Molecular basis of structure and function of the microvillus membrane of intestinal epithelial cells.

Correlation of molecular structure with biochemical functions of the plasma membrane of the microvilli of intestinal epithelial cells has been investigated by biochemical and electron microscopic procedures. Repeating particles, measuring approximately 60 Åin diameter, were found on the surface of the microvilli membrane which had been isolated or purified from rabbit intestinal epithelial cells and negatively stained with phosphotungstic acid. These particles were proved to be inherent components of the microvillus membrane, attached to the outer surface of its trilaminar structure, and were designated as the elementary particles of the microvilli of intestinal epithelial cells. Biochemical and electron microscopic identification of these elementary particles has been carried out by isolation of the elementary particles with papain from the isolated microvillus membrane, followed by purification of the particles by chromatographies on DEAEcellulose and Sephadex columns. The partially purified particles containing invertase and leucine aminopeptidase are similar in size and structure to those of the elementary particles in the microvillus membrane. Evidence indicates that each of the elementary particles coincide with or include an enzyme molecule such as disaccharidase or peptidase, which carry out the terminal hydrolytic digestion of carbohydrates and proteins, respectively, on the surface of the microvillus membrane. Magnesium ionactivated adenosine triphosphatase and alkaline phosphatase cannot be solubilized with papain but remains in the smooth-surface membrane after the elementary particles have been removed. Cytochemical electron microscopic observation revealed that the active site of magnesium ion-activated adenosine triphosphatase is localized predominantly in the inner surface of the trilaminar structure of the microvillus membrane. ∗PMID: 4244045 [PubMed indexed for MEDLINE] Copyright c ©OKAYAMA UNIVERSITY MEDICAL SCHOOL Acta Med. Okayama 23, 357-376 (1969) MOLECULAR BASIS OF STRUCTURE AND FUNCTION OF THE MICROVILLUS MEMBRANE OF INTESTINAL EPITHELIAL CELLS Takuzo ODA, Shuji SEKI, and Sekiko WATANABE Department of Biochemistry, Cancer Institute, Okayama University Medical School, Okayama, Japan (Director: Prof. T. Oda) Received for publication, July 10, 1969 Available evidence suggests that the microvilli of intestinal epithelial cells carry out two principal functions: one is the terminal hydrolytic digestion of carbohydrates and proteins by the action of disaccharidases, peptidases, and some other enzymes; and the other is the absorption, including active transport, of various ions and molecules Iiberated by enzymic digestion, such as certain monosaccharides, amino acids, and others (1-12). The intestinal epithelial microvilli are unique not only for the former function, but also are the prototype for the latter function in all biological membrane systems. These principal functions of the microvilli have been assumed to be closely correlated with and to be carried out by the enzymes or enzyme systems arranged on or in the microvilli. It is the purpose of the present investigation to correlate these functions with the molecular structure of the membrane of the microvilli. Preliminary reports on this work have already been published (4-7). MATERIALS AND METHODS Materials Microvilli of small intestinal epithelial cells from rabbits were mainly used, but in some experiments those of rat, mouse and human origins also served as materials. Isolation of Microvilli (Brush Border) For the isolation of microvillus border the method of MILLER and CRANE (961) (2) was used with a slight modification. Male adult rabbits 0-5 animals/ group) were fasted for 1-2 days, killed by bleeding after stunning by a blow on the head, and the small intestine was taken out. The following procedure was carried out at I_4°C. The intestine was everted and washed several times with an ice cold washing solution containing 140 mM NaCl and 4 mM KC1, pH 7.4. The mucosa was stripped off on the edge of a slide glass, placed in an ice cold homogenizing solution containing 5 mM ethylenediamine tetraacetate (EDTA), pH 7.4, about one liter for one animal, and then homogenized gently in a teflon