No 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 ...

The ability of mice deficient in galactose-1-phosphate uridyltransferase (GALT) to metabolize galactose was determined in animals weaned to a mouse chow diet for a 4-wk period. When given [14 C]galactose intraperitoneally, these animals slowly oxidized the sugar, excreting only 5.5% of the dose as 14 CO 2 in 4 h, whereas normal animals excreted 39.9%. These results mimic those seen in human galactosemic patients given isotopic galactose. When given 10 mol of [1-13 C]galactose, normal animals excrete small amounts of labeled galactose and galactonate but no galactitol in urine whereas GALT-deficient mice excrete significant amounts of all of these as labeled compounds in urine. When challenged with galactose, only about 20% of the dose is excreted in urine, and even on the chow diet, significant amounts of galactose, galactonate, and galactitol are excreted in urine. These compounds are also found to be present in liver, kidney, and brain, except that galactonate is not found in brain. Galactose-1-phosphate accumulates in red blood cells to levels found in humans exposed to large amounts of galactose, and galactose-1-phosphate is found in increased amounts in liver, kidney, and brain of GALT-deficient animals. There was no difference in the hepatic concentration of uridine diphosphate galactose and uridine diphosphate glucose between normal and GALT-deficient mice. The explanation for the presence of galactose and its conversion products in tissues and urine of affected mice appears to be related to the presence of approximately 1.75% of galactose-containing carbohydrates in the chow, which becomes bioavailable to mice. Despite the presence of galactose and its metabolites in tissues and urine and impaired ability to oxidize the sugar, the GALT-deficient animals are indistinguishable from normal animals and do not exhibit the phenotype of humans with GALT-deficiency galactosemia. Abbreviations GALT, galactose-1-phosphate uridyltransferase Gal-1-P, galactose-1-phosphate N/N, normal mice G/N, heterozygous normal and GALT deficient G/G, homozygous for a deficiency of GALT UDPgal, uridine diphosphate galactose UDPglu, uridine diphosphate glucose BSTFA, N,O-bis-(trimethylsilyl)trifluoroacetamide TMCS, trimethylchlorosilane CHOP, Children's Hospital of Philadelphia GC-MS, gas chromatography-mass spectroscopy After more than 60 y of experience with the diagnosis and dietary therapy of galactosemia caused by GALT deficiency, the pathophysiology of the disorder has remained unclear (1). Enigmatic has been the origin of both the rapid development of liver, kidney, eye, and central nervous toxicity occurring in newborns exposed to large amounts of galactose in milk and the long-term diet-independent complications of cognitive impairment, speech and learning disorders, ataxia, and primary ovarian failure in females. To enhance the study of the pathogenesis of GALT deficiency and further our understanding of the cause of the galactosemic phenotype, we made mice deficient in GALT activity using gene-targeting techniques (2).We...

The uptake of cystine by vesicles prepared from rat kidney brush borders occurs by two distinct transport systems. The higher affinity system is inhibited by the dibasic amino acids lysine, arginine, and ornithine. The lower affinity system, unaffected by dibasic amino acids, appears to correspond to that observed by studying uptake of cystine by kidney slices.