Cytochromes in the rat kidney brush border membrane.

Spectrophotometric studies of the brush border membrane fraction of the rat kidney as compared with those of its mitochondria and microsomes were carried out. Occurrence of cytochromes has been demonstrated in the brush border membranes. Either in the brush border membranes and in the mitochondria evidence for the presence of cytochromes of the types $\text{a}\text{,}\text{b}$ and $\text{c}$ was found, whereas in the microsomes only cytochrome $\text{b}$ was demonstrated.     

Binding of lysozyme to brush border membranes of rat kidney

The binding of ¹²⁵I-labelled egg-white lysozyme to isolated brush border membranes of rat kidney cortex was investigated. The lysozyme binding was reversible and saturable. The Scatchard plot revealed a one-component binding type with a dissociation constant of 7.8 μM and 15.6 nmol/mg membrane protein for the number of binding sites. The binding of the basic lysozyme could be reduced by basic amino acids such as l-lysine, l-ornithine or l-arginine, while neutral amino acids such as l-citrulline or l-alanine had no effect. The inhibitory effect of lysine was competitive.     

Cytoskeletal proteins of the rat kidney proximal tubule brush border

Cytoskeletal components backing the brush border of the rat kidney proximal tubule cell were identified and compared with those of the well characterized intestinal brush border by immuneoverlay and immunocytochemistry. Antibodies reactive against the intestinal microvillus core components, villin and fimbrin, as well as against the terminal web components, spectrin (fodrin) and myosin, were used. Proteins of similar molecular weight to these intestinal brush border cytoskeletal components were identified in isolated kidney brush borders by immuneoverlay. Spectrin, a major component of the terminal web region of both cell types, was more concentrated in the kidney brush border relative to both actin and myosin. By immunofluorescence, villin and fimbrin were localized in the microvilli, and spectrin and myosin were localized to the terminal web region of the brush border. In addition, spectrin was found along the basolateral membranes of the proximal tubule cell, and myosin was detected in a punctate staining pattern throughout its cytoplasm. By immunoelectron microscopy using immunogold labeling procedures, fimbrin and villin were localized in the terminal web as well as in microvilli, and spectrin and myosin were localized to fibrils in the terminal web. A key difference between the epithelia of the two organs is the extensive network of clathrin coated pits found in the terminal web region of the kidney but not the intestinal brush border. The clathrin-rich terminal web region of the kidney, like the intestinal brush border, proved to be quite stable and resistant to disruption by non-ionic detergents and harsh mechanical treatment.     

A cryptic meprin-like proteolytic activity in mouse kidney brush border membranes

1. Inbred mouse strains differ markedly in the expression of a kidney brush border metalloendopeptidase, meprin-a. 2. Brush border preparations from mice of the low-meprin-a phenotype (specific activities less than 5% of the high-meprin-a trait) contain a metallo-endopeptidase, meprin-b, that is larger than meprin-a, and which is inactive unless the membrane preparations are treated with trypsin. 3. This cryptic metallo-endopeptidase has been previously postulated to be a stalled precursor of meprin-a. 4. We show here that meprin-b is present in all mice-high and low meprin-a phenotypes--and that this activity is similar in substrate specificity and amount present in the brush border. 5. Meprin-b may therefore be a distinct gene product that is independent of meprin-a phenotype.     

Characterization of rat kidney proximal tubule brush border membrane-associated phosphatidylinositol phosphodiesterase

Isolated rat kidney proximal tubule brush border membrane vesicles exhibit an increase in diacylglycerol levels (20- to 30-fold) and a concomitant decrease in phosphatidylinositol when incubated with [3H]arachidonate-labeled lipids, Ca2+, and deoxycholate. Levels of free arachidonate, triglyceride, and noninositol phospholipids are not altered. These results suggest phosphatidylinositol phosphodiesterase activity is associated with rat proximal tubule brush border membrane. Presence of both deoxycholate and certain divalent cations was necessary to demonstrate enzyme activity. Optimum pH ranged from 7.0 to 8.5. Ca2+, Mg2+, and Mn2+ stimulated diglyceride production while Ba2+, Zn2+, Hg2+, and K+ were ineffective. HgCl2 inhibited Ca2+-stimulated phosphatidylinositol phosphodiesterase. Mg2+ and deoxycholate-dependent enzyme activity was shown to be phosphatidylinositol specific. Sodium lauryl sulfate, tetradecyltrimethylammonium bromide, and Triton X-100 did not activate phosphatidylinositol phosphodiesterase in the presence of Ca2+. In combination with deoxycholate, diglyceride formation was not affected by sodium lauryl sulfate, partially inhibited by Triton X-100, and completely abolished by tetradecyltrimethylammonium bromide. Diglyceride kinase activity was not found associated with brush border membrane phosphatidylinositol phosphodiesterase. ATP (1-5 mM) inhibited Ca2+- or Mg2+-stimulated, deoxycholate-dependent phosphatidylinositol hydrolysis by chelating the required divalent cation.     

Effect of harmaline on rat intestinal brush border sucrase activity

The effect of harmaline, a plant alkaloid has been studied on rat intestinal brush border sucrase activity. Stimulation of sucrase activity by Na+ was found to be pH-dependent. At neutral pH, 20 mM Na+ stimulated sucrase activity by reducing K(m) by 30%, while at acidic pH (5.2), the activity increased 4-fold compared to Na+-free enzyme. At 1.0 mM, harmaline markedly inhibited (67%) the enzyme activity at pH 5.2 in the absence of Na+. However, inhibition was reduced in presence of 20 mM sodium, whereas 4.0 mM harmaline was required to inhibit the enzyme activity by 65%. In the absence of Na+ ions, harmaline inhibition of sucrase activity was of competitive type, but it changed to non-competitive type in presence of 20 mM Na+ at pH 5.2. Sucrase-harmaline interactions as a function of pH, both in presence and absence of Na+ revealed a shift in pH optima of the enzyme towards a higher pH in presence of 4 mM and 1 mM harmaline respectively. The observed inhibition was reversible in nature and was only partially overcome by sodium, lithium, potassium, cesium, rubidium and ammonium ions. These findings suggest that harmaline also inhibits rat brush border sucrase and that the presence of Na+ site is not a pre-requisite for the inhibition.     

myo-Inositol binding and transport in brush border membranes of rat kidney

Using hypotonically treated brush border membranes, binding and transport of myo-inositol were examined.By hypotonic treatment, both total and non-specific uptake decreased significantly, but specific uptake was not affected.myo-Inositol release from membranes preloaded by incubation for 2 min was very rapid and about 98% of preloaded myo-inositol was released in 5 min of incubation. However, myo-inositol release from membranes preloaded by incubation for 20 min was fairly slow and 50% of myo-inositol remained in the membranes even after 10 min of incubation.Uptake of myo-inositol decreased by the increase of osmolarity in the medium. However, effect of osmolarity on the uptake was less significant when myo-inositol concentration was lower.Under conditions in which mainly binding occurred, myo-inositol binding to the membranes was measured. Two binding systems were demonstrated and high affinity site could bind 22 pmol/mg protein at most and the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value was 8.3 μM.Both binding and transport processes were dependent on Na⁺ and enhanced by Na⁺-gradient.     

Isolation and characterization of the brush border fraction from newborn rat renal proximal tubule cells.

A renal brush border fraction was isolated from newborn Sprague-Dawley rats, and its morphological and enzymatic characteristics were studied in comparison to that from the adult. Definite microvillar structures are seen by electron microscopy, and border preparations from the newborn are enriched in known marker enzymes. Though morphological development is more advanced and enzyme specific activities are greater in the adult, polyacrylamide gel electrophoresis of membrane proteins reveals no significant change in pattern with increasing age. These studies suggest that the brush border of the proximal tubule cell is present at birth as a significantly developed structure.     

A high yield preparation for rat kidney brush border membranes. Different behaviour of lysosomal markers

Rat kidney cortex slices were homogenized with a polytron in a isoosmotic medium containing 5 mmol/l EGTA. By two precipitations with MgCl2 (12 mmol/l) and differential centrifugation, brush border membranes were purified. The brush border marker enzymes alkaline phosphatase and aminopeptidase M were found to be enriched 17.0 +/- 5.3-fold and 16.7 +/- 3.7-fold, respectively. By this method, a high yield of brush border membranes was obtained (48.3 +/- 7.9% for alkaline phosphatase; 47.0 +/- 9.5% for aminopeptidase M). The acid phosphatase was enriched 5-fold, whereas other lysosomal enzymes (glucosaminidase, glucuronidase, cathepsin D) were enriched only 0.2-fold. Acid phosphatase activity could not be washed out, but could be separated from alkaline phosphatase and leucine aminopeptidase by means of free flow electrophoresis and sucrose density gradient centrifugation. Vesicles prepared by the presently described Mg/EGTA-method show better transport properties, compared to vesicles prepared by the calcium method of Evers et al. (Evers, C., Haase, W., Murer, H. and Kinne, R. (1978) Membrane Biochem. 1, 203-219), whereas by SDS-polyacrylamide gel electrophoresis, no differences in the protein patterns were observed.     

Studies on the structure of the rabbit kidney brush border.

The effects of salts and non-ionic detergents on renal brush borders have been studied. 2 M sodium chloride, iodide or thiocyanate dissociated up to 40% of the protein from the brush borders, destroying the core filaments and resulting in the formation of membrane vesicles; EDTA had a similar effect on structure but released little protein. Triton X-100 and Nonidet P-40 extracted up to 60% of the protein including the major membrane glycoproteins and the enzymes trehalase, maltase and aminopeptidase (microsomal). Triton exhibited a selective effect on lipids removing phosphatidylserine, phosphatidylethanolamine and sphingomyelin but not the bulk of the phosphatidylcholine or cholesterol. The residual structures after Triton extraction comprised the core filaments associated with vesicles of lipid containing alkaline phosphatase and several other proteins. Treatment of these core-vesicle complexes with 2 M sodium chloride dissociated the filaments, releasing the vesicles which could be recovered as a pellicle on centrifugation. It is suggested that the proteins found in the vesicles might serve to interconnect the core filaments with the lipid bilayer.     

Isolation and characterization of the brush border fraction from newborn rat renal proximal tubule cells

A renal brush border fraction was isolated from newborn Sprague-Dawley rats, and its morphological and enzymatic characteristics were studied in comparison to that from the adult. Definite microvillar structures are seen by electron microscopy, and brush border preparations from the newborn are enriched in known marker enzymes. Though morphological development is more advanced and enzyme specific activities are greater in the adult, polyacrylamide gel electrophoresis of membrane proteins reveal no significant change in pattern with increasing age. These studies suggest that the brush border of the proximal tubule cell is present at birth as a significantly developed structure.