Ostrich intestinal glycohydrolases: distribution, purification and partial characterisation

The International Journal of

PERGAMON

The International Journal of Biochemistry & Cell Biology 30 (1998) 339±352

Biochemistry & Cell Biology

Ostrich intestinal glycohydrolases: distribution, puri®cation and partial characterisation Vaughan Oosthuizen a,*, Durand P. Weldrick a, Ryno J. Naude a, Willem Oelofsen a, Koji Muramoto b, Hisao Kamiya b a

Department of Biochemistry and Microbiology, University of Port Elizabeth, P.O. Box 1600, Port Elizabeth 6000, South Africa b School of Fisheries Sciences, Kitasato University, Sanriku-Cho, Iwate 022-01, Japan Received 19 September 1997; accepted 22 September 1997

Abstract Intestinal glycohydrolases are enzymes involved in assimilating carbohydrate for nutrition. The avian forms of these enzymes, in particular the maltase±glucoamylase complex (MG), are not well characterised. This study encompassed characterisation of these enzymes from ostrich intestines, and the ®rst kinetic analysis of an avian MG. Proteolytically solubilised MG from ileal brush border membrane vesicles was puri®ed by Sephadex G-200 gel ®ltration and Tris-anity chromatography, while jejunal sucrase±isomaltase (SI) and MG were puri®ed by Toyopearl-Q650 and phenyl±Sepharose chromatography. Amino acid sequences and compositions of enzyme subunits, resulting from SDS-PAGE, were determined. Kinetics of hydrolysis of linear oligosaccharides was studied. Ostrich MG and SI showed the highest activity in the jejunum, followed by the ileum and duodenum. No lactase or trehalase activity could be detected. The jejunal MG and SI, resulting from brush-border membrane vesicles, could not be separated during puri®cation. However, a minor form of ileal MG was puri®ed using Sephadex G-200 chromatography. Ileal MG contained three subunits of Mr 145 000, 125 000 and 115 000. Although the N-terminal amino acid sequences bear no homology to SI, the Mr 115 000 subunit shows homology to porcine MG in both sequence and amino acid composition. The pH optimum of maltose-, starch- and isomaltose-hydrolysing activity was 6.5 and that of sucrosehydrolysing activity 5.5. The glycohydrolases were most active at 588C, but were quickly denatured above 608C. Sucrose- and starch-hydrolysing activities were more thermostable than maltose- and isomaltose-hydrolysing activities. Kinetic parameters (Km, kcat and kcat/Km) for the hydrolysis of maltooligosaccharides, starch and glycogen are reported for ileal MG. Maltotriose and maltotetraose displayed partial inhibition of ileal MG. The study revealed large similarities between ostrich SI and MG in charge, size, shape and hydrophobicity, based on their inseparability by several methods. Measurement of the speci®city constants for maltooligosaccharide hydrolysis by ileal MG revealed less ecient hydrolysis of longer substrates as compared to maltose and maltotriose. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Intestinal glycohydrolases; Ostrich; Puri®cation; Kinetics

* Corresponding author. Abbreviations: BB, Brush-border, BBMV, Brush-border membrane vesicles, DABS-Cl, 4-dimethylaminoazobenzene-4'-sulfonylchloride, FITC, ¯uorescein isothiocyanate, MG, maltase±glucoamylase, PITC, phenylisothiocyanate, SI, sucrase±isomaltase. 1357-2725/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 7 ) 0 0 1 2 6 - X

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1. Introduction The brush-border membrane of intestinal enterocytes contains a number of integral hydrolases, including glycohydrolases and proteinases. In the mammalian small intestine four brush-border glycohydrolases have been identi®ed, namely the sucrase±isomaltase complex (EC 3.2.1.48, EC 3.2.1.10), maltase±glucoamylase complex (EC 3.2.1.20, EC 3.2.1.3), lactase-phlorizin hydrolase (EC 3.2.1.23, EC 3.2.1.62) and trehalase (EC 3.2.1.28). The SI and MG complexes are synthesised as single chain polypeptide precursors (Mr~250 000) that are posttranslationally glycosylated, transported to the brush-border membrane and proteolytically cleaved to form heterodimeric hydrolases with the bulk of the protein on the luminal surface of enterocytes. The molecular weight of the subunits of both complexes are nearly identical and both complexes are anchored to the membrane via N-terminal hydrophobic regions on the larger subunit (Mr~140 000). The smaller subunit (Mr~120 000) remains attached by non-covalent forces (for review, see Ref. [1]). This simpli®ed view of the topology of the SI and MG complexes has been complicated by the presence on SDS-PAGE of a third subunit (Mr~110 000) of SI puri®ed from rats [2], humans [3] and chickens [4]. It has been proposed that this subunit originates from proteolytic processing at the N-terminal region of the larger (Mr~140 000) subunit [2] and is thus not a true subunit of these complexes. The SI complex has one active site on each subunit, with the larger subunit having isomaltase activity and the smaller subunit sucrase activity. In addition to this, both subunits hydrolyse maltose [5]. The isomaltase subunit is capable of hydrolysing palatinose and other branched limit a-dextrins [6]. Both subunits of the MG complex split a-1,4-glucopyranosidic bonds from the nonreducing ends of amylose, amylopectin, glycogen and smaller oligosaccharides, including maltose [7]. They also have minimal a-1,6-glucopyranosidase activity [7, 8]. Hence, SI is responsible for all sucrase activity, almost all isomaltase activity and approximately 80% maltase activity. The MG complex contrib-

utes minimal isomaltase activity, about 20% maltase activity and all glucoamylase activity [1]. The full sequence of rabbit [9], human [10] and rat [11] SI has been determined from cDNA clones, but little primary structure knowledge of MG is available. The SI gene in all three species encodes a single polypeptide chain precursor (pro-SI) with signi®cant homology between the isomaltase and sucrase subunits (41% amino acid identity), indicating that pro-SI evolved by partial gene duplication as ®rst proposed by Semenza [12]. A close similarity exists between mammalian and avian SI and MG complexes as witnessed from puri®ed chicken [4] and pigeon [13] complexes, suggesting partial gene duplication of the ancestral genes and subsequent mutations at one of the active sites in each complex occurred prior to the separation of mammals and reptiles [4]. To date little information concerning the avian MG complex exists, especially primary structure data. We have attempted the puri®cation of SI and MG from one of the evolutionary ancestral birds, the ostrich (Struthio camelus), in an attempt to characterise carbohydrate metabolism in the digestive tract of this bird, as well as contribute to the primary structure analysis of avian intestinal glycohydrolases.

2. Experimental 2.1. Materials Isomaltose, maltose, palatinose, sucrose, trehalose, p-hydroxybenzoic acid, 4-aminoantipyrine, peroxidase (Type II from horseradish), glucose oxidase (Type V from Aspergillus niger), p-aminophenyl-a-D-glucopyranoside±agarose were purchased from Sigma Chemical (St. Louis). Starch was purchased from Merck Chemical (Darmstadt). Papain (from Carica papaya) was from Boehringer Mannheim (Mannheim) and lactose from Serva (Heidelberg). All other reagents were of the highest commercial grade available.

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2.2. Ostrich material Ostrich jejuna and ilea from ~20 birds were excised shortly after the birds (aged 14 months and ~100 kg) were killed after one day of fasting. The intestinal contents were gently squeezed out, whereafter the jejuna and ilea were cut open and the mucosa scraped o€ with a glass slide. The mucosa was kept on ice for ~6 h and stored at ÿ208C until required. 2.3. Glycohydrolase assays Intestinal glycohydrolase activities (maltase, sucrase, isomaltase, lactase and trehalase) were assayed according to the procedure of Vasseur [14] using the Tris±glucose oxidase method and substrate concentrations of 28 mM. Lactase was assayed in the presence of p-chloromercuribenzoate to inhibit acid bgalactosidase [15]. Glucoamylase was assayed according to Schlegel±Haueter et al. [16] using starch as substrate (1%, w/v) in the presence of 1 mM EDTA to inhibit pancreatic a-amylase. One unit of activity hydrolyses 1 mmol disaccharide/min at 378C. 2.4. Intestinal distribution of glycohydrolases An ostrich chick (two months old) was sacri®ced and its small intestine removed and stored on ice for 3 h. The intestinal tract was divided into segments of 20 cm in length, cut open and washed in saline. Segments were frozen at ÿ208C until required (no longer than two weeks). Segments of the intestinal tract were thawed individually, the mucosa scraped o€ with a glass slide and homogenised with four parts water (w/ v) in an Ultra-Turrax homogenizer, keeping the homogenate on ice at all times. The homogenates were centrifuged at 480g for 10 min at 48C to remove cell debri and nuclei and the supernatant assayed for glycohydrolase activity. 2.5. Puri®cation procedures BBMV were prepared from 120 g ostrich jejunal mucosa according to Kessler et al. [17].

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Brush-border proteins were solubilised from BBMV by proteolytic cleavage using papain [18] and the digest centrifuged at 105 000g for 60 min at 48C. The resultant jejunal supernatant, containing solubilised glycohydrolases, was loaded onto a Toyopearl-Q650C column (1.6  11 cm) equilibrated in 10 mM potassium phosphate buffer, pH 6.1. The ostrich jejunal glycohydrolases were eluted using a 500 ml linear gradient of potassium phosphate (10±150 mM). Glycohydrolase active fractions (assayed by their maltose-hydrolysing activity, since all four subunits hydrolyse this substrate) were pooled, concentrated using Aquacide IIA and loaded onto a phenyl± Sepharose CL-4B column (1.6  12 cm) following equilibration of both column and enzymes with 10 mM potassium phosphate bu€er, pH 6.8, containing 1 M (NH4)2SO4. The glycohydrolases were eluted by a dual linear 500 ml gradient of (NH4)2SO4 from 1±0 M and ethanol from 0± 30% (w/v). The glycohydrolase active fractions were pooled, dialysed against 10 mM potassium phosphate bu€er, pH 6.0, and stored at 48C after concentration to 3 mg protein/ml. Solubilised BB proteins from 400 g ileum were prepared as described for jejunal tissue. The ileal supernatant from ultracentrifugation was chromatographed on Sephadex G-200 (5  51 cm), equilibrated and developed at room temperature with 10 mM potassium phosphate bu€er, pH 6, resulting in two active peaks. The second peak (MG complex) was loaded onto a Tris± Sepharose 4B anity column (1  19 cm) equilibrated with 10 mM potassium phosphate bu€er, pH 6. MG was eluted with a 150 ml linear gradient of NaCl (0±130 mM). The ®nal active fraction was lyophilised after exhaustive dialysis against water for long term storage of the enzyme.

2.6. Protein determination Protein concentration was determined using the method of Lowry et al. [19] with BSA as standard.

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2.7. SDS-PAGE Gel slabs (7.5% acrylamide) were prepared according to Laemmli [20] as described in the Bio-Rad Mini-Protean1 II Dual slab cell instruction manual. The molecular weight calibration proteins used, with Mr values in parentheses, were myosin (205 000), b-galactosidase (116 000), phosphorylase b (97 000), fructose-6-phosphate kinase (84 000), BSA (66 000), glutamate dehydrogenase (55 000), egg albumin (45 000) and glyceraldehyde-3-phosphate dehydrogenase (36 000). 2.8. Gradient gel electrophoresis In order to calculate the Mr of the intact native enzyme a 5±20% acrylamide gradient gel was cast in a vertical slab gel electrophoresis unit SE 600 from Hoefer Scienti®c Instruments (San Francisco, CA) using a gradient mixer [21]. The molecular weight calibration proteins used, with Mr values in parentheses, were thyroglobulin (670 000), urease (545 000), catalase (240 000) and BSA (66 000 monomer, 132 000 dimer and 198 000 trimer). 2.9. Isoelectric focusing Gel slabs (5% acrylamide) were prepared containing Bio-Lyte ampholytes (pH 3±10) as described by Robertson et al. [22]. Isoelectric focusing markers, with pI values in parentheses, were amyloglucosidase (3.55), trypsin inhibitor (4.55), b-lactoglobulin (5.13), bovine carbonic anhydrase (5.85), human carbonic anhydrase (6.57), myoglobin (6.76), lactic dehydrogenase (8.30) and trypsinogen (9.30). Glucoamylase activity in IEF gels was con®rmed after incubation of gels in 0.125 M MES bu€er, pH 6.5, containing 0.2% (w/v) starch for 2 h at 378C, followed by staining with Gramm's iodine [23]. 2.10. Amino acid composition The puri®ed jejunal glycohydrolase subunits were separated by SDS-PAGE and semidry blotted onto PVDF membranes according to Matsudaira [24]. The blotted subunits were sub-

jected to hydrolysis in 6 M HCl in evacuated pyrex tubes for 22 h at 1108C and dried under vacuum. The hydrolysates were derivatized with DABS-Cl and analysed by RP-HPLC [25]. 2.11. Amino terminal sequencing Amino terminal sequence analysis was performed on puri®ed jejunal glycohydrolase subunits that had been semidry blotted onto PVDF membranes following SDS-PAGE. Sequencing was performed on an automated Shimadzu PSQ1 gas-phase protein sequencer using the FITCPITC double coupling method [26]. 2.12. E€ect of pH The e€ect of pH on puri®ed glycohydrolases was studied using 0.125 M sodium acetate bu€ers (pH 4±5.5), 0.125 M MES-NaOH bu€ers (pH 6 and 6.5), 0.125 M MOPS-NaOH bu€ers (pH 7 and 7.5), 0.125 M HEPES-NaOH bu€er (pH 8) and 0.125 M glycine±NaOH bu€ers (pH 8.5±10). 2.13. E€ect of temperature and heat inactivation The e€ect of temperature on the hydrolysis of dissaccharides by puri®ed glycohydrolases was tested by incubation of enzyme and substrates at the relevant temperatures (15±708C) in 0.1 M sodium maleate bu€er, pH 6.5. The released glucose was assayed at 378C as described above. For heat inactivation studies the glycohydrolases were incubated at 56, 63 and 678C, aliquots removed at 0, 10, 20, 40 and 60 min and kept on ice until they could be assayed for enzyme and protein. BBMV-associated activity was tested in the same fashion at 608C. 2.14. Kinetic parameters Kinetic parameters (Km, kcat, and kcat/Km) were determined by least squares regression analysis of Hanes [27] plots. Linear maltooligosaccharides (maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose) were used as substrates at concentrations of 0± 5 mM, except maltotriose (0±1 mM). Starch and

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glycogen hydrolysis was tested at concentrations of 0±0.5% (w/v). 3. Results The distribution of ostrich glycohydrolases along the intestinal tract is typical of that found in most other vertebrates, where glycohydrolase activity is highest in the jejunum, lowest in the duodenum and moderate in the ileum (Fig. 1a and b). Of note is the relatively high activity of maltose and starch hydrolysis in the ileum (Fig. 1a) where SI activity has declined sharply (Fig. 1b). A similar distribution was found in the human small intestine [28]. No lactase or trehalase activity could be detected in the ostrich intestinal tract, a feature common amongst avian species [29]. Sephadex G-200 chromatography has been used since the 1960s to separate the MG and SI

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complexes due to an anity of isomaltase for the Sephadex backbone [30]. Initial attempts to separate ostrich jejunal SI and MG failed as both complexes eluted at the void volume of such columns (results not shown). Thereafter, numerous attempts to separate the ostrich glycohydrolase complexes using hydroxylapatite, DEAE-cellulose, Tris-Sepharose and p-aminophenyl-a-D-glucopyranoside±agarose chromatography led to coelution of the four enzymes (results not shown). The isolation and puri®cation procedure for jejunal glycohydrolases, summarised in Table 1, proved to be the most successful in that high speci®c activities were obtained, although no separation of MG and SI could be obtained through chromatography on DEAE-Toyopearl or phenyl±Sepharose. Since the SI complex was present in very low levels in the ileum, further attempts to obtain MG complex started with ileal brushborders. The ileal MG and SI complexes still

Fig. 1. Distribution of glycohydrolase activities along the ostrich small intestinal tract. (A) Maltose (*) and starch (q) hydrolysis. (B) Sucrose (Q) and isomaltose (r) hydrolysis. The small intestine was excised at the pyloric valve as the duodenum joins the stomach and extracts prepared as described in the text. Duodenal length was 030 cm, jejunum 0200 cm and ileum 0250 cm.

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Table 1 Summary of the puri®cation of MG and SI complexes from ostrich jejunal mucosa Enzyme activity (U) Step Crude homogenate BBMV Papain solubilisation Toyopearl Q650C Phenyl-Sepharose

M

G

S

I

4252 1162 995 738 118

359 84 117 83 13

272 83 77 57 9

193 53 50 34 6

Total Protein (mg) 12 120 268 216 44.6 6.9

eluted at the void volume of Sephadex-G200 columns, but a minor MG peak appeared to be relatively free of SI contamination (Fig. 2). This MG peak was puri®ed on Tris-Sepharose 4B anity chromatography (Fig. 3), leading to a fraction with only 2% sucrase speci®c activity relative to glucoamylase (Table 2). This enzyme was used

Speci®c activity (U/mg protein) M 0.35 4.34 4.61 16.54 17.1

G

Puri®cation factor S

0.03 0.31 0.54 1.86 1.88

I 0.02 0.31 0.36 1.28 1.3

M 0.02 0.2 0.23 0.76 0.87

1 12 13 47 49

G 1 10 18 63 63

S 1 14 16 59 59

I 1 13 14 48 54

for kinetic analysis of maltooligosaccharide hydrolysis and isoelectric focusing revealed a pI of 4.87 for ostrich ileal MG. Further attempts to separate jejunal SI and MG, as well as studies on subunit structure of the glycohydrolases which eluted from the phenyl-Sepharose column, were performed on gradi-

Fig. 2. Chromatography of papain-solubilised ileal glycohydrolases on Sephadex G-200. A280 nm (w), maltase (*), glucoamylase (q) and sucrase (Q). Arrows show the fractions pooled from tube numbers 47±94 (Peak 1) and 95±125 (Peak 2).

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Fig. 3. Tris-Sepharose 4B anity chromatography of the second peak from Sephadex G-200 showing MG activity. A280 nm (w), maltase (*), NaCl gradient (- - -). Arrows show the fraction pooled from tubes 58±70.

ent PAGE and SDS-PAGE, followed by N-terminal sequencing. On gradient PAGE the glycohydrolases migrated as a single polypeptide chain with an Mr of 420 000 (Fig. 4). SDS-PAGE under reducing conditions resolved three subunits with Mr values of 145 000, 125 000 and 115 000 (Fig. 5), and under nonreducing conditions a single component of Mr 210 000 was detected (result not shown). The three subunits were electroblotted onto PVDF membranes and subjected

to amino acid analysis (Table 3) and N-terminal sequencing (Fig. 6). The amino acid compositions of the three subunits correspond well with those of rat MG complex (Table 3). Only Tyr and Val residues seem to di€er from rat MG, being higher in ostrich glycohydrolase subunits. Threonine residues in ostrich glycohydrolase subunits are marginally lower, but all other amino acids compare favourably. When comparing the amino acid compositions of ostrich glycohydro-

Table 2 Summary of the puri®cation of MG complex from ostrich ileal mucosa Enzyme activity (U) Step Crude homogenate BBMV Papain solubilisation Sephadex G-200 Tris-Sepharose

M

G

6471 3177 901 81 37

1507 626 180 21 10

Total Protein (mg)

S

I

511 280 63 1.1 0.2

364 33 064 170 3198 39 537 0.9 32.7 0.5 5.9

Speci®c activity (U/mg protein) M 0.2 0.99 1.68 2.48 6.27

G

Puri®cation factor S

0.05 0.2 0.34 0.64 1.69

I 0.02 0.09 0.12 0.03 0.03

M 0.01 0.05 0.07 0.03 0.09

1 5.1 8.6 12.7 32

G 1 4.3 7.5 14 37

S

I 1 5.7 7.7 2.2 2.2

1 4.8 6.6 2.5 7.7

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Fig. 4. Gradient PAGE of jejunal glycohydrolase fractions. Lanes 1 and 2, 105 000g supernatant after papain solubilisation at 10 and 20 mg/well, respectively; lanes 3 and 4, Toyopearl-Q650C fraction at 10 and 20 mg/well, respectively; lanes 5 and 6, phenylSepharose fraction at 10 and 20 mg/well, respectively; lanes 7 and 8, BSA; lanes 9 and 10, urease; lanes 11 and 12, catalase; lanes 13 and 14, thyroglobulin.

lases and rabbit SI [33], the similarities were not consistent for all amino acids. From the N-terminal amino acid sequences of ostrich glycohydrolase subunits (Fig. 6) it is apparent that there is signi®cant homology (~70%) between the Mr 125 000 and 145 000 subunits. However, little homology exists with the Mr 115 000 subunit or between the ostrich larger subunits and either subunit of rabbit [9] or human SI [10], or porcine MG [32]. However, the Mr 115 000 subunit shows homology to an N-terminal region of porcine MG from Leu13 to Leu19 [32]. The optimum pH of maltose-, starch- and isomaltose-hydrolysing activity was 6.5 and that of sucrose-hydrolysing activity 5.5 (result not shown). The optimum pH curve followed a typical bell-shaped curve, with the optimum activities (pH 5.5±7) being within normal intestinal pH ranges. They compare favourably with SI and MG activities in other species [7, 13, 16]. The e€ect of temperature on the four glycohydrolase activities was almost identical. There was a linear increase in activity from 15±588C, whereafter a sharp decrease in activity (~50% at 608C) occurred as the enzymes denatured, with no ac-

tivity being measured at 708C (result not shown). The Arrhenius plot for all four enzymes was monophasic, with activation energies being calculated as 23.0 (maltose hydrolysis), 23.2 (starch), 20.3 (sucrose) and 25.2 kcal/mol (palatinose). Heat inactivation studies on the four activities indicate that sucrase and glucoamylase are more thermostable than maltase and isomaltase, since the maltose- and isomaltose-hydrolysing activities decline to ~25% while the sucrose- and starchhydrolysing activities only decline to 60 and 70%, respectively, in one hour (Fig. 7). In accordance with the optimum temperature data the enzymes denature rapidly above 608C, falling to less than 50% activity within 10 min (Fig. 7c). At 678C all four activities were destroyed after 5 min incubation (result not shown). When associated with BBMV all four activities inactivate at the same rate (Fig. 7a) possibly due to protection being a€orded by the membranes. There are also indications of tight associations between membrane anchored subunits and those subunits not anchored to the membrane, since sucrase (normally not anchored to membranes) denatures at the same rate as isomaltase (normally membrane-

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Table 3 Amino acid composition of the ostrich glycohydrolase subunits Mr components of ostrich glycohydrolasesa

Rat MG complexb

Amino acid

115 000

125 000

145 000

D-form

P-form

Asx Thr Ser Glx Pro Cys Gly Ala Val Met Ile Leu Tyr Phe His Lys Arg

12.4 5.8 7.6 11.4 5.7 ND 10.3 7.4 5.5 0.7 4.6 8.3 4.4 5.0 2.8 3.9 4.2

10.9 6.0 7.6 11.1 5.8 ND 8.9 7.3 5.9 1.1 5.0 8.9 4.9 5.1 2.9 4.0 4.5

11.2 6.5 7.2 11.3 6.0 ND 8.7 8.2 5.0 1.0 5.1 8.7 4.7 4.8 3.1 3.1 5.3

11.2 8.1 8.8 11.1 5.8 0.9 10.8 7.3 3.8 1.4 3.0 9.7 2.9 4.4 2.7 4.6 3.5

12.4 6.9 7.7 10.8 6.8 0.2 10.3 4.9 4.0 0.4 4.6 8.0 3.6 5.1 6.1 4.2 4.0

100

100

Total amino 100 acids

100

100

ND, not determined; D-form, detergent-solubilised form; P-form, papain-solubilised form. a Expressed as mol/100 mol. b Lee and Forstner [31].

Fig. 5. SDS-PAGE of puri®ed ileal MG fraction after TrisSepharose 4B chromatography. Lane 1, Reduced MG complex loaded at 12 mg/well and lane 2, Molecular weight marker proteins myosin (205 000), b-galactosidase (116 000), phosphorylase b (97 000), and fructose-6-phosphate kinase (84 000).

anchored). Furthermore, the thermolabile activities of maltase and isomaltase were more stable at 608C when associated with membranes than the puri®ed enzymes were at 568C. The action of ileal MG on linear maltooligosaccharides, starch and glycogen shows the enzyme's preference for short-chain linear substrates (Table 4). The enzyme has a high anity for maltotriose, although the turnover number is greatest for maltose. The low Km for maltotriose,

however, results in a twofold greater catalytic eciency for this substrate over maltose. An observation not represented here was that both maltotriose and maltotetraose displayed inhibition at higher substrate concentrations. The longer, branched polymers of starch and glycogen are poorly hydrolysed by MG as witnessed by markedly lower kcat values, especially for glycogen, and the signi®cantly higher enzyme concentrations required in these hydrolyses to obtain reliable catalytic rates (Table 4).

4. Discussion Ostrich glycohydrolases were abundant in the jejunum, with the exception of lactase and trehalase that could not be detected. Although sucrase and isomaltase activity declined either side of the jejunum, maltose- and starch-hydrolysing activi-

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Fig. 6. Comparison of N-terminal amino acid sequences of ostrich jejunal glycohydrolase subunits and porcine MG. *, NoreÂn et al. [32]; $, ostrich Mr components; X, unidenti®ed residue; -, gap inserted for maximal homology. Identical amino acids of porcine MG and ostrich Mr 115 000 subunit and of ostrich Mr 125 000 and 145 000 subunits, respectively, are blocked.

ties remained substantial in the ileum (Fig. 1a and b). This distribution of intestinal glycohydrolases is typical of most vertebrate species, such as humans [28], rats, rabbits, guinea pigs and pigeons [35], chickens [29], turkeys [36], camels [37] and alligators [38]. The absence of lactase and trehalase seems to be characteristic of avian species [29]. The puri®cation of jejunal SI and MG was not successful since these two complexes coeluted throughout all protocols. The indications are that these two complexes in the ostrich appear to be very closely related in terms of charge, size,

hydrophobicity and catalytic properties. In many species from which these complexes have been puri®ed to date, the use of Sephadex G-200 as an anity matrix to separate MG and SI has been crucial. This is especially true for humans [5], rabbits [30], chickens [4, 29], pigeons [13] and rats [39]. Yet, when ileal material was used, the separation of MG from SI could be achieved in ostriches, although only a minor MG active peak was obtained (Fig. 2). The bulk of ileal MG and SI still coeluted in the void volume of Sephadex G-200. The failure of jejunal SI and MG to separate while a minor ileal MG fraction could be

Fig. 7. Heat inactivation of ostrich glycohydrolases. Maltose (*), starch (q), sucrose (Q) and isomaltose (r) as substrates. (A, B and C) Incubations at 608C (BBMV), 568C (solubilised jejunal enzymes) and 638C (solubilised jejunal enzymes), respectively.

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Table 4 Summary of the kinetic parameters of ostrich ileal MG-catalysed hydrolysis of maltooligosaccharides, starch and glycogen Ostrich MG Substrate

Km (mM)

Maltose Maltotriose Maltotetraose Maltopentaose Maltohexaose Maltoheptaose Starcha Glycogena

0.54 0.09 0.22 0.24 0.26 0.38 0.18 0.36

Human MG [34] ÿ1

kcat (s ) 59.72 22.72 38.24 35.15 30.96 32.51 13.5 2.44

ÿ1 ÿ1

kcat/Km (M
s )

kcat/Km (Mÿ1
sÿ1)

1.10  105 2.58  105 1.74  105 1.48  105 1.18  105 8.56  104 ÿ ÿ

2.67  104 1.35  105 1.95  105 1.58  105 1.00  105 8.00  104 ND ND

Data calculated by least squares regression analysis of Hanes plots from triplicate determinations. r2 values >0.99. The enzyme concentrations used were 7 nM (maltose±maltohexaose), 35 nM (starch) and 75 nM (glycogen) based on a molecular weight of 210 000 for ileal MG. ND, not determined. a Km value expressed as percentage (w/v).

puri®ed free of SI might re¯ect di€erences in glycosylation patterns between these molecules. The Mr of the native complex was 420 000 on gradient PAGE (Fig. 4) and 210 000 on nonreducing SDS-PAGE. It seems likely therefore, that ostrich intestinal glycohydrolases exist as dimers of the a2b2 type, as observed in porcine MG [32] and SI [40]. In most other species the Mr 210 000 form of the intestinal glycohydrolases has been found to be an uncleaved precursor of the mature dimeric enzyme. Such forms have been identi®ed using in vitro labelling studies in explants from pigs, rats, humans and rabbits, as well as human cell lines [32, 41±44]. The existence of the uncleaved precursor form in ostrich intestinal extracts is supported by the presence of an Mr 210 000 component (faint band) in reduced samples (Fig. 5). Reducing SDS-PAGE further resolved three subunits of Mr 145 000 (very faint band), 125 000 and 115 000 (Fig. 5). The subunit Mr values for ostrich glycohydrolases correlate with those from rabbit SI [45] and pig MG [46], whose subunits are 120 000 and 140 000, and 125 000 and 135 000, respectively. The presence of the smaller Mr 115 000 subunit has been found in rat [2], human [3] and chicken [4] SI. The two large subunits of rat SI are believed to be the true subunits, while the smaller subunit (Mr 110 000 in rats) appears to be a product of pro-

teolytic processing of the Mr 140 000 subunit. The N-terminal 185 amino acids are cleaved, resulting in the smaller subunit [2]. The sequence obtained for the ostrich Mr 115 000 subunit seems to have homology with a section of porcine MG from Leu13 to Leu19 though. The two larger subunits show extensive N-terminal homology to each other, but none with the Mr 115 000 subunit (Fig. 6), and the three subunits have similar amino acid compositions (Table 3). The N-terminal sequences of all three subunits are very hydrophobic and might be part of a hydrophobic anchor domain. Therefore, the Mr 115 000 subunit seems likely to be the true N-terminal domain of the MG complex and has arisen from cleavage between Glu12 and Leu13 (porcine MG sequence) when papain digestion was used to free the enzyme from membrane vesicles. This would imply that the BBMV were not sealed though, since the N-terminal regions of intestinal SI and MG are cytosolic. It is clear that the subunits reveal signi®cant similarities to MG complexes from other species in their amino acid compositions (Table 3). Considering the near identical behaviour of ostrich SI and MG throughout puri®cation, they will no doubt bear high levels of structural similarities.

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Two thermostable activities (sucrase and glucoamylase) and two thermolabile activities (`maltase' and isomaltase) were observed when heat inactivation was performed (Fig. 7). All four activities denatured rapidly above 608C. The point of denaturation occurred over a very narrow temperature range, since at 588C all four enzymes displayed maximal activity. From the thermostability studies it could be concluded that the four ostrich glycohydrolase activities reside on separate polypeptide chains, since inactivation occurred at di€erent rates. However, it is imperative to recognise the diculty of ascribing activities to subunits in this case, especially in view of the fact that all four subunits hydrolyse maltose. Thus, the decline of `maltase' activity must be considered in the light that it represents total maltose-hydrolysing activity, and not a speci®c subunit. The denaturation of sucrase and isomaltase activities is a true re¯ection of their thermostability though, since these substrates are speci®c for their subunits. The optimal pH of the enzymes spans a broad pH range from 5.5 to 7, with maximal activity being around pH 6.5. Sucrase activity, however, was optimal at pH 5.5. The ostrich ileal MG isolated in this study shows a clear preference for small, linear substrates (maltose and maltotriose), based on declining kcat values for longer substrates such as maltohexaose and maltoheptaose (Table 4). It must be emphasised though, that the catalytic eciency appears to be greater for maltotriose, rather than maltose, even though kcat is greater for maltose hydrolysis. This phenomenon results from a much higher Michaelis constant (Km) for maltose, in contrast to a particularly low Km for maltotriose. The kinetics of maltotriose hydrolysis appears more complex than for other maltooligosaccharides since substrate inhibition was evident with this substrate and to a lesser extent with maltotetraose. Similar ®ndings were observed by Heymann and GuÈnther [34] with human MG. As was found by these authors, hydrolysis of all other maltooligosaccharides followed simple Michaelis±Menten kinetics. In fact, the catalytic eciencies determined for the ostrich ileal MG are nearly identical to that observed by Heymann and GuÈnther [34]. with

only maltose and maltotriose being slightly higher with ostrich MG. Interestingly, for human MG, the kcat value for maltotriose hydrolysis is almost three times that of maltose hydrolysis, which showed the lowest turnover of all the substrates tested. In this study only starch and glycogen hydrolysis was analysed as branched polymers. Both substrates were hydrolysed very slowly, based on their kcat constants, with high enzyme concentrations being required for reliable activity measurements (Table 4). No ratio of kcat to Km could be determined for these substrates though, since the exact substrate molecular mass was not known. Hence, comparisons of catalytic eciency for linear and branched substrates would not be valid. Future studies could analyse the kinetics of branched oligosaccharide substrates to ascertain the in¯uence of branch points on hydrolytic rates. In conclusion, the ostrich intestinal glycohydrolases (SI and MG) are two closely related multisubunit complexes, probably of the a2b2 type, based on the failure of diverse chromatographic techniques in separating the two complexes. A minor form of ileal MG, however, bears signi®cant similarities to intestinal MG isolated from other species when considering its subunit molecular mass and amino acid composition. Future studies would need to focus on the gene structure to come to a better understanding of the physical nature of these enzymes in ostriches.

Acknowledgements The authors gratefully acknowledge the ®nancial support granted by the South African Foundation for Research and Development and the University of Port Elizabeth, and would also like to express their gratitude to the Klein Karoo Agricultural Co-operative at Oudtshoorn, South Africa, for the experimental material.

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