formed which was recovered by centrifugation (30,000 g for 5 min) and redissolved in 2 M MgC12. The solubilized precipitate contained most of the enzyme activity, which remained in solution following dialysis against Tris buffer containing 0.1% Brij 35 (Sigma). The extract was applied to CM-Sepharose 6B (Pharmacia) equilibrated in Tris buffer/0.1% Brij 35 and a linear gradient (0-0.5 M NaC1) was applied. Pooled active fractions dialysed against Tris buffer/0.1% Brij 35 were applied to a Mono-S cation exchange column (FPLC, Pharmacia) equilibrated in the same Tris buffer and were eluted with a linear gradient (0-I M NaCI).

Enzyme purification jJ'om abomasal tissue Abomasal tissue (60g), homogenized in 180ml of Tris buffer, was centrifuged (30,000g for 30 min) and the pellet was re-homogenized in 100 ml of Tris buffer containing 2 M KC1. After centrifugation (30,000 g, 30 min) the supernatant was dialysed against distilled water. The precipitate which formed was recovered by centrifugation, dissolved in 20 ml of 2 M MgCI z and dialysed against Tris buffer containing 0.1% Brij 35. Further slight precipitation was removed by centrifugation and the supernatant applied to CMSepharose 6B equilibrated in Tris buffer containing 0.1% Brij 35. Fractions eluted with a linear (0q3.5 M NaC1) gradient and containing enzyme were pooled and fractionated on Hydroxylapatite (HTP, Bio-Gel) equilibrated in Tris buffer containing 0.1% Brij 35. Active fractions were pooled and applied to Mono-S equilibrated in Tris buffer/0.1% Brij 35. A linear gradient (O-I M NaCI in Tris buffer containing 0.1% Brij 35) was applied, and the protein peak containing enzyme activity was collected and stored at 20°C.

using Protein A-Sepharose 4B (Pharmacia). The method employed, together with the procedure for coupling IgG to cyanogen bromide-activated Sepharose-4B (Pharmaciat were as described by the manufacturer.

Preparation of antibody and fab .fragments SMCP (400pg) was coupled to 5ml of cyanogen bromide-activated Sepharose-4B and antibody to the proteinase was atfinity-purified from the IgG fraction of rabbit antiserum. Antibody bound to SMCP-Sepharose 4B was eluted with 0.1 M acetic acid containing 0.5 M NaCI, and neutralized with 1 M Tris. Following dialysis against phosphate buffered saline, pH 7.2, aliquots of antibody were stored at -20°C. Fab anti-SMCP was prepared by the method of Wilson and Nakane (1978). The preparation and purification of polyvalent sheep F(ab')~ anti-rabbit Fab were as described previously (Nawa et al., 1978). Sheep F(ab')2 anti-rabbit Fab was coupled to peroxidase according to the method of Nakane and Kawaoi (1974).

lmmunohistochemistry Tissue sections or cytospin smears were pretreated with periodic acid and sodium borohydride to block endogenous peroxidase activity (Heyderman and Neville, 1977) and were incubated in 10% ovalbumin (Sigma, Grade II) dissolved in 0.1 M Tris-HC1 pH 7.5 for l hr. They were incubated overnight at 4"C with affinity purified rabbit IgG or Fab anti-SMCP (1 pg/ml) in 0.1 M Tris-HC1 pH 7.5 containing 10% ovalbumin. Sections washed 3 x l 0 m i n in the Tris-HCl were incubated for 2 hr in sheep F(ab)2 anti-rabbit Fab peroxidase conjugate (25#g/ml) diluted with 10% ovalbumin in 0.1 M Tris-HCl pH 7.5. After three further washes in Tris buffer, peroxidase activity was revealed with 3-amino-9-ethyl-carbazole (Graham et al., 1965). For control purposes, a 1/50 dilution of normal rabbit serum was substituted for antibody. Coverslips were mounted with polyvinyl pyrrolidone (PVP).

Proteinase activity

_

Enzyme purification by immunoaffinity Abomasal tissue (5 g) was homogenized in 15 ml of Tris buffer and the pellet dissolved in 15 ml Tris buffer containing 2 M KC1 as previously described. The salt concentration of the resultant supernatant was reduced to 0.5 M KCI by dilution with Tris buffer containing 0.1% Brij 35. The sample was applied to a 5 ml column of Sepharose-4B equilibrated in Tris buffer containing 0.1% Brij 35 and 0.5 M KCI, to which was bound 50 mg of normal rabbit lgG. The unbound fraction was then applied to a 5 ml column of Sepharose-4B to which was bound 50 mg of rabbit anti-SMCP IgG equilibrated in the same buffer. The bound enzyme, eluted with 0.1 M acetic acid in 0.5 M NaCI, was immediately neutralized with 1 M Tris and the sample applied to a Mono-S column. A linear gradient (0-1 M NaC1 in Tris buffer containing 0.1% Brij 35) was applied, and the protein peak containing enzyme activity was stored at - 20°C.

Raising of antiserum Purified SMCP (15#g) in l m l of Tris buffer was emulsified with an equal volume of Freund's complete adjuvant. Two ml of 2% Tween 80 in phosphate buffered saline was added and mixed to form a water-in-oil-in water emulsion. Each rabbit received two subcutaneous injections of I ml. A further injection was given three weeks later; the amount of antigen and formulation of emulsion being the same except that Freund's complete adjuvant was replaced with incomplete adjuvant. Blood samples were taken weekly after the final injection.

Preparation of IgG and IgG-sepharose 4B IgG was recovered from serum of normal rabbits and from rabbit anti-SMCP serum by affinity chromatography

This was measured by determining the perchloric acidsoluble products of the enzymatic digestion of denatured casein. Denatured casein was prepared by heating to boiling a 5% w/v casein mixture (Hammarsten, BDH) in distilled water and adding 2 M NaOH dropwise until it was dissolved (1-5 drops). It was cooled rapidly, dialysed against large volumes of distilled water, and stored as frozen aliquots. Enzyme activity was measured in an assay consisting of 100#1 of casein solution (7.25mg/ml), 25/tl Tris HC1 (1.0 M, pH 7.5) and 10 pl of enzyme solution. This mixture was incubated at 37°C for 1 hr, and the reaction stopped by the addition of 2.7 ml of 10% perchloric acid. After centrifugation the E 280 was measured. For comparison, the activity of ~-chymotrypsin (Sigma) was also measured in the same assay.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) Discontinuous SDS-PAGE was carried out as described by Laemmli (1970) using 12% gels and electrophoresed for 3 hr at 5°C at a constant current of 20 mA per gel. Protein standards used for calibration were cytochrome c (12,300), myoglobin (17,200), chymotrypsinogen A (25,700), ovalbumin (45,000), albumin (66,250) and ovotransferrin (76-78,000). The silver stain method of Morrissey (1981) was used to detect proteins within the gels.

Western blots Western blots were performed by the method of Towbin and Gordon (1984). Nitrocellulose blots were incubated overnight at 4°C in Fab anti SMCP (1/zg/ml) diluted in 0. I M Tris-HCl pH 7.5 containing 10% ovalbumin. Blots were then developed as described by Gibson and Miller

Sheep mast cell proteinase (1985), using biotinylated donkey anti-rabbit immunoglobin (Sera Labs) and avidin-HRP (Sera Labs) conjugates. Peroxidase activity was detected with diaminobenzidine and hydrogen peroxidase.

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Molecular weight determination The molecular weight of SMCP was determined on a column (l.6 × 54cm) of Sephadex G 150 (Pharmacia) equilibrated with Tris buffer containing 1.0 M NaCI. The column was calibrated with the following protein standards--ferritin (450,000), catalase (240,000) aldolase (158,000), bovine serum albumin (68,000) ovalbumin (45,000), chymotrypsinogen A (25,000) and cytochrome c (12,500). Purified SMCP (50 #g) was applied, the eluate was monitored by absorbance at OD 280 and fractions were tested for activity against CBZ-L-Tyr-4-NPE as previously described.

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RESULTS

Enzyme purification from isolated M M C Following enrichment for M M C on Percoll gradients, a total of 2 × 10s cells was obtained of which the majority (70%) were mast cells, as determined by differential counts on Leishman-stained smears. Little or no esterase activity was observed in the supernatant following homogenization of the cells in 0.02 M Tris buffer and high salt concentration (2 M KCI) was required to extract the enzyme from the cell pellet. Following dialysis to remove the salt, a precipitate formed which contained all of the detectable esterase activity. During subsequent procedures the presence of detergent (0.1% Brij 35) was necessary to maintain the esterase in solution. On CM-Sepharose, esterase was eluted in a broad peak of NaCI concentrations of approx 0.4-0.5 M. When re-chromatographed on Mono-S, enzyme activity was demonstrated exclusively in one major protein peak which eluted at a NaCI concentration of 0.15 M. The purified enzyme was revealed as a major polypeptide band on S D S - P A G E , and was closely associated with a minor band of slightly lower mobility (Fig. 1). The major band was similar in mobility to R M C P I I and chymotrypsinogen (Fig. 1). Because only small amounts ( < 5 / t g ) of S M C P were recovered from isolated mast cells further enzyme was extracted from homogenates of immune abomasal tissue.

Enzyme purification from abomasal tissue The elution profiles, together with the location of esterase activity during the purification of S M C P from tissue are shown in Fig. 2. Preliminary studies had shown that fractionation on hydroxylapatite was necessary for the subsequent separation of enzyme on Mono-S. Following final separation on Mono-S, esterase activity was exclusively in one major protein peak (Fig. 2), which, on S D S - P A G E , produced a major and minor band of identical mobility to that observed for enzyme purified from isolated M M C (Fig. 1). The yield of purified enzyme obtained from 60 g wet wt of tissue was 30 #g.

Isolation of SMCP by immunoaffinity The yields of enzyme at different stages of purification are shown in Table 1. Isolation of S M C P by immunoaffinity produced slightly greater yields than the two other methods, with 78 ttg of enzyme

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FractionNo. Fig.2. Theelution(OD280)profiles(solidline)from(a) CM-Sepharose4B;(b)hydroxylapatiteand(c)Mono-Sion exchange chromatography during the purification of enzyme from a 2 M KCI mucosal extract. The broken lines represent the NaC1 or phosphate concentrations during elution. Enzyme activity was detected with the esterase substrate, CBZ-L-Tyrosine 4-nitrophenyl ester and its location on the profiles is indicated by solid arrow heads.

recovered from 5 g wet wt of immune abomasal tissue. The recovery of S M C P was estimated to be 13% and an almost 1000 fold increase in specific activity (nkat/mg) (Table 1) was demonstrated. On S D S - P A G E , purified S M C P was revealed as a single polypeptide band (Fig. 3) identical in mobility to the major bands previously observed for S M C P recovered from isolated M M C or from tissue homogenates by ion exchange chromatography. However, the mobility of S M C P on this gel was greater than that of Table I. Purification of SMCP from abomasal tissue of immune sheep Total Total Specific protein activity activity Yield Stage (rag) (nkat) (nkat/mg) (%) 1 551 364 0.66 100 2 536 327 0.61 90 3 259 293 1.13 80 4 71 0 --5 72 168 23.3 46 6 0.078 48 610.5 13 The stages of purification were: (1) 0.01 M phosphate tissue homogenate; (2) 2 M KCI extract of tissue pellet; (3) unbound (fall-through) fraction following elution of 2 through a normal IgG-Sepharose 4B column; (4) and (5) non-absorbed and absorbed fractions respectively following elution of 3 through an anti-SMCP-Sepharose4B column; (6) purified SMCP following fractionation of 5 on Mono-S. The values given are those for a single preparation of 5 g of tissue. For stages (1)-(3) protein concentrations were determined by the biuret method and for subsequent stages protein was measured by E280 determinations on the basis of El% (1 cm path length) = 10. Enzyme activities were determined, using CBZ-L-Tyr-4-NPE as substrate, by centrifugal analysis as described by Knox et al. (1985) and are calculated from initial rates. The nkat (nanokatal) is defined as that activity hydrolysing 1 nmol of substrate

per see.

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chymotrypsinogen (Fig. 3), a finding which is at variance with a previous polyacrylamide gel (Fig. 1) where the mobility of SMCP was similar to that of chymotrypsinogen and RMCPII (Fig. 2).

lmmunod~{]usion Rabbit antiserum gave a strong precipitin line in Ouchterlony gel diffusion against SMCP purified from isolated M M C and against an abomasal homogenate in I M KCI Tris buffer (Fig. 4). Single precipitin lines of identity were also observed between SMCP purified from isolated MMC and from tissue, by ion-exchange chromatography or immunoaffinity, demonstrating antigenic homology between the different enzyme preparations.

Proteolytic acth~ity The proteolytic activity of SMCP, measured on denatured casein, increased E280 of the perchloric acid soluble products of hydrolysis equivalent to 1.75/~g/min/#g of enzyme. In the same assay the activity of ~-chymotrypsin was 7.89/~g/min/#g of enzyme.

Molecular weight determination by gel filtration A single protein peak was eluted from Sephadex G150 (Fig. 5) and this corresponded to the peak of esterase activity (Fig. 5). No esterase was detected in other eluted fractions. The estimated molecular weight for SMCP was 19,000.

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Western blots The mono-specificity of rabbit Fab anti-SMCP was confirmed by Western blotting (Figs 3A and B). Single polypeptide bands of identical mobility were detected in all samples except molecular weight standards (Fig. 3B). Furthermore, the relative prominence on SDS-PAGE of SMCP within proteins derived from the M M C extract (Fig. 3A) provides additional evidence of the MMC origin of this enzyme and suggests that SMCP is a major constituent of these cells.

Immunohistochemistry Immunoperoxidase staining for SMCP with whole antiserum, affinity purified anti-SMCP, or Fab antiSMCP was essentially the same in both tissue sections and cytospin smears of isolated cells. In sections of Bouin fixed immune abomasum large numbers of cells were stained within the lamina propria (Fig. 6A). The identity of these cells as M M C was suggested from their morphology, distribution and location within the tissues (Figs 6A and 6B) (Huntley et al., 1982, 1984). Further evidence for their identity was provided when sequential sections of immune and normal abomasum were stained with toluidine blue, pH 0.5, where both the numbers of MMC and their location were consistent with those observed following immunoperoxidase staining. Immunoperoxidase staining of Bouin fixed cytospin preparations of isolated M M C confirmed that the reaction product was localized within mast cell cytoplasm (Fig. 6C). No staining was observed in any tissue when antiSMCP was substituted with normal rabbit immunoglobulins.

0 DISCUSSION

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Fig. 5. Molecular weight determination of SMCP on Sephadex G150. The elution profile (OD 280) ( - - ) is shown, together with enzyme activity ( 0 - - - 0 ) against CBZL-Tyrosine 4-NPE in the eluted fractions (expressed as/~M of 4-nitrophenol released). The arrows represent the eluted positions of the protein calibration standards which were ferritin (450,000), catalase (240,000), aldolase (158,000), albumin 68 (68,000), albumin 45 (45,000), chymotrypsinogen A (25,000) and cytochrome c (12,500). The estimated molecular weight for SMCP was 19,000.

The present paper describes the isolation and purification to homogeneity of a sheep mast cell proteinase (SMCP). The enzyme was initially purified from isolated ovine M M C which, although contaminated with other cell types, nevertheless constituted an enriched source of M M C granule proteins. An important finding was that most, if not all, of the esterase activity was confined to the high salt (2 M KCI) extract and the low solubility of SMCP was exploited during purification of SMCP. Several other proteinases also require high salt concentration for extraction, including RMCPI (Kawiak et al., 1971; Seppa, 1978), elastase (Rindler-Ludwig and Braunsteiner, 1975), and cathepsin G (Starkey and Barrett, 1976). RMCPI has a basic isoelectric point and is rich in lysine residues (Woodbury and Neurath, 1980), and the requirement for extraction in high salt concentration may be due to its association with the highly sulphated glycosaminoglycan within the mast cell granule matrix (Lagunoff and Pritzl, 1976; Yurt and Austen, 1977). That SMCP is also a basic protein was indicated from its behaviour on CM-Sepharose and Mono-S, since the enzyme bound strongly to these cation exchangers. The rat mast cell proteinases RMCPI and RMCPII are now known to be present in different mast cell subsets, RMCPI in connective tissue mast cells and RMCPII in MMC (Gibson and Miller, 1986). Both

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Fig. I. SDS-polyacrylamide gel (silver staining) of purified SMCP from ovine mucosal mast cells or abomasal tissue. (Lan6 1), SMCP purified from whole abomasal tissue by ion-exchange chromatography; (Lane 2), initial 2 M KC1 extract of abomasal tissue, (Lane 3), partially purified rat mucosal mast cell proteinase II and (Lane 4), SMCP purified from isolated mucosal mast cells. Arrows indicate the positions of the protein standards, ovotransferrin (77,000), albumin (66,250), ovalbumin (45,000), chymotrypsinogen (25,700) myoglobin (17,200), and cytochrome c (12,300).

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Fig. 3. SDS-PAGE (a) and comparable Western blot (b) demonstrating the specificity of antibody for SMCP. Samples were molecular weight protein standards (Lane 1), 2 M KCI extract from abomasal tissue (Lane 2), enzyme eluted from an anti SMCP-Sepharose 4B column (Lane 3), 2 M KCI extract from isolated mucosal mast cells (Lane 4), and final preparation of purified SMCP following separation by immunoaflinity and Mono-S cation-exchange chromatography (Lane 5). The presence of salt in samples in lanes 2 and 4 caused some distortion of protein tracks. The nitrocellulose Western blot was incubated with rabbit Fab antibody to SMCP, followed by biotinylated donkey anti-rabbit immunoglobulin and avidin-horse-radish peroxidase. Peroxidase activity was detected with diaminobenzidine, and hydrogen peroxide.

Fig. 4. Gel diffusion of rabbit anti-SMCP against (a) SMCP purified from isolated mucosal mast cells and (b) 2 M KCI mucosal extract. 678

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(c) Fig. 6. (a) Section of abomasal mucosa from an immune sheep, demonstrating the immunohistochemical localization of enzyme within mast cells following staining with rabbit anti-SMCP IgG. The peroxidase reaction product within mucosal mast cells is clearly shown. × 250. (b) Section of abomasal mucosa from a non-infected sheep, stained as for (a). The immunolocalization of SMCP is confined to the relatively few mucosal mast cells present in normal sheep. × 250. (c) Cytocentrifuge preparation of isolated mucosal mast cells stained as for (a). Note the reaction product within the cell cytoplasm. × 400.

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enzymes are very similar but can be distinguished by particularly in the context of nematode parasite their physical, chemical, structural and immunoinfections. logical properties (Woodbury et al., 1978b; Gibson SUMMARY and Miller, 1986). In the present study, SMCP was immuno-localized to mast cells within the mucosa of A mast cell granule proteinase (SMCP) was isonormal and nematode-infected sheep. However, preliminary investigations suggest that SMCP or an lated from the gastric mucosae of sheep hyperantigenically related protein, is also present in sheep immunized with parasitic nematodes. Gastric mastoskin connective tissue mast cells (Huntley, Gibson cytosis was induced by nematode infection and a viable population of cells, enriched for mucosal mast and Miller, unpublished observations). Previous cells, was isolated from the abomasum. Proteinase studies have demonstrated an antigenic cross reaction was purified from the isolated cells by CM-Sepharose between RMCPI and RMCPII (Woodbury et al., and Mono-S cation exchange chromatography. This 1978a; Gibson and Miller, 1986), which can be procedure defined the chromatographic properties of eliminated by immuno-affinity techniques (Gibson the enzyme and facilitated its subsequent purification and Miller, 1986) and further studies are required to from mucosal homogenates. Enzyme preparations determine whether a similar cross-reactivity occurs were homogeneous on a silver-stained SDS-PAGE between enzymes present within sheep mast cell subgel and antibodies raised against them were used in sets. subsequent purification procedures, which yielded The molecular weight of SMCP was estimated to 78 #g of enzyme per 5 g wet wt of abomasal tissue. be 19,000 by Sephadex G150 gel filtration chromaThe enzyme had chymotrypsin-like esterase activity, tography. By SDS-PAGE analysis, SMCP comprised and proteinase activity was determined on denatured one major and a minor polypeptide, and estimates of casein. Immuno-histochemistry with affinity-purified molecular weight of the major polypeptide varied IgG or Fab antibody fractions confirmed that mucobetween 19,000 and 25,000. The reason for this sal mast cells were the source of the enzyme. A anomaly is not known although one possible cause molecular weight of between 19,000 (gel filtration) was the relatively high concentration of salt in the and 25,000 (SDS-PAGE) was determined. electrophoresed samples. The addition of salt prior to S D S denaturation prevented enzyme precipitation Acknowledgements--We thank Dr W. D. Smith for providand/or absorption to the container walls. Only small amounts ( < 5 #g) of SMCP were recov- ing access to parasitized sheep, and Brian Easter and Alan ered from isolated MMC, and further enzyme was Inglis for the preparation of photographs. Dr S. Gibson was supported by a grant from the Wellcome Trust. purified from abomasai tissue by ion-exchange chromatography or by immunoaffinity. The latter REFERENCES method provided the greatest yields of SMCP (78 #g SMCP/5 g wet wt of tissue), although they were Enerback L. (1966) Mast cells in rat gastrointestinal musubstantially less than the yields of RMCPII from cosa. 1. Effects of fixation. Acta path. microbiol, scand. 66, intestines of rats immune to Nippostrongylus brasi289-302. liensis ( > 500 pg of RMCPII/g wet wt; S. Gibson, Gibson S. and Miller H. R. P. (1986) Mast cells subsets in unpublished). This relatively low recovery of SMCP the rat distinguished immunohistochemically by their content of serine proteinases. Immunology. In press. may be a reflection of its concentration in the mucosa, or, alternatively, its loss of activity either by Glenner G. G. and Cohen L. A. (1960) Histochemical demonstration of a species specific trypsin-like enzyme in degradation during purification or through intermast cells. Nature 185, 846-847. action with inhibitors. Graham R. C., Lundholm U. and Karnovsky M. J. (1965) In a histochemical study ovine M M C were shown Cytochemical demonstration of peroxidase activity with to contain an esterase which, because it was inhibited 3-amino-9-ethylcarbazole. J. Histochem. Cytochem. 13, by phenylmethylsulfonylfluoride, was thought to be a 150-152. serine esterase (Huntley et al., 1985a). However, Heyderman E. and Neville A. M. (1977) A shorter immunorecent studies suggest that SMCP is not a typical peroxidase technique for the demonstration of carcinoembryonic antigen and other cell products. J. clin. Pathol. chymotrypsin in that it shares some features of a thiol 30, 138-140. proteinase (Knox et al., 1986), and it remains to be determined whether SMCP constitutes the entire Huntley J. F., Wallace G. R. and Miller H. R. P. (1982) Quantitative recovery of isolated mucosal mast ceils and histochemical esterase activity of ovine MMC, or if globule leucocytes from parasitized sheep. Res. vet. Sci. other esterases are also present. Both trypsin and 33, 58453. chymotrypsin-like enzymes have been isolated from Huntley J. F., Newlands G. and Miller H. R. P. (1984) The mouse mastocytoma cells (Vensel et al., 1971), and a isolation and characterization of globule leucocytes: their tryptase has been demonstrated in human conderivation from mucosal mast cells in parasitized sheep. Parasite Immunol. 6, 371-390. nective tissue mast cells (Glenner and Cohen, 1960) ancl pulmonary mast cells (Schwartz et al., 1981). Huntley J. F., Newlands G. F. J., Gibson S., Ferguson A. and Miller H. R. P. (1985a) Histochemical demonstration However, in the present study no tryptase activity of chymotrypsin like serine esterases in mucosal mast cells was detected in the soluble or high salt extracts of in four species including man. J. clin. Pathol. 38, 375-384. ovine M M C (data not included). Huntley J. F., Newlands G. F. J., Miller H. R. P., McLauchIn summary, a proteinase with chymotrypsin-like lan M., Rose M. E. and Hesketh P. (1985b) Systemic esterase activity has been isolated, purified to homrelease of mucosal mast cell protease during infection with ogeneity, and localized within mucosal mast cell the intestinal protozoal parasite, Eimeria nieschalzi Studgranules by immunohistochemistry. Further work ies in normal and nude rats. Parasite Immunol. 7, will be directed to studies of its role in the mucosa, 489-501.

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