Intestinal disaccharidases in the house musk shrew, Suncus murinus: occurrence of sucrase deficiency

Comp. Biochem. Physiol. Vol. 10311,No. 3, pp. 629-634, 1992 Printed in Great Britain

0305-0491]92$5.00+ 0.00 © 1992PergamonPress Ltd

INTESTINAL DISACCHARIDASES IN THE HOUSE MUSK SHREW, SUNCUS MURINUS: OCCURRENCE OF SUCRASE DEFICIENCY KuNxo YOKOTA, SEN-ICHIODA, SACHIKOTAKESUE* and YOSHIK1TAKESUE~':~ Research Institute of Environmental Medicine, Nagoya University, Chikusa-ku, Nagoya 464, Japan; *College of Medical Technology, Nagoya University, Higashi-ku, Nagoya 461, Japan; and ~'Faculty of General Education, Gifu University, Gifu 501-I I, Japan (Received 7 April 1992; accepted 15 May 1992) Abstract--l. Disaccharidase activities of the small-intestinal brush border membrane were studied in six laboratory lines of the house musk shrew, Suncus murinus. 2. Sucrase activity was detected in all shrews of one line, but not in any shrew of three lines. In the other two lines it was found in some shrews, but not in the others. 3. Maltase, isomaltase, trehalase and lactase activities were found in all shrews of all the lines examined. 4. Sucrase was normally associated with isomaltase to form an enzyme complex. 5. Detergent-solubilized isomaltase, whether associated with sucrase or not, was inhibited by antibodies against rabbit sucrase-isomaltase to almost the same extent as the rabbit one, suggesting that isomaltase is not affected by a mutation(s) in suerase.

INTRODUCTION

physiology of the animal (Oda et al., 1985). In the course of biochemical studies on its intestinal digestion and absorption, we have found hereditary sucrase deficiency in the animal, as will be described below.

Sucrase-isomaltase is an integral enzyme complex of the brush border membrane (BBM) of the small intestine in mammals. It is usually synthesized as a two-active site, single-chain polypeptide precursor, which is then inserted into the BBM and cleaved into the two subunits sucrase and isomaltase by pancreatic proteases in the intestinal lumen (Semenza, 1986). Even after this cleavage, sucrase is tightly associated with isomaltase which is directly anchored into the membrane via its N-terminal hydrophobic peptide segment (Semenza, 1986). Congenital deficiency of sucrase or sucrase-isomaltase has been found in the h u m a n small intestine (Hadorn et al., 1981). It can be classified into several phenotypes, but intolerance to sucrose is a well-known clinical symptom c o m m o n to all the phenotypes (Naim et al., 1988). To elucidate the molecular basis of sucrase(-isomaltase) deficiency or sucrose intolerance, it is desirable to obtain laboratory animals hereditarily deficient in sucrase or sucrase-isomaltase. Mammals of the order Insectivora have received attention as possible laboratory animals which are phylogenetically very different from ordinarily used laboratory animals such as the mouse, rat, hamster and rabbit. Oda and K o n d o (1976) succeeded in rearing and breeding the house musk shrew, Suncus murinus, a m a m m a l belonging to the family Soricidae of the Insectivora. Thereafter, several laboratory lines of the animal which originated from different wild populations in Asian regions have been developed in Japan, and a n u m b e r of studies have been carried out on the morphology, endocrinology and

Preparation of BBM vesicles from the small intestine Shrews were treated individually. They were allowed to freely access feed and water. To avoid disturbances due to possible circadian changes of enzyme and transport activities, all the shrews were killed at the fixed time (10 a.m.) by deep etherizing. The small intestine was quickly removed and flushed with ice-cold isotonic saline, and then the mucosa was scraped. The scraped mucosa (0.3-0.5g) was homogenized in 20ml of 0.3 M mannitol/10mM Tris-Hepes buffer (pH 7.4) using a Polytron homogenizer (Kinematica GembH) for 30 see at the maximum speed. A portion of the homogenate was removed for assay, and BBM vesicles were prepared from the remaining homogenate by a Ca 2+ precipitation method (Yokota et al., 1983). In some experiments where soluble disaccharidase activities were examined, the homogenate to which CaC12 was not added was centrifuged at 105,000g for 30rain to yield the soluble fraction. BBM vesicles from the rabbit small intestine were prepared in a similar way (Yokota etal., 1983).

:~To whom correspondence should be addressed. Abbreviations: BBM, brush border membrane; S/I ratio, ratio between specific activity of sucrase and isomaltase.

Solubilization of BBM vesicles with Triton X-IO0 BBM vesicles (2.5mg/mi protein) were incubated with 1% Triton X-100/0.1 M NaC1/10mM sodium phosphate (pH 7.0) at 25°C for 20 rain, followed by centrifugation at

MATERIALSAND METHODS Animals In this work we used two- or more month-old, either male or female, house musk shrews of three lines originating directly from wild populations (Nem:NAG, Nem:TR and Nem:SRI) and three hybrid lines derived from them (Nem:NJ, Nem:MI and Nem:SNJ) (Fig. 1). They were all bred in the Research Institute of Environmental Medicine, Nagoya University.

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KUNIO YOKOTA et al.

Name of line

O r i g i n a l s h r e w s c a p t u r e d in

Nagasaki, Japan

Nero:NAG

West Java, Indonesia

Nem:NJ

Okinawa, Japan

Nem:Ml

Tarama, Japan

Nem:TR

Nem:SNJ Nem:SRI

Koralawella, Sri L a n k a

Fig. 1. The origin and mutual relationships of the laboratory lines of the house musk shrew, Suncus murinus, used in this study. 105,000 g for 30 min. The solubilization of enzyme activity was expressed as described previously (Takesue et al., 1989). Incubation o f Triton X-lOO-solubilizate with antibodies against sucrase-isomaltase The Triton X-100-solubilizate was incubated with various amounts of antibodies against rabbit sucrase-isomaltase (Nishi and Takesue, 1978) in 1% Triton X-100/0.1 M NaCI/10 mM phosphate (pH 7.0) in a total volume of 0.4 ml for 1 hr at 25°C and then for 2 days at 4°C, after which the mixture was centrifuged at 3000 g for 30 min to precipitate antigen-antibody aggregates. Enzyme activities in the whole mixture and the supernatant were assayed. Enzyme assays Sucrase, isomaltase, maltase, trehalase and lactase were assayed at 37°C in 50 mM sodium phosphate (pH 6.5). Lactase was assayed in the presence of 0.2 mM p-chloromercuribenzoate to completely inhibit contaminating soluble and lysosomal lactases (Koldovsky et al., 1969). The concentration of substrate was 100 mM (sucrose) or 50 mM (isomaltose, maltose, trehalose and lactose). Glucose produced was determined by a modified Tris-glucose oxidase--peroxidase method (Dahlqvist, 1964; Messner and Dahlqvist, 1966). Aminopeptidase N and alkaline phosphatase were assayed as described previously (Takesue et al., 1989). Measurement o f Na + -dependent D-glucose transport activity Na + -dependent transport of D-glucose into BBM vesicles was measured at 0.I mM [t4C]-D-glucose in the presence of an initial concentration gradient across the membrane of 100 mM NaSCN by a rapid membrane filtration method (Kessler et al., 1978; Yokota et al., 1983). Protein determination Protein was determined by the method of Lowry et aL (1951) as modified by Peterson (1983) using bovine serum albumin as standard.

SDS-polyacrylamide gel electrophoresis SDS-polyacrylamide gel electrophoresis was carried out using the system of Laemmli (1970) with 8.4% polyacrylamide gel and Coomassie Brilliant Blue as a stain. Electron microscopy Isolated BBM vesicles were negatively stained with 2% potassium phosphotungstate (pH 7.0) (Nishi and Takesue, 1978), and observed in an electron microscope (Hitachi Model H-7000). In some experiments BBM vesicles were stained with I% uranyl acetate (Kessler et al., 1978). RESULTS AND DISCUSSION Intestinal shrews

disaccharidase

activities

of

Nem:NAG

Disaccharidase activities were assayed in the h o m o g e n a t e a n d the B B M vesicles p r e p a r e d from the small intestine o f N e m : N A G shrews (Table 1). Isomaltase, maltase, trehalase a n d lactase activities were f o u n d in all the shrews examined; lactase activity was m u c h lower t h a n the others, as usually seen in adult m a m m a l s . O n the o t h e r h a n d , sucrase activity was n o t always detected; some shrews of either sex showed n o activity either in the h o m o g e n ate or in the B B M vesicles. Thus, N e r o : N A G shrews can be divided into two groups of sucrase-endowed ( n o r m a l ) a n d sucrase-deficient (deficient) shrews. The B B M vesicles from the two groups were very similar to each o t h e r in specific activity a n d e n r i c h m e n t of the disaccharidascs except for sucrase. In b o t h groups sucrase (if present), isomaltase, a n d maltase activities were enriched ca 10-fold, a n d n o n e o f t h e m was virtually detected in the soluble fraction. O n average, they were recovered by ca 3 0 % in the B B M vesicles Trehalase activity was a little lower enriched, which

Table 1. Specific activity and enrichment of disaccharidascs in intestinal BBM vesicles isolated from Nero: NAG shrews Groups Sucrase Isomaltas¢ Maltase Trehalase Lactase Normal (9) Specific activity 0.275 _+0.128 0.467 + 0.080 1.585 + 0.284 0.307 + 0.096 0.019 _+0,006 Enrichment 12.0 _ 2.8 10.4 __ 1.9 10.4 + 2.1 5.8 + 1.1 6.0 _+1.9 Deficient (7) Specificactivity ND 0.434 + 0.090 1.502 + 0,241 0.364 + 0,109 0.036 + 0.009 Enrichment -9.5 +_1.2 10,3 + 1.5 6.6 -_k_1.1 7.1 + 1.5 Specific activity is given in #tool substrate hydrolyzexl/min/mgprotein; figures are the mean _+SD from triplicated assays on BBM vesicles individually isolated from shrews, the number of which is given in parentheses. Enrichment was calculated by dividing the specificactivity in the BBM vesicles by that in the homogenate; figures represent the mean + SD. ND: not detected.

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Sucrase deficiency in shrew Table 2. Disaccharidaseactivitiesin intestinal BBM vesiclesisolated from shrews of various lines Specific activity (/lmol/min/mg) Lines Sucrase Isomaltase Maltase Trehalase Nero:TR (3) ND 0.340 __.0.057 1.239-1-0.282 0.495 +_.0.156 Nem:SRI (5) 0.768 + 0.296 0.652 + 0.248 2.687 + 0.879 0.683 + 0.347 Nero:NJ (9) ND 0.464 _.+0.069 1.535+ 0.201 0.414 + 0.163 Nem:MI (13) ND 0.360 + 0.133 1.135 + 0.402 0.384 + 0.239 Nem: SNJ (4) ND 0.387 + 0.079 1.222 + 0.371 0.299 + 0.186 - - (11) 0.373 __.0.145 0.539 __.0.217 2.192 + 0.886 0.410 _ 0.176 Figures are the mean+ SD from triplicatedassays on BBM vesiclesisolated individuallyfrom shrews, the number of which is given in parentheses. ND: not detected.

may be due to the presence of soluble trehalase in a significant a m o u n t (ca 8% of the homogenate activity).

Intestinal disaccharidase activities of shrews of the other lines BBM vesicles were isolated from shrews of the other lines and examined for the disaccharidase activities (Table 2). In any line the enrichment of each activity in BBM vesicles was very similar to that of the line N e m : N A G (data not shown). The disaccharidases except sucrase were always found in all the shrews examined, although their average specific activity varied considerably with the line. All Nem: SRI shrews showed sucrase activity, which was even higher than isomaltase activity. On the other hand, none of N e m : T R , N e m : N J and Nem: MI shrews had sucrase activity either in the homogenate or in the BBM vesicles. The line N e m : S N J was similar to the line Nem: N A G ; that is, sucrase activity was found in some shrews but not in others, independently of the sex. Interestingly, in normal N e m : N A G and N e m : S N J shrews sucrase activity was distinctly lower than isomaltase activity, in contrast to the above N e m : S R I shrews. This problem will be discussed later.

Morphology of negatively stained B B M vesicles When isolated shrew BBM vesicles were negatively stained with phosphotungstate and examined in an electron microscope, they were largely elongated and their surface was studded with a great n u m b e r of particles, irrespectively of whether suerase was present or not (Fig. 3), as reported for the hamster and rabbit intestinal BBMs (Johnson, 1967; Nishi et al. 1968; T a m u r a et aL, 1982). The particles have been proposed to be membrane-associated hydrolases such as sucrase-isomaltase (Nishi and Takesue, 1978; Nishi et al., 1968) and maltase-glucoamylase (Norrn et al., 1986). Therefore, one can expect to find some differences in number and/or distribution of the particles between the BBM of a normal and a deficient shrew under better conditions. When negatively stained with uranyl acetate, the BBM vesicles showed a vesicle form of the type often described in the literature (Kessler et al., 1978) (data not shown).

SDS-polyacrylamide gel electrophoresis The protein composition of BBM vesicles isolated from a normal and a deficient N e m : N A G shrew was analyzed by SDS-polyacrylamide gel eleetrophoresis (Fig. 2). The two preparations showed almost the same pattern, which was, however, different from that of rabbit intestinal BBM vesicles, particularly in the range of > 90 kDa. The shrew vesicles gave two strong bands of 96 and 106 kDa, while the rabbit ones showed those of 120 and 140kDa. Rabbit intestinal sucrase and isomaltase are known to migrate as a protein of 120 and 140 kDa, respectively, under the same conditions as in this study (Semenza, 1986). On the other hand, sucrase-free isomaltase which was solubilized with Triton X-100 and purified from N e m : M I shrew BBM vesicles gave a single band of 96 k D a under the same conditions (Takesue et al., in preparation). Therefore, the shrew 96 k D a band is manifested at least partly by isomaltase. The 106 kDa band was seen even in the deficient shrew. Considering the results of Sephadex gel filtration mentioned later together, we tentatively suggest that shrew maltase migrates as a protein of 106 kDa. At present we cannot decide whether or not shrew sucrase and isomaltase have the same relative molecular weight. CBPB 103/3--[

106 96

1

2

3

Fig. 2. SDS-polyacrylamide gel electrophoresis of intestinal BBM vesicles isolated from a normal and a deficient Nero: NAG shrew and a rabbit. Lane 1, deficient shrew (45#g protein); lane 2, normal shrew (70pg protein; S/I ratio, 0.46); lane 3, rabbit (100#g protein). Protein bands: 96, 96kDa; 106, 106kDa; I, rabbit isomaltase; S, rabbit sucrase; V, villin; A, actin.

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Fig. 3. Electron micrographs of BBM vesicles negatively stained with potassium phosphotungstate. In either of the BBM vesicle preparations from a normal (A; S/I ratio, 0.72) or a deficient (B; C, part of B at a higher magnification) Nem:NAG shrew are seen many elongated fragments of microvilli, the surface of which is studded with a great number of particles. Bars represent 100 (A, C) or 200 (B) nm. Association o f sucrase with isomaltase

Sucrase and isomaltase activities were solubilized with 1% Triton X-100 from isolated BBM vesicles to the same extent, which was different from those of the other BBM enzymes tested (Table 3). The data on rabbit BBM vesicles where sucrase is tightly associated with isomaltase are presented for comparison; here also the extent of solubilization of the two activities was the same. When the Triton Table 3. Solubilization of enzymes with Triton X-100 from BBM vesicles Solubilization (%) Enzymes Sucrase Isomaltase Maltase Trehalase Aminopeptidase N Alkaline phosphatase

Shrew

Rabbit

61.3 + 5.1 61.6 +_ 6.9 86.6 ± 2.7 83.0 + 1.8 84.2 + 4.5 14.5 ± 7.5

69.1 71.4 71.8 80.4 84.6 40.2

BBM vesicles were incubated with 1% Triton X-100 at 25°C for 20rain, followed by centrifugation at 105,000g. Solubilization represents the activity in the supematant relative to that in the whole mixture as expressed in percentage. Figures are the mean + SD from assays on three independent preparations (shrew) or the mean from assays o n two preparations (rabbit).

X-100-solubilizate from normal shrews was gelfiltered on a Sephadex G-200 column, sucrase and isomaltase activities were adsorbed on the column at 4°C; upon raising the elution temperature to 30°C, the adsorbed sucrase was eluted together with isomaltase (data not shown). Rabbit sucrase is well known to be adsorbed on Sephadex G-200 in such a temperature-dependent manner, owing to its association with isomaltase (Kolinskfi and Semenza, 1967; Takesue and Kashiwagi, 1969). These results indicate that in the shrew also sucrase is normally associated with isomaltase to form an enzyme complex as in other mammals (Semenza, 1986). A brief mention should be made of maltase activity. Its extent of solubilization was virtually the same as sucrase and isomaltase in the rabbit, but not in the shrew (Table 3). The shrew maltase activity was largely unadsorbed on Sephadex G-200 even at 4°C (data not shown), in contrast to the rabbbit one, which is mostly adsorbed (Kolinskfi and Semenza, 1967). The activity ratio of maltase to isomaltase is three or more, even in sucrase-deficient shrews (see Tables 1 and 2), whereas it is ca 0.1 in the purified shrew isomaltase (Takesue et al., in preparation). These results indicate that in the shrew BBM vesicles maltase activity is largely manifested by another enzyme(s) than sucrase and isomaltase, in contrast to

Sucrase deficiencyin shrew

633 CONCLUSIONS

1oo

<

E

I o

I

I

5 10 15 Antibodies ( m g / U isoma)tase)

I 2O

Fig. 4. Inhibition of isomaltase activity by antibodies against rabbit sucrase-isomaltase.Triton X-100-solubilizate from shrew or rabbit intestinal BBM vesicleswas incubated with various amounts of antibodies against rabbit sucraseisomaltase antibodies. Resulting antigen-antibodiy aggregates were precipitated by low speed ccntrifugation. Isomaltase activity in the whole mixtures and the supernatants were assayed. The activity found is expressed as a percentage of that in the whole mixture without antibodies added. The amount of antibodies used is expressed in mg of -immunoglobulin per unit of isomaltase activity initially used; 1 U = 1 gmol isomaltose hydrolyzed/min, solid symbols, activity in whole mixture; open symbols, activity in supernatant. &, A: Nem:SNJ shrew (S/I ratio, 0.36); O, ©: Nem:MI shrew; V, V: rabbit.

the rabbit where maltase activity is mostly due to the enzyme complex sucrase-isomaltase (Kolinsk/t and Semenza, 1967; Takesue, 1969). Inhibition o f isomaltase activity by antibodies against sucrase-isomaltase Isomaltase activity of rabbit sucrase-isomaltase complex is greately inhibited by antibodies against rabbit sucrase-isomaltase (Takesue et al., 1977). Here we examined effects of the antibodies on isomaltase activity of the shrew (Fig. 4). Triton-solubilized shrew isomaltase was inhibited by the antibodies to almost the same extent as the rabbit enzyme, though much higher amounts of the antibodies were required. The inhibition was the same between isomaltase from normal and deficient shrews. These results suggest that shrew isomaltase has an active site similar to that of the rabbit one and that it is not affected by a mutation(s) in sucrase. Sucrase activity was not inhibited at all by the antibodies, as in the rabbit (Takesue et al., 1977). Na +-dependent D-glucose transport activity BBM vesicles from normal and deficient shrews showed an overshoot uptake of D-glucose, which was dependent on a Na + gradient across the membrane and inhibited by phlorizin with a ~ of 3/~M, as seen with rabbit intestinal BBM vesicles (Yokota et al., 1983). Therefore, active intestinal absorption of D-glucose across the BBM obviously occurs even in deficient shrews.

The present study shows that with respect to disaccharidase activity in the small-intestinal BBM the house musk shrew, Suncus murinus, is the same as most mammals usually used in laboratories (Semenza, 1986). The shrew BBM normally had sucrase associated with isornaltase to form an enzyme complex. However, hereditary sucrase deficiency was found in some laboratory lines of the shrew which, however, always showed isomaltase activity. Since the isomaltase moiety is most unlikely to be modified by a mutation(s) in the sucrase moiety, sucrase deficiency may be expressed in terms of a ratio between specific activity of sucrase and isomaltase (S/I ratio) in BBM vesicles. From the data in Tables 1 and 2, it is calculated to be ca 1.2 in Nem: SRI shrews, ca 0.6 in normal N e m : N A G and Nem: SNJ shrews, and 0.0 in Nem:TR, Nem:NJ and Nem:MI, and in deficient Nem: N A G and Nem: SNJ shrews. These data suggest that in respect to the sucrase gene normal Nero: N A G and Nem: SNJ shrews are heterozygous, while Nem:SRI shrews are homozygous. Detailed hereditary analysis (Oda et al., in preparation) shows that sucrase deficiency in the shrew is controlled by an autosomai recessive gene, as in the human (Hardon et al., 1981). It is interesting from a clinical point of view whether or not sucrase-deficient shrews are intolerant to sucrose. In fact, a significant decrease in body weight or death in the extreme is caused by dosing with a 10% aqueous solution of sucrose, but not of maltose (unpublished data). From the results described above, the shrew is considered as a laboratory animal suitable for studying sucrase deficiency and intolerance to sucrose at the molecular level.

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