Proline and glycine uptake by renal brushborder membrane vesicles.

Abstract

Uptake of L-proline and glycine by rat renal brushborder membrane vesicles was seen to be osmotically sensitive, pH dependent, and occurred in the absence of proline and glycine metabolism. The uptake system for proline was Na+ gradient dependent, and exhibited a dual system for entry, Km, = 0.067 mM and Km2 = 5.26 mM. The uptake of glycine was also Na+ gradient dependent, and exhibited a two Km system, Km, -0.22 mM and Kin = 4.00 mM. Studies of proline and glycine interactions indicate a shared site which has a lower affinity and higher capacity for glycine than for proline. The high affinity glycine site and low affinity proline site do not appear to be shared.The occurrence of inherited iminoglycinuria in man as well as a hyperexcretion of proline and glycine in the normal human neonate prompted numerous studies to delineate the mechanism of renal tubular reabsorption of proline and glycine. These employed rat renal cortical slices or isolated rabbit renal tubules in vitro and resulted in the concept of multiple systems for tubule cell uptake of glycine and proline (1-3). Inherent, however, in the use of such model systems is the difficulty of interpretation of transport data because of involvement of the two types of membranes of the proximal tubule cell, and the rapid metabolism of the substrates by these cells. Amino acid uptake by the tubule cells of the cortical slice or isolated tubule may take place either from the luminal brushborder or the basal smooth membrane, thereby making it difficult to discern the contribution of these membranes independently. In addition, proline taken into such cells was observed to be largely converted to intracellular glutamate, a process which may be a regulatory factor in the transport of proline across the membrane (4).Recently, Kinne and his associates described the preparation of purified rat kidney brushborder membranes and reported the uptake of phenylalanine (5) Membrane vesicles, suspended in Na+-free THM buffer at pH 7.4, were used for the transport studies. The standard uptake experiment, which is under conditions of a Na+ gradient unless otherwise stated, consisted of 0.5 ml of freshly prepared membrane vesicles in THM buffer at 220, which was added at the starting time to a disposable 10 X 75 mm test tube containing 0.1 ,Ci of 14C-labeled amino acid, 0.1 1ACi of 3-O-[methyl-3H]methyl-D-glucose, 50,umol of NaCl, and unlabeled amino acid to bring the incubation mixture to the desired final concentration of amino acid. The mixture was stirred on a Vortex genie for 6 sec. Membrane vesicles in THM buffer were preincubated with 100 mM NaCl for 40 min in experiments where uptake in the absence of Na+ gradient was to be studied.At various times ranging from 15 sec to 20 min, the incubation mixture was transferred by pasteur pipette to a Millipore filter apparatus. Uptake was stopped by rapid filtration of the mixture through a Millipore filter (HAWP, 0.45 nm) and washed once with 5 ml of 154 mM NaCI in 1 mM Tris-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic ...

Role of Na + in α-aminoisobutyric acid uptake by membrane vesicles from mouse fibroblasts transformed by simian virus 40

“…It seems reasonable to suggest that Na+ is essential because it is involved in the formation of a Na+-carrier-AIB ternary complex and that this complex functions in the cotransport of Na+ and AIB across the membrane. A similar conclusion regarding the role of Na+ in transmembrane transport has been reached in studies of amino acid and sugar transport by membrane vesicles from mammalian intestinal and renal epithelial cells (29)(30)(31)(32)(33)(34)(35)(36)(37).…”

Developmental aspects of proline transport in rat renal brush border membranes.

“…These preparations of intact cells with solute fluxes at brush border and basal lateral membranes, the ability to pump sodium and maintain standing electrochemical potential gradients, are, indeed, complex model systems with inherent difficulties in the interpretation of transport data. The advent of methods to prepare purified brush border membrane vesicles (12)(13)(14) has permitted studies of transport systems under defined conditions that reflect solute and membrane carrier interactions at one renal tubule reabsorptive surface. Although reports have appeared characterizing the transport of many solutes by renal brush border membranes from adult animals, little is known about the function of renal membranes from newborn and young animals despite the description of their preparation from rat kidney by Qoldmann et al a decade ago (19).…”

“…Membrane preparations were made according to the method of PockrandtHemstedt et al (13) as modified by McNamara et al (14), starting from whole kidney in all groups. The final pellet was resuspended in THM buffer (100 mM mannitol/2 mM Hepes, adjusted to pH 7.4 by addition of Tris) to a concentration of -=0.8 mg of protein per ml.…”

Transport of β-Hydroxy-β-Methyl-Glutarate and β-Hydroxbutyrate by Renal Brushborder Membrane Vesicles

“…All measurements of uptake were performed using Millipore filtration on HAWP filters (0.45 pm) according to the technique described by McNamara et al (14). The vesicle preparation was osmotically active and did not metabolize substrate.…”

“…Brushborder membrane vesicles were then prepared using the method of Booth and Kenny (2) modified as described by Weiss et al (21). The final membrane pellet was suspended in T H M buffer, pH 7.4 (2 mM Tris/HEPES + 100 mM mannitol) to a protein concentration of 0.3-0.4 mg/ml as determined by the method of Lowry et al (13).All measurements of uptake were performed using Millipore filtration on HAWP filters (0.45 pm) according to the technique described by McNamara et al (14). The vesicle preparation was osmotically active and did not metabolize substrate.…”