Yoshimi Nishi

Solubilization of trehalase from rabbit renal and intestinal brush-border membranes by a phosphatidylinositol-specific phospholipase C

Trehalase (EC 3.2.1.28) associated with renal and intestinal brush‐border membranes was solubilized by highly purified phosphatidylinositol‐specific phospholipase C (EC 3.1.4.10) from Bacillus thuringiensis, but not by phosphatidylcholine‐hydrolyzing phospholipase C (EC 3.1.4.3) from Clostridium welchii or phospholipase D (EC 3.1.4.4) from cabbage. The solubilized trehalase was not adsorbed on phenyl‐sepharose, indicating that it was hydrophilic. Phosphatidylinositol‐specific phospholipase C also converted Triton X‐100‐solubilized amphipathic trehalase into a hydrophilic form. These results suggest that trehalase is bound to the membrane through a direct and specific interaction with phosphatidylinositol.

Electron microscope studies on the structure of rabbit intestinal sucrase

Abstract Negatively stained preparations of sucrase purified from rabbit small-intestinal mucosal cells were found to consist of doughnut-shaped structures with outer and inner diameters of about 110 and 40 A, respectively, at a concentration of 10 mg/ml. At higher magnification, a subunit structure was seen in electron micrographs of purified sucrase. When diluted to 1 mg/ml., the structure dissociates into paired smaller units each of which has a dimension of 45 A × 65 A. These were converted into separated smaller units upon further dilution to 0.1 mg/ml. This process is reversed by concentrating the diluted preparation. Negatively stained specimens of isolated microvilli were characterized by the presence on their surfaces of a great number of ring particles, the dimension and shape of which resembled closely the doughnut structure of purified sucrase. A possible relationship between the two structures is discussed.

Immunochemical studies on the subunits of rabbit-intestinal sucrase-isomaltase complex

Purified sucrase-isomaltase complex sucrose alpha-glucohydrolase, EC 3.2.1.48 - dextrin 6-alpha-glycanohydrolase, EC 3.2.1.10) solubilized by papain from rabbit intestine was dissociated by citraconylation into its subunits, sucrase and isomaltase, which were then isolated in a form active immunologically as well as enzymatically by affinity chromatography on Sephadex G-200 and gel-filtration on Bio-gel P-300. Antibodies against the purified complex inhibited isomaltase but not sucrase and formed precipitation lines, crossing each other, with isolated sucrase and isomaltase, showing that the two enzymes differ in antigenicity from each other. By absorbing the antibodies with isolated sucrase and isomaltase, antibodies specific for isomaltase and sucrase, respectively, were obtained. Like the original antibodies, both of the specific antibodies quantitatively agglutinated microvillous vesicles. Sucrase was inhibited by neither of the antibodies. In contrast, isomaltase was greatly inhibited by the isomaltase-specific antibodies, but not by the sucrase-specific ones.

Purification and characterization of amphiphilic trehalase from rabbit small intestine.

Rabbit intestinal trehalase (alpha,alpha-trehalose glucohydrolase, EC 3.2.1.28) was solubilized with Triton X-100 and purified in the presence of EDTA. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis in the presence of Triton X-100 or SDS. It showed amphiphilic properties on gel filtration. polyacrylamide gel electrophoresis, charge-shift electrophoresis and phenyl-Sepharose chromatography. Its molecular weight was estimated to be about 330 000 by gel filtration under nondenaturing conditions and in the presence of Triton X-100, the value being in satisfactory agreement with the sum of the weight of one Triton X-100 micelle and twice the molecular weight (105 000) of purified hydrophilic trehalase which had been deprived of the anchor segment. The two purified trehalases gave almost the same molecular weights (about 75 000) on SDS-polyacrylamide gel electrophoresis. These results suggest that intestinal trehalase consists of two subunits with a molecular weight of 75 000 and that its anchor segment is small (less than 5000). Triton X-100 extracts freshly prepared from intestinal microvilli essentially showed one form of trehalase, which behaved on phenyl-Sepharose and Con A-Sepharose chromatography in the same manner as purified amphiphilic trehalase.

Electron microscope studies on triton-solubilized sucrase from rabbit small intestine

Triton-solubilized sucrase from rabbit intestine was examined by negative-staining electron microscopy. It has the dimeric structure in which two subunits similar in shape and size (about 45 × 65 A) are united to each other at their long axes. In aqueous solution a certain number (10–15 in most cases) of the enzyme molecules gather together to form an aggregate usually 400 to 435 A in diameter, in which they align radially around a core, the identity of which is unknown at present. Neither of Triton-solubilized sucrase digested by papain nor papain-solubilized sucrase forms any aggregates. The detergent-solubilized sucrase is adsorbed on the hydrophobic surface of fragmented polystyrene latex, and the adsorbed sucrase is released by papain. These results indicate that the Triton-solubilized sucrase has a hydrophobic portion at or near an end of its elongated molecule. Anti-sucrase immunoglobulin G and monovalent fragments from it bind to the aggregate and enlarge its diameter by at most 120 A.

Topographical studies on intestinal microvillous leucine β-naphthylamidase on the outer membrane surface

SummaryThe location of leucine β-naphthylamidase on the outer surface of the microvillous membrane of rabbit small intestine was examined by analyzing the interaction of antibodies against leucine β-naphthylamidase or another microvillous enzyme, sucrase-isomaltase complex, with microvillous vesicles having different relative amounts of these enzymes, in respect to vesicle agglutination, inhibition of enzyme activity, and electron-microscopic morphology. The results obtained indicate that leucine β-naphthylamidase, or at least its antigenic sites, protrude about 10 nm from the outer surface of the microvillous membrane.

Immunolocalization of the 33 kD protein in the microvilli of rabbit small-intestinal epithelial cells☆

Rabbit small-intestinal microvilli isolated by a Ca2+ precipitation method contain a 33 kD protein, which has not been observed in microvilli isolated in the presence of Ca2+-chelators. The intracellular localization of this protein in rabbit intestinal epithelial cells was studied by immunofluorescence and immunoperoxidase microscopy, and was compared with that of aminopeptidase M, a well-known microvillus membrane-bound enzyme. The results obtained show that the 33 kD protein is located in the inside of the microvillus, but not in the terminal web of the epithelial cell. The protein may also be located on the basolateral surface of the cell.

Intestinal sucrase-isomaltase complex: morphological identification of the subunit directly bound to the microvillar membrane.

The rabbit-intestinal sucrase-isomaltase complex is an intrinsic protein of the microvillar membrane. It protrudes about 150 A from the membrane surface. In the present work the subunit directly bound to the membrane was identified by electron microscopy using nonlabeled antibodies specific for sucrase and isomaltase, respectively. When microvillar vesicles were reached with the antibodies specific for sucrase, a new electron-opaque layer of 193 ± 27 A wide appeared on the outer surface of the membrane, which was similar to that formed by antibodies against sucrase-isomaltase ( Nishi and Takesue, 1978 J. Cell Biol. 79 , 516–525) except that the electron opaqueness of the former layer near the membrane was different from that away from the membrane. On the other hand, isomaltase-specific antibodies gave a layer of 155 ± 21 A ide in which there was apparently no such difference in electron opaqueness as observed with the layer by anti-sucrase antibodies. These results indicate that the isomaltase part or at least its bulk is located nearer the membrane surface than the sucrase or its bulk. The present results, together with those with the Triton- or papain-solubilized sucrase-isomaltase and their subunits (Nishi and Takesue, 1978 , J Ultrastruct. Res. 62, 1–12), lead to the conclusion that sucrase-isomaltase is associated with the microvillar membrane exclusively or predominantly through the isomaltase.

Topography of intestinal microvillar membrane proteins as revealed by electron microscopy.

Recent ultrastructural studies have revealed certain topographical characteristics of intestinal microvillar membrane proteins. When viewed by electron microscopy of conventional thin sections prepared from isolated microvilli, the external surface of the membrane is very poorly overlaid with fuzzy structures. However, negative staining electron microscopy shows that the external surface is studded with numerous dimeric particles and sometimes with doughnut-shaped particles, which are known to be hydrolytic enzymes abundant in the microvillar membrane. The freeze-fracture faces of the microvillar membrane numerous intramembrane particles, which are presumed to represent integral membrane proteins, e.g. transporters. The large part of the present review is concerned with the hydrolytic enzymes, especially sucrase-isomaltase complex, the enzyme most studied ultrastructurally. The hydrolytic enzymes have certain common structural features. They consist of two similar or dissimlar subunits, though a few exceptions are known. The major hydrophilic part of the molecule contains the enzymic active site and protrudes from the external surface. The minor hydrophbic domain is responsible for anchoring the molecule to the membrane and located near the amino terminus of the polypeptide chain. It is likely that for the symmetric enzymes, e.g. pig aminopeptidases A and M both subunits are associated with the membrane, while for the asymmetric enzymes, e.g. sucrase-isomaltase complex and gamma-glutamylpeptidase only one subunit is bound to the membrane. The relationship between the microvillar membrane proteins and the core cytoskeleton remains to be elucidated