Brush Border Plasma Membrane Research Articles

Renal Membrane Transport of Glutathione in Toxicology and Disease

Membrane transport processes, at both the plasma membranes and intracellular membranes, play critical roles in renal function and are a determining factor in the susceptibility of renal epithelial cells to blood-borne drugs and toxic chemicals. Proximal tubular epithelial cells possess a large array of transport proteins for organic anions, organic cations, and peptides on both basolateral and brush-border plasma membranes. Although these transporters function in excretion of waste products and reabsorption of nutrients, they also play a role in the susceptibility of the kidneys to drugs and other toxicants in the blood. The proximal tubules are typically the primary target cells because they are the first epithelial cell population exposed to such chemicals in either the renal plasma or glomerular filtrate and because of their large array of membrane transporters. Besides transport across the basolateral and brush-border plasma membranes, transport across intracellular membranes such as the mitochondrial inner membrane is a critical determinant of metabolite distribution. To illustrate the function of these transporters, carrier-mediated processes for transport of the tripeptide and antioxidant glutathione across the basolateral, brush-border, and mitochondrial inner membranes of the renal proximal tubule are reviewed. Studies are summarized that have identified the involvement of specific carrier proteins and characterized the role of these transporters in glutathione metabolism and turnover, susceptibility of the proximal tubules to oxidative and other stresses, and modulation in disease and other pathological processes.

Renal glutathione transport: Identification of carriers, physiological functions, and controversies

Glutathione (GSH) is an endogenous tripeptide composed of the amino acids L-glutamate, L-cysteine, and glycine. It is found in virtually all aerobic cells and plays critical roles in maintenance of cellular redox homeostasis and drug metabolism. An important component of its regulation is transport across biological membranes. Because GSH is a charged, hydrophilic molecule, transport occurs via catalysis by specific carrier proteins rather than by simple diffusion. Although it has been clearly understood that efflux of GSH across membranes such as the canalicular and sinusoidal plasma membranes in hepatocytes and the brush-border plasma membrane in renal proximal tubules is a key step in GSH turnover and interorgan metabolism, the existence and physiological functions of uptake of GSH across various epithelial plasma membranes has been subject to some debate. Besides transport across plasma membranes, GSH transport across intracellular membranes, most notably the mitochondrial inner membrane, has received some attention in recent years because of the importance of mitochondrial redox status and the mitochondrial GSH pool in cellular physiology and pathology. This commentary will focus on renal transport processes for GSH and will discuss some of the controversies that have existed and still seem to exist in the literature, specifically regarding uptake of intact GSH by basolateral membranes of renal proximal tubular cells and uptake of intact GSH by the mitochondrial inner membrane.

Rat kidney MAP17 induces cotransport of Na-mannose and Na-glucose in Xenopus laevis oocytes.

Renal reabsorption is the main mechanism that controls mannose homeostasis. This takes place through a specific Na-coupled uphill transport system, the molecular identity of which is unknown. We prepared and screened a size-selected rat kidney cortex cDNA library through the expression of mannose transport in Xenopus laevis oocytes. We have identified a membrane protein that induces high-affinity and specific Na-dependent transport of d-mannose and d-glucose in X. laevis oocytes, most likely through stimulation of the capacity of an endogenous transport system of the oocyte. Sequencing has revealed that the cDNA encodes the counterpart of the human membrane-associated protein MAP17, previously known by its overexpression in renal, colon, lung, and breast carcinomas. We show that MAP17 is a 12.2-kDa nonglycosylated membrane protein that locates to the brush-border plasma membrane and the Golgi apparatus of transfected cells and that it is expressed in the proximal tubules of the kidney cortex and in the spermatids of the seminiferous tubules. It spans twice the cell membrane, with both termini inside the cell, and seems to form homodimers through intracellular Cys55, a residue also involved in transport expression. MAP17 is responsible for mannose transport expression in oocytes by rat kidney cortex mRNA. The induced transport has the functional characteristics of a Na-glucose cotransporter (SGLT), because d-glucose and alpha-methyl-d-glucopyranoside are also accepted substrates that are inhibited by phloridzin. The corresponding transporter from the proximal tubule remains to be identified, but it is different from the known mammalian SGLT-1, -2, and -3.

Hepatocyte growth factor activates phosphoinositide 3-kinase C2 beta in renal brush-border plasma membranes.

Upon stimulation of renal cortical slices with hepatocyte growth factor (HGF), inositol lipid metabolism was studied in basal-lateral plasma membranes (BLM) and brush-border plasma membranes (BBM). Whereas in BLM rapid increases in 1,2-diacylglycerol, PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) were observed, suggesting that in BLM HGF activates both phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K), in BBM only HGF-induced transient accumulation of PtdIns3P was seen, which was temporarily delayed from signalling events in BLM and could be blocked by the PtdIns-specific-PLC inhibitor ET-18-OCH(3) and the calpain inhibitor calpeptin, suggesting that 3-kinase activation in BBM lies downstream of PLC activation in BLM and is a calpain-mediated event. Moreover, the increase in immunoprecipitable PI3K-C2 beta activity, which is sensitive to wortmannin (10 nM) and shows strong preference for PtdIns over PtdIns4P as a substrate, was observed only in BBM upon stimulation of renal cortical slices with HGF and could be mimicked by the Ca(2+) ionophore A23187 and blocked by the cell-penetrant Ca(2+) chelator BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)]. On Western blots PI3K-C2 beta revealed a single immunoreactive band of 180 kDa in BLM and BBM, while after stimulation with HGF a gel shift of 18 kDa was noticed only in BBM, suggesting that the observed enzyme activation is achieved by proteolysis. When BBM were subjected to short-term (15 min) exposure to mu-calpain, a similar gel shift together with an increase in PI3K-C2 beta activity was observed, when compared with the BBM harvested after HGF stimulation. The above-mentioned gel shift and increase in PI3K-C2 beta activity could be prevented by the calpain inhibitor calpeptin. The data presented in this report show that in renal cells there is a spatial separation of the inositol lipid signalling system between BLM and BBM, and that HGF causes activation of PLC and PI3K primarily in BLM, which leads to calpain-mediated activation of PI3K-C2 beta in BBM with a concomitant increase in PtdIns3P.

Nutritional regulation of nucleoside transporter expression in rat small intestine

Background & Aims: Concentrative nucleoside transporters CNT1 (pyrimidine preferring) and CNT2 (purine preferring) may be involved in the uptake of nucleoside-derived drugs used in antiviral and chemical therapies. The possibility that nucleoside carrier isoform expression is modulated by nutrient availability has been studied. Methods: CNT1 and CNT2 tissue distribution was determined by Western blot analysis. The effect of 48-hour starvation on CNT expression was then studied. Nucleoside transporter expression and uptake activity were measured in jejunal brush border plasma membrane vesicles from fed and starved rats. The expression of nucleoside transporters was later determined in a second model of nutrient deficiency: rats fed a purified diet with or without nucleotides for 10 days. Results: CNT1 and CNT2 nucleoside transporters were expressed in a wider variety of tissues than expected from messenger RNA distribution analysis. CNT1 was sensitive to nutrient availability in small intestine and, accordingly, jejunal brush border membrane vesicles from 48-hour–fasted rats showed increased expression of CNT1 and enhanced Na+-dependent thymidine and gemcitabine uptake. This effect was mimicked by feeding semipurified diets lacking nucleotides. Conclusions: Substrate availability modulates nucleoside transporter expression (CNT1) in rat jejunum in vivo.GASTROENTEROLOGY 2000;119:1623-1630

1,25-Dihydroxyvitamin D3Targets PKC-βII but Not PKC-α to the Basolateral Plasma Membranes of Rat Colonocytes

Prior studies by our laboratory have shown that 1,25-dihydroxyvitamin D3activated PKC-α, but not PKC-δ, -ϵ, or -ζ, in normal rat colonocytes. In the present studies we demonstrate for the first time that this secosteroid also activated PKC-βII, another DAG- and Ca2+-dependent PKC isoform recently shown to be present in these cells. Moreover, this activation of PKC-βII by 1,25-dihydroxyvitamin D3treatment of isolated colonocytes was shown to be lost in cells from vitamin D-deficient rats and, at least partially, restored by repleting these animals with this secosteroid for 7 days. Under basal conditions, the expression of PKC-α and -βII in brush-border membranes was comparable to their respective expression in basolateral plasma membranes of rat colonocytes. In contrast, the expression of PKC-δ was significantly greater in brush-border membranes, whereas PKC-ϵ and -ζ were enriched in the basolateral plasma membranes. Furthermore, 1,25-dihydroxyvitamin D3specifically induced the translocation of PKC-βII, but not PKC-α, to the basolateral, but not brush-border plasma membranes of rat colonocytes, via a pp60c-src-dependent mechanism.

Quantitation and immunolocalization of glucose transporters in the human placenta

The subcellular distributions of the mammalian passive glucose transporter isoforms GLUT1, GL UT3 and GL UT4, in the human placenta, there investigated using isoform-specific anti-peptide antibodies. On western blots of both basal and brushborder plasma membranes isolated from the syncytiotrophoblast, antibodies specific for GLUT1 labelled a broad band (apparent Mr 55000) that co-migrated with the human erythrocyte GLUT1 glucose transporter. In contrast, no labelling was detectable when blots were probed with antibodies specific for the GLUT3 or GLUT4 isoforms. Densitometric analysis of blots showed that GLUT1 accounts for approximately 90 and 65 per cent of the d-glucose-sensitive cytochalasin B binding sites present in brush-border and basal membranes, respectively. Confocal immunofluorescence microscopy of fixed placental tissue showed that GLUT1 is abundant at both maternal- and fetal facing surfaces of the syncytiotrophoblast whereas it was undetectable at the fetal capillary endothelium. In parallel experiments, no staining by antibodies against either the GLUT3 or the GLUT4 isoforms was detected in placental tissue. These results indicate that GLUT1 is the major isoform responsible for glucose transfer from mother to fetus. The absence of GLUT4 is consistent with the lack of insulin-sensitive glucose transport across the placenta.

Evidence for a G protein system in the tegumental brush border plasma membrane of Hymenolepis diminuta

Guanine nucleotide-binding regulatory proteins (G proteins) mediate the transduction of signals from cell-surface receptors to intracellular effector enzymes. G protein α-subunits are routinely identified (and partially characterized) on the basis of their susceptibility to NAD +-dependent ADP-ribosylation catalysed by cholera and/or pertussis toxins. Analysis of purified tegumental brush border plasma membrane from Hymenolepis diminuta by relevant methodologies has revealed the presence of a 42 kDa putative G protein α-subunit that is susceptible to ADP-ribosylation by both cholera and pertussis toxins. This polypeptide shows no definite resemblance to any of the four major mammalian G protein classes on the basis of M r, and toxin-susceptibility. These results provide evidence for the existence of a tegumental G protein-linked signal transduction system in H. diminuta

Differential effect of 1,25-dihydroxycholecalciferol on phosphoinositide turnover in the antipodal plasma membranes of colonic epithelial cells

1,25-dihydroxycholecalciferol stimulates membrane phosphoinositide turnover in colonic epithelial and other cells, but the effects of this hormone on phosphoinositide metabolism in specific antipodal plasma membranes has not been examined. In the present studies, addition of 10 −8M 1,25-dihydroxycholecalciferol to rat colonic crypts for 90 seconds decreased the phosphatidylinositol-4,5-bisphosphate content and increased the diacylglycerol content of the baso-lateral, but not the brush border plasma membrane. Using Caco-2 cells grown as tight polarized monolayers, 1,25-dihydroxycholecalciferol reduced cellular phosphatidylinositol-4,5-bisphosphate and increased cellular inositol-1,4,5-triphosphate and diacylglycerol when added to the buffer bathing the baso-lateral, but not the brush border membrane surface. These data indicate, therefore, that 1,25-dihydroxycholecalciferol activates the phosphoinositol signal transduction cascade specifically in the baso-lateral cell membrane of colonic cells.

Antibodies to mammalian and plant V-ATPases cross react with the V-ATPase of insect cation-transporting plasma membranes.

In immunobiochemical blots, polyclonal antibodies against subunits of plant and mammalian vacuolar-type ATPases (V-ATPases) cross-react strongly with corresponding subunits of larval Manduca sexta midgut plasma membrane V-ATPase. Thus, rabbit antiserum against Kalanchoe daigremontiana tonoplast V-ATPase holoenzyme cross-reacts with the 67, 56, 40, 28 and 20 kDa subunits of midgut V-ATPase separated by SDS-PAGE. Antisera against bovine chromaffin granule 72 and 39 kDa V-ATPase subunits cross-react with the corresponding 67 and 43 kDa subunits of midgut V-ATPase. Antisera against the 57 kDa subunit of both beet root and oat root V-ATPase cross-react strongly with the midgut 56 kDa V-ATPase subunit. In immunocytochemical light micrographs, antiserum against the beet root 57 kDa V-ATPase subunit labels the goblet cell apical membrane of both posterior and anterior midgut in freeze-substituted and fixed sections. The plant antiserum also labels the apical brush-border plasma membrane of Malpighian tubules. The ability of antibodies against plant V-ATPase to label these insect membranes suggests a high sequence homology between V-ATPases from plants and insects. Both of the antibody-labelled insect membranes transport K+ and both membranes possess F1-like particles, portasomes, on their cytoplasmic surfaces. This immunolabelling by xenic V-ATPase antisera of two insect cation-transporting membranes suggests that the portasomes on these membranes may be V-ATPase particles, similar to those reported on V-ATPase-containing vacuolar membranes from various sources.

Ontogenic development of lamb intestinal sodium‐glucose co‐transporter is regulated by diet.

1. The ontogenic development of the intestinal Na(+)-glucose co-transporter was measured in lambs as a function of diet. Transport activity was assayed in brush-border membrane vesicles and the expression of transport protein in the brush-border membrane determined by Western analysis. 2. Na(+)-dependent D-glucose transport increased to a maximum (300-700 pmol mg-1 s-1) within the first 2 weeks of birth and then declined to negligible amounts (less than 10 pmol mg-1 s-1) over the next 8 weeks. There was no further change over the next 2-3 years. Early changes were associated with modifications in both the maximum velocity Vmax for transport and expression of carrier protein in the brush-border plasma membrane. 3. Maintaining lambs on a milk replacer diet beyond the normal weaning period prevented the normal decline in the expression of Na(+)-glucose co-transport. At 5 weeks the transport rate was 433 +/- 150 pmol mg-1 s-1 in lambs maintained on milk replacer, but only 79 +/- 40 pmol mg-1 s-1 in normally reared control lambs. 4. Infusing the proximal intestine of 2- to 3-year-old sheep with 30 mM-D-glucose for four days increased the rate of transport 40- to 80-fold above that found in control animals perfused with mannitol. A similar but smaller increase was observed in one animal perfused with the non-metabolizable sugar alpha-methyl-D-glucopyranoside. The induced increase in glucose transport was correlated with the expression of the co-transporter protein in the brush-border plasma membrane. 5. It is concluded that the age-related decline in Na(+)-glucose co-transport in the sheep intestine is directly due to the decrease in D-glucose (and D-galactose) reaching the small intestine after development of the rumen. These results further suggest that luminal sugar substrates for the co-transporter promote both the maintenance and the up-regulation of the brush-border transport protein and it is the intact sugar itself which controls gene expression during enterocyte maturation.

Activation and inhibition of the brush-border membrane-bound alkaline phosphatase activity of Hymenolepis diminuta (Cestoda) by divalent cations

In the absence of exogenous divalent cations, the isolated brush-border plasma membrane of Hymenolepsis diminuta possesses alkaline phosphatase activity (APA). APA is stimulated in the presence of exogenous Mg2+ and inhibited by low concentrations of Zn2+ or high concentrations of Ca2+, and inhibition of APA by Zn2+ is reversed by both Mg2+ and Ca2+. APA is inhibited by ethylenediamine tetraacetic acid (EDTA), ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid, and 1,10-phenanthroline in time- and concentration-dependent fashions, with EDTA being the most effective inhibitor. Following treatment with EDTA, APA is restored by Mg2+ and, to a lesser extent, by Ca2+, but not by Zn2+. Thus, APA represents a Mg2(+)-dependent enzyme that can be partly activated by Ca2+ but only in the absence of Mg2+.

Iron uptake by human upper small intestine microvillous membrane vesicles. Indication for a facilitated transport mechanism mediated by a membrane iron-binding protein.

To investigate the hypothesis that iron absorption in man involves a carrier-mediated cellular uptake mechanism, influx velocity (Vo) of 59Fe3+ by isolated human microvillous membrane (MVM) vesicles of the upper small intestine was examined. Vo revealed saturation kinetics (Km = 315 nM; Vmax = 361 pmol Fe3+ x min-1 x mg protein-1) was temperature dependent and inhibited by pronase pretreatment of MVM. In the presence of an inwardly directed Na(+)-gradient a typical overshoot phenomenon with maximal uptake at 30-40 s was observed. The suggestion of an active, carrier-mediated uptake mechanism for iron was pursued by isolation of a 160-kD iron-binding protein from solubilized human MVM proteins. This glycoprotein was assembled as a trimer composed of 54-kD monomers. A monospecific antibody against the 54-kD subunit inhibited vesicular influx of Fe3+ into MVM by greater than 50%. Immunofluorescence and immunoblot analysis confirmed the localization of the protein in brush border plasma membranes. It was detectable in human intestinal mucosa and liver, but not in esophagus. These data indicate that the translocation of Fe3+ across human MVM represents a facilitated transport mechanism which is, at least in part, mediated by a membrane iron-binding protein.

Isolation and characterization of dipeptidyl aminopeptidase IV from human kidney cortex

Intact dipeptidyl aminopeptidase IV (DAP IV) was solubilized by bromelain treatment from human kidney brush border plasma-membranes. Purification of DAP IV was performed by a 3-step method, applying lectin-affinity chromatography on WGA-Sepharose, gel filtration and anion-exchange chromatography. DAP IV from human kidney cortex showed a pH optimum of 8.7 and was totally inhibited by 1 mmol/l Zn 2+. Isolated DAP IV revealed a relative molecular mass of 250 kDa as determined by the native-PAGE method and of 220 kDa by the gel filtration method. Analytical isoelectric focussing of DAP IV revealed an isoelectric point of pH 5.3. Ultrastructural analysis of isolated DAP IV fractions, using the negative staining technique, disclosed the presence of numerous globular particles with an average diameter of 5 nm which correspond to the structural substrate of the purified protein.

Uptake of fatty acids by jejunal mucosal cells is mediated by a fatty acid binding membrane protein.

The previous identification of a membrane fatty acid binding protein (MFABP) in brush border plasma membranes of the jejunum suggested that mucosal cell uptake of fatty acids might represent a carrier-mediated transport system. For evaluation of this hypothesis cellular influx kinetics (V0) of [3H]-oleate were examined in isolated rat jejunal mucosal cells. With increasing unbound oleate concentration in the medium V0 was saturable (Km = 93 nM; Vmax = 2.1 nmol X min-1 per 10(6) cells) and temperature dependent with an optimum at 37 degrees C. Pretreatment of the cells with a monospecific antibody to MFABP significantly inhibited V0 of oleate, other long-chain fatty acids, and D-monopalmitin, but not of L-alanine. Moreover, in the in vivo system of isolated perfused jejunal segments the physiologic significance of MFABP in the directed overall intestinal absorption process of fatty acids was documented. In the presence of the anti-MFABP oleate absorption was markedly reduced, whereas uptake of L-alanine remained unaltered. By antibody inhibition studies it was suggested that this membrane carrier also reveals transport competence for various other long-chain fatty acids, D-monopalmitin, L-lysophosphatidylcholine, and cholesterol. These data support the hypothesis that absorption of fatty acids is mediated by a fatty acid binding membrane protein.

Nucleotide hydrolysis by solubilized membrane-bound enzymes of the brush border plasma membrane of Hymenolepis diminuta

Pyrophosphate, p-nitrophenyl phosphate and a variety of pyrimidine and purine nucleotides are hydrolyzed by the solubilized membrane-bound enzymes of the brush border plasma membrane of Hymenolepis diminuta. The pH optima (or ranges) for hydrolysis of substrates are 8.0 (pyrophosphate), 8.8 ( p-nitrophenyl phosphate), 8.4–8.9 (nucleoside monophosphates), and 7.1–8.1 (nucleoside triphosphates); all substrates, with the exception of nucleoside triphosphates, have a higher affinity for the solubilized enzyme at pH 7.4 than at their optimal pH for hydrolysis. ATP is degraded completely by the enzyme preparation to adenosine and inorganic phosphate, but since neither ADP nor ATP accumulate in the incubation medium it is not known whether ATP hydrolysis involves the sequential hydrolysis of terminal phosphate groups. Isoelectric focusing and various chromatographic procedures (gel permeation, ion-exchange and hydrophobic interaction chromatography) fail to separate the alkaline phosphatase, phosphodiesterase, 5′-nucleotidase, adenosine triphosphatase and ribonuclease activities associated with the solubilized membrane preparation. Additionally, inhibitor studies indicate that only a single enzyme with low substrate specificity is involved in the hydrolysis of nucleotides, p-nitrophenyl phosphate, pyrophosphate and hexose phosphate esters. Purines and pyrimidines and their nucleosides interact with the active site, and in some instances activity of the enzyme is stimulated by an unknown mechanism.

Characterization of cobalamin receptor sites in brush-border plasma membranes of the tapeworm Spirometra mansonoides.

The interaction of receptor sites in microtriches (brush-border) membranes isolated from the sparganum (larva) of the cestode (tapeworm) Spirometra mansonoides with [57Co]cyanocobalamin (CN-[57Co]Cbl) has been characterized on the basis of the following criteria: time dependence, reversibility, high-affinity binding, finite binding capacity, and ligand specificity. The association of CN-[57Co]Cbl to isolated microtriches membranes at 22 degrees C reached equilibrium in approximately 90 min. The initial rate of association follows second order kinetics with an association rate constant (ka) of 1.1 X 10(6) M-1 S-1. In the presence of an excess of unlabeled CN-Cbl, the receptor CN-[57Co]Cbl complex dissociated according to first order kinetics with a dissociation constant (kd) of 4.4 X 10(-4) S-1. An equilibrium dissociation constant (KD) of 0.4 nM was calculated on the basis of rate constants (KD = kd/ka). Scatchard analysis of equilibrium-binding data revealed a single class of high affinity binding sites with a KD of 0.3 nM and a maximum binding capacity for CN-Cbl of 221 fmol/mg of membrane protein. The stereo-specificity of CN-Cbl binding to receptor sites in isolated microtriches membranes was determined using various Cbl analogs. Receptor-site binding affinity was lowered by the following modifications: deamination of the b-, d-, and e-propionamide side chains to the corresponding monocarboxylate derivatives; modification of the B pyrrole ring to lactam or lactone; substitution of the benzimidazole moiety by a purine; modification of the benzimidazole; or deletion of the entire nucleotide moiety. On the basis of the action of trypsin and pronase, this CN-Cbl receptor may contain a relatively exposed membrane protein. The physiological role of this CN-Cbl receptor in the binding, internalization, and metabolism of Cbl is discussed in relation to cestodes and their mammalian hosts.

51] Nonspecific lipid transfer protein from rat and beef liver: Purification and properties

This chapter discusses the purification and properties of nonspecific lipid transfer protein from rat and beef liver. The nonspecific lipid transfer proteins accelerate the intermembrane transfer of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, sphingomyelin, cholesterol, neutral glycosphingolipids, and gangliosides. The transfer proteins are useful tools for the study of the distribution and movement of phospholipids in artificial vesicles, microsomes, intact erythrocytes, and Semliki Forest virus and of cholesterol in the brush-border plasma membranes of the rabbit intestine. The assay of transfer activity is based on the ability of nonspecific lipid transfer proteins to accelerate the transfer of labeled phosphatidylethanolamine from phosphatidylethanolamine–phosphatidylcholinc small unilamellar vesicles to beef heart mitochondria. A trace of labeled triolein is included in the small unilamellar vesicles during preparation to correct for vesicle recovery. After the incubations, the mitochondria are pelleted and aliquots of the supernatants, containing the vesicles, are counted. A measure of the transfer activity is given by the decrease in the phosphatidylethanolamine/triolein ratio of the supernatants.

Hymenolepis diminuta: Partial characterization of the membrane-bound and solubilized alkaline phosphohydrolase activities of the isolated brush border plasma membrane

The membrane-bound and solubilized (using Triton ×-100 or sodium dodecyl sulfate (SDS)) alkaline phosphohydrolase (APase) activities of the isolated brush border membrane of Hymenolepis diminuta require a divalent cation for maximum activity. Highest rates of substrate ( p-nitrophenyl phosphate) hydrolysis are obtained with low concentrations of Mg 2+ (1 m M), although low concentrations of Mn 2+, Ca 2+, or Zn 2+ will also partially satisfy this requirement; higher concentrations of Mg 2+ and Mn 2+, and other divalent cations (Cu 2+, Fe 2+, and Pb 2+), inhibit the membrane-bound APase activity. Solubilization of the membrane-bound enzyme in either Triton or SDS results in an increase in specific activity and K m , but has little effect on thermal stability of the APase activity. Phosphate, pyrophosphate, adenosine 5′-triphosphate, adenosine 5′-monophosphate, glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-diphosphate inhibit substrate hydrolysis, and the relative affinities of these inhibitors for the APase enzyme are altered only slightly upon solubilization. Graphic analyses of data from inhibitor studies indicate that all eight inhibitors will inhibit membrane-bound and solubilized APase activities 100% at high inhibitonsubstrate ratios. Molybdate, F −, 2-mercaptoethanol, cysteine, and p-chloromercuribenzoate inhibit membrane-bound APase activity. Inhibitor data indicate that if more than one enzyme is responsible for the APase activity of the brush border membrane of H. diminuta, the enzymes cannot be differentiated on the basis of substrate specificity.

Solubilization of the Membrane-Bound Enzymes of the Brush-Border Plasma Membrane of Hymenolepis diminuta (Cestoda) Using Nonionic Detergents

The ability of nonionic detergents to solubilize the membrane-bound enzymes of the brush-border plasma membrane of Hymenolepis diminuta was investigated. Of the detergents tested (Triton X-100, Tween 80, Brij 35, Lubrol PX and WX, W-1, and beta-octyl-D-glucoside), only Triton was an effective solubilizing agent. Optimal solubilization was achieved by incubating an isolated fraction of the brush-border membrane in the presence of 1% Triton X-100 for 60 min at 37 C, followed by centrifugation at 100,000 g for 60 min at 25 C. This treatment resulted in solubilization of 94% of the alkaline phosphohydrolase, 91% of the phosphodiesterase and ribonuclease, and 88% of the 5'-nucleotidase activities. The pH optima for enzymes solubilized in nonionic and ionic detergents (Triton and sodium dodecyl sulfate, respectively) did not differ. Isoelectric focusing of the Triton-solubilized material demonstrated the presence of at least 14 polypeptides, a majority of which had isoelectric points below pH 7.