Postinsertional processing of sucrase-a-dextrinase precursor to authentic subunits: multiple step cleavage by trypsin GARY L. SHAPIRO, SETH LAWRENCE A. SCHEVING,

D. BULOW, KENNETH AND GARY M. GRAY

A. CONKLIN,

Division of Gastroenterology, Department of Medicine and Digestive Disease Center, Stanford University School of Medicine, Stanford, California 94305

SHAPIRO, GARY L., SETH D. BULOW, KENNETH A. CONKLIN, LAWRENCE A. SCHEVING, AND GARY M. GRAY. PostinsertionaZ processing of sucrase-a-dextrinase precursor to authentic subunits: multiple step cleavage by trypsin. Am. J. Physiol. 261

(Gastrointest. Liver Physiol. 24): G847-G857, 1991.-Sucrasear-dextrinase,a hybrid digestive carbohydraseof the intestinal brush border, is initially synthesized and transported to the surface membraneas a single-chain glycoprotein, P, which is then cleaved to cy-and P-subunits,presumably by one or more pancreatic proteases.However, efforts to convert P under controlled conditions to authentic cyand ,0have been unsuccessful. Sucrase-dextrinase immunoprecipitates from rats intraintestinally labeled with [3H]leucine or [35S]methionine without presenceof biliary-pancreatic secretionsrevealed only the 230kDa P precursor. Restoration of intestinal flow converted the brush border P to the cy-(140 kDa) and ,8- (125 kDa) subunits. Biliary plus pancreatic secretions facilitated this postinsertional cleavage, but bile alone played no role in conversion. When isolated brush borders, prelabeledin vivo, were exposed to a mixture of pancreatic proteasesat physiological concentrations, P was converted to authentic a! and p, but only trypsin was responsiblefor the conversion. Kinetic analysis in prelabeled isolatedbrush-border vesiclesrevealedthe appearanceof severalintermediate species(205-145 kDa) produced either by endogenousmembraneproteasesor by trypsin itself. Reconstituted duodenal luminal contents yielded a fragmentation pattern identical to that produced by trypsin alone. Trypsin was necessary and sufficient for processing of the intermediate precursorsto the final authentic cy-and ,&subunits. Based on the a-to-p radioactivity ratio and the known amino acid composition of the subunits, differential cleavage occurred with relatively greater production of the P-subunit (a-to-@ molar ratio = 0.77). The conversion of P to the cy-and ,&units, rather than occurring in a single step after membrane insertion, is differentially catalyzed by trypsin trimming to unequal amounts of the subunitsinvolving a complex seriesof cleavage steps. intestinal; membranehydrolase; ar-glucosidase; sucrase-isomaltase;posttranslational processing;pancreatic protease;trypsin; brush border

(S-D), a hybrid hydrolase of the intestinal brush border essential for the surface digestion of oligosaccharide nutrients (9)) is initially synthesized in association with the endoplasmic reticulum and Golgi as a large glycoprotein precursor called pro-sucrase-cudextrinase (P) (10) . After its insertion into the brushSUCRASE-CPDEXTRINASE

0193-1857/91

$1.50 Copyright

border membrane, P is eventually converted to two major noncovalently associated subunits, one carrying an active site for sucrase and the other for dextrinase. Although a single cleavage by luminal pancreatic elastase has been assumed, the combined mass of the cy- (-140 kDa) and p- (-125 kDa) subunits produced in the intact animal is 20-30 kDa greater than that of the P precursor (-230 kDa) (13, 16, 17, 20), and it has not been possible to convert P to authentic CYand p in vitro. (Because the assignment of sucrase or dextrinase active sites to a particular postinsertional subunit is uncertain (4, 6, 7, 20), we have designated the putative subunits according to their relative migration on sodium dodecyl sulfate (SDS) acrylamide electrophoresis: CY,slowest migrating, and ,8, faster migrating. A third unit, y, also commonly identified, is the most rapidly migrating species). In studies in the intact rat, Hauri et al. (10) identified P in Golgi membranes 15 min after administering [3H]fucose intravenously and found the single-chain form at low levels transiently in brush border at l-3 h. Yet the small quantity of newly synthesized P in brush border did not account for the much greater amount of the radioactive S-D hybrid present in that surface membrane even at early periods (15 min) after the pulse. Although this finding might indicate a very rapid conversion of P to its subunits after brush-border insertion, exposure of isolated brush-border membranes to purified elastase did not yield the authentic S-D doublet on SDS electrophoresis, the more slowly migrating upper or a band localizing to a lower position than the authentic mature subunit (10). Similarly, fetal rat intestine that was transplanted and allowed to mature free of pancreatic secretions beneath the skin of an adult animal produced the P precursor that was cleaved by elastase to a doublet whose larger component migrated faster than that of the authentic S-D hybrid (15). Labeling of human intestine biopsy specimens with [35S]methionine in vitro produced a 245-kDa single-chain species of S-D, which was converted to a l45- to 130-kDa doublet by trypsin but not by other pancreatic proteases (16). However, there was no direct comparison with the native doublet of the mature S-D hybrid in that study. When pancreatic ducts were ligated in the pig, S-D was isolated from brushborder membranes as the large single-chain form possessing full hydrolytic activity (17). Yet treatment of this purified P with pancreatic proteases rapidly inactivated

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both active sites and cleaved it to one or more products that were larger on SDS gels than the authentic subunits (17) Because in no instance has the putative P precursor been shown to be converted to the authentic postinsertional CY-and ,&subunits, there is considerable doubt about the mechanism of postinsertional processing of the P precursor. Besides luminal pancreatic proteases, other luminal factors such as bile acids or integral brush-border proteases may be required for proper physiological cleavage. We have established a model in the intact rat in which newly synthesized P, radiolabeled with amino acid precursors, is synthesized and transferred to the luminal surface membrane still in the single-chain form. This has allowed detailed analysis of the intraluminal factors required for the conversion of the P to the hybrid S-D doublet after its transfer to the brush-border membrane both in the intact rat in vivo and in isolated brush-border membranes in vitro. We now find that luminal trypsin is the essential luminal protease for postinsertional P processing, which appears to involve a series of trimming steps yielding either the cy or p mature products, usually in unequal amounts. EXPERIMENTAL

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Intestinal perfusion experiments. Male Sprague-Dawley rats (250-350 g) were housed in a central animal facility and given regular rat food ad libitum until 16 h prior to the experiment when only water was allowed. For intraintestinal labeling with radioactive amino acid, the animal was anesthetized with pentobarbital sodium (50 mg/kg) intraperitoneally and a jejunal loop was prepared with catheters placed at the duodenal-jejunal junction and 40 cm distally as previously described (18). In some animals, a Swan-Ganz type catheter (model 93116-4F, Edwards Laboratories, Santa Ana, CA) with an inflatable balloon near the inflow tip was passed through the wall of the distal stomach and advanced to the duodenal-jejunal junction to permit temporary occlusion of the lumen proximal to the infusion site. The isolated loop was rinsed gently with 145 mM NaCl at 37°C to remove residual debris, and 5 mCi of [3H]leucine (50-65 Ci/pmol) or 1 mCi of [35S]methionine (~800 Ci/mmol) in 5 ml of 145 mM NaCl was instilled in the loop for 5 min. After the radiolabeled pulse, the amino acid was chased by flushing the loop with 1 mM nonradioactive amino acid and 145 mM NaCl and by perfusing for an additional 20 min with the same solution at 1 ml/min. The perfusion was then continued with 145 mM NaCl without the amino acid for varying periods, depending upon the individual experiment. Under these conditions, the vast majority of radiolabeled S-D was in the doublechain cu-p hybrid form. However, after preliminary experiments revealed that intraintestinal perfusion with 145 mM NaCl at 1 ml/min for 20 min prior to the pulsechase produced maximal incorporation of the [35S]methionine into the single-chain form of S-D, a ZO-min preperfusion with saline was used in most of the experiments examining the fate of the S-D precursor. After the

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appropriate chase period, the distal half (20 cm length) of the intestinal segment was removed, placed in 145 mM sodium chloride at 4”C, and rapidly processed as described below; this was the untreated or control segment. The residual half of the in vivo segment (test segment) was then exposed to endogenous pancreatic and biliary secretions or perfused with mixtures of pure pancreatic proteases for the periods specified in individual experiments. In some experiments, the proximal half of the perfused segment served as the control segment, and the distal 20 cm was the test segment. Preliminary experiments revealed no differences in sucrase activity, radiolabeled amino acid incorporation into protein, or immunoprecipitated S-D in the paired half-segments. The in vivo rat studies shown in Figs. l-3 and Table 1 each represent an experiment in a single rat with an adjacent jejunal loop serving as the control. Preparation of intestinal mucosa and brush-border vesicles. After the in vivo experiment, intestinal loops were placed directly into chilled 145 mM NaCl and processed

at 4°C. The loop was rinsed with 1 mM dithiothrietol (DTT) and 145 mM NaCl, cut longitudinally, and the mucosa was removed from the underlying serosa by scraping with glass microslides. The mucosa was weighed and homogenized in 2 mM tris(hydroxymethyl)aminomethane (Tris) and 50 mM mannitol, pH 7.5, with a Polytron (model PTlO, Brinkman) at setting no. 4 using two bursts of 30 s each. This procedure left no intact cells as monitored by phase-contrast microscopy. Brushborder vesicles were prepared by modification of the calcium chloride method. Solid calcium chloride was added to the homogenate to achieve a final concentration of 10 mM, and, after incubation at 4°C for 1 h, the treated homogenate was centrifuged at 3,000 g for 15 min. The supernatant containing brush-border vesicles was centrifuged at 27,000 g for 30 min, and the resulting pellet was suspended in 10 mM sodium-potassium phosphate and 140 mM NaCl, pH 7.5, recentrifuged at 27,000 g for 30 min, and taken up in 2 ml of the same buffer. This procedure yielded brush-border vesicles purified an average of 12-fold over the original homogenate as estimated from measurement of sucrase (5) or aminooligopeptidase activities (2). The brush-border vesicle preparation was solubilized by addition of Triton X-100 to 2% (vol/vol) and sonicated in a Branson apparatus (model W350) at setting 5, 50% duty cycle for 20 pulses; the solution was then mixed on a tube rotator at 4°C for 12 h and centrifuged at 105,000 g for 60 min. The final supernatant containing solubilized brush-border glycoproteins was assayed for sucrase, cu-dextrinase (isomaltose as substrate), and aminooligopeptidase as previously described (2,5) and for protein by the Bio-Rad method (3). The average yield of these membrane glycoprotein hydrolases in the final supernatant was 75% compared with the original brushborder vesicle starting material. Immunoprecipitation. S-D was specifically immunoprecipitated by reaction with monospecific polyvalent anti-S-D as previously detailed (5). The incubation mixture, containing solubilized brush borders (100 mU suerase), anti-S-D (200 ~1; aggregating capacity, 150 mU

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sucrase), and sufficient buffer A (10 mM sodium-potassium phosphate, 140 mM NaCl, 0.5% Triton X-100, 0.02% SDS, pH 7.5) to yield a final volume of 1 ml, was maintained at 37°C for 60 min. Staphylococcal protein A [ 100 ~1, Pansorbin (Calbiochem), 10 mg/ml, prewashed three times with buffer A] was then added and the final mixture incubated for 16 h at 4°C. The immune pellet was recovered after centrifugation for 5 min in a Beckman Microfuge. Assay of the postprecipitin supernatants revealed that 87-97% of sucrase but no alkaline phosphatase, aminooligopeptidase, or lactase was precipitated. Monospecificity was confirmed both by presence of a single precipitin band on immunodiffusion and immunoelectrophoresis and by the presence of only P and its subunit products on SDS-electrophoresis (cf. RESULTS). The immune pellet was suspended in 10 mM Tris . HCl, 0.05% Triton X-100,0.01% SDS, and 300 mM NaCl, pH 6.8, recentrifuged in the microfuge, and then resuspended and recovered three times from 62.5 mM Tris. HCl, pH 6.8. The final pellet was solubilized and analyzed by SDS-acrylamide electrophoresis as detailed below. Quantitative polyacrylamide gel electrophoresis. The immune pellet containing 100 mU brush-border sucrase was solubilized in 62.5 mM Tris HCl, 2% SDS, 10% glycerol, 1 mM EDTA, and 10 mM DTT, pH 6.8 at lOO”C, for 5 min and applied to cylindrical acrylamide gels of 6% total acrylamide with 2% cross-linking according to Wycoff et al. (21). Each gel was stained with Coomassie Blue and scanned at 595 nm in a Gilford 250 spectrophotometer. The gel was sliced by a single cut with a multi-blade device (Bio-Rad) yielding 2.2-mm disks. Each gel disk was added to a glass liquid scintillation vial and the protein solubilized with 10 ml of 89% lipofluor, 9% solulyte (J. T. Baker), and 1% HZ0 for 16 h at room temperature. Radioactivity was determined by scintillation spectrometry with automatic quench correction. The radioactivity of each gel segment was determined from its total radioactivity minus the comparable gel slice obtained when normal rabbit globulin instead of specific anti-S-D globulin was reacted with the solubilized brush-border vesicles; this nonspecific activity was uniform along the gel (-40 dpm per 2.2-ml disk). Recovery of radioactivity applied to the gels was 85-105%. The relative migration of the protein bands was measured directly from these scans compared with the bromphenol blue at the gel front. The distance migrated was determined from the sum of the slices of known length beginning at the origin to the midpoint of the slice that was plotted. In addition, alignment of Coomassie protein peaks and radioactivity peaks was checked by determination of relative migration (Rf) with respect to the bromphenol blue front. In vitro experiments with radiolabeled brush-border vesicZes.Brush-border vesicles were prepared as described

above from rats that had been pulse labeled intraluminally with the radioactive amino acid and chased for 3 h so that the radioactive S-D was in the single-chain P form of the enzyme (cf. Figs. 1, 5, and 7). These membrane vesicles (50-250 mU sucrase activity) were incubated with purified pancreatic proteases for up to 3 h,

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washed three times in cold 0.02 M sodium-potassium phosphate and 0.14 M NaCl, pH 7.0, and recovered from a 40,000 g 30-min centrifugation. The brush-border proteins were solubilized by Triton X-100-sonication, immunoprecipitated, and treated with SDS as detailed above. The samples were then subjected to SDS acrylamide electrophoresis in cylindrical gels as detailed above or in a slab gel system as previously described (1) except that the separation gel was 6% total acrylamide instead of 7.5%. For the slab gel experiments, brush-border vesicles were either used directly or solubilized with Triton X-100 in Krebs-Ringer phosphate (KRP) buffer (18). The original vesicles or the solubilized supernatant were diluted to 4 ml with KRP buffer, and 1 ml of 50 pg/ml trypsin was added to each with vigorous mixing. Aliquots (1 ml) were removed immediately (time 0) and mixed with 1 ml aprotinin (1,250 KIU, Sigma) in the same buffer on ice. The remainder of the sample was incubated at 37°C and l-ml aliquots were removed and transferred to the cold aprotinin solution at 15, 30, 75, and 120 min. The recovered brush-border vesicles were then pelleted and solubilized as described above. The solubilized vesicle material and presolubilized enzyme were then immunoprecipitated and processed for acrylamide electrophoresis-autoradiography as detailed above. All experiments on prelabeled brush-border vesicles (cf. Figs. 47), done on at least two occasions several weeks apart, showed a precision of &lo%. RESULTS

Synthesis of brush-border P precursor (P) in vivo and the effect of intraintestinal secretions. In preliminary ex-

periments with anesthetized rats, we found that administration of an amino acid precursor either intraperitoneally or intraluminally into a jejunal loop produced mainly the double-chain form of the S-D hybrid, >80% of the labeling being identified 3 h later in the QI- and psubunits (data not shown). This confirmed the findings of Hauri et al. (10) when [3H]fucose was used as the precursor. In contrast, when the jejunal segment was preperfused prior to the intraluminal [ 3H] leucine pulse with 145 mM NaCl for 20 min to remove any residual intraintestinal secretions, 80% of the newly synthesized brush-border S-D was consistently in the single-chain P species at 3 and 6 h. Table 1 provides quantitative reference for the in vivo rat studies. Notably, experiment A shows the relatively stable pattern of radioactivity in the molecular forms of S-D for adjacent intestinal segments of the same animal 3 and 6 h after the intraintestinal pulse. A typical quantitative gel electrophoretic analysis of a 3-h pulse-chase experiment is shown in Fig. 1 where the relative protein mass of P and its putative cy- and ,&products of the S-D hybrid are shown by the Coomassie protein scan and the newly synthesized S-D immunoprotein species are identified from the radioactivity in individual gel slices. Whereas the cy- and ,& subunits accounted for the majority of the steady-state S-D mass (as estimated from protein staining), the vast majority of the radioactivity in newly synthesized immunoreactive S-D was confined to the larger P species

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1. Summary of in vivo experiments on conversion of single-chain (P) to double-chain (cy-@)S-D in rat brush border

TABLE

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Control reanastamosis

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Radioactivity, P

a+P

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9,140

10,890 1,940 11,080

81 82

7,860 310

1,660 4,450

83 7 81 28 16 72 40

8,770

2,120

9,520 4,760 16,860 15,800 5,380 4,320 4,150

C. Control (-) 5 13,680 3,180 Bil. Pant. Secretion (+) 5 4,480 11,320 D. Bile duct ligation 3 880 4,500 E. Control 3 3,130 1,190 Trypsin and elastase 6 1,670 2,480 A 5-min intraintestinal pulse of 5 mCi [3H]leucine was given to a single rat and the segment was examined under differing conditions in each experiment except for D, where pulse was given intraperitoneally. Each experiment compared data on adjacent segments of intestine in one animal. Brush borders, prepared at times indicated, were solubilized and immunoprecipitated (100 mU sucrase), and radioactivity in each molecular species was quantified from acrylamide gels as detailed in EXPERIMENTAL PROCEDURES. Bil. Pant. Secretion; biliary-pancreatic secretions. Because preliminary experiments in 10 different rats revealed 80-85% of radiolabeled S-D in cy+ p species and 15-20% in the P precursor after intraperitoneal labeling, no control animal was used in experiment D.

at 3 and 6 h of chase when intraintestinal secretions were absent (Table 1, experiments A and B, and Fig. 1). Such experiments in which the precursor was preferentially synthesized and inserted into the surface membrane involved catheterizing a jejunal loop and preperfusing with saline to remove residual biliary and pancreatic secretions from the segment. To study the possible effect of intraluminal secretions, we carried out a 3-h pulse-chase experiment and removed half of the perfused segment to serve as the control (free of intraluminal secretions) and then reestablished continuity of the intestine for the residual portion of the jejunal loop and returned the animal to its cage where it was allowed to drink and eat. Notably, 18 h after reinstitution of intestinal continuity and restoration of luminal biliarypancreatic flow, most of the radiolabeled P had been converted to smaller molecular weight forms that migrated identically to the authentic cy- and ,&components as monitored by the S-D doublet on the Coomassie scan (Fig. 1,21 h, and Table 1, experiment B). Whereas about half of the original radioactivity identified in the control gel was still associated with immunoprecipitable S-D at 21 h, 93% of this was associated with the cy- and psubunits, while only 7% remained with the single-chain P. Thus the reinstitution of intestinal flow appeared to promote the conversion of the single-chain P to the mature hybrid sucrase-dextrinase at the intestinal surface. Studies were designed to determine whether biliary or pancreatic factors or both might be responsible for this conversion. Role of the biliary-pancreatic secretions in the conversion of single-chain to hybrid enzyme. To determine di-

rectly the effect of luminal biliary-pancreatic secretions on the single-chain P, we installed a Swan-Ganz catheter with a penultimate balloon that prevented biliary-pancreatic secretions from entering the test segment while

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1. Effect of intraintestinal contents on molecular forms of newly synthesized S-D. After preperfusion with 145 mM NaCl to remove intraluminal biliary-pancreatic secretions, a 5-min pulse with [3H]leucine, and 3-h chase (cf. EXPERIMENTAL PROCEDURES), the proximal half of perfused intestinal segment was removed and processed. The distal half of the segment was reconnected to restore continuity of bowel, and the rat was returned to its cage for 18 h. Distal segment was then recovered, brush borders were solubilized, and S-D immunoprecipitates were subjected to SDS-acrylamide electrophoresis and stained with Coomassie blue. Gels were scanned for protein (solid line, arbitrary scale) and radioactivity (solid circles, dotted line) quantified in each 2.2-mm cut disk. Note apparent conversion of P precursor (3 h, left) to radioactive peaks coinciding with those of authentic cy- and P-subunits as a consequence of restoration of in vivo luminal secretions (21 h, right). Ab, antibody; BPB, bromphenol blue front. FIG.

still allowing its use as an inflow catheter. A luminal pulse-chase experiment was carried out as described in EXPERIMENTAL PROCEDURES, and, after 3 h of chase when the newly synthesized S-D was in the single chain form (cf. Fig. l), the balloon was deflated and the biliarypancreatic secretions were allowed to enter the test segment. When the intestine was recovered 2 h later, the proximal half of the segment (20 cm length) displayed a typical yellow color produced from the adherent mixture of biliary and pancreatic secretions, but, fortuitously, the distal half remained a pink-gray, indicating it had not been exposed to biliary-pancreatic secretions. These two time-matched segments were processed separately, as detailed in EXPERIMENTAL PROCEDURES. As shown in Fig. 2 and Table 1, experiment C, in the segment devoid of biliary-pancreatic secretions, more than three-fourths of immunoprecipitable radioactivity was associated with the P precursor; in marked contra st, the presence of lum inal biliary-pancreatic set retions was associated w ith a marked reduction of the radioactivity in the P precursor (28%) and the appearance of substantial labeling (72%) of the authentic cy-and ,&doublet. Notably, this apparent conversion of P to its products was not associated with any significant loss of radioactivity (Table 1, experiment

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tions or intraluminally without prior saline preperfusion, the P precursor was immediately converted to its cyCY-and ,&units despite the absence of intraintestinal biliary secretions. Because this P to cy + p conversion occurred despite bile diversion, intraintestinal bile did not appear to be responsible for the processing of P to its subunits in the brush-border membrane, and pancreatic proteases appeared to be the primary candidates for the postinsertional processing of P to its cy- and ,&subunits. Role of pancreatic proteases in the conversion of P to the two-chain S-D. To assess whether pancreatic pro-

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2. Effect of biliary-pancreatic secretions on molecular species of S-D. After lumen was occluded at the duodenal-jejunal junction by inflating balloon of Swan-Ganz catheter, [3H]leucine was instilled via catheter tip for 5 min and chased for 3 h (cf. EXPERIMENTAL PROCEDURES). The balloon was then deflated and proximally accumulated biliary and pancreatic secretions were allowed to enter jejunum. The entire jejunum was harvested 2 h later and processed for quantitative immunoprecipitation and acrylamide electrophoresis. Left: S-D pattern from distal half of segment, which fortuitously had not become bile stained (-); right: proximal segment, which had visible staining with bile (+) indicating restoration of flow of biliary-pancreatic secretions. Symbols are given in Fig. 1 legend and quantitative data are provided in Table 1, experiment C. FIG.

C, total radioactivity), suggesting that cleavage to the subunits under these conditions did not involve concomitant removal of S-D from the brush-border membrane. This study involving two adjacent segments of intestine from a single animal documents that exposure of the small intestinal surface in vivo to biliary-pancreatic secretions for only 2 h evokes a dramatic change in the structure of newly synthesized S-D. Whether this conversion was due to a particular component in bile or pancreatic secretions is considered further below.

teases are responsible for the cleavage of the single-chain enzyme, we performed intraluminal pulse-chase experiments with [3H]leucine as described under EXPERIMENTAL PROCEDURES. After a 3-h chase, a portion of the intestinal loop was removed and processed as the control (Fig. 3, left). The remaining segment was then perfused with physiological concentrations of trypsin (50 pg/ml) plus elastase (1.25 pg/ml) in KRP buffer at 2 ml/h for an additional 3 h. (The concentrations of proteases in intestinal contents from normal adult rats after feeding were found to be 50-100 pg/ml for trypsin and chymotrypsin and 1.25-5.0 pg/ml for elastase.) Notably, there was appreciable conversion of the newly synthesized P to smaller molecular forms that migrated in the positions of the authentic cy- and ,&subunits (localized by Coomassie staining of the same gel; Fig. 3, right). Comparative analysis of the radioactivity in the control and protease-treated adjacent segments revealed that nearly 50% of the P had been converted to the double-chain hybrid by action of the proteases, while the total radioactivity in all molecular forms of immuno-S-D remained unchanged (Table 1, experiment E). This experiment

Biliary secretions are not required for the conversion of brush-border single-chain P to the S-D hybrid enzyme.

To determine whether biliary secretions are required for the conversion of P to the cy- and ,&dimer, we prevented bile from entering the intestine by ligating the bile duct at its point of exit from the liver in a rat under pentobarbital anesthesia, as described under EXPERIMENTAL PROCEDURES. The pancreatic outflow to the intestine was maintained. The rat was returned to its cage, and 14 h later, [ 3H] leucine was administered intraperitoneally. Three hours after the radioactive pulse, the animal was killed and the intestine was processed as described under EXPERIMENTAL PROCEDURES. There was no yellow bile staining of intestinal mucosa, verifying that biliary obstruction was complete. Yet, 84% of radioactivity associated with S-D was localized to the cy- and ,&subunits when analyzed by SDS electrophoresis (Table 1, experiment 0). Thus, as uniformly found for rats labeled intraperitoneally with intact biliary-pancreatic secre-

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FIG. 3. Role of trypsin + elastase in conversion of single-chain P to double-chain S-D. After an intraintestinal pulse chase with [3H]leucine as detailed in text and previous figures, half of intestinal segment was removed and served as control (left) and the remaining half-segment was perfused with trypsin plus elastase in KRP buffer (pH 7.4) at 2 ml/h for an additional 3 h. Trypsin and elastase perfusion converted a substantial portion of newly synthesized P to cy- and ,&species. Note differences in ordinate scales. Symbols are given in Fig. 1 and quantitative data are in Table 1, experiment E.

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suggested that trypsin, elastase, or both may be primarily responsible for specific cleavage of P at the intestinal surface to the sucrase and dextrinase components of the mature hybrid enzyme. Conversion of P to the authentic S-D hybrid dimer in isolated brush-border vesicles. These experiments in the

intact rat strongly suggested that the single-chain enzyme is converted to the hybrid S-D dimer by virtue of intraluminal trypsin, elastase, or both. However, in intact animal studies, it was not possible to control precisely other factors such as nonpancreatic luminal fluids or hormonal factors that could play a role in this conversion. Therefore we examined brush-border vesicles isolated from rats metabolically labeled intraintestinally under conditions favoring preservation of the newly synthesizedPprecursor(see EXPERIMENTAL PROCEDURES). Brush-border vesicles were prepared from rats that had received a 5-min intraluminal [3H]leucine pulse and 4-h chase to allow the vast majority of newly synthesized SD to be maintained in the single-chain P form (cf. Fig. 1, left). Vesicles were suspended in KRP buffer and exposed to physiological concentrations of pancreatic proteases for 3 h at 37°C followed by solubilization, specific immunoprecipitation, and analysis by SDS acrylamide electrophoresis. As shown in Fig. 4, appreciable conversion to authentic cy- and P-subunits was evoked only by the combination of trypsin plus elastase or by trypsin alone. Elastase or chymotrypsin alone had minimal action on the single-chain P and did not yield peptides in the positions of the authentic a- and ,8subunits. During the incubation of brush-border vesicles under physiological conditions, minimal amounts of sucrase activity were released into the medium (OOCcontrol l.l%, 37°C control 4.5%; trypsin 11%; chymotrypsin 6%; elastase . . 5%;. . all proteases _. . .13%). While. direct analysis of gel of slices allows quantitative comparison ofn radiolabeling TRYPSIN I

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Conversion of P by total duodenal luminal secretions vs. trypsin or elastase. Because other components of duo-

denal secretory contents besides the pancreatic proteases may also modify the P precursor, we recovered duodenal intraluminal contents from normal rats and incubated them with jejunal brush-border membranes that had been prelabeled intrajejunally for 7 h in a rat with diverted pancreatic and biliary secretions. Because of the higher concentrations of pancreatic proteases in previous reports (10,16), we increased the concentrations of trypsin (150 fig/ml) and elastase (50 pg/ml) in these experiments. As shown in the gel autoradiograph of the S-D immunoprecipitates (Fig. 5), there was heavy labeling of the P precursor over this period. The gel was intentionally overexposed to enhance the analysis of the fragmentation products. The most prominent of these were positioned between the P and the faint cy- and ,&species. Exposure of these brush-border membranes to physiological amounts of duodenal contents recovered from normal rats (containing bile and pancreatic secretions) produced a marked increase in both the a-species and particularly the P-species (Fig. 5, lanes 3, 4, 8, and 9). The qualitative fragmentation pattern was identical to that produced by purified trypsin alone (150 pg/ml; Fig. 5, lanes 5 and 10) and persisted after longer (90 min) exposure to duodenal fluid or trypsin. Elastase, even at relatively high (50 pg/ml) concentrations, did not convert the membrane-bound P to smaller species. Instead, in contrast to the intramembrane processing by trypsin, elastase removed the P and its cy- and ,&products from the membrane (Fig. 5, lanes 6 and 11). The presence of species intermediate between P and the final cy- and ,8products raised the possibility that there may be endogenous brush-border proteases that initiate the processing

FIG. 4. Effect of purified pancreatic proteases on newly synthesized single-chain S-D protein (P). Isolated brush-border vesicles, prepared from rats pulsed with [3H] leucine intraintestinally and chased with 1 mM leucine for 3 h, were exposed to purified proteases (cf. EXPERIMENTAL PROCEDURES) prior to solubilization and immunoprecipitation with anti-S-D. Immunoprecipitates were subjected to SDS-acrylamide electrophoresis, and radioactivity was quantified. Only trypsin (50 pg/ ml) plus elastase (5 pg/ml) or trypsin alone produced significant quantities of products migrating to position of cy-and P-species. (Concentrations of proteases in intestinal contents from normal adult rats after feeding were found to be 50-150 pg/ml for trypsin and chymotrypsin and 1.25-5.0 pg/ml for elastase.) Neither chymotrypsin (50 pg/ml) nor elastase (5 pg/ml) yielded authentic CY-and /?products. Arrows indicate positions of authentic CY and fi localized by Coomassie staining of same gel.

60 DISTANCE

(mm)

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(mm)

CHYMOTRYPSIN :::;I

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particular species, the qualitative protein and radioactivity patterns can be more precisely correlated by analysis of intact SDS-acrylamide slab gels.

TRYPSIN

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PROCESSING 30 min Luminal contents Proteases

1 C

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St @Da) 200-

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116FIG. 5. Comparison of duodenal luminal contents and purified pancreatic proteases on P in brush-border vesicles. After flushing of a 20cm upper jejunal segment in an anesthetized rat and proximal ligation to prevent entry of biliary and pancreatic secretions, [3JS]methionine (1.5 mCi) was instilled into lumen and allowed to remain for 7 h to label P maximally. Adjacent proximal segment, bathed with pancreaticbiliary secretions, served as control (C) to define positions of authentic P, o, and @.Brush-border vesicles were incubated with luminal contents (pooled intraduodenal secretions) or purified pancreatic proteases. After 30 and 90 min, aliquots (200 mu) of S-D were solubilized, immunoprecipitated, and analyzed by acrylamide electrophoresis-autoradiography. Try, trypsin (150 pg/ml); Ela, elastase (50 pg/ml); +, present in incubation; ++, present at, 2 times concentration of +.

of P. But, trypsin appears to be the sole luminal factor required for the processing to authentic CYand /?. No additional intermediate or final membrane-associated species was produced by other components in duodenal fluid (Fig. 5, lanes 4 vs. 5 and 8 vs. 9). Stoichiometry of tryptic conversion of P to CYand p in isolated brush-border vesicles. Although the experiments shown in Figs. 4 and 5 established that only trypsin is essential for the P to Q: + p conversion, the presence of intermediate products and the apparent unequal quantities of the final cy- and P-products suggested that the processing of P may not occur in a single-cleavage step. To examine this further, we carried out sequential incubation of trypsin with isolated brush-border vesicles that had been prelabeled metabolically with [35S]methionine in intact animals. Labeled brush-border vesicles were either exposed directly to trypsin (50 pg/ml; Fig. 6, left lanes) or first solubilized with Triton X-100 prior to trypsin exposure (Fig. 6, right lanes) as detailed in EXPERIMENTAL PROCEDURES. Aliquots were removed and the reaction was stopped by addition of aprotinin and transferred to a tube on ice either immediately (time 0) or after incubation for 15-120 min. The cold samples were then assayed for sucrase and cu-dextrinase, solubilized, immunoprecipitated, denatured, and analyzed by SDS acrylamide electrophoresis. Initially (0 min), nearly all the newly synthesized enzyme was in the single-chain P (230 kDa) form in both intact and solubilized vesicles (Fig. 6A, center). In addition, there were several more rapidly migrating bands with apparent masses ranging between 205 and 145 kDa. The most rapidly migrating of these localized just above the authentic 140-kDa a-species. This can be readily discerned by analyzing the sequential progression in the vesicle study; the 145-kDa band rather

Brurh130

75

30

15

0

0

15

30

75

120

P’ OLRY’-

FIG. 6. Kinetics of trypsin effect on S-D precursor in isolated brushborder vesicles. Brush-border vesicles, prepared from rats 4 h after intraintestinal pulse-labeling with [?S]methionine, were exposed, either intact or after presolubilization with 1% Triton X-100, to 50 hg/ ml purified Trypsin in KRP buffer for O-120 min. After incubation times shown, samples (1 ml) were removed, mixed immediately with 1 ml of KRP buffer containing 50 pg/ml aprotinin, centrifuged, solubilized with 1% Triton X-100, immunoprecipitated with anti-S-D, and run on SDS-acrylamide gels. The same amount of original brush-border material was applied to each lane. A: autoradiogram showing effect of trypsin on newly synthesized P from treated vesicles (left lanes) and on Triton-solubilized P from the same membrane preparation (right lanes). B: protein pattern by Coomassie staining of same gel (prominent band near front is antibody fragment). P. predominated initially when trypsin action was immediately stopped (time O), but intermediately migrating species (most prominent designated as P’ and P”) were also detected and persisted at relatively constant levels as authentic (Y-and @-subunits were generated. Triton-solubilized P yielded authentic 0 but upper band migrated appreciably farther than authentic (Y and is denoted as (Y’.

than the (Ywas present initially (0 min), merged with the upper edge of the CY(140 kDa) band at 15 min, and then waned as the cy predominated at later times. The most prominent of these intermediate bands, designated P’ (205 kDa) and P” (185 kDa), appeared to be intermedi-

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ate species produced by initial cleavage of P either by endogenous membrane proteases or by trypsin in the short time required to stop the reaction. This conclusion is supported by densitometric scanning because the intermediate species persisted at a relatively constant (0.10-0.15) fraction of the parental P precursor until it became depleted late in the reaction (Fig. 6A). We interpret this to reflect a steady-state pool of intermediates that are being formed from P breakdown and then rapidly degraded to the final cy- and ,&units by trypsin. Substantial quantities of radiolabeled cy- and ,&subunits appeared rapidly in the intact vesicles after only 15 min of trypsin exposure; by 75 min, the radioactivity in P was noticeably diminished and the bands corresponding to the authentic cy-and ,&subunits became prominent in the autoradiogram (Fig. 6A). Authenticity was established by the fact that the subunits migrated to the exact position of the cy-and ,&species in the stable protein pool identified by Coomassie staining (compare Fig. 6, A and B ). The protein-stained gel revealed considerable amounts of the P in brush-border vesicles due to the 6h chase period without luminal pancreatic secretions; P’ and P” could also be identified, even at 0 min. Because only a few seconds of tryptic action could have occurred during the time required to add aprotinin to the test sample, the initial cleavage of P could also have occurred by action of endogenous brush-border membrane proteases. As estimated from protein staining (Fig. 6B), the quantity of P declined over the course of trypsin exposure, and the amounts of cy- and ,&species remained relatively constant until 120 min, when they declined somewhat, and an additional band (-135 kDa) appeared at the lower edge of the a-band. Of particular note was the presence of the y band on the Coomassie gel but not on the autoradiogram, indicating that it is a component of the S-D protein derived from a pool of P or its subunit products synthesized prior to the pulse chase. When the Triton-solubilized supernatant was treated with trypsin (Fig. 6, A and B, right lanes), cleavage of solubilized P was more rapid than that found for intact membrane vesicles (Fig. 6, A and B, right lanes), yielding a predominantly smaller fragment CY’(-130 kDa), rather than CY.Thus additional trypsin cleavage sites appear to be made available by virtue of removal of P from the membrane. In contrast, the P-product of trypsin reaction on the soluble P was indistinguishable from that found for the vesicles. On the protein-stained gel, the cybecame relatively less intense than the p and was replaced completely by CY’by 120 min (Fig. 6B, right lanes). The quantitation of the autoradiograph by densitometry revealed a continuous conversion of the P to cy and ,0 within the brush border over 75-90 min with a slight decline in the major products at 120 min (Fig. 7). Also of interest was the production of the relatively larger quantity of the ,&subunit, which exceeded that of the cysubunit (Fig. 7) throughout the 120-min period. The relatively stable Q-to-P ratio of 0.56-0.59 cannot be explained by differential radiolabeling because, based on the amino acid sequences deduced from the rabbit cDNA (U), equal labeling of all methionine residues would produce a higher a-to-p ratio of 0.75.

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a+B

P a n

u-p-

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40 Incubation

II

II

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Time (min.)

FIG. 7. Quantitative conversion of P to CY + ,8 in brush-border membranes. Densitometric scan of SDS-autoradiogram shown in Fig. 6A. As radioactivity in P precursor decreased, there was a differential conversion to cy- and P-species. a-to-@ ratio was 0.56-0.59 throughout experiment, despite expected ratio of 0.75 based on probable methionine content of cy- and P-units. (See text for elaboration.)

DISCUSSION

Synthesis of S-D as the precursor in the intact rat and conversion to the authentic cu-p doublet in the brush border. S-D is a most interesting and possibly unique

membrane glycoprotein for several reasons. First of all, it is expressed only in the brush-border surface of the polarized intestinal enterocyte and in colon carcinoma cell lines that mimic this tissue morphologically and to some extent functionally (11). Second, after being assembled and glycosylated in association with endoplasmic reticulum and Golgi, rather than being cleaved intracellularly, the P is vectorially transported intact only to the brush-border surface; third, it undergoes postinsertional cleavage to a- and ,&subunits. Finally, these subunits remain noncovalently associated and anchored to the surface membrane by the hydrophobic NH2-terminus of the a-unit. Our experiments demonstrate that a single luminal pancreatic protease, trypsin, is necessary for postinsertional conversion of P or at least of the intermediate P’ products and that the cleavage process is much more complex than previously assumed. In vivo experiments established that the single-chain P macromolecule is the predominant S-D species synthesized in intact adult intestine in the absence of intraluminal intestinal contents, including biliary and pancreatic secretions (Fig. 1, Table 1, experiment A). This might have been predicted from cell culture and organ transplant experiments where, in the absence of intraintestinal secretions including trypsin, only the singlechain precursor accumulated (11,15). Restoration of the flow of intestinal contents produced nearly complete disappearance of newly labeled P and replacement with the CY-and P-subunits (Fig. 2, Table 1, experiment B). While the presence of biliary and pancreatic secretions facilitated the postinsertional cleavage to the cy- and ,& subunits (Fig. 2, Table 1, experiment C), bile alone played no role in this conversion (Table 1, experiment 0). Only luminal trypsin, with or without the presence of elastase, acted to cleave the newly synthesized P precursor to the authentic LX-~ doublet in the brush border (Figs. 3-7). In

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the in vivo studies, the amount of radioactivity incorporated into the P precursor appeared to be quantitatively transferred to the a- and P-species over several hours with very high efficiency (cf. Table 1). Because trypsin alone yielded the identical cleavage pattern to that produced by complete intraduodenal luminal secretions (Fig. 5), it appears to be the sole luminal factor responsible for the postinsertional processing of the P precursor. While other secretory substances may influence the rate of cleavage, only trypsin appears necessary and sufficient for the proper processing of P to the CY-and P-units. Previous studies in both intact animals (10) and in fetal intestinal transplants (15) had suggested elastase was the primary protease responsible for cleavage, but the doublet product consistently revealed an a-subunit that migrated more rapidly than the authentic a of the mature doublet. Notably, elastase has a peptide bond specificity similar to papain, a protease that has been used to solubilize S-D from the brush-border membrane by cleaving it from the NH2-terminal anchor of the asubunit; previous studies had not ascertained whether the hybrid dimer product remained attached to the surface membrane after its cleavage by elastase. Rather than being involved in processing, relatively high concentrations of elastase (20 mg/ml) appear to remove P and its cy- and ,&products from the membrane. Naim et al. (16) observed cleavage of P in human intestinal explants by trypsin rather than elastase but were not able to establish the relative position of the doublet product to that of the authentic CY-and ,&subunits. Whereas these authors attributed the differences in protease requirement in the human cell line to possible species variation in peptide structure or glycosylation (16), our results support the concept that luminal trypsin is specific for P cleavage in all mammalian species. Indeed, the major cleavage site between an Arg and Ile residue of the P precursor (14) yielding the ,&subunit with the Ile at its NH2-terminus is a trypsin-specific site that is not attacked by either elastase or chymotrypsin, the other endoproteases secreted by the pancreas. The P to cy + p conversion is quantitative, involves the appearance of intermediate products, and yields unequal quantities of the subunits. When the stoichiometry of the

conversion of P to cy+ ,8 was examined in isolated brushborder preparations in vitro (Figs. 5-7), the consistent presence of several degradation products (predominantly P’ and P’ ‘) that represented a relatively constant radiolabeled fraction throughout the trypsin kinetic experiments is of particular interest (Fig. 6). It has been generally accepted that there is a single cleavage site near the center of the P precursor that leads to the production of cy- and P-subunits. Although cy and ,8 are clearly final products, the several species of intermediate mass (205-145 kDa) migrating between them and the parental P are strong candidates for transient species that are differentially modified to yield one or the other of the final subunits. Such a hypothesis could explain the observations that the combined masses of cy + p are 30 kDa greater than that of the parental P precursor and that unequal quantities of cy and ,8 are frequently observed by protein staining and quantitative autoradiog-

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G855

raphy (12, 15). Documentation of this will require structural evidence that the COOH-terminus of the a-subunit contains an amino acid sequence identical with the NH2terminus of the ,&unit. In our experiments in rats, the P-subunit nearly always predominated both in vivo (Figs. l-3) and in vitro (Figs. 5-7); this was true even early in the reaction (Fig. 6, 15min lane). Indeed, the a-to-0 radioactive ratio of 0.56-0.59 (cf. Fig. 7) is appreciably lower than the 0.75 predicted by the methionine content of the subunits (14) and indicates that the actual D-to-P molar ratio is 0.77 rather than the expected 1.0 that would define equal production of subunits. While a differential trimming process could be relatively inefficient, leading to the loss of up to 50% of the P as small fragments from the trimmed half of the molecule, the loss of label from P was accounted for by the gain of label in the cy- and ,&subunits both in vivo (Table-l) and in vitro (Fig. 7). Although it is also possible that the intermediate species are relatively stable forms that represent a minor fractional degradation route for P (243% of the radioactivity in the 230-kDa precursor was released into the medium during the incubation of brushborder vesicles with trypsin), it seems likely that an appreciable portion of the postinsertional processing involves a series of trimming steps producing unequal quantities of the cy- and ,&units, which then reassociate into the mature S-D. We considered the possibility that the a-subunit might be subsequently degraded to smaller fragments that are not recognized by the polyvalent antiserum or that migrate in the exact position of the punit on acrylamide gels. However, amino acid sequence studies of the species isolated from the ,&band blotted to polyvinylidine difluoride revealed only a single amino acid per cycle that had the expected ,&sequence (J.-S. Zhu, K. A. Conklin, L. A. Scheving, A. J. Smith, and G. M. Gray, unpublished observations). Furthermore, the fact that the polyvalent antiserum recognizes several different domains of the S-D protein makes it likely that fragments produced from a would be detected by the antiserum. However, differential removal of the cu-subunit by rapid cleavage to small peptides could also contribute to the unequal a-to-@ labeling ratios. By use of specific immunoassay with monoclonal antibodies directed against sucrase or a-dextrinase, Goda et al. (8) found a two- to threefold apparent molar excess of dextrinase (S-to-D ratio = 0.3-0.5) in rat jejunum. The S-to-D ratio was restored to unity after pancreaticbiliary duct obstruction, presumably because the P was not postinsertionally processed. Gel filtration of papainsolubilized brush borders revealed not only the expected peak containing both sucrase and dextrinase activities but also a second peak having only dextrinase activity. They interpreted these findings to reflect degradation of the S-D hybrid induced by biliary-pancreatic secretions with destruction of sucrase and release of the dextrinase unit. Notably, these studies depended upon papain treatment, which may itself have modified the native S-D species. Although unequal degradation of the cy- and ,& subunits may have been responsible for their findings, Goda et al. (8) did not quantify these components individually. Both differential postinsertional processing of

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P by trypsin and enhanced removal of the a-unit from the membrane may contribute to the relative abundance of the ,&unit [which we have found in other studies (22) to harbor the dextrinase active site] and reduction of the sucrase-containing a-unit. A comprehensive study of the degradation of the individual subunits will be required to determine whether there is selective accelerated destruction of the sucrase and preservation of dextrinase. Other observations are compatible with the variable S-to-D activity ratios reported by Goda et al. (8); partial denaturation and renaturation not involving membrane solubilization of S-D has revealed activity in at least two oligomeric forms (-330 and -260 kDa) that appear to have very different S-to-D activity ratios (22). Overall, there is now ample evidence that the postinsertional processing and native association of these two a-glucoside units are much more complex than the simple 1:l subunit molar ratio generally assumed. Perhaps the differential postinsertional cleavage of the P precursor by luminal trypsin and subsequent unequal degradation of the two subunits constitute complementary processes that together define the subunit structure and consequent oligomeric association of S-D at the enterocyte’s surface. Whether trypsin, alone or in combination with an endogenous brush-border membrane protease, produces the intermediate products in the initial stages of P cleavage is uncertain. Because the major P’ and P” products could be identified in in vivo metabolic labeling experiments after biliary-pancreatic diversion (Fig. 5) and in the protein-stained pool by SDS gels even after only the few seconds of exposure to trypsin required for processing of the time 0 sample (Fig. 6), it seems tenable that they antedated the trypsin exposure and hence may be products of endogenous membrane protease action during the pulse-chase experiment. Indeed, an integral brush-border endopeptidase that might have such an action has been identified (19). Notably however, studies of S-D synthesis in intestinal organ culture systems devoid of pancreatic proteases have revealed only the single P species, suggesting that integral brush-border peptidases may not be capable of the initial trimming of the P precursor (15, 16). In any case, trypsin is clearly essential for the major trimming necessary to produce the authentic cy- and psubunits. The y unit, frequently observed on SDS gels of brushborder solubilized immunoprecipitates or on direct gel immunoblots (15), was not present on the autoradiogram after the pulse-chase experiments (Fig. 7A) and hence is unlikely to be a newly synthesized species derived directly from P cleavage. However, because it was present consistently on the protein-stained gel (Fig. 7B) and has high homology with a sequence near the NH2-terminus of the a-subunit (J.-S. Zhu, K. A. Conklin, L. A. Scheving, A. J. Smith and G. M. Gray, unpublished observations), it is likely to be a product of additional but delayed cleavage of CY.Interestingly enough, although its cleavage separates it from the hydrophobic transmembrane anchor close to the NH2-terminus of the cy, it continues to remain associated with the brush-border membrane. Whether it remains attached to its original ,&partner

AUTHENTIC

SUBUNITS

and what its functional plored.

role may be remain

to be ex-

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants (NIDDK) DK-11270, DK-35033, and DK-38707. G. L. Shapiro was supported by NIDDK Training Grant in Academic Gastroenterology DK-07056. Present address for G. L. Shapiro: 1 Lynxholm Ct., Hyannis, MA 02601. Address for reprint requests: G. M. Gray, Digestive Disease Ctr., S069, Stanford Univ. Medical Center, Stanford, CA 943055100. Received

15 February

1991; accepted

in final

form

12 June

1991.

REFERENCES D. J., A. K. MIRCHEFF, N. A. SANTIAGO, C. YOSHIOKA, 1. AHNEN, AND G. M. GRAY. Intestinal surface aminooligopeptidase. Distinct molecular forms during assembly on intracellular membranes in vivo. J. Biol. Chem. 258: 5960-5966, 1983. 2. AHNEN, D. J., N. A. SANTIAGO, J-P. C~?ZARD, AND G. M. GRAY. Intestinal aminooligopeptidase. In vivo synthesis on intracellular membranes of rat jejunum. J. BioL. Chem. 257: 12129-12135,1982. 3. BRADFORD, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976. 4. BRUNNER, J., H. HAUSER, H. BRAUN, K. J. WILSON, H. WACKER, B. O’NEILL, AND G. SEMENZA. The mode of association of the enzyme complex sucrase-isomaltase with the intestinal brush border membrane. J. Biol. Chem. 254: 1821-1828, 1979. B. C. DAS, AND G. M. GRAY. 5. C~ZARD, J-P., K. A. CONKLIN, Incomplete intracellular forms of intestinal surface membrane sucrase-isomaltase. J. Biol. Chem. 254: 8969-8975, 1979. 6. COGOLI, A., A. EBERLE, H. SIGRIST, C. Joss, E. ROBINSON, H. MOSIMANN, AND G. SEMENZA. Subunits of the small-intestinal sucrase-isomaltase complex and separation of its enzymatically active isomaltase moiety. Eur. J. Biochem. 33: 40-48, 1973. 7. FRANK, G., J. BRUNNER, H. HAUSER, H. WACKER, G. SEMENZA, AND H. ZUBER. The hydrophobic anchor of small-intestinal suerase-isomaltase: NHz-terminal sequence of isomaltase subunit. FEBS L&t. 96: 183-188, 1978. 8. GODA, T., A. QUARONI, AND 0. KOLDOVSKY. Characterization of degradation process of sucrase-isomaltase in rat jejunum with monoclonal-antibody-based enzyme-linked immunosorbent assay. Biochem. J. 250: 41-46, 1988. 9. GRAY, G. M., B. C. LALLY, AND K. A. CONKLIN. Action of intestinal sucrase-isomaltase and its free monomers on an a-limit dextrin. J. Biol. Chem. 254: 6038-6043, 1979. 10. HAURI, H. P., A. QUARONI, AND K. J. ISSELBACHER. Biogenesis of intestinal plasma membrane: posttranslational route and cleavage of sucrase-isomaltase. Proc. Natl. Acad. Aci. USA 76: 5183-5186, 1979. 11. HAURI, H. P., E. E. STERCHI, D. BIENZ, J. A. FRANSEN, AND A. MARXER. Expression and intracellular transport of microvillus membrane hydrolases in human intestinal epithelial cells. J. Cell Biol. 101: 838-851, 1985. 12. HAURI, H. P., H. WACKER, E. E. RICKLI, B. BIGLER-MEIER, A. QUARONI, AND G. SEMENZA. Biosynthesis of sucrase-isomaltase. Purification and NHg-terminal amino acid sequence of the rat sucrase-isomaltase precursor (prosucrase-isomaltase) from fetal intestinal transplants. J. Biol. Chem. 257: 4522-4528, 1982. 13. Hu, C., M. SPIESS,, AND G. SEMENZA. The mode of anchoring and precursor forms of sucrase-isomaltase and maltase-glucoamylase in chicken intestinal brush-border membrane. Phylogenetic implications. Biochim. Biophys. Acta 896: 275-286, 1987. 14. HUNZIKER, W., M. SPIESS, G. SEMENZA, AND H. F. LODISH. The sucrase-isomaltase complex: primary structure, membrane-orientation, and evolution of a stalked, intrinsic brush border protein. Cell 46: 227-234, 1986. 15. MONTGOMERY, R. K., M. A. SYBICKI, A. G. FORCIER, AND R. J. GRAND. Rat intestinal microvillus membrane sucrase-isomaltase is a single high molecular weight protein and fully active enzyme

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in the absence of luminal factors. Biochim. Biophys. Acta 661: 346349,198l. 16. NAIM, H.

Y., E. E. STERCHI, AND M. J. LENTZE. Biosynthesis of the human sucrase-isomaltase complex. Differential O-glycosylation of the sucrase subunit correlates with its position within the enzyme complex. J. Biol. Chem. 263: 7242-7253,1988. 17. SJOSTROM, H., 0. NoR~IN, L. CHRISTIANSEN, H. WACKER, AND G. SEMENZA. A fully active, two-active-site, single-chain sucraseisomaltase from pig small intestine. Implications for the biosynthesis of a mammalian integral stalked membrane protein. J. Biol. Chem. 255: 11332-11338,198O. 18. SMITHSON, K. W., AND G. M. GRAY. Intestinal assimilation of a tetrapeptide in the rat. Obligate function of brush border aminopeptidase. J. Clin. Invest. 60: 665-674, 1977. 19. STERCHI, E. E., H. Y. NAIM, M. J. LENTZE, H. P. HAURI, AND J.

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A. FRANSEN. N-benzoyl-L-tyrosyl-P-aminobenzoic acid hydrolase: a metalloendopeptidase of the human intestinal microvillus membrane which degrades biologically active peptides. Arch. Biochem. Biophys. 265: 105-118,1988. 20. TAKESUE, Y., R. TAMURA, AND Y. NISHI. Immunochemical studies on the subunits of rabbit-intestinal sucrase-isomaltase complex. Biochim. Biophys. Acta 483: 375-385,1977. 21. WYCKOFF, M., D. RODBARD, AND A. CHRAMBACH. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate-containing buffers using multiphasic buffer systems: properties of the stack, valid Rfmeasurement, and optimized procedure. And. Biochem. 78: 459482,1977. 22. ZHU, J.-S., K. A. CONKLIN, L. A. SCHEVING, A. J. SMITH, AND G. M. GRAY. Structural and functional correlates of sucrase-at-detrinase in intact brush border membranes. Biochemistry. In press.

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