Intestinal Brush Border Revisited R HOLMES and R W LOBLEY (This Article Is One Ofa Series Linked with the Festschrift for Christopher Booth

Gut: first published as 10.1136/gut.30.12.1667 on 1 December 1989. Downloaded from

Gut, 1989, 30, 1667-1678 Alimentary tract Intestinal brush border revisited R HOLMES AND R W LOBLEY (This article is one ofa series linked with the Festschrift for Christopher Booth. See Gut Festschrift 1989; 30.) The intestinal brush border is situated at the luminal microvilli overlying a transverse fibrillar meshwork, pole of the enterocyte where it constitutes a func- the terminal web. Ultrastructurally and functionally, tional organelle subserving terminal digestion and however, the brush border is better considered as absorption of the end products of ingested food. Its comprising two different substructures, the surface component microvillus membrane forms a digestive- (microvillus) membrane and the underlying brush absorptive surface the functions of which are com- border cytoskeleton, each with its own distinctive plementary to and integrated with those of the molecular composition (Figure, Table 1). basolateral membrane of the enterocyte, ensuring effective transfer of food products across the entero- MICROVILLUS MEMBRANE cyte from the intestinal lumen to the interstitial fluid The microvillus membrane consists of a lipid bilayer, and blood. the external (luminal) face of which is lined with a Since a previous progress report' there has been a carbohydrate-rich 'fuzzy coat' or glycocalyx.1' The virtual explosion of information concerning the intes- membrane possesses a large complement of integral tinal brush border, with an almost exponential in- membrane proteins, many of which are glycoproteins crease in the number of publications appearing and have been identified with specific brush border annually. In 1960 Crane and coworkers isolated enzymes (Table 1).5X-"' As most, if not all, of the brush borders from hamster small intestine and latter have also been localised to the external face of showed in two classic papers that it was the location the microvillus membrane,'9 it is possible that they of the enzymes sucrase and maltase,' and also of account for at least part of the glycocalyx. the sodium dependent transport of glucose and The brush border enzymes which have been inves- http://gut.bmj.com/ galactose.3 Twenty five years later, over 22 enzymes tigated in detail share a number of common features and 19 transport or binding functions had been (Table 1). All are relatively large (70-320 kDa) localised to the brush border. Much of this work was glycoproteins and most are apparently composed of carried out using intestinal preparations from small two or more subunits. They appear to be essentially animals, but since 1972 human tissue obtained at globular structures which are attached to the external operation or by peroral biopsy has been increasingly face of the membrane by a small (2-5 kDa) anchoring

used.45 Subcellular fractionation techniques for segment embedded in the lipid bilayer. 19 In negatively on September 27, 2021 by guest. Protected copyright. isolating brush borders and microvillus membranes stained preparations, the membrane surface appears have been refined` and microvillus membrane to be covered with numerous particles of approxi- vesicles are now used almost routinely in the investi- mately 50 nm diameter.' These can be largely re- gation of intestinal enzyme and transport functions,7 moved by proteolytic treatment with papain or elas- while definitive information on the structure and tase, with the concomitant release of several of the function of the microvillus membrane has been glycosidases and peptidases from the membrane in a obtained through the use of electrophoresis,1'" soluble form.47 Careful study of renal microvilli has monoclonal antibodies,1'3 gene sequencing,'1" and indicated that before papain treatment, the mem- the study of colon cancer cell lines in vitro." brane is coated with particles attached by 'stalks' of This review is focused on newer, molecular aspects between 2.5 and 9 nm in length whereas after treat- of the structure, biosynthesis and function of the ment only those particles with stalks 2-3 nm in length human brush border and its cytoskeleton rather than remain attached.' This suggests that brush border the traditional, though no less important, areas of enzyme release by papain is largely a function of digestive and absorptive function. whether the stalk is long enough to allow access of the papain molecule between the globular portion of the Brush border structure and composition protein and the membrane surface. For most enzymes studied (sucrase-isomaltase, aminopeptidase From a morphological viewpoint, the intact brush N, dipeptidyl aminopeptidase IV, y-glutamyl trans- border is composed of numerous fingerlike apical ferase) the anchoring segment is characterised by a 1667 Gut: first published as 10.1136/gut.30.12.1667 on 1 December 1989. Downloaded from

1668 Holmes and Lobley Table 1 Brush border enzymes and related proteins ofthe microvillus membrane

Function Protein Molecular Carbohydrate DNA Primary Abundance References mass (kDa) content sequence deficiency (On) Glycosidase Maltase-glucoamylase 330/125+ 135§ + 9 11 20 21 Sucrase-isomaltase 145+151 + + + 10 911 1420(120 Lactase-phlorizin hydrolase 160 (x2) + + + 9 11 1522-24 12( Trehalase 80 (x2) + + 0(2 25 26 120 Peptidase Aminopeptidase A 170 (x2) + 27-29 60 Aminopeptidase N 162 (x2) + 4 9 11 2829 Aminopeptidase W 130 + 1 13 Carboxypeptidase P 130 + 3( 6(0 Dipeptidyl aminopeptidase IV 136 (x2) + 9 11 29 3(1 Peptidyl dipeptidase* 180 11 29 31 Pteroyl polyglutamate hydrolaset 91 32 Enteropeptidase 300§ + + <01 33-36 12(1 Endopeptidase-24. 11 96 (x 2) + (01-1§ 37 87 Endopeptidase-2t 100 (x 2) + 38 39 y-Glutamyl transferase 62+21 + + + 16 40 41 Phosphatase Alkaline phosphatase 86 (x 2) + + (01 17 42 43 Phosphodiesterase-1 51 Unknown '14OkDa Glycoprotein' 140 + 1 53 54 Regulatory Guanylate cyclase 44 Phospholipase A2 45

*Angiotensin converting enzyme; t'folate conjugase'; V'PABA peptidase'; §variable, dependent on species. Where possible reference has been made to human data. single hydrophobic sequence of about 20 amino acids cholesterol, phospholipids and various neutral lipids, which spans the membrane only once in an a-helical together with an assortment of glycolipids, and to conformation.'9 This hydrophobic domain is located differ from the basolateral membrane in its lipid adjacent to a short, hydrophilic N-terminal sequence which is exposed at the cytoplasmic face of the

membrane. http://gut.bmj.com/ A second mode of anchorage of brush border enzymes was recently discovered in which the protein is anchored to the membrane not by an N-terminal hydrophobic peptide but through the diacylglycerol moiety of a phosphatidylinositol glycan attached covalently to the C-terminal amino acid.49" Proteins anchored in this way are released only slowly or not at all by papain, but are readily released from the on September 27, 2021 by guest. Protected copyright. membrane by treatment with phospholipase. Brush border alkaline phosphatase, ` phosphodiesterase-l' and trehalase52 have so far been shown to have this mode of anchorage. These structural features are not unique to the microvillar enzymes. Thus Gorvel et al have identified a 140 kDa microvillar glycoprotein which is apparently devoid of enzyme activity but is, like the brush border enzymes, attached externally to the membrane surface.`3-9 The function of this glycoprotein remains to be elucidated but, unlike the brush border enzymes, it is present in immature crypt cells as well as in mature enterocytes. Although the lipid composition of intestinal brush borders was investigated over 20 years ago, much less is known about the lipids of the brush border Figure Schematic diagram ofthe molecular architecture membrane than about the proteins. The microvillus and ultrastructural organisation ofthe intestinal brush membrane has, however, been reported to contain border. Adaptedfrom references 63 64 65. Gut: first published as 10.1136/gut.30.12.1667 on 1 December 1989. Downloaded from

Intestinal brush border revisited 1669 composition.7 In the early 1980s, it was reported C YTOS K E L EIOON that the microvillus membranes of the enterocyte The brush border cytoskeleton, comprising the were much less fluid (more rigid) than their baso- microvillus core and terminal web, was long regarded lateral counterparts"s and, since a lower membrane as a relatively simple, inert support for the micro- fluidity implies a lower passive membrane perme- villus membrane. Recent advances in cell biology ability and an increased mechanical stability, this have stimulated great interest in this structure, which may have functional implications.'"" The lower is now known to be a highly organised, dynamic fluidity of the apical membrane has been attributed to complex of at least three inter-relating filamentous its distinctive lipid composition and, specifically, to networks, each with its own complement of special- a high cholesterol:phospholipid ratio." A recent ised, filament forming proteins (Figure). This not analysis of this topic, however, has emphasised that, only serves as a supporting structure to maintain the when due allowance is made for all the major classes architecture of the entire apical pole of the entero- of membrane lipid, the principal difference between cyte, but also may play an active role in regulating the microvillar and basolateral lipids is a replacement in uptake of some nutrients into the cell and in the the microvillus membrane of most of the phos- control of paracellular permeability. phatidylcholine by glycosphingolipids, to the extent In transmission electron micrographs of well pre- that these account for one third of the total micro- pared specimens, each microvillus can be seen to villar lipid.5`'7 Because glycosphingolipids are more contain an ordered bundle of 2(-3() microfilaments highly ordered than phospholipids because of their composed predominantly of the P and y -isoforms of extensive hydrogen bonding capacity, and are also actin and running longitudinally from the microvillus invariably located to the external face of cellular tip into the underlying terminal web (Figure).""'-- The membranes, it seems likely that the lower fluidity of core is attached laterally to the membrane through the microvillus membrane is essentially a property of spirally arranged bridges containing a complex of an these components of the outer leaflet of the lipid unnamed 110 kDa protein, the 17 kDa Ca' -binding bilayer.'6 ` protein calmodulin, and a 140 kDa membrane bind- The protein composition of the microvillus mem- ing glycoprotein. The portion of the microvillus core brane varies both during development'"" and along embedded in the terminal web, the core rootlet, lacks the length of the intestine,"" as might be expected in the 11) kDa-calmodulin complex but contains in- view of the temporal and regional variations that are stead another actin binding protein, tropomyosin.

known to occur in intestinal function. Thus consider- The axial core bundle of,actin microfilaments is http://gut.bmj.com/ able changes were observed in the clectrophoretic cross linked into a regular hexagonal array through profile of microvillar proteins of neonatal rats during short, cross-bridging filaments which are organised intestinal maturation and weaning,`" some of which longitudinally in an et-helical arrangement.-""' Two could be correlated with functional development - actin binding proteins, fimbrin and villin, are in- for example, the appearance of sucrase activity and volved in the interfilament cross links. Fimbrin is an the postweaning decline in lactase. Differences be- ubiquitous 68 kDa globular protein which, in the tween duodenal and ileal microvillar protein profiles presence of K' and divalent cations, cross links actin have also been recorded for human""""' and animal"2 filaments in lvitro into highly ordered bundles closely on September 27, 2021 by guest. Protected copyright. intestine, but correlation with functional differences resembling the native microvillus core. Villin is a 95 has been more difficult, possibly owing to a low kDa globular protein of restricted tissue distribution, abundance of the relevant proteins (transport-rela- and may prove to be useful as a tumour marker." It ted proteins such as the Na'-glucose cotransporter or has three Ca"t-binding sites per molecule and inter- the ileal vitamin B,2 intrinsic factor receptor are acts with actin in a Ca'n-dependent manner, pro- minor components of the microvillus membrane, moting filament bundling at submicromolar Ca' each representing 0-2% or less of the total micro- concentrations but causing fragmentation at higher villar protein). Differences in glycosylation between Ca" levels. proximal and distal microvillus membranes have also Within the terminal web two distinct regions can be been recorded, as shown by altered binding of lectins distinguished (Figure). The inter-rootlet zone con- to isolated brush border membranes and changes in sists of a dense meshwork of fine, non-actin filaments the electrophoretic mobility of such enzymes as which appear to attach the core rootlets of adjacent lactase, aminopeptidase N and dipeptidyl amino- microvilli."-' These filaments contain non-muscle peptidase IV.`2 These differences could be explained myosin (200 kDa) and fodrin, a calmodulin binding, on the basis of less complete glycosylation in the spectrin-related protein comprising 240 and 235 kDa proximal intestine and substantially greater sialic subunits. At the level of the zonula adherens (ZA), acid incorporation distally, but the physiological the junctional complex at the lateral margin of the significance of this is unclear. cell consists of a circumferential bundle of actin Gut: first published as 10.1136/gut.30.12.1667 on 1 December 1989. Downloaded from

1670 Holnes and Lobley filaments, beneath which lies a network of thicker, notably sucrase-isomaltase`" and aminopeptidase N, intermediate filaments containing cytokeratin. The as well as on microvillar glycolipids. Finally the ZA bundle filaments, which resemble the stress fibres mature enzyme is transported by an undefined mech- of cultured fibroblasts, contain myosin, tropomyosin anism to the brush border and incorporated into the and ca-actinin and appear to be closely associated with microvillus membrane, where it remains anchored by the ZA tight junctions through adhesion plaque like the N-terminal hydrophobic signal sequence (see structures containing a-actinin and vinculin."6' Like above). There is some evidence that transfer of at stress fibres, the ZA bundle is contractile through an least aminopeptidase N to the brush border from the ATP-dependent process," and this provides an Golgi may be indirect and involve transient passage attractive candidate mechanism to control the open- through the basolateral membrane," but at present ing of the tight junctions between adjacent en- this interpretation is controversial. terocytes and thereby regulate paracellular perme- Superimposed on this basic biosynthetic theme are ability.""" a number of variations. So far as is known, all brush Two other proteins of the brush border cyto- border enzymes are synthesised as single polypeptide skeleton are of interest, although their structural and chains, each having a single anchoring peptide. functional roles are unclear. One, a relatively minor Many, especially peptidases, appear to dimerise at 80 kDa component of the microvillus core, appears to somc point after synthesis and there is dual anchorage be immunologically related to a substrate for the of the dimer to the microvillus membrane." ` Other tyrosine kinase of the epidermal growth factor recep- enzymes, notably sucrase-isomaltase, are agalin syni- tor.' The other, a 36 kDa protein named calpactin 1, thesised as large, single chain polypeptides but, after is confined to the terminal web.-' Calpactin-1 is insertion into the microvillus membrane, are cleaved a calmodulin-, phospholipid-, and actin-binding into two subunits through the action of luminal protein which is also a major substrate of the proteases, particularly clastase. "' The resulting dual oncogene-related pp6O src tyrosine kinase. The enzyme is thus attached to the membrane via a single presence of these proteins raises the intriguing possi- anchor which, in the case of sucrase-isomaltase, bility of functional regulation of the brush border resides in the sucrase subunit while the isomaltase cytoskeleton through hormone or growth factor subunit is anchored indirectly via the sucrase moiety. receptor-induced protein phosphorylation. Essentially the same basic biosynthetic scheme applies to maltase-glucoamylase and lactase-phlorizin Biosynthesis of brush border proteins hydrolase, both of which are double enzymes with a single anchor,"' and also to y-glutamyl transferase http://gut.bmj.com/ The past decade has seen considerable progress in which differs from the disaccharidases only in that the understanding the biosynthesis of brush border pro- larger of its two subunits is enzymatically inactive.4' teins and it now appears that most, if not all, A slightly different biosynthetic route must be microvillar enzymes are produced in a fundamentally postulated for alkaline phosphatase, phosphodiester- similar fashion.'' ""'- Translation of the appro- ase-l and trehalase which possess a phospholipid priate mRNA takes place in the rough endoplasmic anchor instead of a hydrophobic anchoring peptide. reticulum, beginning with an N-terminal, hydro- In these cases, the original N-terminal signal sequence on September 27, 2021 by guest. Protected copyright. phobic 'signal sequence' whose function is to provide required for membrane attachment during transla- anchorage to the endoplasmic reticular membrane tion is presumed to be cleaved intracellularly and while the nascent polypeptide chain passes across the replaced by covalently attached phospholipid at the membrane into the cisternal space. The first and C-terminus.1'5""The subsequent processing and mem- apparently co-translational phase of N-linked glyco- brane insertion of these enzymes is likely to resemble sylation of the molecule takes place with the addition that of the aminopeptidases. en bloc of oligomannosyl glycans at asparagine The action of luminal proteases on newly syn- residues. The resulting 'high mannose' product is a thesised microvillar enzymes can be regarded either transient form which undergoes further processing in as a late biosynthetic event, as described above the Golgi apparatus. There, the linear, N-linked for sucrase-isomaltase, or as an early event in the high-mannose oligosaccharide chains are converted enzyme's degradation, as for example in the case of into the complex, branched structures of the mature porcine aminopeptidase N."' Both organ culture enzyme and 0-linked glycosylation at serine and experiments and studies in animals with pancreatic threonine residues also takes place. In several mam- duct diversion have clearly shown that the mature, malian species, including man," rabbit and dog,"b brush border form of this enzyme is a 160 kDa the brush border of secretors is rich in blood group glycoprotein, whereas porcine microvillus mem- determinants and the carbohydrate antigens respon- branes which have been exposed to normal levels sible are expressed on the microvillar enzymes, of pancreatic enzymes in Oivo contain substantial Gut: first published as 10.1136/gut.30.12.1667 on 1 December 1989. Downloaded from

Intestinal brush border revisited 1671 amounts of 120 and 60 kDa breakdown products. absorption.)' Recent studies have clearly shown, Pancreatic proteases also appear to be involved in however, instances where two or more brush border brush border catabolism through their action in peptidases may function in concert in the degradation solubilising microvillar enzymes and releasing them of oligopeptides. Thus the degradation of gliadin from the membrane into the intestinal lumen.' This peptides was shown to be dependent on the con- release mechanism has been demonstrated in viv'o for certed action of angiotensin converting enzyme and disaccharidases and alkaline phosphatase and, most aminopeptidase N,'8 and the combined action of notably, for enteropeptidase.7" For disaccharidases, endopeptidase-2 and aminopeptidase N` on various this appears to be a major factor in regulating their substrates has also been demonstrated. The vexed turnover and in determining their mucosal activity, question of the function of intestinal alkaline phos- since subtotal pancreatectomy or pancreatic bypass phatase has also been partly resolved: a recent causes a 50% increase in disaccharidase activity and a investigation of its role in phosphate absorption decrease in the rate of turnover in rats.77 79 Turnover concluded that the enzyme had no direct role in the rates of brush border proteins are known to be rapid absorption of phosphate but was involved indirectly (half-life of approximately 18 h) when compared with through the vectorial release of inorganic phosphate the whole mucosal homogenate (t½/=31 h) and from dietary organic phosphates,88 a role analogous turnover of disaccharidases is even more rapid to that of sucrase in glucose absorption from sucrose. (t½/2=11.5 h), as larger brush border proteins generally have shorter half-lives than the smaller TJRANSPORT FUNCTIONS components.7 In 1961 Crane and coworkers made the definitive Compared with the microvillus membrane, rela- proposal that monosaccharides such as glucose and tively little is known about the biosynthesis of the galactose were transported across the brush border brush border cytoskeleton although its assembly has membrane by a carrier mechanism which cotrans- been extensively investigated and reviewed. That the ports Na-.` The energy required for sugar transport cytoskeleton is not a static structure is indicated by is derived from the flux of Na' at high concentration the transient microvillus shortening which occurs, for example, during fasting.`' In 'it'o pulse chase experiments have indicated that turnover of actin and other Table 2 Maljor tran.sport functions ofthe bi-usli bor-tder components of the cytoskeleton takes place at rela- - tively low rates," but in vitro studies have shown that .Subsraute(x) Nau rimarY Referetwes http://gut.bmj.com/ villin and the 110 kDa-glycoprotein are synthesised depenletwne l(eficitencl' very rapidly, approximately five times faster than for Monosaccharides glucose. brush border membrane proteins.' galactose + + 9091 93 fructose 92) Functions of the brush border Amino acids acidic; - glutarnic acid + + The primary functions of the brush border undoubt- netitral, edly relate to the terminal digestion and absorption - alanine + Hartnup 85 93 on September 27, 2021 by guest. Protected copyright. basic -lysine + Cvstinuria of nutrients. While these have, for the most part, imino: been well documented over many years, newly - proline + discovered enzymes and transport functions have to phenvlalanine + be integrated into the overall picture. In addition Peptides dipeptidels ? 94 Fatty acids long chilin there are newly recognised functions, mainly of a - olcic acid ? 91n regulatory nature, such as the possible modulation of Bile salts such as. paracellular permeability through the brush border taurocholate + + 96 97 cytoskeleton, and of ion transport by receptors and Vitamins ascorbic acid + biotin + other regulatory proteins located in the brush border. folic acid + + inositol + 98 DIGESTIVE/ENZYMATIC FUNCTIONS pantotlicnic The importance of the disaccharidases in carbo- acid + thianiinc + hydrate digestion has long been recognised.)' It is Ions Ca 99 widely assumed that the brush border peptidases Fe , Fe 11(X) have an analogous function in protein digestion but it Na /1' + 1(11 1(21 has proved difficult to define the role of individual Cl /iCOa + 11)3 104 phosphate. peptidases, possibly because of their overlapping sulphate + 105 106 specificities and the existence of intact peptide Gut: first published as 10.1136/gut.30.12.1667 on 1 December 1989. Downloaded from

1672 Holmes and Loblev outside to low concentration inside the cell, the several G-proteins including the oncogene-related gradient being maintained by the Na+-ATPase pump p21 ras; and phospholipase A`...4'"." There is thus in the basolateral membrane. As shown in Table 2, the potential for both direct regulation - for example, Na -dependent transport has been demonstrated for by the cyclic AMP- or GMP-stimulated phosphoryla- a wide variety of substances including sugars, amino tion of a component of a transporter molecule, and acids, bile salts, and some vitamins and ions. Although for indirect processes mediated through the release much of this information was obtained from studies of second messengers such as Ca" acting, for using animal tissues, steady progress has been made example, via protein kinase C and the phospha- in confirming the findings using human preparations, tidylinositol cascade system.'"-'" Except in certain particularly microvillus membrane vesicles. " "h 101 10k enteropathic diarrhoecas, however (see below), it is Support for the existence of some of these transport unclear which of these mechanisms operate in rivi'o. pathways comes from the recognition of specific clinical conditions in which a congenital absence or Brush borders and disease inactivity of the membrane carrier protein leads to inability to absorb the corresponding substrate PRIMARY DISORDERS (Table 2). Primary alteration in the structural or functional The structure and biochemical characteristics of organisation of the brush border membranc diue to most of the carrier proteins are unknown, but con- the congenital or acquired abscice or maytit' of a siderable effort has been directed to the isolation and specific functional component providec\ . t .'! t ional characterisation of the Na+-glucose co-transporter. explanation of certain clinical condition's ol ''y tired This appears to be a large protein comprising three 75 digestion and absorption (Tables 1 aind 22). \ itli the kDa subunits, each with an active site for glucose and exception of primary acquired lactase deficieciey, Na'.W"' "" Expression of the transporter mRNA in which is common and of racial origin,'"' these con- Xenopus oocytes has been studied and the gene ditions are rare and most of them manifest by sequenced."' "'" Work is in progress to determine malabsorption of the specific substrate involved. which biochemical features of the molecule are Specific malabsorption syndromes have been associa- responsible for its transport properties, and to un- ted with absence or inactivity of the following enizyme ravel the nature of the molecular defect in primary proteins: sucrase-isomaltase, lactase, trehalase, glucose-galactose malabsorption. Some progress has enteropeptidase, and y-glutamyl transferase.""-"' In been made in a 100 kDa patients with disaceharidase deficiency - for example, also identifying microvillar http://gut.bmj.com/ polypeptide as a component of the proline carrier."' lactase, avoiding the specific substrate (lactose) brings Transport of some macromolecules is effected by about clinical remission; alternatively this has been receptor-mediated endocytosis. Thus vitamin B12- achieved by feeding the appropriate enzyme orally intrinsic factor complex binds in a Ca"'-dependent - for example, lactase (of microbial origin) ini hypo- manner to an ileal microvillar receptor protein"' ll lactasia. . before absorption by endocytosis."4 A similar mech- Of the primary enzyme deficiencies, oiily in anism is thought to be responsible for the absorption sucrase-isomaltase deficiency have detailed bio- of immunoglobulin-G in the neonate."' chemical investigations been carried out."'""' Three on September 27, 2021 by guest. Protected copyright. distinct phenotypes were recently identified with the RI.GUI-ATORY FUNCIIONS aid of monoclonal antibodies."' In phenotype I Although a fast growing discipline, investigation of there was overproduction of an intracellular, high- the regulation of brush border function is still in its mannose precursor of the enzyme which accumula- infancy and accounts of the processes involved are ted in the endoplasmic reticulum and was degraded largely hypothetical. Nevertheless the brush border intracellularly without reaching the brush border; is known to contain at least two Ca"- and cyclic this precursor was poorly converted into the mature, nucleotide-sensitive transport systems, viz Na-Cl complex glycosylated form, apparently because of cotransport (by linked Na/H' and Cl /IHCO3 ex- its inability to be properly transported in the endo- change) and an electrogenic Cl channel,"` while a plasmic reticulum. Phenotype 11 was characterised by number of potential regulatory proteins have been apparently normal synthesis of the high-mannose found there. These include guanylate cyclase; cyclic precursor but without conversion to the complex AMP-dependent protein kinase; a brush border form; the precursor accumulated in the Golgi specific, 86 kDa G-kinase; 25 and 21 kDa phospho- apparatus, where it was degraded without reaching proteins acting as cosubstrates for the A- and the brush border. In phenotype III, biosynthesis of G-kinases; several phosphoprotein substrates for a both high-mannose and complex precursors appeared Ca - and calmodulin-dependent kinase and the normal and the mature enzyme protein was incor- Ca '- and phospholipid-dependent protein kinase C; porated into the microvillus membrane and cleaved Gut: first published as 10.1136/gut.30.12.1667 on 1 December 1989. Downloaded from

Intestinal brush border revisited 1673

into separate subunits; the absent sucrase activity occurred the A subunit penetrates the membrane and in the presence of normal isomaltase indicated a activates adenylate cyclase, thereby initiating the mutation affecting the sucrase active site alone, secretory diarrhoea."' 3'9 E coli and Yersinia heat although the presence of immunoreactive enzyme at stable toxins bind to a specific microvillar glycopro- the basolateral membrane suggested that the intra- tein and provoke secretion by activating guanylate cellular sorting responsible for directing the enzyme cylase,"I 139 140 although Ca+, calmodulin and the to its correct cell surface destination was also affected. phosphoinositol cascade appear also to be involved. Patients with primary deficiencies of transport Binding of Clostridial enterotoxin A to a microvillar carrier proteins have also been described showing glycoprotein has also been shown'4' and recent work malabsorption of glucose-galactose, glutamic acid, suggests that it may induce diarrhoea partly by alanine, lysine, bile salts, folic acid, Na+ and C1- altering paracellular permeability through an effect (Table 2). In none of these disorders, however, has on the cytoskeleton."9 an abnormal transporter been characterised. In the rare condition congenital microvillus Conclusion atrophy the brush border may appear hypoplastic or even invaginated into the Cytosol.'24 ,2 Electro- The immense amount of work carried out over the phoretic analysis of microvillar proteins has shown a past 30 years has enabled much of the structure and marked reduction in the myosin band and this function of the intestinal brush border to be des- disorder may therefore be caused by a defect of the cribed in molecular terms. It is now apparent that the brush border cytoskeleton.'29 microvillus membrane and the subjacent cytoskeleton interact functionally and there can be little doubt SECONDARY MALABSORPTION that further details of this interaction will be forth- In secondary malabsorption there is structural or coming. Rapid progress is being made concerning functional damage to the brush border membrane as the mode of production, regulation and biological a consequence of other disease. Thus in coeliac function of individual proteins of the microvillus disease the microvilli are abnormally short, irregular membrane, and it is anticipated that the isolation and and sparse and the terminal web is incompletely molecular characterisation of additional transport developed.'27 Brush border enzymes are reduced to proteins will not be long delayed. The gene sequence around 10% of normal but recover on gluten with- of several brush border proteins is already known and drawal, except for 3-glucosidase and lactase which others are doubtless being determined. The intestinal http://gut.bmj.com/ show a slower and less complete response.1'28 brush border has thus truly come of age: it is no The microvillus membrane composition is severely longer solely of interest to gastroenterologists and deranged in untreated disease but returns virtually to gut physiologists but is now playing a central role in normal after treatment.1'29 13 In postinfective tropical answering fundamental questions at the heart of cell malabsorption damage to the microvilli is less severe biology itself. and microvillar enzyme activities are less affected.'3'

In extensive small intestinal Crohn's disease total University Department ofGastroenterology, on September 27, 2021 by guest. Protected copyright. microvillar membrane function can be severely Manchester Royal Infirmary, reduced. In Crohn's disease not involving the Oxford Road, jejunum, brush border disaccharidase activities were Manchester reduced'32 but, in contrast, subcellular fractionation of diseased ileal mucosa has shown an essentially normal distribution of marker enzymes. 131 References Bacterial overgrowth of the small intestine appears I Holmes R. The intestinal brush border. Gut 1971; 12: to involve adherence of sugar binding proteins 668-77. (lectins) of the microorganism to specific oligosac- 2 Miller R, Crane RK. The digestive function of the charides of the microvillus membrane.'34 '^ Brush epithelium of the small intestine. II. Localization of border enzyme activities may then be reduced by the disaccharide hydrolysis in the isolated brush border action of bacterial proteases and this may contribute portion of intestinal epithelial cells. Biochim Biophys to the malabsorption found. 131 131 Similarly in Acta 1961; 52: 293-8. Giardiasis, there may be a significant reduction in 3 McDonald DB, Little KD. Crane RK. Studies on the mechanism of intestinal absorption of sugars. IV. lactase, sucrase and aminopeptidase activities which Localisation of galactose concentrations within the is reversible with effective treatment.'38 In cholera, intestinal wall during active transport in vitro. Biochim the B subunits of the enterotoxin bind with high Biophys Acta 1960; 45: 483-9. affinity to specific glycolipid receptors (GM1-ganglio- 4 Schmitz J, Preiser H, Maestracci D, Ghosh BK, Cerda side) on the microvillus surface. After binding has JJ, Crane RK. Purification of the human intestinal Gut: first published as 10.1136/gut.30.12.1667 on 1 December 1989. Downloaded from

1674 Holmes and Lobley

brush border mcmbrane. Biochim Biophys Acta 1973; alkaline phosphate. Proc Natl Acad Sci USA 1987: 84: 323: 98-112. 1234-8. 5 Freiburghaus AU, Hauri HP, Green J, Hadorn B. 18 Ito S. Structure and function of the glycocalyx. Fed A micromethod for separation and identification of Proc 1969: 28: 12-25. digestive enzymes in brush border mcmbrane frag- 19 Semenza G. Anchoring and biosynthesis of stalked ments of single human intestinal biopsies. Clin Cliim brush border membrane proteins: glycosidases and Acta 1978; 86: 227-34. peptidases of enterocytes and renal tubuli. Ann,l Rev 6 Kessler M, Acuto 0, Storelli C, Murer H, Muller M, Cell Biol 1986: 2: 255-313. Semenza G. A modified procedure for the rapid 20 Kelly JJ, Alpers DH. Blood group antigenicity of preparation of efficiently transporting vesicles from purified human intestinal disaccharidases. J Biol Chemi small intestinal brush border membranes their use in 1973: 218: 8216-21. investigating some properties of D-glucose and choline 21 Kelly JJ, Alpers DH. Properties of human intestinal transport systems. Biochim Biop/iys Acta 1978; 506: glucoamylase. Biochimn Bioph'xs Acta 1973: 315: 113- 136-54. 20. 7 Stevens BR, Kaunitz JD, Wright EM. Intestinal trans- 22 Lau HKF. Physicochemical characterization of human port of amino acids and sugars: advances using intestinal lactase. Biochemn J 1987; 241: 567-72. membrane vesicles. Ann Rev Physiol 1984; 46: 417-33. 23 Skovbjerg H, Sjostrom H, Noren 0. Purification 8 Maestracci D, Schmitz J, Preiser H, Crane RK. Pro- and characterisation of amphiphilic lactase/phlorizin teins and glycoproteins of the human intestinal brush hydrolase from human small intestine. Elur J Biochemi border membrane. Biocliim Biophys Acta 1973; 323: 1981: 114: 653-61. 113-24. 24 Potter J, Ho MW, Bolton H. Furth AJ, Swallow DM, 9 Skovbjerg H, Sjostrom H, Noren 0, Gudmand-H0yer Griffiths B. Human lactase and the molecular basis E. Immunoclectrophoretic studies on human small of lactase persistence. Biochetin Getiet 1985: 23: 423- intestinal brush border proteins. A quantitative study 39. of brush border enzymes from single intestinal biopsies. 25 Galand G. Purification and characterization of kidney Clin Chim Acta 1979; 92: 315-22. and intestinal brush border membrane trehalases from 10 Lobley RW, Pemberton PW, Holmes R. Protein the rabbit. Biochim Biophys Acta 1984: 789: 10-19. mapping of the human intestinal brush-border mem- 26 Yokota K, Nishi Y, Takesue Y. Purification and brane by two-dimensional electrophoresis. In: characterization of amphiphilic trehalase from rabbit Alvarado F, van Os CH, eds. Ion gradient-coupled small intestine. Biochimn Biophvys Acta 1986: 881: 405- transport (INSERM Symp 26). Amsterdam: Elsevier, 14. 1986: 37-40. 27 Benajiba A. Maroux S. Purification and characteriza- 11 Hauri HP, Sterchi EE, Bienz D. Fransen JAM, Marxer tion of an aminopeptidase A from hog intestine brush A. Expression and intracellular transport of microvillus border membrane. EiurJ Biochein 1980; 107: 381-8. http://gut.bmj.com/ membrane hydrolases in human intestinal epithelial 28 Tobey N, Heizer W, Yeh R, Huang TI, Hoffner C. cells. J Cell Biol 1985; 101: 838-51. Human intestinal brush border peptidases. Gastro- 12 Quaroni A. Use of monoclonal antibodies in the study enterology 1985: 88: 913-26. of intestinal structure and function. In: Porter R. 29 Naim HY, Sterchi EE, Hauri HP, Lentze MJ. Purifica- Collins GM, eds. Bruish border membranes (Ciba tion and biosynthesis of the brush-border hydrolases of Foundation Symp 95). London: Pitman Books. 1983: the human small intestine. In: Alvarado F, van Os CHR 113-31. eds. Ion gradient-colupled tranisport (INSERM Symp

13 Kenny AJ, Gee NS. Exploring the brush border 26). Amsterdam: Elsevier, 1986: 71-4. on September 27, 2021 by guest. Protected copyright. membrane with monoclonal antibodies: the discovery 30 Auricchio S, Greco L, de Vizia B, Buonocore V. of aminopeptidase W. In: Alvarado F, Van Os CH, Dipeptidylaminopeptidase and carboxypeptidase eds. Ion gradient-coupled transport (INSERM Symp activities of the brush border of rabbit small intestine. 26). Amsterdam: Elsevier, 1986: 49-59. Gastroenterology 1978; 75: 1073-9. 14 Green F, Edwards Y, Hauri HP, et al. Isolation 31 Ward PE, Sheridan MA, Hammon KJ, Erdos EG. of a cDNA probe for a human jejunal brush border Angiotensin I converting enzyme (kininase II) of the hydrolase, sucrase-isomaltase, and assignment of the brush border of human and swine intestine. Biochein gene locus to chromosome 3. Gene 1987: 57: 101-10. Pharmacol 19800; 29: 1525-9. 15 Mantei N, Villa M, Enzler T, et al. Complete primary 32 Reinsenauer AM, Halstead CH. Human jejunal brush structure of human and rabbit lactase-phlorizin hydro- border folate conjugase. Characteristics and inhibition lase: implications for biosynthesis, membrane anchor- by salicylazosulfapyridine. Biochimn Biophys Acta 1981; ing and evolution of the enzyme. EMBO J 1988: 7: 659: 62-9. 2705-13. 33 Schmitz J, Preiser H, Maestracci D, Crane RK, Troesch 16 Laperche Y, Bulle F, Aissani T, et al. Molecular V, Hadorn B. Subcellular localisation of enterokinase cloning and nucleotide sequence of rat kidney y- in human small intestine. Biochimn Biophys Acta 1974; glutamyl transpeptidase cDNA. Proc Natl Acad Sci 343: 435-9. USA 1986; 83: 937-41. 34 Lobley RW, Franks R, Holmes R. Subcellular localisa- 17 Henthorn PS, Raducha M, Edwards YH, et al. Nucleo- tion of enterokinase in human duodenal mucosa. Clini tide and amino acid sequences of human intestinal Sci Mol Med 1977; 53: 551-62. alkaline phosphatase: close homology to placental 35 Anderson LE, Walsh KA, Neurath H. Bovine entero- Gut: first published as 10.1136/gut.30.12.1667 on 1 December 1989. Downloaded from

Intestinal brush border revisited 1675

kinase. Purification, specificity, and some molecular 51 Nakabayashi T, Ikezawa H. Alkaline phosphodiester- properties. Biochemistry 1977; 16: 3354-60. ase-l release from eukaryotic plasma membranes by 36 Magee AI, Grant DAW, Hermon-Taylor J. Further phosphatidylinositol-specific phospholipase C. 2. The studies on the subunit structure and oligosaccharide release from brush border membranes of porcine moiety of human enterokinase. Clin Chim Acta 1981; intestine. Toxicon 1986; 24: 975-81. 115: 241-54. 52 Takesue Y, Yokota K, Nishi Y, Taguchi R, Ikesawa H. 37 Kenny AJ, Fulcher IS. Microvillar endopeptidase, an Solubilization of trehalase from rabbit renal and intes- enzyme with special topological features and a wide tinal brush border membranes by a phosphoinositol- distribution. In: Porter R, Collins GM, eds. Bruish specific phospholipase C. Febs Lett 1986; 201: 5-8. border membranes (Ciba Foundation Symp 95). 53 Gorvel JP, Rigal A, Olive D, Mawas C, Maroux S. London: Pitman Books, 1983; 12-33. Identification of an early expressed marker of the 38 Sterchi EE, Naim HY, Lentze MJ, Hauri HP, Fransen luminal membrane of rabbit small intestinal columnar JAM. N-Benzoyl-L-tyrosyl-p-aminobenzoic acid cell. Presence of a homologous antigen in kidney. Biol hydrolase - a metalloendopeptidase of the human Cell 1986; 56: 121-6. intestinal microvillus membrane which degrades bio- 54 Gorvel JP, Rigal A, Bernadac A, Maroux S. Further logically active peptides. Arch Biochem Biophvs 1988; characterization of an early expressed glycoprotein of 265: 105-18. the rabbit small intestinal brush border. EurJ Biochem 39 Sterchi EE. Naim HY, Lentze MJ. Biosynthesis of 1986; 159: 499-505. N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase: 55 Schachter D. Lipid fluidity of rat enterocyte plasma disulfide-linked dimers are formed at the site of membranes in relation to membrane function. In: synthesis in the rough endoplasmic reticulum. Arch Alvarado F, van Os CH, eds. Ion gradient-coupled Biochem Biophys 1988; 265: 119-27. transport (INSERM Symp 26) Amsterdam: Elsevier, 40 Auricchio S, Ciccimarra F, Vegnente A. Andria G. 1986: 23-32. Vetrella M. Enzyme activity hydrolysing y-glutamyl-p- 56 van Meer G. How epithelia grease their microvilli. naphthylamide in human intestine during adult and Trends Biochem Sci 1988; 13: 242-3. fetal life. Pediatr Res 1973; 7: 95-9. 57 Patton JS. Is the intestinal membrane bilayer freely 41 Tate SS, Meister A. Subunit structure and isozymic permeable to lipophilic molecules? In: Alvarado F, van forms of y-glutamyl transpeptidase. Proc Natl Acad Sci Os CH, eds. Ion gradient coupled transport (INSERM USA 1976; 73: 2599-603. Symp 26) Amsterdam: Elsevier, 1986: 33-6. 42 Hirano K, Sugiura M, Miki K, lino S, Suzuki H, Uda T. 58 Seetharam B, Yeh K, Moog F, Alpers DH. Develop- Characterization of tissue-specific isoenzyme of ment of intestinal brush border membrane proteins in alkaline phosphatase from human placenta and intes- the rat. Biochim Biophys Acta 1977; 470: 424-36.

tine. Chem Pharm Blull (Tokyo) 1977; 25: 2524-9. 59 Bailey DS, Cook A, McAllister G, Moss M, Mian N. http://gut.bmj.com/ 43 Komoda T, Sakagishi Y. The function of carbohydrate Structural and biochemical differentiation of the moiety and alteration of carbohydrate composition in mammalian small intestine during development. J Cell human alkaline phosphatase isoenzymes. Biochim Sci 1984; 72: 195-212. Biophys Acta 1978; 523: 395-406. 60 Skovbjerg H. Immunoelectrophoretic studies on 44 de Jonge HR. The localization of guanylate cyclase in human small intestinal brush border proteins - the rat small intestinal epithelium. FEBS Lett 1975; 53: longitudinal distribution of peptidases and disacchari- 237-42. dases. Clin Chim Acta 1981; 112: 205-12. 45 Pind S, Kuksis A. Isolation and purification of brush 61 Triadou N, Bataille J, Schmitz J. Longitudinal study of on September 27, 2021 by guest. Protected copyright. border membrane from rat jejunum containing a Ca>- the human intestinal brush border membrane proteins. independent phospholipase A2. Biochim Biophys Acta Distribution of the main disaccharidases and pepti- 1987; 901: 78-85. dases. Gastroenterology 1983; 85: 1326-32. 46 Oda T, Seki S. Molecular structure and biochemical 62 Morita A, Miura S, Erickson RH, Sleisenger MH, Kim function of the microvilli membrane of intestinal YS. Comparison of brush border membrane glyco- epithelial cells with special emphasis on the elementarv proteins and glycoenzymes in the proximal and distal particles. J Electron Micr 1965; 14: 210-7. rat small intestine. Biochim Biophys Acta 1986; 883: 47 Maestracci D. Enzymic solubilization of the human 506-16. brush border membrane enzymes. Biochim Biophys 63 Mooseker MS. Organisation, chemistry and assembly Acta 1976; 433: 469-81. of the cytoskeletal apparatus of the intestinal brush 48 Kenny AJ, Fulcher IS, McGill K, Kershaw D. Proteins border. Ann Rev Cell Biol 1985; 1: 209-41. of the kidney microvillar membrane. Reconstruction of 64 Bretscher A. The molecular structure of the microvillar endopeptidase in liposomes shows that it is a short- cytoskeleton. In: Alvarado F, van Os CH, eds. Ion stalked protein. Biochem J 1983; 211: 755-62. gradient-coupled transport (INSERM Symp 26) 49 Low MG, Ferguson MAJ, Futerman AH, Silman I. Amsterdam: Elsevier, 1986: 13-21. Covalently attached phosphatidylinositol as a hydro- 65 Maroux S, Coudrier E, Feracci H, Gorvel JP, Louvard phobic anchor for membrane proteins. Trends Biochem D. Molecular organisation of the intestinal brush Sci 1986; 11: 212-5. border. Biochimie 1988; 70: 1297-306. 50 Low MG. Biochemistry of the glycosyl-phosphatidy- 66 West AB, Isaac CA, Carboni JM, Morrow JS, inositol membrane anchors. Biochem J 1987; 244: Mooseker MS, Barwick KW. Localisation of villin, a 1-13. cytoskeletal protein specific to microvilli, in human Gut: first published as 10.1136/gut.30.12.1667 on 1 December 1989. Downloaded from

1676 Holmes and Lobley

ileum and colon and colonic neoplasms. Gastroenter- 82 Misch P, Giebel P. Faust R. Intestinal microvilli ology 1988; 94: 343-52. responses to feeding and fasting. Eur J Cell Biol 1980; 67 Keller TCS, Conzelman KA. Mooseker MS. The role 21: 264-79. of myosin in terminal web contraction in isolated 83 Stidwell RP, Wysolmerski T, Burgess DR. The brush intestinal epithelial brush borders. J Cell Biol 1985; border cytoskeleton is not static: in vivo turnover of 100: 1647-55. proteins. J Cell Biol 1984: 98: 641-5. 68 Madara JL, Barenberg D, Carlson S. Effects of 84 Cowell GM, Danielsen EM. Biosynthesis of intestinal cytochalasin D on occluding junctions of intestinal microvillar proteins: rapid expression of cytoskeletal absorptive cells: further evidence that the cytoskeleton components in microvilli of pig small intestinal explants. may influence paracellular permeability and junctional FEBS Lett 1984; 172: 309-14. charge selectivity. J Cell Biol 1986; 102: 2125-36. 85 Alpers DH. Digestion and absorption of carbohydrates 69 Hecht G. Pothoulakis C, La Mont JT, Madara JL. and proteins. In: Johnson LR, ed. Plhysiology of the Clostridium difficile toxin A perturbs cytoskeletal gastrointestinal tract. New York: Raven Press, 1987: structure and tight junction permeability of cultured 1469-87. human intestinal epithelial monolayers. J Clin Invest 86 Bruce GT, Woodley JF. The breakdown of gliadin 1988; 82: 1516-24. peptides by rat intestinal brush-borders in titro. 70 Gould KL, Cooper JA, Bretscher A, Hunter T. The Biochem Soc Trians 1984; 12: 1116-7. protein-tyrosine kinase substrate, p81. is homologous 87 Song IS, Yoshioka M, Erickson RH, Miura S, Guan D, to a chicken microvillar core protein. J Cell Biol 1986; Kim YS. Identification and characterization of brush- 102: 660-9. border membrane-bound neutral metalloendopepti- 71 Gerke V, Weber K. Identity of a p36K phosphorylated dase from rat small intestine. Gastroenterology 1986; upon Rous sarcoma virus transformation with a protein 91: 1234-42. purified from brush borders: calcium dependent 88 Roubaty C, Portmann P. Relation between intestinal binding to nonerythroid spectrin and F-actin. EMBO J alkaline phosphatase activity and brush border mem- 1984; 3: 227-33. brane transport of inorganic phosphate, D-glucose and 72 Forgacs 1. Oncogenes and gastrointestinal cancer. Gut D-glucose-6-phosphate. Pflugers Arch 1988; 412: 482- 1988; 29: 417-21. 90. 73 Sjostrom H, Noren 0, Danielsen EM, Skovbjerg H. 89 Crane RK, Miller D, Bihler I. The restrictions on the Structure of microvillar enzymes in different phases of possible mechanisms of intestinal transport of sugars. their life cycles. In: Porter R, Collins GM, eds. Brush In: Kleinzeller A, Kotyk A, eds. Membrane transport border membranes (Ciba Foundation Symp 95). and metabolism. London: Academic Press, 1961: 434- ILondon: Pitman Books. 1983: 50-72. 50. 74 Triadou N, Audran E, Rousset M, et al. Relationship 90 Hopfer U, Nelson K, Perrotto J, Isselbacher KJ. between the secretor status and the expression of ABH Glucose transport in isolated brush border membranes http://gut.bmj.com/ blood group antigenic determinants in human intestinal from rat small intestine. J Biol C'lem 1973; 248: 25-32. brush-border membrane hydrolases. Biochim Bioplhys 91 Booth IW, Patel PB, Sule D, Brown GA, Buick R, Acta 1983; 761: 231-6. Beyreiss K. Glucose-galactose malabsorption: 75 Gorvel JP, Rigal A, Sarles J, Maroux S. Amino- demonstration of specific jejunal brush border mem- peptidase N- and human blood group A-antigenicity brane defect. Gut 1988; 29: 1661-5. along the digestive tract and associated glands in the 92 Sigrist-Nelson K, Hopfer U. A distinct D-fructose rabbit. Cell Tissue Res 1985; 239: 241-8. transport system in isolated brush border membrane.

76 Lobley RW, Pemberton PW, S0rensen SH, Batt RM. Bioclhim Biopliys Acta 1974; 367: 247-54. on September 27, 2021 by guest. Protected copyright. Analysis of canine intestinal microvillar membranes by 93 Hopfer U. Membrane transport mechanisms for two-dimensional electrophoresis. Biochem Soc Trans hexoses and amino acids in the small intestine. In: 1987; 15: 861-2. Johnson LR, ed. Physiology of the gastrointestinal 77 Alpers DH, Tedesco FJ. The possible role of pancre- tract. New York: Raven Press 1987: 1499-526. atic proteases in the turnover of intestinal brush border 94 Hoshi T. Proton-coupled transport of dipeptides across proteins. Biochim Biophys Acta 1975; 401: 28-40. renal and intestinal brush border membranes. In: 78 Nordstrom C. Release of enteropeptidase and other Alvarado F, van Os CH, eds. Ion gradient-coupled brush-border enzymes from the small intestinal wall in transport (INSERM Symp 26). Amsterdam: Elsevier the rat. Biochim Bioplhys Acta 1972; 289: 367-77. 1986: 183-91. 79 Riby JE, Kretchmer N. Participation of pancreatic 95 Stremmel W. Uptake of fatty acids by jejunal mucosal enzymes in the degradation of intestinal sucrase- cells is mediated by a fatty acid binding membrane isomaltase. J Pediatr Gastroenterol Nutr 1985; 4: 971-9. protein. J Clin Invest 1988; 82: 2001-10. 80 James WPT, Alpers DH, Gerber JE, Isselbacher KJ. 96 Barnard JA, Ghishan FK. Taurocholate transport by The turnover of disaccharidases and brush border human ileal brush border membrane vesicles. Gastro- proteins in rat intestine. Biochim Biophys Acta 1971; enterology 1987; 93: 925-33. 230: 194-203. 97 Heubi JE, Ballestieri WF. Fondacano JD, etal. Primary 81 Forstner G, Galand GT. The influence of hydro- bile acid malabsorption: defective in vivo active bile cortisone on the synthesis and turnover of microvillus acid transport. Gastroenterology 1982; 83: 804-11. membrane glycoproteins in suckling rat intestine. Can 98 Rose RR. Intestinal absorption of water soluble J Biochem 1976; 54: 224-32. vitamins. In: Johnson LR, ed. Physiology of the Gut: first published as 10.1136/gut.30.12.1667 on 1 December 1989. Downloaded from

Intestinal brush border revisited 1677

gastrointestinal tract. New York: Raven Press 1987: 115 Mackenzie N. F,-receptor-mediated transport of 1581-96. immunoglobulin across the intestinal epithelium of the 99 Ghishan FK, Arab N, Nylander W. Characterization of neonatal rodent. Itnmunol Todbay 1984; 5: 364-70. calcium uptake by brush border membrane vesicles of 116 Donowitz M. Walsh MJ. Regulation of mammalian human small intestine. Gastroenterology 1989; 96: 122- small intestinal electrolyte secretion. In: Johnson LR, 129. ed. Plhysiology of tlie gastrointestinal tract. New York: 100 Peters JT, Raja KB, Simpson RJ. In vivo absorption Raven Press 1987: 1351-88. and in vitro uptake of Fe" and Fe" by mouse 117 De Jonge HR. Lohmann SM. Mechanisms by which duodenum. In: Alvarado F. van Os CH, eds. Ion cyclic nucleotides and other intracellular mediators gradient-coupled transport (INSERM Symp 26). regulate secretion. In: Fvered D, Whelan J, eds. Amsterdam: Elsevier 1986: 325-8. Microbial toxins an(l disease. (Ciba Foundation Symp 101 Kikuchi K, Abumrad NN, Ghishan FK. Na'/H' 112). London: Pitman Books 1985: 116-38. exchange by brush border membrane vesicles of human 118 De Jonge HR. van den Bcrghe N. Bijman J. (Dys) ileal small intestine. Gastroenterology 1988; 95: 388-93. regulation of epithelial chloride channels. Biochem Soc 102 Booth IW, Murer H, Stange G, Fenton TR, Milla PJ. Tr-ans 1989. (In press). Defective jcjunal brush-border Na'/H+ exchange: a 119 Simoons FJ. Geographic patterns of primary adult cause of congenital secretory diarrhoea. Lancet 1985; lactase malabsorption. In: Paige DM. Bayless TM. eds. i: 1066-9. Lactose digestion. Baltimore: Johns Hopkins Univ 103 Knickelbein RG, Aronson PS, Dobbins JW. Press. 1981: 23-48. Membrane distribution of sodium-hydrogen and 120 Hadorn B, Green JR. Sterchi EE. Hauri HP. Bio- chloride-bicarbonate exchangers in crypt and villus cell chemical mechanisms in congenital enzyme deficien- membranes from rabbit ileum. J Clin Invest 1988; 82: cies of the small intestine. Clin Gastroenterol 1981: 10: 2158-63. 67 1-90. 104 Holmberg C. Congenital chloride diarrhoea. Clin 121 Lloyd ML, Olsen MD. A study of the molecular Gastroenterol 1986; 15: 583-602. pathology of sucrase-isomaltase deficiency. A defect in 105 Murer H. Transport of sulphate and phosphate in small the intacellular processing of the enzyme. N EnglJ Med intestine and renal proximal tubule: methods and basic 1987; 316: 438-42. properties. Comp Biochem Physiol (Pt A) 1988; 90: 122 Naim HY. Roth J, Sterchi EE. et al. Sucrase- 749-55. isomaltase deficiency in humans. Different mutations 106 Borowitz SM, Ghishan FK. Phosphate transport in disrupt intracellular transport, processing and function human jejunal brush border membrane vesicles. of an intestinal brush border enzyme. J Clin Invest Gastroenterology 1989; 96: 4- 10. 1988; 82: 667-79.

107 Peerce BE, Wright EM. Conformational changes in 123 Moskovitz M. Curtis C. Gavaler J. Does oral enzyme http://gut.bmj.com/ the intestinal brush border sodium-glucose cotransport replacement therapy reverse intestinal lactose malab- labelled with fluorescein isothiocyanate. Proc Natl sorption? Am J Gastroenterol 1987; 82: 632-5. Acad Sci USA 1984; 81: 2223-6. 124 Davidson GP, Cutz Es HamiltonJR. Gale DG. Familial 108 Peerce BE, Wright EM. Evidence for tyrosyl residucs enteropathy: a syndrome of protracted from birth. at the Na'/glucose cotransporter. J Biol Clhem 1984; failure to thrive and hypoplastic villus atrophy. Gastr-o- 260: 6026-31. enterology 1978; 75: 783-90. 109 Hediger MA, Ikeda T, Coady M, Gundersen CB, 125 Phillips AD. Jenkins P. Raafat F. Walker-Smith JA. Wright EM. Expression of size selected mRNA Congenital microvillous atrophy: specific diagnostic encoding the intestinal Na/glucose cotransporter in features. Archl Dis Clhild 1985; 60: 135-40. on September 27, 2021 by guest. Protected copyright. Xenopus laevis oocytes. Proc Natl Acad Sci USA 1987; 126 Carruthers L. Phillips AD. Dourmashkin R. Walker- 84: 2634-7. Smith JA. Biochemical abnormality in brush border 110 Hediger MA, Coady MJ, Ikeda TS, Wright EM. membrane protein of a patient with congenital micro- Expression, cloning and cDNA sequencing of the Na'/ villus atrophy. J Pediatr Gastroenterol Nutr 1985; 4: glucose co-transporter. Nature 1987; 330: 379-81. 902-7. 111 Wright EM, Peerce BE. Identification and conforma- 127 Rubin W. Ross LL. Sleisenger MHz Weser E. An tional changes of the intestinal proline carrier. J Biol electron microscopic study of adult celiac disease. Lab Chem 1984; 259: 14993-6. Invest 1966; 15: 1720-47. 112 Mackenzie IL, Donaldson RM. Effect of divalent ions 128 Peters TJ. Jones PE. Wells G. Analytical subcellular and pH on intrinsic factor-mediated attachment of subfractionation of jejunal biopsy specimens: enzyme vitamin B 12 to intestinal microvillus membranes. J Clin activities, pathology and response to gluten withdrawal Invest 1972; 51: 2464-71. in patients with coeliac disease. Clin Sci Mol Med 1978; 113 Kuovonen I, Grasbeck R. A simplified technique to 55: 285-292. isolate porcine and human ileal intrinsic factor recep- 129 Pemberton PW. Lobley RW. Holmes R. Electro- tors and studies on their subunit structures. BiochIem phoretic analysis of microvillus membranes in coeliac Biophy Res Commun 1979; 86: 358-64. disease. [Abstracti Gut 1987: 28: A 1335. 114 Robertson JA, Gallagher ND. In vivo evidence that 130) Bailey DS, Freedman AR. Price SC. Chescoe D. cobalamin is absorbed by receptor-mediated endo- Ciclitira PJ. Early biochemical responses of the small cytosis in the mouse. Gastroenterology 1985; 88: intestine of coeliac patients to wheat gluten. Gut 1989: 908-12. 30: 78-85. Gut: first published as 10.1136/gut.30.12.1667 on 1 December 1989. Downloaded from

1678 Holmes and Lobley

131 Petcrs TJ, Jones PE, Wcils G, Cook GG. Sequential disaccharidascs from brush border membrancs by cnzymc and subccilular fractionation studies on jcjunal cxtracts of bacteria obtained from intestinal blind loops biopsy spccimens from paticnts with post-infcctive in rats. Gastroetnterologv 1978; 75: 791-5. tropical malabsorption. Cliti Sci 1979'; 56: 479-86. 137 Riepe SP. Goldstein J. Alpers DH. Effcct of sccrcted 132 Dunne WT, Cooke WT. Allen RN. Enzymatic and Bactcroides proteascs on human intestinal brush morphometric evidence for Crohn's diseasc as a diffuse border hydrolases. J C/in Ini,est 1980; 66: 314-22. lesion of the gastrointestinal tract. Gutt 1977; 18: 290-4. 138 Hartong WA, Gourley WK, Arvanitakis C. 133 Hcuman R, Boervd B, Bolin T, et il. Subecilular Giardiasis: clinical spectrum and functional-structural fractionation of human intestinal mucosa by large scale abnormalities of the small intestinal mucosa. castro- zonal centrifugation. Acta C'/iir Scanid 1980X; 146: 195- eniterologs' 1979; 77: 6 1-9. 20(1 139 Moriarty KJ, Turnberg LA. Bacterial toxins and 134 Cheney CP, Boedeker EC. Adherence of an entero- diarrhoea. Clin Gasutoeiterol 1986; 15: 529-43. toxigenic Escherichia coli strain, serotypc 078:H 11. to 140 Rao MC. Toxins which activate guanyl cyclasc: hcat- purified human intestinal brush borders. Infect Imilmuntl* stable enterotoxins. In: Evcred D, Whelan J, eds. 1983; 39: 1280-4. 1Wicrobial toXins an(1 diarrhlioeeal (lisease. (Ciba Found- 135 Jones GW, Fretcr R. Adhesive properties of Vibrio ation Symp 112). ILondon: Pitman Books, 1985: 74-93. cholerae: nature of the intcraction with isolated rabbit 141 Wilkins T, Krivan H, Stiles B, Carmen R, Lycrly D. brush border membranes and human erythrocytes. Clostridial toxins active locally in the gastrointestinal Infect Immun 1976; 14: 240-5. tract. In: Evered D, Whelan I. eds. Microbiol tovins 136 Jonas A, Krishnan C. Forstncr Ci. Pathogencsis of an(/ diarrIhloeal disease. (Ciba Foundation Symp 112). mucosal injury in the blind loop syndromc. Release of London: Pitman Books, 1985: 230-41. http://gut.bmj.com/ on September 27, 2021 by guest. Protected copyright.