scholarly journals Preparation and characterization of basolateral membrane vesicles from pig and human colonocytes: the mechanism of glucose transport

1993 ◽  
Vol 294 (2) ◽  
pp. 529-534 ◽  
Author(s):  
S A Pinches ◽  
S M Gribble ◽  
R B Beechey ◽  
A Ellis ◽  
J M Shaw ◽  
...  
Keyword(s):  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Enzyme Analysis ◽  
Marker Enzyme ◽  
Independent Manner ◽  
Basolateral Membranes ◽  
Luminal Membrane ◽  
C Terminus ◽  
Normal Human

Membrane vesicles were isolated from the basolateral domains of pig and normal human colonocytes. The activity of the ouabain-sensitive K(+)-activated phosphatase, the basolateral membrane marker, was enriched 13-fold in these membrane vesicles over the original homogenate. The membranes displayed cross-reactions with antibodies to the (Na+/K+)ATPase and the RLA class I major histocompatibility antigen, both known indicators of the basolateral membrane. There was negligible contamination by other organelles and the luminal membrane, as revealed by marker-enzyme analysis and Western blotting, using an antibody to villin. The vesicles transported D-glucose in a cytochalasin B-inhibitable Na(+)-independent manner, with a Km of 28.1 +/- 0.8 mM and Vmax. of 3.1 +/- 0.4 nmol/s per mg of protein. The transport was inhibited by 2-deoxy-D-glucose and 3-O-methyl-D-glucose, but not by L-glucose or methyl-alpha-D-glucose. Probing the colonocyte basolateral membranes with an antibody against the C-terminus of the human liver GLUT 2 produced a cross-reaction at 52 kDa. These properties indicate the presence of a GLUT 2 isoform on the basolateral membranes of human and pig colonocytes.

Characterization of Cl-HCO3 exchange in basolateral membrane of rat distal colon

2003 ◽  
Vol 285 (4) ◽  
pp. C912-C921 ◽  
Author(s):  
Mutsuhiro Ikuma ◽  
John Geibel ◽  
Henry J. Binder ◽  
Vazhaikkurichi M. Rajendran
Keyword(s):  
Anion Exchange ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Distal Colon ◽  
Basolateral Membranes ◽  
Exchange Inhibitor ◽  
Rat Distal Colon ◽  
Different Types ◽  
Voltage Clamping

Sodium-independent Cl movement (i.e., Cl-anion exchange) has not previously been identified in the basolateral membranes of rat colonic epithelial cells. The present study demonstrates Cl-HCO3 exchange as the mechanism for 36Cl uptake in basolateral membrane vesicles (BLMV) prepared in the presence of a protease inhibitor cocktail from rat distal colon. Studies of 36Cl uptake performed with BLMV prepared with different types of protease inhibitors indicate that preventing the cleavage of the COOH-terminal end of AE2 protein by serine-type proteases was responsible for the demonstration of Cl-HCO3 exchange. In the absence of voltage clamping, both outward OH gradient (pHout/pHin: 7.5/5.5) and outward HCO3 gradient stimulated transient 36Cl uptake accumulation. However, voltage clamping with K-ionophore, valinomycin, almost completely (87%) inhibited the OH gradient-driven 36Cl uptake, whereas HCO3 gradient-driven 36Cl uptake was only partially inhibited (38%). Both electroneutral HCO3 and OH gradient-driven 36Cl uptake were 1) completely inhibited by DIDS, an anion exchange inhibitor, with a half-maximal inhibitory constant ( Ki) of ∼26.9 and 30.6 μM, respectively, 2) not inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid(NPPB), a Cl channel blocker, 3) saturated by increasing extravesicular Cl concentration with a Km for Cl of ∼12.6 and 14.2 mM, respectively, and 4) present in both surface and crypt cells. Intracellular pH (pHi) was also determined with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetomethylester (BCECF-AM) in an isolated superfused crypt preparation. Removal of Cl resulted in a DIDS-inhibitable increase in pHi both in HCO3-buffered and in the nominally HCO3-free buffered solutions (0.28 ± 0.02 and 0.11 ± 0.02 pH units, respectively). We conclude that a carrier-mediated electroneutral Cl-HCO3 exchange is present in basolateral membranes and that, in the absence of HCO3, Cl-HCO3 exchange can function as a Cl-OH exchange and regulate pHi across basolateral membranes of rat distal colon.

Calcium transport by rat duodenal villus and crypt basolateral membranes

1987 ◽  
Vol 252 (2) ◽  
pp. G170-G177 ◽  
Author(s):  
J. R. Walters ◽  
M. M. Weiser
Keyword(s):  
Vitamin D ◽  
Calcium Transport ◽  
Calcium Uptake ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Calcium Pump ◽  
Cell Fraction ◽  
Basolateral Membranes ◽  
Pump Activity ◽  
Crypt Cells

Rat duodenal cells were isolated sequentially to give fractions enriched for villus and crypt cells. From each of these fractions, basolateral-enriched membrane vesicles were prepared and ATP-dependent calcium uptake was studied. Calcium uptake was sensitive to temperature, was inhibited by vanadate and by A23187, and was lower in vitamin D-deficient animals. In normal animals, calcium transport was approximately twofold greater in villus-tip than in crypt cell-fraction basolateral membranes though the affinity of the uptake for calcium was similar (Km = 0.3 microM). In vitamin D-deficient animals, the crypt-to-villus gradient was reduced, and in all fractions, calcium transport was similar to or lower than that in the crypts of normal animals. Six hours after vitamin D-deficient animals were repleted with 1,25-dihydroxycholecalciferol, a significant increase in calcium transport by everted gut sacs was present; however, basolateral calcium transport was significantly increased in only the mid-villus fractions, and no change was seen in the villus-tip fractions. Thus vitamin D appears necessary for the development of increased basolateral membrane calcium pump activity in duodenal villus cells, but not all cells in vitamin D-deficient rats are able to respond to 1,25-dihydroxycholecalciferol.

Taurocholate transport by basolateral plasma membrane vesicles isolated from developing rat liver

1985 ◽  
Vol 248 (6) ◽  
pp. G648-G654
Author(s):  
F. J. Suchy ◽  
S. M. Courchene ◽  
B. L. Blitzer
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Marker Enzyme ◽  
Marker Enzymes ◽  
Plasma Membrane Vesicles ◽  
Adult Rat ◽  
Basolateral Plasma Membrane ◽  
Taurocholate Transport

Taurocholate transport was characterized in basolateral plasma membrane vesicles prepared from the livers of 14-day-old Sprague-Dawley rats using a self-generating Percoll gradient method. Liver plasma membrane protein yield, intravesicular volume, and enrichments of various marker enzymes were similar to those obtained for vesicles from adult rat liver. The basolateral marker enzyme Na+-K+-ATPase was enriched 26-fold in the suckling rat basolateral membrane fraction while the bile canalicular marker enzymes alkaline phosphatase and Mg2+-ATPase were enriched only 3- and 5-fold, respectively. The activities of marker enzymes for endoplasmic reticulum, mitochondria, or lysosomes were not enriched compared with homogenate. In the presence of an inwardly directed 100 mM Na+ gradient, vesicle accumulation of taurocholate transiently reached a concentration 1.5- to 2-fold higher than that at equilibrium ("overshoot") in suckling and adult membrane vesicles, but the initial rate of taurocholate entry and peak intravesicular accumulation were markedly decreased in suckling compared with adult membrane vesicles. In the presence of an inwardly directed 100 mM K+ gradient, the rate of uptake was slower, and no overshoot occurred in either suckling or adult rat vesicles. The decreased rate of Na+-coupled taurocholate uptake by membrane vesicles from suckling rat liver could not be explained on the basis of more rapid dissipation of the transmembrane Na+ gradient. Kinetic studies demonstrated saturable, Na+-dependent taurocholate uptake for both suckling and adult vesicles. However, the Vmax for taurocholate uptake in suckling rat vesicles was less than half of the adult rate (2.46 +/- 0.13 vs. 5.25 +/- 0.22 nmol X mg prot-1 X min-1, respectively, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a distinct Na(+)-H+ exchanger in basolateral membranes of human jejunum

1993 ◽  
Vol 264 (1) ◽  
pp. G45-G50
Author(s):  
S. Acra ◽  
W. Dykes ◽  
W. Nylander ◽  
F. K. Ghishan
Keyword(s):  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Kinetic Studies ◽  
Maximal Velocity ◽  
Nacl Transport ◽  
Human Organ ◽  
Basolateral Membranes ◽  
Plot Analysis ◽  
Gradient Technique ◽  
Na Uptake

Kinetically distinct Na(+)-H+ exchangers exist on the apical and basolateral membranes of rabbit ileal enterocytes. The apical Na(+)-H+ exchanger appears to function in electroneutral NaCl transport, whereas the basolateral Na(+)-H+ exchanger may function in homeostatic intravesicular pH (pHi) regulation and volume regulation. This study is designed to determine the presence and characteristics of the Na(+)-H+ exchanger in basolateral membrane vesicles (BLMV) prepared from jejunal tissues of human organ donors. A well-validated Percoll-gradient technique was used to prepare BLMV. An outwardly directed H+ gradient [pHi/extravesicular pH (pHo) = 5.2/7.5] resulted in a Na+ uptake overshoot (1.45 +/- 0.21 nmol/mg protein) 2.5-fold above equilibrium values (0.59 +/- 0.13 nmol/mg protein). Na+ uptake at equilibrium represented transport into an osmotically sensitive intravesicular space as validated by an osmolality study. Na+ uptake represented an electroneutral process, as shown by studies in which negative membrane potentials were induced by K+ and the ionophore valinomycin. Na+ uptake was linear for the first 15 s of transport as depicted by y = 0.042x + 0.002, r2 = 0.98. Dixon plot analysis of amiloride sensitivity revealed an ID50 value for amiloride of 29 microM (fourfold lower than ID50 for brush-border Na(+)-H+ exchanger). Kinetic studies of amiloride-sensitive Na+ uptake revealed a maximal velocity = 1.53 +/- 0.19 nmol.mg protein-1.5 s-1 and Michaelis constant = 9.83 +/- 3.5 mM. By varying pHi a sigmoidal effect of internal H+ on Na+ uptake was noted consistent with an internal modifier site for protons. To confirm this finding, the effect of pHi on Na+ efflux and Na(+)-Na+ exchange was studied.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The lipoprotein lipase of white adipose tissue. Studies on the intracellular distribution of the adipocyte-associated enzyme

1986 ◽  
Vol 236 (3) ◽  
pp. 749-756 ◽  
Author(s):  
A A Al-Jafari ◽  
A Cryer
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Guinea Pig ◽  
Lipoprotein Lipase ◽  
Membrane Vesicles ◽  
Capillary Endothelium ◽  
Enzyme Analysis ◽  
Marker Enzyme ◽  
Igg Antibodies ◽  
Plasma Membrane Vesicles

The separation of rat epididymal adipocytes into plasma-membrane, mitochondrial, microsomal and cytosol fractions is described. The fractions, which were characterized by marker-enzyme analysis and electron-micrographic observation, from the cells of fed and 24 h-starved animals were used to prepare acetone/diethyl ether-dried powders for the measurement of lipoprotein lipase activities. The highest specific activities and proportion of recovered lipoprotein lipase activity were found in the plasma-membrane and microsomal fractions. The two fractions from the cells of fed rats showed similar activities and enrichments of the enzyme, these activities being higher than the plasma-membrane and lower than the microsomal activities recovered from the cells of starved animals. Chicken and guinea-pig anti-(rat lipoprotein lipase) sera were prepared, and an indirect labelled-second-antibody cellular immunoassay, using 125I-labelled rabbit anti-(chicken IgG) or 125I-labelled sheep anti-(guinea-pig IgG) antibodies respectively, for the detection of cell-surface enzyme was devised and optimized. The amount of immunodetectable cell-surface lipoprotein lipase was higher for cells isolated from fed animals than for cells from 24 h-starved animals, when either anti-(lipoprotein lipase) serum was used in the assay. The amount of immunodetectable cell-surface lipoprotein lipase fell further when starvation was extended to 48 h. The lipoprotein lipase of plasma-membrane vesicles was shown to be a patent activity and to be immunodetectable in a modification of the cellular immunoassay. Although the functional significance of the adipocyte surface lipoprotein lipase is not known, the possibility of it forming a pool of enzyme en route to the capillary endothelium is advanced.

Proton-lactate co transport in basolateral membrane vesicles from rat jejunum

1996 ◽  
Vol 16 (6) ◽  
pp. 521-527
Author(s):  
Maria Novella Orsenigo ◽  
Marisa Tosco ◽  
Umberto Laforenza ◽  
Alide Faelli
Keyword(s):  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Rat Jejunum ◽  
Basolateral Membrane Vesicles ◽  
Strong Inhibition ◽  
Lactate Transport ◽  
Basolateral Membranes ◽  
Lactate Uptake ◽  
Stimulation Of

Proton-coupled lactate transport across the basolateral membrane of rat jejunal enterocyte was studied using well purified membrane vesicles. L-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP and by pCMBS; unlabelled L-lactate causes a strong inhibition, whilst furosemide is uneffective. The H+ gradient-dependent stimulation of L-lactate uptake is significantly inhibited also by SCN−: this finding could explain results recently reported in the literature in which H+-lactate symport was not evidenced in basolateral membranes from rat jejunum.

Inorganic anion transport in kidney and intestinal brush border and basolateral membranes

1980 ◽  
Vol 238 (6) ◽  
pp. F452-F460 ◽  
Author(s):  
S. Grinstein ◽  
R. J. Turner ◽  
M. Silverman ◽  
A. Rothstein
Keyword(s):  
Brush Border ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Anion Transport ◽  
Significant Inhibition ◽  
Kidney Cortex ◽  
Basolateral Membrane Vesicles ◽  
Inorganic Anion ◽  
Transport Pathways ◽  
Basolateral Membranes

The efflux of inorganic anions from purified brush border and basolateral membrane vesicles from dog kidney cortex was measured under equilibrium exchange conditions. Marked differences in temperature sensitivity and effects of inhibitors were found between the Cl and SO4 transport pathways and between the two types of membranes. SO4 transport in both brush border and basolateral membranes was markedly reduced by cooling, but significant inhibition by 4,4'–diisothiocyano-2,2'–disulfonic stilbene (DIDS) was only observed in basolateral vesicles. In contrast, Cl efflux from both types of vesicles was neither substantially inhibited by DIDS nor by lowering the temperature to 0 degrees C. Phosphate efflux from basolateral membrane vesicles was found to be only partially sensitive to DIDS. Attempts to label the stilbene-sensitive SO4 pathway in basolateral vesicles using [3H2]DIDS as a marker were unsuccessful due to the nonspecific labeling of many membrane components. The asymmetry in inorganic anion transport behavior exhibited by brush border and basolateral membrane vesicles from dog renal proximal tubule was also observed in equivalent vesicles prepared from rat small intestine.

scholarly journals Identification and Characterization of Aminopeptidase-N as a Binding Protein for Cry3Aa in the Midgut of Monochamus alternatus (Coleoptera: Cerambycidae)

2020 ◽  
Vol 113 (5) ◽  
pp. 2259-2268
Author(s):  
Yajie Guo ◽  
Rebeca Carballar-Lejarazú ◽  
Liangjing Sheng ◽  
Yan Fang ◽  
Shaozhen Wang ◽  
...  
Keyword(s):  
Insect Pests ◽  
Binding Protein ◽  
Phosphatidyl Inositol ◽  
In Silico Analysis ◽  
Membrane Vesicles ◽  
Aminopeptidase N ◽  
Monochamus Alternatus ◽  
Cry Proteins ◽  
C Terminus

Abstract Bacillus thuringiensis Cry proteins have been widely used over the past decades for many different insect pests, which are safe for users and the environment. The coleopteran-specific Cry3Aa toxin from B. thuringiensis exhibits toxicity to the larvae of Monochamus alternatus. Receptors play a key role in the mechanisms underlying the toxic action of Cry. However, the binding receptor for Cry3Aa has yet to be identified in the midgut of M. alternatus larvae. Therefore, the aim of this study was to identify the receptor for Cry3Aa toxin in the brush border membrane vesicles (BBMVs) of M. alternatus larvae. Our results indicate that the Cry3Aa toxin binds to the BBMVs (Kd = 247 nM) of M. alternatus via a 107 kDa aminopeptidase N (APN) (Kd = 57 nM). In silico analysis of the APN protein predicted that an 18 amino acid sequence in the N-terminal acted as a signal peptide, and that the Asn residue, located at position 918 in the C-terminus is an anchored site for glycosyl phosphatidyl inositol. Further analysis showed that M. alternatus APN exhibits 75% homology to the APN from Anoplophora glabripenis. Our work, therefore, confirmed that APN, which is localized in the BBMVs in the midgut of M. alternatus larvae, acts as a binding protein for Cry3Aa toxins.

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