Suzanne Maroux

The aminopeptidase from hog intestinal brush border

Abstract The aminopeptidase bound to the brush border membrane of porcine jejunum and ileum has been purified. After treatment of the mucosa with Triton X-100, the enzyme was found to be still attached to very small membrane fragments (the aminopeptidase complex) from which it was liberated by trypsin. The free enzyme could be further purified to homogeneity by using classical techniques. The high molecular weight complex was shown to contain, besides aminopeptidase, at least one other protein inactive on the substrates of this enzyme. The free aminopeptidase was observed to have a molecular weight of about 280 000, and to contain 23% sugars and 2 atoms of zinc per mole. Gel electrophoresis in sodium dodecyl sulfate showed 4 bands. One, however, is probably an artefact arising from trypsin treatment. The other 3 suggest the existence in aminopeptidase of 3 subunits with molecular weights of 130 000, 97 000, 49 000 and which were also obtained from the complex. The aminopeptidase which has been purified accounts for all, or almost all, of the peptidase activity of the brush border membrane. It also accounts for all of the arylamidase activity, all of the activity against an heptapeptide substrate, about half the tripeptidase activity and a small but apparently significantly part of the dipeptidase activity of the whole jejunal and ileal mucosa.

Topological studies on the hydrolases bound to the intestinal brush border membrane. I. Solubilization by papain and Triton X-100.

Papain digestion of closed, right side out vesicles from pig, rat and rabbit jejunum brush border induces the release of the hydrolases bound to the membrane without grossly affecting the lipid bilayer limiting the vesicles. This observation definitely proves that intestinal hydrolases are surface components attached to the external side of the membrane. All proteins released by papain could be identified by electrophoresis and immunoelectrophoresis to already known intestinal hydrolases, with the exception of an unidentified substance strongly stained by the Schiffs reagent. The early observation that the aminopeptidase form released from pig bursh border by Triton X-100 is different from that released by papain was extended to other hydrolases from pig, rat and rabbit. In some cases, the Triton-released form could be converted by further proteolytic digestion into a new form similar to that liberated by papin. These facts may be related to the existence of hydrophobic anchors retaining the intestinal hydrolases to the membrane surface.

On the preparation and some properties of closed membrane vesicles from hog duodenal and jejunal brush border

Abstract Closed and nearly spherical vesicles were obtained from both hog duodenum and jejunum after mucosa homogenization in the absence of EDTA and a series of fractional centrifugations. The vesicles were found to contain large amounts of two of the characteristic enzyme markers of the brush border membrane (aminopeptidase and alkaline phosphatase). They were seen by electron microscopy on thin sections or after negative staining to be composed of an apparently intact, 90–100 A-thick membrane overlaid by the fuzzy coat and to be partly filled by a fibrous material tentatively identified with the cross-filaments of the microvilli. This filling was not removed by 5 mM EDTA or/and 1 M Tris unless the structure of the vesicles was largely destroyed. Very few empty vesicles were obtained at the end of these treatments. The vesicles from hog duodenum and jejunum were observed to contain nearly 2 molecules of cholesterol for 1 molecule of phospolipids. Specific differences were noted between both types of vesicles at the level of their sugar composition and associated enzyme activities. For instance, the jejunal vesicles contained no sialic acid and no enterokinase. They contain, respectively, 2 and 4 times as much alkaline phosphatase and aminopeptidase as duodenal vesicles.

On the hydrophobic part of aminopeptidase and maltases which bind the enzyme to the intestinal brush border membrane

The intestinal brush border aminopeptidase and unfractionated maltases M2+M3 are composed of a hydrophilic, sugar containing and enzymatically active part, and a smaller hydrophobic part presumably binding the catalytic part of the lipid matrix of the membrane. Hydrophobic parts detaced by trypsin from the detergent forms of aminopeptidase and the maltases were purified and shown to have molecular weights ranging from 8000 to 10000. All are rich in hydrophobic residues and contain no disulfide bridges. However, their overall amino acid composition is different. The hydrophobic parts appear to be N-terminal in the detergent forms of the enzymes.

Simultaneous isolation of brush border and basolateral membrane from rabbit enterocytes. Presence of brush border hydrolases in the basolateral membrane of rabbit enterocytes

By a slight modification of the procedure described by Gratecos et al. (Gratecos, D., Knibiehler, M., Benoit, V. and Sémériva, M. (1978) Biochim. Biophpys. Acta 512, 508-524), the basolateral and brush border membranes of rabbit enterocytes have been purified concomitantly from the same aliquot of mucosa. The two types of membrane have been obtained with the same yield (15%) and enrichment of specific markers (18-fold). The presence in the basolateral membrane of hydrolases known to be specific of the brush border membrane has been confirmed by using immunological techniques.

Topological studies on the hydrolases bound to the intestinal brush border membrane: II. Interactions of free and bound aminopeptidase with a specific antibody☆

The position of the intestinal brush border aminopeptidase with respect to the lipid bilayer has been investigated with the aid of right side out vesicles prepared from the brush border and an immunological technique using an unlabelled or peroxidase-labelled antibody specific for aminopeptidase. The finding that the bound form of the enzyme was almost as readily inhibited and agglutinated as the free form during incubation with the antibody was consistent with the view that the majority of the aminopeptidase surface emerged from the bilayer. This finding was entirely corroborated by the observation that only a few antigenic determinants were not free to react with the antibody in bound aminopeptidase. This immunological technique may be applied to other membrane proteins provided that preparations of the pure proteins and of specific antibodies are available.

Structural and topological homology between porcine intestinal and renal brush border aminopeptidase

A method for the preparation of closed, right-side-out vesicles from the brush border membrane of the kidney proximal tubules is described. The aminopeptidase known to be bound to this membrane was investigated in order to compare its properties with those already reported for the intestinal enzyme. Both are composed of a hydrophilic, catalytically active part lying on the external side of the membrane and a short hydrophobic domain probably located in the N-terminal region of one of the subunits ensuring fixation to the lipid matrix. The enzyme were also found to be clinically similar. Moreover, a quantitative immunological technique showed that they contained 6 cross-reacting determinants, consistent with a very high degree of homology. Four of these determinants were accessible in the bound form of the enzymes in the region of the active site. The other two, probably related to the junction between the hydrophilic moiety and the hydrophobic anchor were completely masked in the bound form. The remainder (6 in the intestinal and 4 in the renal enzyme), were heterologous. The accessibility of two well determinants in this latter group was substantially reduced, perhaps by the proximity of the lipid and/or of other enzyme molecules.

The brush-border intestinal aminopeptidase, a transmembrane protein as probed by macromolecular photolabelling.

Abstract Aminopeptidase has been previously shown (Louvard et al., 1975b) to be present at least in part at the outer surface of the brush-border membrane of enterocytes. In order to show that this hydrolase was also exposed at the inner face of the membrane, the reagent 4-fluoro-3-nitrophenyl azide was covalently attached to the Fab fragment of a human myeloma protein to produce a photo-generated macromolecular reagent. This latter was trapped into closed and sealed right-side-out vesicles, then photolyzed in situ to generate a nitrene capable of reacting with a large variety of chemical bonds. After extraction of the membrane proteins with detergent the aminopeptidase was recognized by its specific antibody and the extent of labelling was determined by titrating the Fab bound with a monovalent anti-Fab labelled with peroxidase. By this general methodology aminopeptidase was found to be a transmembrane protein which was exposed at both the outer and the inner face of the vesicles. Furthermore, in this latter case, the label was recovered in the “so-called” hydrophobic part of the molecule, which remains in the membrane after the removal of the external and hydrophilic part of aminopeptidase by a papain treatment of the vesicles. Thus three distinct regions in aminopeptidase can be delineated. The principal one is located at the outer surface of the membrane. A hydrophobic part is embedded within the lipid interior of the membrane. Finally, the last region, attached to the latter one, is situated at the inner face of the membrane.

On the distribution of enterokinase in porcine intestine and on its subcellular localization

Abstract Enterokinase, which is responsible for the in vivo activation of pancreatic zymogens, was shown in the pig to be exclusively synthetized by the duodenal mucosa. High amounts of the free enzyme were also encountered in the ileal content. By contrast, other intestinal enzymes, such as aminopeptidase and alkaline phosphatase, were found to be essentially synthetized by the mucosa of jejunum and ileum, thus demonstrating a specialization at the enzymatic level of the 3 characteristic regions of small intestine. Enterokinase was not observed to separate to any detectable extent from aminopeptidase and alkaline phosphatase, 2 known markers of the brush border membrane, during a number of fractionation steps leading to apparently pure preparations of closed membrane vesicles from duodenal brush border. The enzyme was removed very fast from the vesicles by papain, but not by trypsin, chymotrypsin and subtilisin. These results confirm that enterokinase is attached to the external side of the duodenal brush border membrane.

On porcine enterokinase. Further purification and some molecular properties.

Abstract Enterokinase has been purified more than 1000-fold from porcine duodenal mucosa. The final preparations appear to be homogenous by disc electrophoresis and immunodiffusion assays, and to be entirely free of aminopeptidase activity. Starting with 20 kg of duodenum (5 kg of mucosa), about 75 mg (0.4 μmole) of the pure enzyme can be obtained, with a 26% yield. Since enterokinase is a glycoprotein with an unusually high sugar content (neutral sugars, 20%; amino sugars, 15%; sialic acids, 2%; total, 37%), several techniques were applied for the determination of its molecular weight. Although all techniques gave similar values, the most likely was considered to be 195 000. The only visible effect of the bulky sugar part was to significantly lower the partial specific volume of the protein (0.705 g/ml). The enterokinase molecule (about 1100 amino acid residues) is not composed, like other proteins of similar molecular weight, of several subunits linked by non-covalent bonds. However, upon reduction it gives rise to 2 chains, the “heavy” and the “light” chain, with molecular weights of 134 000 and 62 000, respectively (about 700 and 350–400 amino acid residues). The sugar content of both chains is approximately the same. One active serine per mole can be titrated by [ 32 P]DFP in pure enterokinase preparations. This serine is located in the light chain.