A Fully Active, Two-active-site, Single-chain Sucrase OIsomaltase

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 255, No. 23, Issue of December 10, pp. 11332-11338, 1980 Printed in U. S. A.

A Fully Active, Two-active-site, Single-chain Sucrase OIsomaltase from Pig Small Intestine IMPLICATIONS FOR THE BIOSYNTHESIS OF A MAMMALIAN INTEGRAL STALKED MEMBRANE PROTEIN*

(Received for publication, June 13,1980)

Hans Sjostrom$, Ove Noren$, Leif Christianseng, Hans Wackery, and Giorgio Semenzal From the +Department of Biochemistry C, The Panum Institute, University of Copenhagen, the $Department of Surgical Gastroenterology C, Rigshospitalet, Copenhagen, Denmark and the VLaboratorium fur Biochemie der Eidgemkwischen Technischen Hochschule, Zurich, Switzerland

Detergent-solubilized pig intestinal sucrase isomaltase (EC 3.2.1.48-EC 3.2.1.10) was purified 40 to 100 times with a yield of 10 to 20% by a rapid immunoadsorbent technique. The purified enzyme was shown to be homogeneous by immunoelectrophoresis and was essentially free of other known brush border pepti- dases and disaccharidases.

Intestinal sucrase isomaltase isolated from pigs with intact pancreatic ducts consisted of two polypeptide chains with apparent molecular weights of 140,000 and 150,000, respectively. In contrast, the enzyme isolated from pigs in which the pancreas was completely disconnected from the duodenum 3 days before killing migrated in polyacrylamide gel electrophoresis in dodecyl sulfate as a single polypeptide chain with an apparent molecular weight of 260,000. Treatment with pancreatic proteases in vitro converted the large polypeptide chain into bands with molecular weights equal to or somewhat larger than those of sucrase isomaltase purified from normal pigs. No increase of enzymatic activity could be detected during this transformation.

It is suggested that the single-chain sucrase-isomaltase represents a precursor, which is converted to the final sucrase-isomaltase in vivo by pancreatic proteolytic enzymes. This is one of the few examples in vertebrates of a single polypeptide chain carrying two enzymatically active sites. The significance of the result for the mechanism of the biosynthesis of sucrasemiso- maltase is discussed.

Sucrase - isomaltase, consisting of two polypeptide chains (sucrose-a-glucohydrolase, EC 3.2.1.48; and isomaltase, EC 3.2.1.10), is one of the major integral proteins of the intestinal brush border membrane. The activity of the sucrase polypeptide is generally assayed with sucrose, and that of the isomaltase polypeptide is assayed with palatinose (isomaltu- lose) as the substrate. Both polypeptides are maltases and indeed account for much of the maltase activity of the small intestine, and thus play an important role in the final digestion of starch.

The sucrase-isomaltase complex has been isolated from

* This work was supported by a grant from the Danish Medical Research Council (Project 512-15590). The synthesis of glycyl-L-proline-Cnitroanilide was aided by a grant from the Danish Natural Research Council (Project 511-8065). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

rabbit (1, 2), human (3), and rat (4) small intestine, and has been characterized extensively in a number of properties (for a recent review, see Ref. 5). The rabbit sucrase.isomaltase is anchored to the membrane via a highly hydrophobic segment near the NHz-terminal region of the isomaltase polypeptide (6). The COOH-terminal regions of both polypeptides and the NHn-terminal region of sucrase are not involved in the anchoring to the membrane fabric, and in fact the sucrase polypeptide seems to interact with the membrane via the isomaltase polypeptide only (7).

In order to explain this particular mode of insertion in the membrane, it was suggested on theoretical grounds (6, 8, 9) that sucrase-isomaltase is synthetized and inserted into the membrane as a single, long polypeptide chain, presumably starting with the isomaltase portion. After folding, extracellular proteolysis would split the single-chain pro-sucrase - isomaltase into the final “normal” polypeptide chains.

Indications for the existence of a high molecular weight, single-chain precursor of rat intestinal sucrase .isomaltase have been presented recently (10). In contrast, soluble free sucrase and free isomaltase of smaller molecular weights have been demonstrated (11). Furthermore, in 3H incorporation experiments, the soluble sucrase was more rapidly labeled than was the membrane-bound form. On this basis, it was suggested that these small forms represent intracellular pre- cursors of the final brush border membrane enzyme (11).

We have purified a high molecular weight, single-chain, membrane-bound sucrase - isomaltase in amounts allowing its further chemical characterization from pig small intestinal mucosa not exposed to pancreatic proteolytic enzymes. The results of the present paper have been presented in a prelim- inary form (59-61).

MATERIALS AND METHODS

Chemicals Trypsin (type III), elastase (type I), human transferrin, egg albumin

(grade V), palatinose, and maltose were purchased from Sigma. Mal- tose was purified before use (12). ~-Alanine-4-nitroanilide, lactose, Folin-Ciocalteau’s reagent, a-methylmannoside, and phenylmethanesulfonyl fluoride (PhCHZSOzF) were obtained from Merck (Darms- tadt, F. R. G.). /?-Galactosidase (Escherichia coli) was delivered by Boehringer. Concanavalin A-Sepharose 4B and molecular weight markers (HMW Calibration Kit) were purchased from Pharmacia. Agarose (type HSA) was obtained from Litex (Glostrup, Denmark), Triton X-100 was from Roth (Karlsruhe, F. R. G.) , bovine serum albumin was from Armour, incomplete Freund‘s adjuvant was from Statens Seruminstitut (Copenhagen, Denmark), GLOX (glucose assay kit) was from Kabi Diagnostica (Stockholm, Sweden), and sucrose was from BDH. Glycyl-~-proline-4-nitroanilide was synthesized by the Institute of Protein Chemistry (Hprsholm, Denmark). Chymo-

11332

Two-active-site, Single-chain Sucrase. Isomaltase

trypsin and aprotinin (Kunitz trypsin inhibitor) were kind gifts from Novo (BagsvEerd, Denmark).

All other chemicals were of analytical grade and the water was purified by a Meg-0-Lite ZD-30.230.00 system from Millipore.

Assays Sucrase, palatinase, maltase, and lactase activities were measured

using either sucrose, palatinose, maltose, or lactose as substrates principally according to Dahlqvist (13). The liberated glucose was quantified by a glucose reagent kit, GLOX.’

The activities of microvillus aminopeptidase (EC 3.4.11.2) and dipeptidyl peptidase IV (EC 3.4.14.-) were determined spectropho- tometrically as described previously (14) using ~-alanine-4-nitroanilide and glycyl-~-proline-4-nitroanilide, respectively, as substrates.

The amount of enzyme activity measured under the assay conditions is expressed in katals according to the recommendations of the Commission on Biochemical Nomenclature.

Protein concentration was determined according to Wang and Smith (15) using crystalline bovine serum albumin as a standard.

Source of Enzyme Pig small intestines were kindly delivered by the Department of

Experimental Pathology (Rigshospitalet, Copenhagen, Denmark). The intestine was taken out, washed, and frozen as described previously (14). Pieces of ileum were used, and 2.8 mg of aprotinin per liter were added to all solutions.

Disconnection of Pancreas The abdomen was opened with a median line incision. The pancreas

was completely dissected from the duodenum. The major pancreatic duct was ligated and cut. This procedure secured a total disconnection between the pancreas and the duodenum. At the end of the operation, the duodenum had normal color and peristalsis. After the operation, the pig was nourished with 5% glucose and the fluid balance was maintained with 0.9% saline solution intravenously. The pig was given 4 g of ampicillin per day. Reoperation after 70 h revealed a normal duodenum. The pig was bled to death and the intestine was removed and treated as described previously (14). For this investigation, intestines from two pigs, operated upon at different times, were used.

Preparation and Solubilization of a Microvillus Membrane Fraction

t i d y according to the Ca2+ precipitation method (16) as modified by The preparation of microvillus membranes was performed essen-

Kessler et al. (17). All buffers contained 2.8 mg of aprotinin per liter. Intestinal pieces (150 g), equivalent to approximately 2.5 m of pig small intestine, were used. The prepared membrane fraction was washed in 30 rnl of a 50 mM Tris-HC1 buffer, pH 8.0, followed by centrifugation at 48,000 X g for 1 h. To solubilize the proteins, the sediment was suspended in 115 ml of the same buffer containing 1% Triton X-100 and stirred gently for 1 h at 4°C. Finally, the suspension was centrifuged at 48,000 X g for 1 h and the supernatant (solubilized microviuus fraction) was used for immunization and further purification.

Immunization and Isolation of the Immunoglobulin Fraction The antigen was mixed with an equal volume of incomplete

Freund’s adjuvant. Over a period of 6 weeks, 200 pg of the solubilized microvillus fraction in 100 pl or 20 pg of purified sucrase.isomaltase in 300 p1 was injected intracutaneously at intervals of 2 weeks (18). One week after the last injection, the rabbits were bled for 40 ml. The immunizations were continued with booster injections every 6th week, 40 rnl of blood was collected after each injection, and the immuno- globulins were isolated (19).

Preparation of Immunoglobulin-Sepharose The prepared immunoglobulin was coupled to Sepharose 4B by a

cyanogen bromide method (20) using 0.2 M sodium citrate buffer, pH 6.5, as coupling buffer. The immunoglobulin concentration was about 15 mg/ml and the ratio of the volume of the antibody solution to the packed Sepharose was 1:l. Based on the absorbance at 280 nm before and after the coupling step, the amount of immunoglobulin bound to the Sepharose was about 90% of the initial amount.

’ N.-G. Asp, personal communication.

Solubilized microvillus fraction

Affiity chromatography on 1

Sephadex G-200 1

Antigen I

Antiserum I

Immunoadsorbent I

.L

1

I

11333

Solubilized microvillus fraction

Antiaminopeptidase-Sepharose 1

chromatography 1

Concanavalin A-Sepharose chromatography

1 Antiaminopeptidase-Sepharose

chromatography 1

raphy 1

1

Immunoadsorbent I chromatog-

Sucrase. isomaltase

Anti-sucrase isomaltase antise- Turn

1 Anti-sucrase-isomaltase-Sepha-

rose

SCHEME 1. Preparation of anti-sucrase-isomaltase-Sepha- rose.

Preparation of Anti-sucrase. isomaltase Antiserum The preparation of a specific anti-sucrase . isomaltase antiserum is

summarized in Scheme 1. The amphiphilic form of pig intestinal sucrase. isomaltase was purified by aftinity chromatography on Seph- adex G-200 (1). The antiserum (Antiserum I) against this preparation, which was not totally specific, was immobilized on Sepharose as an immunoadsorbent in combination with other chromatographic steps. The purification procedure for sucrase.isomaltase for the second immunization was as follows. Microvillus aminopeptidase was removed from a solubilized microvillus fraction by chromatography on a column of antiaminopeptidase-Sepharose (14). The unadsorbed material was further purified by chromatography on a column of concanavalin A-Sepharose as described earlier (21). The eluate contained mainly sucrase. isomaltase and traces of microvillus aminopeptidase. The latter was totally removed by chromatography on the antiaminopeptidase-Sepharose (14). The final purification was achieved by immunoadsorption to a column of Sepharose to which Antiserum I had been coupled. Sucrase a isomaltase was eluted with 2 mM Tris-HC1 buffer, pH 8.0, containing 0.1% Triton X-100 and used for the preparation of anti-sucrase. isomaltase antiserum.

Purification of Sucrase. Isomaltase Unless stated otherwise all buffers contained 0.1% Triton X-100

and 2.8 mg/liter of aprotinin, and the procedures were performed at 4°C.

A solubilized microvillus fraction was applied to a column (1.2 X 4.8 cm) of the anti-sucrase. isomaltase-Sepharose equilibrated with 50 nm Tris-HC1 buffer, pH 8.0, containing 0.15 M NaCI. The flow rate was 5 ml X cm-’ X h-’ and fractions of 5 ml were collected. After washing with 100 ml of the equilibration buffer, the sucrase.isomaltase was eluted at room temperature with 2 mM Tris-HC1 buffer, pH 8.0 (immunoadsorbent eluate).

Treatment with Proteolytic Enzymes Prior to Polyacrylamide Gel Electrophoresis-The immunoad-

sorbent eluate (sucrase activity, 35 pkat X liter”) was dialyzed against 0.1 M sodium phosphate buffer, pH 7.2, containing 0.1% Triton X-100. Trypsin, chymotrypsin, or elastase (0.5 mg/rnl, final concentration) were added to aliquots of this sample and the mixture was incubated at 37°C. After suitable intervals (0 to 3 h), samples were withdrawn and boiled immediately for 5 min at 100OC.

Prior to Sucrase or Palatinase Assays-To the immunoadsorbent eluate dialyzed against 0.025 M sodium phosphate buffer, pH 8.0, containing 0.1% Triton X-100 (sucrase activity, 2 pkat X liter” for sucrase and 10 pkat X liter” for palatinase activity studies), trypsin, chymotrypsin, or elastase was added and the mixture was incubated at 37°C. After suitable intervals (0 to 60 min), aliquots of 10 pl were withdrawn and 1 p1 of 2.8 mg X ml” of aprotinii (trypsin) or 1 4 40 m~ phenylmethanesulfonyl fluoride in 2-butanol (chymotrypsin and elastase) was added. Sucrase and palatinase activities were determined after adding 10 pl of the appropriate substrate (final concentration, 28 m).

11334 Two-active-site, Single-chain Sucrase - Isomaltase

Immunoelectrophoresis Electroimmunoassay (22) and fused rocket immunoelectrophoresis

(23) were performed in 0.038 M Tris, 0.1 M glycine buffer, pH 8.7, containing 0.1% Triton X-100 (24). The electrophoresis was run overnight a t 2 V X cm". Enzymatic staining of the sucrase and palatinase precipitate was performed as described previously (25) using sucrose or palatinose as substrate.

Polyacrylamide Gel Electrophoresis in Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis was performed in tubes using a

gel concentration of 5% (26) for molecular weight estimations up to 180,000. Higher molecular weights were estimated from electrophoresis on glass plates using a gel gradient between 4 and 10% (27). Before the electrophoresis, sodium dodecyl sulfate (final concentration, 1.4%). 2-mercaptoethanol (final concentration, 4%), bromphenol blue (final concentration, 0.01%). and glycerol (final concentration, 75%), were added to all samples, which were then boiled for 5 min. As standards, thyroglobulin subunit (M, = 330,000), femtin subunit (220,000), P-galactosidase subunit (13O,OOO), transferrin (76,000). serum albumin (67,000), catalase subunit (60,000). ovalbumin (43,OOO), and lactate dehydrogenase (36.000) were used.

RESULTS

Preparation of Anti-sucrase. isomaltase Antiserum The purification of sucrase. isomaltase for immunization

purposes resulted in a preparation essentially free from other brush border hydrolases. A specific activity of 185 mkat x kg of protein" with an overall yield of 9% was obtained. This rather low yield was probably related to the many purification steps.

Fig. 1 demonstrates the specificity of the anti-sucrase-isomaltase antiserum obtained. In this experiment, a solubilized microvillus fraction was used as antigen. Only one precipitate was seen by protein staining. It was also stained histochemi- cally when sucrose or palatinose was used as substrate. It can thus be concluded that the prepared antiserum is highly specific against sucrase isomaltase.

Purification of Sucrase. Isomaltase from Normal Pigs After immobilization on Sepharose, the anti-sucrase. iso-

maltase antiserum was used for a one-step purification of

FIG. 1. Rocket immunoelectrophoresis: a solubilized micro- vi l lus f ract ion against the sucrase a isomaltase antiserum. Ten microliters of a solubilized microvillus fraction containing 300 pkat of sucrase.isomaltase were applied and run against the sucrase.isomaltase antiserum prepared.

EFFLUENT(ml1 FIG. 2. Elution of sucrase- isomaltase of a norma l pig (0) or

pig I1 with disconnected pancreatic ducts (0) from a column of anti-sucrase-isomaltase-Sepharose.

TABLE I Purification ofpig intestinal sucrase. isomaltase

Figures are given for 150 g of intestine (wet weight). Pig I with discon- Pig I1 with dis- nected pancreatic connected pan-

Fraction Normal pig ducts" creatic ducts

Total Specific Total Specific Total Specifc activity activity activity activity activity activity

mkat X mkat x mkat X pkal k g p y pkal kgprq- @at kgpro-

tein tein- tein"

Homogenate 7.09 1.76 1.87 0.873 10.5 3.36 Solubilized 3.07 19.4 0.437 14.6 4.81 33.3

microvillus fraction

sorbent eluate "The small intestine of this pig had been stored a t -20°C for 2

Immunoad- 1.19 155.6 0.165 83.7 1.85 142.1

years prior to purification.

sucrase isomaltase from a solubilized microvillus fraction. As seen from Fig. 2 and Table I (normal pig), the activity could be eluted as a sharp peak with acceptable recovery (32%) using 2 m~ Tris-HC1 buffer, pH 8.0, containing 0.1% Triton X-100. This principle has been used in our laboratory earlier for the purification of dipeptidyl peptidase IV (28) and microvillus aminopeptidase (14). The total purification factor was 88 and the total recovery of the same magnitude was as has been reported earlier for the purification of microvillus pep- tidases (14, 28).

The purity of the preparation was high as judged from its negligible contamination of other known brush border enzymes (Table 11, normal pig). The activities on maltose and palatinose relative to that on sucrose are of the same magnitude as has been described for rabbit sucrase. isomaltase (29, 30).

In the immunoelectrophoresis against a microvillus membrane antiserum, only one precipitate was conspicuous (Fig. 3, right arch). This precipitate stained for both sucrase and palatinase activities.

In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (Fig. 4, S I ) , three bands corresponding to molecular weights of 140,000, 150,000, and 260,000 were seen. The purified Triton-solubilized rabbit sucrase. isomaltase displayed two bands in a corresponding polyacrylamide gel electrophoresis which were assigned to sucrase and iso- matlase polypeptides with molecular weights of 140,000 and

Two-active-site, Single-chain Sucrase. Isomaltase

TABLE I1 Activity of the different preparations against typical substrates for

microvillus enzymes Figures are given as a percentage of the activity (kat X liter")

against sucrose.

Substrate Pig I with dis- Pig I1 with dis-

Normal Din connected Dan- connected oan- . - creatic ducts creatic ducts

R acfiuify D-Sucrose 100 100 100 D-Palatinose 19 9 10 D-Maltose 260 210 250 D-hctose 0.4 0.1 0.1 ~-Alanine-4-nitroani- 4.0 0.8 1.6

Glycyl-~-proline-4- 0.5 0.6 0.1 lide

nitroanilide

FIG. 3. Fused rocket immunoelectrophoresis of normal and single-chain sucrase-isomaltase. Sucrase. isomaltase (40 pkat) from an ordinary pig in 10 pI was applied in the left well and 90 pkat of sucrase.isomaltase from a pig with disconnected pancreatic ducts in 10 1.11 was applied in the right well. The plate was allowed to diffuse for 1 h before it was run against a microvillus membrane antiserum.

160,000, respectively (7). It is probable that the pig polypeptides with molecular weights of 140,000 and 150,000 are sucrase and isomaltase, respectively, but this point was not investi- gated further. The polypeptide with a molecular weight of 260,000 may be either a noncovalent aggregate of sucrase and isomaltase or trace amounts of a possible precursor (see be- low).

From the enzymatic analyses, immunoelectrophoresis, and polyacrylamide gel electrophoresis, it could be concluded that the prepared immunoadsorbent is an efficient and specific tool for the rapid purification of the pig sucrase - isomaltase.

Purification and Characterization of Sucrase Zsomaltase from Pigs with Disconnected Pancreatic Ducts

The elution profile of the immunoadsorbent chromatography is shown in Fig. 2, and Table I shows the results of the purification. The total purification factors were 95 and 41, and the total yields were 9 and 18%. The preparation was pure as judged by immunoelectrophoresis (Fig. 3, left arch) and essentially free from other known brush border enzymes (Table 11, pigs I and I1 with disconnected pancreatic ducts). In

11335

260 000 1 a0 000 165 000 150 000 140 000

c3"h' 2h 3h Ela Ela

FIG. 4. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the two preparations and the single-chain sucrase isomaltase after treatment with different proteolytic enzymes. About 10 pg of protein were applied to each tube except for the normal sucrase.isomaltase, where 20 pg were applied. SZ, sucrase. isomaltase; proSZ, single-chain sucrase. isomaltase; Try, trypsin; Chy, chymotrypsin; Ela, elastase.

polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, one band with an apparent molecular weight of about 260,000 was highly prominent (Fig. 4, pro-SI).

Apart from the molecular weight, no significant difference between the results of the purification of sucrase-isomaltase from normal pigs and from pigs with disconnected pancreatic ducts was detected: the elution patterns (Fig. 2) from the immunoadsorbent chromatographies were very similar; there were no striking differences in behavior during the purification procedure (Table I); and the ratios between sucrase, maltase, and palatinase activities were essentially the same (Table 11). The most important evidence for the sucrase- isomaltase identity of the high molecular weight polypeptide came from the immunological comparison (Fig. 3). Using a polyvalent antiserum against microvillus membrane proteins, total immunological identity (31) was obtained between high molecular weight polypeptide and normal sucrase - isomaltase. Further- more, all parts of the fused precipitate stained with the same intensity after histochemical staining using sucrose or palatinose as substrate. We thus conclude that the sucrase-isomaltase from the brush border membrane of those pigs in which the pancreas was disconnected from the duodenum (and in which the villus cells, whose life is limited to 2 to 3 days, which thus were never exposed to pancreatic juice) consists of a single polypeptide chain.

Treatment of the High Molecular Weight Form with Pancreatic Endopeptidases

Changes in Molecular Weight-The results on changes in molecular weight after treatment with proteolytic enzymes are shown in Fig. 4. In all experiments, bands appeared in the region of the normal sucrose-isomaltase. The exact pattern and position of the normal sucrase.isomaltase could not be produced, as the new bands often migrated somewhat slower. A somewhat larger band (Mr = 165,000) was conspicuous in almost all experiments.

Changes in Enzymatic Activity-The sucrase activity of the small intestinal homogenates from pancreas-disconnected pigs was comparable to that of normal pigs (Table I) as were also the specific activities of the different purified preparations. There is thus no indication of altered specific activity of the precursor.

In rocket immunoelectrophoresis, the ratio between the area under the sucrase precipitate and the amount of enzy-

11336 Two-active-site, Single-chain Sucrase -Isomaltase

a b

FIG. 5. The effects of different pancreatic proteolytic enzymes on the activity of the single-chain sucrase.isomaltase. a, sucrase activity: 0, trypsin, 0.005 mg X rn"; 0, trypsin, 0.25 mg X rn"; U, elastase, 0.1 mg x rn-I; A, chymotrypsin, 0.1 mg X IC'. 6, palatinase activity: 0, trypsin, 0.1 mg X I&'; W, elastase, 0.1 mg X d"; A, chy-

0 3 Ln 6

LL motrypsin, 0.1 mg X rn". 0 20 LO 0 20 LO 60

0

TIME (rnin) TIME(min1

matic activity applied of single-chain sucrase. isomaltase was similar to that of normal sucrase isomaltase.

Treatment of the single-chain sucrase. isomaltase with pancreatic endopeptidases (Fig. 5) never produced any increase in either sucrase or palatinase activity (much lower amounts of proteases than in the experiments of Fig. 5 were also tested). Rather, with the exception of elastase on sucrase activity, most treatments led to a decrease in enzymatic activity. In conclusion, we have no indication that the single-chain sucrase isomaltase has alowerspecific activity than thenormalsucrase - isomaltase.

DISCUSSION

The data presented here can be summarized as follows. 1. The small intestine of pigs in which the pancreas was

disconnected from the duodenum a few days prior to killing contains a brush border-bound sucrase. isomaltase which has the same catalytic and immunological properties as those of the sucrase-isomaltase isolated from pigs with intact pancreatic ducts. However, the sucrase - isomaltase from operated animals differs in one important respect from that of the intact animals: it is composed of a single, long polypeptide chain with an apparent molecular weight of approximately 260,000. The sucrase. isomaltase from normal pigs is composed of two polypeptide chains with apparent molecular weights of 140,000 and 150,000, respectively.

2. Appropriate treatment of sucrase.isomaltase from the operated animals with pancreatic proteases in vitro leads to the appearance of bands with molecular weights similar to or somewhat larger than those of the two polypeptides of normal sucrase. isomaltase. The differences in band pattern between the proteolytically treated single-chain sucrase isomaltase and the normal enzyme may be caused by different conditions in vivo and in vitro such as a possible cooperation between different proteases in uiuo, including also, e.g., pancreatic and intestinal exopeptidases. In spite of this, the conclusion seems warranted that the sucrase-isomaltase of normal animals arises from an enzymatically active protein consisting of a single polypeptide chain.

The proteolytic conversion does not seem to lead to drastic changes in the secondary or tertiary structure (and thus in the major interactions between the two polypeptides), since it is not accompanied by any drastic change in enzymatic or immunological properties. Furthermore, although not quite identical, the fragmentation patterns obtained with the three endopeptidases are similar (Fig. 4). Since the specificities of the three proteases are quite different, this observation indicated that they are all attacking a similar exposed area of the folded polypeptide chain. This implies that the single polypeptide chain is folded in such a way as to form at least two

domains, presumably one corresponding to the isomaltase and the other corresponding to the sucrase subunit. Our data, therefore, agree with the independent observa-

tions by Hauri et a1.' that subcutaneous transplants of small intestine from fetal rats into adult rats produce a fast fucose- labeled high molecular weight protein which is immunologi- cally similar to sucrase-isomaltase. By treatment with elastase, it is converted into two bands with nearly the same apparent molecular weight values as the normal sucrase. isomaltase polypeptides.

The essentially identical specific activities and substrate specificities of the single-chain sucrase e isomaltase and of the two-chain sucrase a isomaltase (Tables I and 11) make it very likely that sucrase and palatinase activities in single-chain sucrase.isomaltase are due to two independent sites, as they are in the two-chain enzyme (29). This conclusion, moreover, is borne out directly by the observations of Fig. 5: in single- chain sucrase * isomaltase, sucrase and palatinase activities are sensitive to a different degree to the action of some of the proteases used (compare particularly the effect of elastase on sucrase and palatinase activities. Fig. 5, a and b) . Sucrose and palatinose must clearly be split at different sites. It therefore follows that single-chain sucrase - isomaltase carries two enzymatically active sites on the same polypeptide chain. Ex- amples of this situation are exceptionally rare in vertebrates (e.g. Refs. 32-38). Moreover, the case of single-chain sucrase. isomaltase, contrary to the examples in the above references, is almost unique, in that the two enzyme activities are nearly identical. The only other case known to us is that of sea lion isomaltase (39); which is composed of a single polypeptide chain carrying two identical (at least at the present state of knowledge) active sites.

Pancreatic Proteases and Intestinal Sucrase. Isomal- tase-Our data are not at variance with those of Alpers and Tedesco (40). Working with rats with only the major pancreatic duct ligated (i.e. with still some 5% of the pancreatic juice flowing into the intestinal lumen), these authors found a sucrase-isomaltase complex composed of two polypeptide chains but having a slower turnover rate than the enzyme of nonoperated rats. Pancreatic elastase was found to solubilize sucrase. isomaltase from the brush border membrane and to cause some degradation (40). These results indicated that pancreatic elastase may play a role in the degradation of sucrase. isomaltase.

It may be that the single-chain sucraseeisomaltase is the most active form and that the sucrase-isomaltase normally

* H.-P. Hauri, A. Quaroni, and K. Isselbacher, manuscript in prep-

H. Wacker, R. Aggeler, N. Kretchmer, and G. Semenza, manu- aration.

script in preparation.

Two-active-site, Single-chain Sucrase. Isomaltase 11337

isolated represents the first step in the catabolism of the enzyme. The possibility of a postmortem proteolysis of the one polypeptide chain sucrase - isomaltase during the isolation of normal sucrase. isomaltase might also be considered.

We suggest that the pancreatic proteases play dual role in the turnover of small intestinal brush border sucrase-isomaltase: they catalyze the transformation of the single-chain sucrase. isomaltase into the final sucrase. isomaltase and they initiate the fist steps of its degradation.

In analogy with this view, and until there are more definite experiments on this problem, we wish to suggest the name pro-sucrase. isomaltase for the single-chain sucrase. isomaltase which is present in the brush border membrane prior to proteolytic conversion. This is in analogy with proinsulin (41), which has essentially equal activity as insulin and with other zymogens (42), which have an albeit small biological activity.

Physiological Importance-There are some clinical indications that pancreatic proteolytic enzymes are of importance for proper intestinal function. Thus, Berg et al. (43) recently described morphological and enzymatic abnormalities in intestinal mucosa of both cystic fibrosis and Shwachman syn- drome patients and suggested that exocrine pancreatic insuf- ficiency predisposes to the development of small intestinal dysfunction. The results of this paper and our earlier results on the connection between trypsin and the quaternary stmc- ture of the microvillus aminopeptidase (14) favors this sug- gestion.

Biosynthetic Implications-In nearly all normal mammals studied thus far, the sucrase - isomaltase of the small intestinal brush border is composed of two polypeptides of similar sue. Most of the information available refers to the enzyme from rabbit small intestine, but it can probably be extended to the enzyme from most other species (for species distribution of sucrase and isomaltase activities, see Ref. 44). The sucrase and isomaltase polypeptides have largely overlapping substrate specificities; they are inhibited fully competitively by the same compounds (e.g. Tris (45, 46), lanthanides: nojiri- mycin, deoxynojirimycin (47), and D-glucose (46, 48)); both are activated by Na’ (49); both are inactivated by the same affinity label (50); and both have identical amino acid sequences in the active sites (51), at least judged from the (limited) information available. In addition, they are subjected to the same or to related biological control mechanism(s), as they are usually simultaneously absent in sucrase. isomaltase deficiency (52, 53), by their simultaneous appearance in development (54, 55), and by the constancy of the sucrase, isomaltase activity ratios in samples of human intestine (56). Thus, the conclusion seems warranted that sucrase and isomaltase (i) have sizeable homology and (ii) have arisen in phylogenesis by gene duplication.

One more important fact must be considered. Of the two polypeptides of the enzyme, isomaltase alone interacts with the membrane fabric (7), a highly hydrophobic segment at the NH2-terminal region being involved (6). Sucrase apparently has a more peripheral position, and interacts with the membrane primarily or only via the isomaltase polypeptide (7). The COOH-terminal regions of both polypeptides and the NH2-terminal region of sucrase are located at the luminal, extracellular side of the membrane (7), and it seems probable (6) that so is the NHderminal part of isomaltase.

In order to accomodate all these observations into a single framework, the “one polypeptide-chain-precursor” hypothesis has been put forward (6-9) which can be formulated as follows: (i) sucrase and isomaltase have arisen by gene duplication; (ii) the two genes give rise to a single two-cistron mRNA (via

P. Vanni, H. Braun, and G. Semenza, manuscript in preparation.

mRNA splicing or because the two cistrons are still close to one another with no stop signal in between); (iii) translation of this long mRNA gives rise to a single-chain pro-sucrase- isomaltase; and (iv) extra- or intracellular proteolytic process- ing of pro-sucrasevisomaltase leads to the final two-polypeptide sucrase. isomaltase. The data presented above provide direct evidence to point (iv) and make point (iii) likely, although its final proof must await in vitro translation experiments. Points (i) and (ii) are made likely by the similarities between sucrase and isomaltase which were quoted at the beginning of this section, and also by the existence of a high molecular weight isomaltase (a “double isomaltase?”) in some cases of sucrase. isomaltase malabsorption (57) and in the sea lion (39).3 Also, pig intestinal maltase, recently purified, occurs as one long polypeptide chain, which may be split into two polypeptides of similar size.‘

It is tempting to suggest a similar synthesis mechanism for other brush border hydrolases of about the same molecular weight and quaternary structure (pig microvillus aminopeptidase, 2 X 162,000, though often further degraded (14); pig dipeptidyl peptidase IV, 2 X 137,000 (28)). However, experiments parallel to those described in this paper using the specific antiaminopeptidase-Sepharose (14) or the specific anti-dipeptidyl-peptidase IV-Sepharose (28) for purification failed to give any indication of a similar mechanism in the case of microvillus aminopeptidase or dipeptidyl peptidase IV.

The sequence of segments in the pro-sucrase. isomaltase polypeptide chain and its mode of insertion in the membrane fabric are the object of present and future investigation. Both sequences are equally likely: (i) N-(hydrophobic)-isomaltase- ?-N-sucrase-C; and (ii) N-sucrase-?-N-(hydrophobic)-isomaltase-C. Sequence (i) may (but need not) make a pre-piece unnecessary, the hydrophobic segment in the NH2-terminal region of isomaltase possibly acting as a “signal” (6, 8, 9; for the signal hypothesis, see Refs. 27 and 58). In sequence (ii), the hydrophobic segment may be of importance in anchoring the pro-sucrase isomaltase chain approximately in its middle at the membrane. It is possible that the somewhat larger polypeptides obtained by in vitro proteolysis are explained by the presence of signal or anchor sequences unnecessary for the final enzyme.

Acknowledgments-We are indebted to Ms. Jette Hauberg Nielsen and Ms. Jette Mgller for skillful technical assistance.

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A Fully Active, Two-active-site, Single-chain Sucrase OIsomaltase