Microvilli, alkaline phosphatase

Sites of alkaline phosphatase activity are frequently in endothelial cells of blood capillaries, mucous glandular cells (F3), microvilli of intestine (C6, CIO, D2, H21, P8, W7), bile canaliculi (D21, F27, W2), and placenta (W3), as well as in the brush border of the lumenal surface of epithelial cells of the proximal convoluted renal tubules (M22, Wl). The location of L-phenylalanine-sensitive alkaline phosphatase in human intestine and placenta is illustrated in Fig. 30. Electron micrographs (Fig. 31) show the details of the alkaline phosphatase, and illustrate the... 

Fig. 31. (A) Rat duodenum stained for alkaline phosphatase with azo dye method (B5a). Semidense particles of azo dye are deposited in the plasma membranes of the microvilli X70,000. (B) Placental labyrinth cell reacted for alkaline phosphatase with the cadmium method (M22). Deposits are related only to the cell surface X56,000. (C) Brush border of proximal tubule cell of the kidney showing deposits of final product related to the surface of the microvilli. Cadmium method of Mizutani and Barrnett (M22) X 35,000. Electron micrographs, courtesy of R. J. Barrnett.

Extensive studies by Moog (Ell, G19, N16, M23-M26) have described the nature of the alkaline phosphatase in the microvilli of the duodenum during development in the mouse, rat, and chick. There is a rise in... 

In the pregnant subject, the placenta and intestine become the two major sources of serum alkaline phosphatase. Like intestine, placenta is a rich source of alkaline phosphatase and it enriches the outermost cell membranes of the microvilli of the syncytiotrophoblast, which is bathed by the maternal circulation. The properties of the placental enzyme differ sufficiently from those of the intestinal isozyme to permit their differentiation. ... 

In general, it would seem that osteoblasts could not compare as a source of alkaline phosphatase with intestine or placenta. In pregnancy the level of alkaline phosphatase is rarely increased above 10 Bodansky or Shinowara units, and yet the placenta s microvilli, which are extremely rich in alkaline phosphatase, are directly immersed in the ample and efficient maternal blood supply (H3). In the nonpregnant individual, therefore, before hyperphosphatasemia can be attributed to bone it would appear necessary to evaluate the intestinal contribution that is evident as heat-sensitive non-LPSAP protein. 

The active toxin has two main functional entities, responsible for receptor binding and ion channel activity, respectively. The activated toxin binds to receptors, which seem to be of different types, on the midgut microvilli of the susceptible insects. Different toxins seem to bind to different receptor proteins that may be an enzyme such as aminopeptidase or alkaline phosphatase, or a cadherin-like membrane protein. (The cadherins are proteins that are important in keeping the cells together by mediating Ca+-dependent cell-cell adhesion in animal tissue.) The toxins are anchored to the outer epithelial cell membrane in such a way that the membrane is perforated by pores or channels where ions can freely pass. This model proposes that an influx of water, along with ions, results in swelling and lysis. The epithelium is destroyed and the insect rots. 

The apical plasma membrane of epithelial cells of small intestinal and renal proximal tubules is characterised by the presence of many microvilli (brush border). These membranes can be isolated relatively easily by centrifugation and free flow electrophoresis techniques. Kinne-Saffran and Kinne [15] found that after free-flow electrophoresis of a rat kidney-cortex membrane preparation, the anion-sensitive ATPase co-migrated with the alkaline phosphatase activity but was separated from the (Na + K )-ATPase activity, which is assumed to be a marker of basolateral plasma membranes. This suggests that the brush-border membrane of the proximal tubule contains an anion-sensitive ATPase. The same conclusion was reached by Liang and Sacktor [17] for a brush-border preparation from rabbit kidney. 

Fia 6. Electronmicrograph of alkaline phosphatase reaction in a cross section of the tips of the microvilli of an absorbing cell of the duodenum of adult male hamster. The reaction product is localized on the external leaflet of the double membrane, but some of it appears also on the internal leaflet. X 135,000. (Hugon, 1972.)... 

However, it is the association of alkaline phosphatase with membranes which is of greatest interest here. Miller and Crane (1961) and Holt and Miller (1962) were the first to demonstrate that alkaline phosphatase was present in the microvilli which had been separated from the epithelial cells of the intestine by techniques of tissue disruption and ultracentrifugation. Later, Overton (1965) was able to separate the fibrous cores from the covering membranes of the microvilli by sonication and density gradient centrifugation and proved that the covering membranes contained the alkaline phosphatase. This evidence certainly qualifies alkaline phosphatase as an integral membrane protein. 

In viewing alkaline phosphatase, it is important to consider that it is a glycoprotein and it shares its location on the microvilli with other glycoproteins as is developed in the following section. 

Crane (1966) has theorized that the brush border plasma membrane is the site of a mosaic of the enzymes associated with the microvillus. This was based on experiments of Eichholz and Crane (1965) who recovered a fraction of pure microvillous membranes by density gradient centrifugation of brush border homogenate which possessed the total activities of alkaline phosphatase, maltas and sucrase and various peptidases. Evidence in favor of this idea was also collected by Johnson (1967) who demonstrated the presence of knobs 60 A in diameter on the glycocalyx of the luminal side of the plasma membrane which contained the brush border invertase and maltase (see Fig. 6). These knobs could be removed entirely from the microvilli of hamster intestine by papain digestion the remaining membrane, however, still has the alkaline phosphatase incorporated into it (Eichholz, 1969 Oda and Seki, 1966). 

Actinomycin D, puromycin, and cycloheximide reduce alkaline phosphatase in the kidneys of those animals whose microvilli of the duodenal epithelial cells of the mouse undergoes an increase in activity (Moog, 1971a,b Moog and Grey, 1966). This is evidence that de novo enzyme... 

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