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A Rigal

Publications and source records attributed to A Rigal.

At least 37 records · Page 2Linked to original sources
PubMed1989

Conformational change of rabbit aminopeptidase N into enterocyte plasma membrane domains analyzed by flow cytometry fluorescence energy transfer.

Membrane vesicle preparations are very appropriate material for studying the topology of glycoproteins integrated into specialized plasma membrane domains of polarized cells. Here we show that the flow cytometric measurement of fluorescence energy transfer used previously to study the relationship between surface components of isolated cells can be applied to membrane vesicles. The fluorescein and rhodamine derivatives of a monoclonal antibody (4H7.1) that recognized one common epitope of the rabbit and pig aminopeptidase N were used for probing the oligomerization and conformational states of the enzyme integrated into the brush border and basolateral membrane vesicles prepared from rabbit and pig enterocytes. The high fluorescent energy transfer observed in the case of pig enzyme integrated into both types of vesicles and in the case of the rabbit enzyme integrated into basolateral membrane vesicles agreed very well with the existence of a dimeric organization, which was directly demonstrated by cross-linking experiments. Although with the latter technique we observed that the rabbit aminopeptidase was also dimerized in the brush border membrane, no energy transfer was detected with the corresponding vesicles. This indicates that the relative positions of two associated monomers differ depending on whether the rabbit aminopeptidase is transiently integrated into the basolateral membrane or permanently integrated into the brush border membrane. Cross-linking of aminopeptidases solubilized by detergent and of their ectodomains liberated by trypsin showed that only interactions between anchor domains maintained the dimeric structure of rabbit enzyme whereas interactions between ectodomains also exist in the pig enzyme. This might explain why the noticeable change in the organization of the two ectodomains observed in the case of rabbit aminopeptidase N does not occur in the case of pig enzyme.

Aminopeptidases↗
PubMed1989

The mouse T-lymphocyte-activating molecule THAM is associated to ECTO-aminopeptidase activity.

In the mouse there are three non-CD3/T-cell receptor T cell-activating molecules, Thy-1, Ly-6 antigens, and the CD28 homologue. Using a mitogenic rat monoclonal antibody, we identified a novel thymocyte-activating molecule (THAM). Here we review the main characteristics of this heterodimeric glycoprotein with respect to its expression on lymphoid and non-lymphoid cells, and its biochemical and T-cell-activating properties. THAM shares a number of structural features with surface peptidases of epithelial cell brush borders, and is in fact associated with a neutral aminopeptidase activity. The biologic significance of this observation is discussed in the context of recent data on the involvement of several ecto-enzymes in T-cell activation and differentiation.

Aminopeptidases↗
PubMed1987

Evidence for the transit of aminopeptidase N through the basolateral membrane before it reaches the brush border of enterocytes.

In vivo pulse-chase labeling of rabbit jejunum loops was used in conjunction with subcellular fractionation and quantitative immunoprecipitation to determine whether or not the newly synthesized aminopeptidase N transits through the basolateral membrane before it reaches the apical brush border, its final localization. The kinetics of the arrival of the newly synthesized enzyme in the Golgi complex, basolateral and brush border membrane fractions strongly suggest that on leaving the Golgi aminopeptidase N is transiently integrated into the basolateral domain before reaching the brush border.

Aminopeptidases↗
PubMed1986

Further characterization of an early expressed glycoprotein of the rabbit small intestinal brush border. Its interaction with some hydrolases.

Mouse 23 B 921 monoclonal antibody recognized a rabbit brush-border antigen with an apparent molecular mass 140 kDa (140-kDa Ag) which, unlike most hydrolases, is expressed in the poorly differentiated crypt cells of the small intestine. Immunoelectron microscopy of brush-border vesicles showed that the 23 B 921 bound to an epitope localized on the outside of the membrane. As is the case with hydrolases the external domain of the 140-kDa Ag constitutes the main part of the molecule, which can be released by papain treatment of brush-border vesicles. The presence of a small hydrophobic domain, anchoring the molecule into the membrane and responsible for its amphipatic character, was shown by its affinity for Triton-X114 micelles. The topological organization in the membrane of 140-kDa Ag seemed to be identical to that of hydrolases. Unlike hydrolases, however, the native structure of the antigen was found from its sensitivity to proteolysis to be very dependent on its integration into the lipid bilayer. Nevertheless, detergent-extracted 140-kDa Ag can be purified by immunoaffinity chromatography although it cannot be stocked for more than 48 h even in the presence of protease inhibitors. The carbohydrate moiety of 140-kDa Ag, bearing the human blood group A determinant, amounts to 20% of the total molecular mass. The existence of some privileged relationship was established between 140-kDa Ag and hydrolases: in the membrane, hydrolases protect the 140-kDa Ag from papain action; after detergent extraction, 140-kDa Ag is strongly associated with several hydrolases particularly disaccharidases.

Amino Acids↗
PubMed1986

Subcellular fractionation and subcellular localization of aminopeptidase N in the rabbit enterocytes.

A fast and easy procedure is proposed for preparing concomitantly from the same sample of intestinal mucosa of A+ rabbits, four fractions high enriched in the brush-border and basolateral plasma membrane domains, rough endoplasmic reticulum, and smooth endoplasmic reticulum plus Golgi apparatus membranes, respectively. This is the first time the technique of flow fluorometry has been applied to characterize the brush-border and basolateral membrane fractions using polyclonal or monoclonal antibodies against antigens common to or specific for these two plasma membrane domains. This technique definitely proves the presence of aminopeptidase in at least 60% of the basolateral membrane vesicles, where its level is about 4.5% of that in the brush-border membrane vesicles. The endoglycosidase H-sensitive intermediate of glycosylation of aminopeptidase N in the steady state is accumulated in both the rough and smooth endoplasmic reticulum membranes. Although the rough membrane is more extensive it contains only about 40% of this transient form.

Aminopeptidases↗
PubMed1986

Identification of an early expressed marker of the luminal membrane of rabbit small intestinal columnar cells. Presence of a homologous antigen in kidney proximal tubules and glomeruli.

Four monoclonal antibodies have been produced that specifically react with a rabbit intestinal brush-border glycoprotein which migrates in SDS-polyacrylamide gel electrophoresis as a protein of molecular weight 140,000. Contrarily to brush-border hydrolases, it is expressed in poorly differentiated crypt cells of the small intestine. Absent from colon columnar cells, it can be considered to be an early marker of differentiation of absorbing cells of the small intestine. Immunofluorescence technique showed the presence of a homologous antigen in kidney proximal tubule brush-border and podocytes of kidney glomeruli.

Animals↗
PubMed1986

Evidence for selective transport of two brush-border glycoproteins from endoplasmic reticulum to Golgi complex in rabbit enterocytes.

In vivo pulse-chase labeling of rabbit jejunum loops was used in conjunction with subcellular fractionation and quantitative immunoprecipitation to compare the intracellular transport kinetics of aminopeptidase with that of a 140 kDa brush-border antigen not belonging to the hydrolase class. As judged by the maturation kinetics of Asn-linked glycans, these glycoproteins were found to be transported from the endoplasmic reticulum into the Golgi apparatus at different rates (t1/2 = 25-50 min). The transport from the Golgi complex to the brush-border was rapid and seemed to occur at the same rate for both glycoproteins. In keeping with these kinetic data, the steady-state levels of aminopeptidase and the 140 kDa antigen in the Golgi complex were low, although that of aminopeptidase was significantly higher.

Aminopeptidases↗
PubMed1985

Aminopeptidase N- and human blood group A-antigenicity along the digestive tract and associated glands in the rabbit.

The cellular localization of an aminopeptidase N homologous to the brush-border intestinal enzyme and that of human blood group A-substances were investigated using the immunofluorescence technique on thin frozen sections (200 nm) of the digestive tract and associated glands of A+ and A- rabbits. Aminopeptidase N was found to be a common specific marker of both the apical region of plasma membrane of acinar cells in submaxillary and parotid glands and pancreas and the brush border of jejunum and colon absorbing cells. In hepatocytes, the enzyme was localized in the sinusoidal domains. Soluble A-substances were present in mucus secretory granules of intestinal goblet cells and those of stomach and gall bladder mucous cells. In contrast, the mucous acini of sublingual and submaxillary glands were devoid of A-antigenicity. The columnar cells of striated ducts of these glands exhibited A-antigenicity. Soluble A-substances were also found in zymogen granules of parotid and pancreas acinar cells and those of stomach chief cells. Moreover, in all cells secreting A-substances, and in the non-secreting absorbing intestinal cells, the glycoproteins of the plasma membrane bore A-determinants. Aminopeptidase N was one of the membrane-bound glycoproteins that bore A-determinants in cells that expressed A-antigenicity.

ABO Blood-Group System↗
PubMed1985

Biosynthesis and intracellular pool of aminopeptidase N in rabbit enterocytes.

A papain treatment at 15 degrees C and pH 7.3 of a microsomal fraction from rabbit enterocytes quantitatively releases the aminopeptidase N integrated in the plasma membranes without solubilizing the enzyme integrated in the intracellular membranes. Working on A+ rabbits, characterized by the presence on the brush-border hydrolases of glycans corresponding to the human blood group A-determinant structure, it was possible to separate the intracellular aminopeptidase into two major molecular forms with or without these determinants. The molecular form devoid of human blood group A antigenicity corresponds to the only stable intermediate of glycosylation, bearing N-linked high mannose oligosaccharides. This endoglycosidase H-sensitive form is fully active and represents in the steady state about 1% of the total cellular aminopeptidase. It contains a cytoplasmic sequence of about 3000 daltons that has not yet been detected in the mature form. The A antigenicity is acquired simultaneously with processing of high mannose glycans to complex glycans. Pulse chase labeling of jejunum loops with [35S]-methionine showed that the complete processing of the transient form synthesized during 10 min takes 1 hr. During the last 30 min of processing, all the newly transformed molecules are transported to the plasma membrane.

Aminopeptidases↗
PubMed1983

Analysis of two chloride requirements for sodium-dependent amino acid and glucose transport by intestinal brush-border membrane vesicles of fish.

Intestinal brush border vesicles of a Mediterranean sea fish (Dicentrarchus labrax) were prepared using the Ca2+-sedimentation method. The transport of glucose, glycine and 2-aminoisobutyric acid is energized by an Na+ gradient (out greater than in). In addition, amino acid uptake requires Cl- in the extravesicular medium (2-aminoisobutyric acid more than glycine). This Na+- and Cl--dependent uptake is electrogenic, since it can be stimulated by negative charges inside the vesicles. The specific Cl- requirement of glycine and 2-aminoisobutyric acid transport is markedly influenced by pH, a change from 6.5 to 8.4 reducing the role played by Cl-. In the presence of Cl-, the Km of 2-aminoisobutyric acid uptake is reduced and its Vmax is enhanced. Cl- affects also a non-saturable Na+-dependent component of this amino acid uptake. Amino acid transport is also increased by intravesicular Cl- (2-aminoisobutyric acid less than glycine). This effect is more concerned with glucose uptake, which can be then multiplied by 2.3. A concentration gradient (in greater than out) as well as the presence of Na+ in the incubation medium seems to enter into this requirement. This intravesicular Cl- effect is not influenced by pH between 6.5 and 8.4.

Amino Acids↗
PubMed1983

Further studies on intestinal accumulation of glycine during fasting in rainbow trout (Salmo gairdneri Richardson).

Using the sliced intestine method, we studied in vitro the effects of long-term fasting (4 and 8 weeks) on the accumulation of 0.5 and 10 mM glycine in the mid- and hindgut of the trout. Increased glycine accumulation during fasting was noted when accumulation was calculated per gram of intestinal dry weight, per milliliter of cellular water, but especially per gram of mucosal tissue. The glycine tissue to medium ratios are also higher in fasted intestines, revealing an enhancement of the active pathways of the amino acid transport. After 4 weeks of fasting, the increased glycine accumulation is greater in hindgut, especially for the lowest glycine concentration (0.5 mM). With the highest concentration (10 mM), the effects of fasting similarly decrease in intensity and affect in the midgut and hindgut. After 8 weeks of fasting, the differences between fed and fasted intestines tend to be fewer, probably because of a progressive attenuation of the effect of fasting on the active pathways of glycine accumulation.

Animals↗
PubMed1981

A chloride requirement for Na+-dependent amino-acid transport by brush border membrane vesicles isolated from the intestine of a Mediterranean teleost (Boops salpa).

The uptake of D-glucose, 2-aminoisobutyric acid and glycine was studied with intestinal brush border membrane vesicles of a marine herbivorous fish: Boops salpa. The uptake of these three substances is stimulated by an Na+ electrochemical gradient (Cout greater than Cin). For glucose, an increase of the electrical membrane potential generated by a concentration gradient of the liposoluble anion, SCN-, increases the Na+-dependent transport. This responsiveness to the membrane potential was confirmed by valinomycin. Differently from glucose, uptake of glycine and 2-aminoisobutyric acid requires, besides the Na+ gradient, the presence of Cl- on the external side of the vesicles. In the absence of Cl-, amino acid uptake is not stimulated by the Na+ gradient and is not influenced by an electrical membrane potential generated by SCN- gradient (Cout greater than Cin) or by a K+ diffusion potential (Cin greater than Cout). This Cl- requirement differs from the Na+ requirement, since a Cl- gradient (Cout greater than Cin) does not result in an accumulation of glycine or 2-aminoisobutyric acid similar to that produced by an Na+ gradient.

Amino Acids↗
PubMed1979

Isolation of brush border membranes in vesicular form from the intestinal spiral valve of the small dogfish (Scyliorhinus canicula).

A simple, rapid method for the preparation of purified brush border membranes in vesicular form from rabbit kidney proximal tubules has been applied with closely similar results to the intestinal spiral valve of the small dogfish (Scyliorhinus conicula). Since the dogfish belongs to one of the most ancient species of fish, it may be suggested that the method is generally applicable to all species later evolved which possess a brush border membrane at the mucosal surface of the cells of the intestine or kidney.

Animals↗