Microvillus Membrane Proteins Research Articles

Detergent-resistant domains in Spodoptera frugiperda midgut microvillar membranes and their relation to microapocrine secretion.

The midgut from lepidopteran insects has a particular way to release proteins to the lumen, named microapocrine secretion that could be an adaptation to release secretory contents into the lumen at water absorbing regions. In this process small vesicles (microapocrine vesicles) bud from the midgut microvilli as double membrane vesicles, where the inner membrane comes from the secretion vesicle and the outer one from the microvillar membrane. The molecular machinery associated with this process may be recruited by specific midgut microvilli membrane domains. To address to this, Spodoptera frugiperda midgut microvillar membranes, prepared by magnesium treatment and free from cytoskeleton with the hyperosmotic Tris procedure, were submitted to detergent extraction and fractionated by density gradient ultracentrifugation. Detergent-resistant membrane domains (DRM) were recovered and their proteins identified by proteomics. Microapocrine vesicles were isolated by washing the luminal surface of the midgut epithelium, followed by freezing and thawing plus centrifugation to recover only membranes. Proteins from purified microvillar membranes and microapocrine vesicle membranes were identified by proteomics. Comparison of the two populations suggests that the budding of microapocrine vesicles surrounded by microvillar membrane is not a random process, because only around 50% of the microvillar membrane proteins are in the microapocrine vesicles. From the 16 proteins from DRM, 14 were enriched in the microapocrine membrane vesicles. These results suggest that on budding, the microapocrine vesicle membrane is enclosed by DRM and a surrounding area of the microvillar membrane. It is proposed that the DRMs somehow recruit the proteins composing the secretory machinery.

PDZ interactions regulate rapid turnover of the scaffolding protein EBP50 in microvilli

Scaffolding proteins containing PDZ (postsynaptic density 95/discs large/zonula occludens-1) domains are believed to provide relatively stable linkages between components of macromolecular complexes and in some cases to bridge to the actin cytoskeleton. The microvillar scaffolding protein EBP50 (ERM-binding phosphoprotein of 50 kD), consisting of two PDZ domains and an ezrin-binding site, retains specific proteins in microvilli and is necessary for microvillar biogenesis. Our analysis of the dynamics of microvillar proteins in vivo indicated that ezrin and microvillar membrane proteins had dynamics consistent with actin treadmilling and microvillar lifetimes. However, EBP50 was highly dynamic, turning over within seconds. EBP50 turnover was reduced by mutations that inactivate its PDZ domains and was enhanced by protein kinase C phosphorylation. Using a novel in vitro photoactivation fluorescence assay, the EBP50-ezrin interaction was shown to have a slow off-rate that was dramatically enhanced in a PDZ-regulated manner by addition of cell extract to near in vivo levels. Thus, the linking of relatively stable microvillar components can be mediated by surprisingly dynamic EBP50, a finding that may have important ramifications for other scaffolding proteins.

A shift in microvillus membrane fucosylation to sialylation by ethanol ingestion in rat intestine

The luminal surface of enterocytes is covered with glycocalyx which is rich in glycoproteins. Ethanol ingestion is shown to induce morphological and biochemical changes in the intestine. In this study, the effect of ethanol ingestion on membrane glycoproteins has been investigated. Chemical analysis of microvillus membranes revealed an increase in hexose and sialic acid contents, but a reduction in fucose levels in ethanol-fed rats compared with controls. The observed changes were apparent in animals fed with ethanol for 35-56 days compared with controls. Lectin-binding assay indicated an increase in Wheat germ agglutinin (affinity for GlcNAc/sialic acid) and a decrease in Aleuria aurantia (affinity for alpha-L: -fucose) reactivity of brush borders in ethanol-fed animals for 4-8 weeks. Western blot analysis using biotin-labeled Wheat germ agglutinin revealed increased binding to proteins of M(r) 66-205 kDa in ethanol-fed rats compared with controls. The binding of Aleuria aurantia to membrane proteins of M(r) 97-185 kDa was reduced in ethanol-fed animals. These findings suggest that long-term ethanol feeding modulates the sialylation and fucosylation processes of microvillus membrane proteins in rat intestine. This could affect the intestinal digestive and absorptive functions in chronic alcoholism.

Regulation of Expression of Microvillus Membrane Proteins by Estrogen in Baboon Fetal Ovarian Oocytes1

We previously demonstrated that the number and height of oocyte microvilli were reduced in baboon fetuses deprived of estrogen in utero and restored to normal in animals supplemented with estradiol. Phosphorylated ezrin and Na+/H+ exchange regulatory factor 1 (NHERF, now termed SLC9A3R1) link f-actin bundles to the membrane, whereas alpha-actinin cross-links f-actin to form microvilli. Therefore, we determined whether these proteins were expressed in oocytes of the fetal baboon ovary and whether expression and/or localization were altered between mid and late gestation in association with an increase in estrogen and in late gestation in animals in which estrogen was suppressed (>95%) or restored by treatment with an aromatase inhibitor with or without estradiol. Expression of alpha-actinin was low at mid gestation, increased on the surface of oocytes of primordial follicles in late gestation, and was negligible in the ovaries of estrogen-suppressed fetuses and normal in animals treated with estrogen. Ezrin (total and phosphorylated) and SLC9A3R1 expression was localized to the surface of oocytes at mid and late gestation in estrogen-replete baboons and to the cytoplasm in late gestation after estrogen suppression. These results are the first to show that the fetal baboon oocyte expressed ezrin, SLC9A3R1, and alpha-actinin, and that these proteins were localized to the oocyte surface consistent with their role in microvilli development in epithelial cells. The current study also showed that the developmental increase in oocyte expression of alpha-actinin is regulated by estrogen and correlated with the estrogen-dependent increase in oocyte microvilli demonstrated previously. Therefore, we propose that development of oocyte microvilli requires expression of alpha-actinin and that expression of alpha-actinin and localization of ezrin-phosphate and SLC9A3R1 to the oocyte membrane are regulated by estrogen.

Lipid raft organization and function in brush borders of epithelial cells (Review)

Polarized epithelial cells of multicellular organisms confront the environment with a highly specialized apical cell membrane that differs in composition and function from that facing the internal milieu. In the case of absorptive cells, such as the small intestinal enterocyte and the kidney proximal tubule cell, the apical cell membrane is formed as a brush border, composed of regular, dense arrays of microvilli. Hydrolytic ectoenzymes make up the bulk of the microvillar membrane proteins, endowing the brush border with a huge digestive capacity. Several of the major enzymes are localized in lipid rafts, which, for the enterocyte in particular, are organized in a unique fashion. Glycolipids, rather than cholesterol, together with the divalent lectin galectin-4, define these rafts, which are stable and probably quite large. The architecture of these rafts supports a digestive/absorptive strategy for nutrient assimilation, but also serves as a portal for a large number of pathogens. Caveolae are well-known vehicles for internalization of lipid rafts, but in the enterocyte brush border, binding of cholera toxin is followed by uptake via a clathrin-dependent mechanism. Recently, 'anti-glycosyl' antibodies were shown to be deposited in the enterocyte brush border. When the antibodies were removed from the membrane, other carbohydrate-binding proteins, including cholera toxin, increased their binding to the brush border. Thus, anti-glycosyl antibodies may serve as guardians of glycolipid-based rafts, protecting them from lumenal pathogens and in this way be part of an ongoing 'cross-talk' between indigenous bacteria and the host.

Identification and characterization of a novel peritrophic matrix protein, Ae-Aper50, and the microvillar membrane protein, AEG12, from the mosquito, Aedes aegypti

Immuno-screening of an adult Aedes aegypti midgut cDNA expression library with anti-peritrophic matrix antibodies identified cDNAs encoding a novel peritrophic matrix protein, termed Ae. aegypti Adult Peritrophin 50 (Ae-Aper50), and the epithelial cell-surface membrane protein, AEG12. Both genes are expressed exclusively in the midguts of adult female mosquitoes and their expression is strongly induced by blood feeding. Ae-Aper50 has a predicted secretory signal peptide and five chitin-binding domains with intervening mucin-like domains. Localization of Ae-Aper50 to the peritrophic matrix was demonstrated by immuno-electron microscopy. Recombinant Ae-Aper50 expressed in baculovirus-infected insect cells binds chitin in vitro. Site-directed mutagenesis was used to study the role that cysteine residues from a single chitin-binding domain play in the binding to a chitin substrate. Most of the cysteine residues proved to be critical for binding. AEG12 has a putative secretory signal peptide at the amino-terminus and a putative glycosyl-phosphatidylinositol (GPI) anchor signal at its carboxyl-terminus and the protein was localized by immuno-electron microscopy to the midgut epithelial cell microvilli.

Escherichia coli strain RDEC-1 AF/R1 endogenous fimbrial glycoconjugate receptor molecules in rabbit small intestine.

Escherichia coli strain RDEC-1 causes a diarrheagenic infection in rabbits with AF/R1 fimbriae, which have been identified as an important colonization factor in RDEC-1 adherence leading to disease. The AF/R1-mediated RDEC-1 adherence model has been used as a model systems for E. coli diarrheal diseases. In this study, RDEC-1 adhered specifically to small intestinal brush borders, with both sialic acid and beta-galactosyl residues apparently involved. The AF/R1-mediated adherence activity of [(14)C]-labeled RDEC-1 was analyzed quantitatively by using 24-well plates coated with purified brush borders and purified microvilli. Two microvillus membrane proteins (130 and 140 kDa) were individually isolated, and chicken antibody raised to each protein inhibited bacterial adherence. These same two proteins, previously shown to be recognized by AF/R1, were individually digested with trypsin, and the amino acid sequences of peptides were determined by reversed-phase capillary liquid chromatography-mass spectrometry tandem mass spectrometry (LC-MS). This LC-MS analysis indicated that these proteins are subunits of the rabbit sucrase-isomaltase protein (SI) complex. Guinea pig serum raised to purified rabbit SI complex inhibited bacterial adherence to microvilli. Additionally, as determined by high-performance thin-layer chromatography and autoradiography, RDEC-1 adhered selectively, via AF/R1 fimbriae, to a glycolipid tentatively identified as galactosylceramide (Gal beta 1-1Cer) in the lipid extract of rabbit small intestinal brush borders. RDEC-1 adherence to Gal beta 1-1Cer was partially inhibited in the presence of galactose. These combined results indicate that the endogenous receptor molecule for AF/R1 fimbriae of RDEC-1 is each individual component of the SI complex, although binding to glycolipid may be responsible for an additional adherence mechanism.

Overview

This symposium entitled, "Bacterial Colonization of the Gut and the Use of Pre- and Probiotics" was organized by the Shanghai Children's Medical Center of the Second Shanghai Medical University in conjunction with the Combined Program in Pediatric Gastroenterology and Nutrition at Harvard Medical School to underscore a 10-year agreement between both medical centers to provide training of academic pediatricians from China in basic and translational research. The training program is supported by a training grant from the National Institute of Child Health and Development (NICHD)/Fogarty Center (1-U01A145441-01), and research projects are, in part, supported by a research grant from Wyeth Nutritionals International, Inc., (Philadelphia, PA, U.S.A.) The symposium, itself, was sponsored from an educational grant from Wyeth Nutritionals International, Inc. for which the the organizers are grateful. The topic for this initial Sino-American symposium represents an important area of research in pediatrics and a major research interest of the two organizing groups. Dr. W. Allan Walker, a pediatric gastroenterologist with a strong research interest in the development of host defense, introduces the topic of the symposium by considering the importance of pre- and probiotics in the development of intestinal host defense in the extrauterine environment. He reviews the effects of initial colonization of the gut on the development of the effector immune response to antigen stimulation. At birth, the gut's lymphoid tissue has the capacity to respond to microbial-food antigens. However, the initial colonization of the newborn gut with indigenous microflora provides the appropriate "adjuvant effect" to activate antibody production and T-cell activation. Furthermore, the nature of initial oral feedings (breast-milk vs. formula) helps to determine the nature of colonizing bacteria (lactobacilli and bifidobacteria vs. enterobacteria and Escherichia coli). In addition, specific nutrients such as nucleotides, omega-3 fatty acids, and lactoferrin can enhance the effects of indigenous flora on strengthening the mucosal barrier and minimizing inflammatory responses to pathologic bacteria colonization. Dr. David S. Newburg, a glycobiologist from Harvard Medical School, reviews the importance of oligosaccharides in human milk on the attachment of pathologic bacteria to glycoconjugates and glycolipids and proteins in the intestinal microvillus membrane. Oligosaccharides, a natural prebiotic, has an influence on the nature of colonizing bacteria. He notes that enterocyte cell surface glycoconjugates are pathogen receptors and help to influence pathologic colonization of the newborn gut. In contrast, soluble oligosaccharides in breast milk can attach to "adhesions" on pathogens and thereby block their interaction with the intestinal surface and reduce their colonization. He cites examples of breast milk protection by oligosaccharides for Campylobacter and rotaviral infection. These studies may help to identify additives to future infant formula. Dr. Michael S. Caplan, a neonatologist from North-western University with a strong research interest in necrotizing enterocolitis, and Dr. Tamas Jilling reviews the possible role of probiotics in the treatment of necrotizing enterocolitis. The immature human enteric surface has an immature glycosylation pattern favoring colonization with pathologic organisms. In the premature neonate, this process may contribute to the pathogenesis of necrotizing enterocolitis. If probiotics are introduced into the diet of the newborn at birth, their presence could block pathologic colonization and thereby reduce the incidence of necrotizing enterocolitis. The authors provide evidence in an experimental model for necrotizing enterocolitis that the use of probiotics (Lactobacillus GG) can prevent the upregulation of platelet-activating factor, an inflammatory cytokine and indicator of early necrotizing enterocolitis. Dr. Dingwei Dai, an investigator at the Shanghai Children's Medical Center and a research fellow in the Developmental Gastroenterology Laboratory of the Combined Program in Pediatric Gastroenterology and Nutrition at Harvard Medical School, and colleagues from the United States present exciting new data to support the notion that glycosylation of microvillus membrane lipids and protein through glycosyltransferase activity is developmentally regulated and that cortisone treatment of pregnant or newborn animals can activate the maturation of this process. They also provide preliminary evidence that glycosylation in the human fetus is also under developmental regulation. These observations help explain pathologic colonization of the human newborn and the potential for treatment of this process with growth factors such as cortisone. Finally, Dr. Jon A. Vanderhoof, a pediatric gastroenterologist from the University of Nebraska Medical Center, reviews the use of probiotics in the treatment inflammatory disorders in infants and children. He reviews clinical studies to suggest that Lactobacillus GG administered orally to children who have rotaviral infection reduces the severity and length of time of the clinical illness. He also reviews its use in the treatment of allergic dermatitis of children and its preliminary use in the treatment of inflammatory bowel disease. These presentations provide an excellent review of the current state of our knowledge of the role of nutrients in bacterial colonization in health and disease of infants and children.

Role of Nutrients and Bacterial Colonization in the Development of Intestinal Host Defense

In this introduction to the supplement on the use of pre- and probiotics in the health and disease of pediatric patients, I have summarized factors affecting the initial colonization of the neonatal intestine. The term bacterial-epithelial cross-talk was defined, and examples of the enterocyte response to both pathologic and indigenous flora stimulation illustrated. Immaturities in the human neonatal intestinal response to bacteria and their toxins were reviewed in the context of the pathogenesis of age-specific, bacterial gastrointestinal infectious diseases. Finally, the importance of pre- and probiotics as measures to strengthen the neonate's intestinal host defenses in the prevention and treatment of specific age-related disease were considered.

Effects of sulfate and chloride on three separate oxalate transporters reconstituted from rabbit renal cortex.

Understanding the mechanism of sulfate-dependent, oxalate-stimulated chloride reabsorption in the mammalian proximal tubule is complicated by the presence of multiple oxalate and sulfate transport pathways. Accordingly, we developed a method of reconstituting functional oxalate transport from the rabbit renal cortex so that the individual transporters might be examined. Solubilized microvillus membrane proteins were separated by hydroxyapatite chromatography and then reconstituted into proteoliposomes. Two peaks of oxalate/oxalate exchange activity were observed. Sulfate (10 mM) cis-inhibits oxalate transport in the early peak by 93% and in the later peak by 41%. In contrast, 20 mM chloride inhibits oxalate/oxalate exchange by only 32% in the early peak but inhibits oxalate exchange by 70% in the later peak. Oxalate-stimulated sulfate uptake was observed in the early fractions but not in the later fractions. These data are consistent with the recovery of the sulfate/oxalate exchanger in the early hydroxyapatite fractions and the chloride/oxalate exchanger in the later fractions. The basolateral membrane sulfate/oxalate exchanger was also reconstituted. The reconstituted basolateral and apical membrane sulfate/oxalate exchangers demonstrate nearly identical patterns of substrate specificities. However, 98% of apical sulfate/oxalate exchange activity is lost following exposure to octylglucoside at room temperature, whereas the basolateral sulfate/oxalate exchange activity was reduced 67% (P < 0.05). In conclusion, functional reconstitution of solubilized membrane proteins demonstrates that apical membrane chloride/oxalate exchange and sulfate/oxalate exchange are mediated by different transport proteins. Apical and basolateral sulfate/oxalate exchange may also represent transport on two separate exchangers.

Gluten-sensitive enteropathy in Irish setter dogs: characterisation of jejunal microvillar membrane proteins by two-dimensional electrophoresis

This study investigated whether gluten-sensitive enteropathy ( gse) in Irish setter dogs was associated with underlying structural abnormalities of microvillar membrane proteins. Jejunal biopsies taken from eight-month-old gse-affected dogs reared on a normal, gluten-containing diet exhibited partial villous atrophy and contained more intra-epithelial lymphocytes than controls. The morphological abnormalities were reversed by feeding a gluten-free diet for five months and the changes were accompanied by an increase in the mucosal activity of the microvillar hydrolases, particularly aminopeptidase N and dipeptidyl aminopeptidase IV, which reverted to pre-treatment levels after a gluten challenge. Two-dimensional electrophoresis of microvillar membrane proteins isolated from gse-affected dogs revealed an essentially normal protein map that was comparable to controls. The exception was an intense 85 kDa protein spot that diminished when the affected dogs were fed a gluten-free diet and re-intensified after a gluten challenge.

Effects of concanavalin A and pokeweed lectins on microvillar membrane proteins during the organ culture of rabbit intestinal mucosa

The effects of pokeweed lectin ( PWL) and concanavalin A (Con A) on microvillar membrane ( MVM) proteins during the organ culture of rabbit ileal explants for 24 hours were compared with the known effects of enteropathogenic Escherichia coli ( EPEC). PWL resulted in the accelerated release of brush border enzymes into the culture medium, accompanied by decreased tissue activities and an increase in the total activity present in the tissue and culture medium. Con A had less effect on the release and tissue activities of brush border enzymes and the total activity was not increased. Sucrose density gradient centrifugation of the culture medium showed that MVM enzymes were predominantly particulate, consistent with their release as vesicles. Centrifugation of ileal explants showed that PWL, but not Con A, resulted in a decrease in the modal density of the brush border which was consistent with a lower glycoprotein-to-lipid ratio. These findings suggest that PWL, in common with EPEC, may cause the disruption and vesiculation of microvilli and the compensatory stimulation of MVM protein synthesis.

Application of Organ Culture of Small Intestine to the Investigation of Enterocyte Damage by Equine Rotavirus

SummaryWe used organ culture of jejunal mucosal explants obtained from ponies aged between 2 and 12 months to study enterocyte damage by group A strains of equine rotavirus. Electron microscopy of jejunal explants maintained for 48 h in the presence of organ culture medium alone showed that entcrocytes were structurally intact and had a densely packed brush border and overlying mucus. Similarly, examination of explants maintained in the presence of rotavirus for 48 h revealed no apparent ultrastructural abnormalities. However, obvious replication and assembly of virus in enterocytes had occurred following exposure to equine rotavirus strains H2 (three of eight occasions), trypsinactivaled H2 (two of four), L338 (five of five), and EQ124 (two of two) although brush borders and intracellular organelles ap peared unaltered. Despite the absence of obvious ultra structural damage, analytical subcellular fractionatior showed that rotavirus infection can result in decreasec activities of microvillar membrane enzymes, with a par ticular loss of the main brush border peak of modal den sity 1.21 g/ml. These findings demonstrate that organ cul ture of small intestine may be applied to the investigatior of rotavirus pathogenicity; they further suggest that cer tain strains impair enterocyte function by affecting the turnover of microvillar membrane proteins.

Application of organ culture of small intestine to the investigation of enterocyte damage by equine rotavirus.

We used organ culture of jejunal mucosal explants obtained from ponies aged between 2 and 12 months to study enterocyte damage by group A strains of equine rotavirus. Electron microscopy of jejunal explants maintained for < or = 48 h in the presence of organ culture medium alone showed that enterocytes were structurally intact and had a densely packed brush border and overlying mucus. Similarly, examination of explants maintained in the presence of rotavirus for 48 h revealed no apparent ultrastructural abnormalities. However, obvious replication and assembly of virus in enterocytes had occurred following exposure to equine rotavirus strains H2 (three of eight occasions), trypsin-activated H2 (two of four), L338 (five of five), and EQ124 (two of two), although brush borders and intracellular organelles appeared unaltered. Despite the absence of obvious ultrastructural damage, analytical subcellular fractionation showed that rotavirus infection can result in decreased activities of microvillar membrane enzymes, with a particular loss of the main brush border peak of modal density 1.21 g/ml. These findings demonstrate that organ culture of small intestine may be applied to the investigation of rotavirus pathogenicity; they further suggest that certain strains impair enterocyte function by affecting the turnover of microvillar membrane proteins.

Characterization of microvillar membrane proteins of dog small intestine by two-dimensional electrophoresis

A method for analysing microgram amounts of microvillar membranes by two-dimensional electrophoresis (protein mapping) is described, and has been used to characterize the microvillar proteins of the small intestine of German shepherd, corgi, and beagle dogs. Detergent-solubilized microvillar membranes were radiolabelled with 14C and separated by isoelectric focussing followed by SDS-PAGE. Proteins were detected fluorographically and glycoproteins by lectin-affinity staining. The microvillar hydrolases alkaline phosphatase and dipeptidyl aminopeptidase IV were identified by active-site labelling and aminopeptidase N by immunoprecipitation. Changes following pancreatic duct diversion were consistent with accumulation of pro-sucrase-isomaltase and diminished expression of the sucrase and isomaltase subunits. Cytoskeletal proteins were concentrated in the core fraction remaining after extraction of microvillar membranes with Triton X-100. There were no consistent differences between dogs of different breed, and the canine protein maps were similar to the human.

Possible Changes in Luminal Surface Charge Densities of Small Intestine Membrane in the 4-7.4 pH Range Exhibit Varied Influence on the Absorption Rate Constants of the Ionized and Un-Ionized Species of Sulfadiazine in Rats

The overall apparent first-order rate constants (Kab) of small intestinal absorption of sulfadiazine were determined in rats in situ at various pHs (4.01-7.42) of the recirculation fluids at 32 degrees. The purpose of the study was to verify our hypothesis that the rate-determining step for the absorption of sulfonamides involves the formation of an "activated complex" consisting of a transient association of the sulfonamide molecules with the surface protein of the microvillus membrane. The Kab versus Fu (where Fu is the fraction of un-ionized sulfadiazine at a given pH) profile prepared according to the equation Kab = Ki+Fu(Ku-Ki), where Ki and Ku are the first-order rate constants of absorption of the ionized and un-ionized species, respectively, was clearly resolvable into two linear segments: one based on the data in the 4.01-6.43 pH range and the other based on the data in the 6.43-7.42 pH range. In view of the fact that the combined molar concentration of aspartic acid and glutamic acid in the microvillus membrane protein is about twice that of arginine and lysine, the resolution of the data into two linear segments suggested that the negative charge density of the microvillus surface membrane protein in the 6.43-7.42 pH range is greater than that in the 4.01-6.43 pH range. The Ku and Ki values of sulfadiazine were determined in each pH range. It was noted that Ku was greater than Ki in each pH range, and that Ki in the 4.01-6.43 pH range was greater than that in the 6.43-7.42 pH range.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a secretase activity which releases angiotensin-converting enzyme from the membrane.

Angiotensin-converting enzyme (ACE; EC 3.4.1.15.1) exists in both membrane-bound and soluble forms. Phase separation in Triton X-114 and a competitive e.l.i.s.a. have been employed to characterize the activity which post-translationally converts the amphipathic, membrane-bound form of ACE in pig kidney microvilli into a hydrophilic, soluble form. This secretase activity was enriched to a similar extent as other microvillar membrane proteins, was tightly membrane-associated, being resistant to extensive washing of the microvillar membranes with 0.5 M NaCl, and displayed a pH optimum of 8.4. The ACE secretase was not affected by inhibitors of serine-, thiol- or aspartic-proteases, nor by reducing agents or alpha 2-macroglobulin. The metal chelators, EDTA and 1,10-phenanthroline, inhibited the secretase activity, with, in the case of EDTA, an inhibitor concentration of 2.5 mM causing 50% inhibition. In contrast, EGTA inhibited the secretase by a maximum of 15% at a concentration of 10 mM. The inhibition of EDTA was reactivated substantially (83%) by Mg2+ ions, and partially (34% and 29%) by Zn2+ and Mn2+ ions respectively. This EDTA-sensitive secretase activity was also present in microsomal membranes prepared from pig lung and testis, and from human lung and placenta, but was absent from human kidney and human and pig intestinal brush-border membranes. The form of ACE released from the microvillar membrane by the secretase co-migrated on SDS/PAGE with ACE purified from pig plasma, thus the action and location of the secretase would be consistent with it possibly having a role in the post-translational proteolytic cleavage of membrane-bound ACE to generate the soluble form found in blood, amniotic fluid, seminal plasma and other body fluids.

The potential value of integral membrane proteins in the vaccination of lambs against Haemonchus conortus

An extract of adult Haemonchus contortus enriched in the parasite's intestinal microvillar membrane protein H11 and other integral membrane proteins but free of the protein contortin was evaluated as a potential vaccine in two breeds of sheep. The worm burdens of Clun Forest sheep injected with the extract and challenged with 25,000 infective larvae were reduced 89% by weight compared to the average for the controls. The worm burdens of Dorset sheep (challenged with 10,000 infective larvae) were reduced 72%. In both breeds the reduction in the number of female worms, 92 and 71.8%, respectively, was greater than the reduction in the males (86.5 and 46%). Parasite egg output, determined only for the Dorsets, was reduced 92%. protection correlated with serum antibody titre. Most of the antibodies were directed against H 11.

Effects of enteropathogenic Escherichia coli on microvillar membrane proteins during organ culture of rabbit intestinal mucosa.

This study examines the effects of an enteropathogenic Escherichia coli on microvillar membrane proteins during organ culture of rabbit ileal explants. Explants maintained with enteropathogenic E coli showed brush border effacement affecting approximately 50% of enterocytes, and where enteropathogenic E coli were closely adherent to the enterocyte surface microvilli were apparently being shed as vesicles. The microvillar membrane enzymes alkaline phosphatase, aminopeptidase N and alpha-glucosidase were released into the culture medium during organ culture, and this process was significantly enhanced by enteropathogenic E coli. This increased loss of microvillar membrane enzymes into the culture medium was associated with decreased tissue activities of microvillar membrane enzymes in enteropathogenic E coli infected ileal explants compared with control. For aminopeptidase N in particular, however, total enzyme activities in the tissue plus culture medium were increased comparing enteropathogenic E coli with control, suggesting that there might be an increase in the rate of synthesis of certain microvillar membrane proteins. Reorientating sucrose density gradient centrifugation of culture medium showed that alkaline phosphatase, aminopeptidase N and alpha-glucosidase were predominantly associated with particles of peak modal density 1.19 g/ml in both groups, confirming that enteropathogenic E coli accelerate release of microvillar membrane enzymes as vesicles. Analytical fractionation of ileal explants showed that enteropathogenic E coli resulted in a loss of microvillar membrane enzyme activities from the main brush border peak of modal density 1.21 g/ml present in controls. The density of the remaining smaller and lighter peak increased from 1.19 g/ml to 1.23 g/ml after homogenisation in digitonin, confirming association of these proteins with cholesterol containing membranes and not endoplasmic reticulum. These findings suggest that enteropathogenic E. coli accelerate the normal shedding of microvillar membrane proteins as vesicles, and may stimulate a compensatory increase in microvillar membrane protein synthesis.

Cloning of cDNAs encoding a rabbit renal brush border membrane protein immunologically related to band 3. Sequence similarity with microsomal dipeptidase

Distinct anion transport processes have been identified in the mammalian renal proximal tubule, but none of the responsible proteins or genes have been isolated. A 43 kDa rabbit microvillus membrane protein that is immunologically related to the erythroid anion exchanger (band 3) was a candidate for a renal anion transporter. To examine the structural relationship with band 3, we cloned cDNAs encoding the 43 kDa protein. The 43 kDa band-3-like protein was purified, and a novel sequence of 24 amino acids was obtained from the N-terminus. Degenerate oligonucleotides were synthesized based on this sequence, and the polymerase chain reaction with single-sided specificity was used to amplify and clone a 1330 bp cDNA from rabbit renal cortex. Additional overlapping 272 bp and 1123 bp cDNAs were obtained by synthesizing and screening a rabbit renal cortical cDNA library. The composite sequence was 1483 bp, terminated with (A)16, and was similar in size to the principal transcript expressed in rabbit renal cortex. The single long open reading frame was predicted to encode a protein composed of 410 amino acids with a molecular mass of 45,193 Da; 15 amino acids predicted to reside at the N-terminus were absent in the mature protein and may constitute a signal peptide. There was only limited sequence similarity with human erythroid band 3. Rather, the sequence was highly similar to microsomal dipeptidase, including the presence of a signal peptide and a consensus sequence for covalent linkage to glycosylphosphatidylinositol. In summary, the 43 kDa protein from rabbit renal cortex that is recognized by a monospecific antibody to erythroid band 3 is most likely a microvillus membrane dipeptidase.