A rapid method for the preparation of microvilli from rabbit kidney

Abstract

A simple method for the isolation of microvilli from kidney brush border is described. The method depends on the preferential aggregation of other subcellular structures by bivalent metal ions. MgCl(2) is added to a homogenate of cortical tissue prepared from frozen rabbit kidneys. Aggregated material is removed by a low-speed centrifugation and the supernatant centrifuged at 15000g to yield a pellet enriched in microvilli. This is resuspended and given a second treatment with Mg(2+). The purified preparation is obtained after four short differential centrifugations. The six brush-border enzymes that were monitored were enriched 11-17-fold compared with the original homogenate and were obtained in about 10% yield. Marker enzymes for other subcellular components showed the preparation to be essentially free of mitochondria and to be less contaminated with endoplasmic reticulum and baso-lateral plasma membranes than are conventional brush-border preparations. The main contamination was of lysosomal origin, about half of which was attributable to adsorbed acid hydrolases rather than to intact lysosomes. The aggregated components in the low-speed pellet bound less Mg(2+) than did the microvillus fraction. A possible mechanism for the role of Mg(2+) is discussed.

Amyloid precursor protein, although partially detergent-insoluble in mouse cerebral cortex, behaves as an atypical lipid raft protein

“…Pig kidney microvillar membranes were prepared as described previously [41]. The final membrane pellet was resuspended in MBS to a protein concentration of 8 mg\ml.…”

Biosynthesis of microvillar proteins

“…This has undoubtedly to do with the fact that these enzymes, contrary to viral proteins, each represent only a tiny portion (0.1-1%) of the total mass of protein ofthe cell and that their biosynthesis therefore inevitably must be studied against a high background. By using immunofluorescence labelling, Feracci et al (1982) (Schmitz et al, 1973) and kidney (Booth & Kenny, 1974) tissue and has been used in preparative scale in the purification of several microvillar enzymes (Kenny & Maroux, 1982). Using a modification of the procedure of Kessler et al (1978), it was possible to obtain a Ca2 +-precipitated membrane fraction that contains only 2-4% of the total activity of the microvillar enzymes, indicating a minimal contamination with microvillar membranes (Danielsen et al, 1981a).…”

“…In addition, specific antibodies, polyclonal (for references see as well as monoclonal (Hauri et al, 1980;Gee et al, 1983) have been raised against these enzymes. Furthermore, effective methods for isolating microvillar membranes from both intestine and kidney have been described (Schmitz et al, 1973;Booth & Kenny, 1974), as have methods for organ culture of tissue explants for long enough periods to study biosynthetic events (Moscona et al, 1965). Lately, preparations of translatable mRNA from both small intestine (Wacker et al, 1981;Danielsen et al, 1982b) and kidney (Nash & Tate, 1984) have become available, and thus the scientific tools and model systems necessary for a thorough study of the biosynthesis of microvillar proteins are at hand.…”

An immunoradiometric assay for endopeptidase-24.11 shows it to be a widely distributed enzyme in pig tissues

“…Endopeptidase-24.11 is an ectoenzyme that is most abundant in the brush border of renal proximal tubules (George & Kenny, 1973;Booth & Kenny, 1974;Gee et al, 1983). It is capable of hydrolysing a wide range of peptides, including hormones (such as the chains of insulin and glucagon; Kerr & Kenny, 1974) and many neuropeptides (Matsas et al, , 1984a.…”