Rat Kidney Brush-border Membrane Research Articles

Some kinetic properties of γ-glutamyltransferase from rabbi liver

γ-Glutamyltransferase ((5-glutamyl)-peptide: amino-acid 5-glutamyltransferase, EC 2.3.2.2) of rabbit liver (detergent form) was purified 1100-fold in order to study its kinetic properties. Kinetic studies were conducted from pH 6.0 to 12.0 in the absence and presence of the acceptor substrate glycylglycine using γ-glutamyl-3-carboxy-4-nitroanilide as the donor. The existence of more than one binding site for both donor and acceptor is postulated on kinetic evidence such as donor substrate activation, donor substrate inhibition and acceptor substrate activation. Homotropic interaction is also observed, in the form of negative cooperativity, in donor substrate binding, in the absence of acceptor at pH less than 9.0 and positive cooperativity ( n=2), in the absence or presence of acceptor at pH greater than 9.0. Hydrolase reaction reaches a maximum of activity at pH 10 (p K 8.6). Transferase activity under conditions of maximal velocity is maximal at pH 9.0 (p K 7.1). The ratio of transferase activity/hydrolase activity is maximal at pH 7.0–7.5. At low donor substrate concentrations, maximal activity is attained at pH 7.5. Autotransfer is not thought to play a significant role in the conditions used for the study. Studies done with rat kidney brush border membrane at pH 6 and 7.5 corroborate the findings with the purified enzyme.

Occurrence of 3-hydroxybutyrate dehydrogenase activity in the rat kidney brush border membrane.

3-Hydroxybutyrate dehydrogenase activity was found in both mitochondria and brush border membrane fractions of the rat kidney. Differences in properties provide evidence of a nonmitochondrial 3-hydroxybutyrate dehydrogenase in the brush border membranes.

Further evidence for the existence of an intrinsic bicarbonate-stimulated Mg2+-ATPase in brush border membranes isolated from rat kidney cortex

The aim of this study was to provide further evidence for the existence of a nonmitochondrial becarbonate-stimulated Mg2+-ATPase in brush border membranes derived from rat kidney cortex. A plasma membrane fraction rich in brush border microvilli and a mitochondrial fraction were isolated by differential centrifugation. Both fractions contain a Mg2+-ATPase activity which can be stimulated by bicarbonate. The two Mg2+-ATPases are stimulated likewise by chloride, bicarbonate, and sulfite or inhibited by oligomycin and aurovertin, though to different degrees. In contrast to these similarities, only the Mg2+-ATPase activity of the mitochondrial fraction is inhibited by atractyloside, a substance which blocks an adenine nucleotide translocator in the inner mitochondrial membrane. On the other hand, filipin, an antibiotic that complexes with cholesterol in the membranes inhibits exclusively the Mg2+-ATPase of the cholesterol-rich brush border membranes. Furthermore it could be demonstrated by the use of bromotetramisole, an inhibitor of alkaline phosphatase activity, that the Mg2+-ATPase activity in the membrane fraction is not due to the presence of the highly active alkaline phosphatase in these membranes. These results support the assumption that an intrinsic bicarbonate-stimulated Mg2+-ATPase is present in rat kidney brush border membranes.

Transport of glutamine by rat kidney brush-border membrane vesicles.

Transport of glutamine by brush-border vesicles prepared from the renal cortex was studied. The transport system had both Na+-dependent and Na+-independent components. The presence of Na+ in the incubation resulted in an 'overshoot' at 30s at which time the rates of transport were approx. 8 times the values obtained in the absence of Na+. Variation of the glutamine concentration showed that the system obeyed Michaelis-Menten kinetics with Km and Vmax. values for the Na+-dependent system of 0.86 mM and 9.6 nmol/min per mg of protein respectively. Vesicles obtained from chronically acidotic rats showed similar kinetic characteristics. The Km and Vmax. values for the Na+-dependent system were 0.76 mM and 9.6 nmol/min per mg of protein respectively. There was increased uptake of glutamine by vesicles from acidotic rats and this increase was associated with increased activity of gamma-glutamyltransferase in these preparations. Vesicles from acidotic rats, however, showed no increase in glucose transport and no increase in the activity of maltase, another brush-border enzyme.

L-proline transport by newborn rat kidney brush-border membrane vesicles

The transport of L-proline was studied in brush-border membrane vesicles isolated from the kidneys of newborn rats. In contrast with the rapid initial uptake with an 'overshoot' observed in adult vesicles, uptake by the newborn vesicle was slow, showed no 'overshoot', and proline continued to accumulate at a time when the adult vesicle had already equilibrated. L-Proline transport in the newborn rat occurs by Na+-dependent and independent mechanisms. There appeared to be essentially no uptake by anti-luminal vesicles isolated from newborn rat kidney. These observations may help to explain the prolinuria that occurs in the newborn animal.

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 a, b and c was found, whereas in the microsomes only cytochrome b was demonstrated.

On the maleic acid induced Fanconi syndrome: Effects on transport by isolated rat kidney brushborder membrane vesicles

The association of maleic acid with isolated rat kidney brushborder membranes and the effect of maleic acid on uptake of amino acids and α-methyl-D-glucoside by brushborder membrane vesicles has been studied. Although maleic acid associates with the membrane, no alteration of sodium dependent transport of these substrates into the vesicles was observed. These findings indicate that the maleic acid induced Fanconi syndrome in the rat with aminoaciduria and glycosuria is not due to a direct, adverse effect on the renal tubule brushborder membrane per se .

Incorporation of D-glucose-, L-alanine- and phosphate-transport systems from rat renal brush-border membranes into liposomes.

An extract of soluble proteins was prepared from a rat kidney brush-border membranes by Triton X-100 solubilization followed by centrifugation for 1 h at 100000g. Its protein composition was markedly different from that of the brush-border membranes. Proteoliposomes were formed by co-sonication of the Triton X-100-free extract with a naturally occurring mixture of phospholipids extracted from rat kidney. These proteoliposomes were shown to contain Na+-stimulated D-glucose-, L-alanine- and phosphate-transport systems.