RESUMO
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.
Assuntos
Arginina/metabolismo , Cistina/metabolismo , Túbulos Renais/metabolismo , Lisina/metabolismo , Ornitina/metabolismo , Animais , Sistema Livre de Células , Cinética , RatosRESUMO
The sodium-dependent entry of proline and glycine into rat renal brush-border membrane vesicles was examined. The high Km system for proline shows no sodium dependence. The low Km system for glycine entry is strictly dependent on a Na+ gradient but shows no evidence of the carrier system having any affinity for Na+. The low Km system for proline and high Km system for glycine transport appear to be shared. Both systems are stimulated by a Na+ gradient and appear to have an affinity for the Na+. The effect of decreasing the Na+ concentration in the ionic gradient is to alter the Km for amino acid entry and, at low Na+ concentrations, to inhibit the V for glycine entry.
Assuntos
Membrana Celular/metabolismo , Glicina/metabolismo , Rim/metabolismo , Microvilosidades/metabolismo , Prolina/metabolismo , Sódio/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Cinética , Masculino , Microvilosidades/efeitos dos fármacos , RatosRESUMO
Isolated rat renal cortical tubules were used to study the nature of homocystine entry into the tubule cell and its transport interactions with cystine and the dibasic amino acids. The uptake of homocystine with time was progressive, reaching a steady state after 60 min. of incubation. Analysis of the intracellular pool after 5 and 30 min. of incubation revealed that virtually all of the transported homocystine had been converted to other metabolites of the transsulfuration pathway. The major metabolite was cystathionine with a somewhat lesser, but still significant amount as S-adenosylhomocysteine. A kinetic analysis showed that two systems for cellular entry of homocysteine existed with a Km1 of 0.17 mM and a Km2 of 7.65 mM. Arginine and lysine inhibited homocystine uptake via the low Km, high affinity system, but appeared not to inhibit the high Km, low affinity system. Cystine inhibited the low Km, high affinity system, but had an indeterminate effect on the high Km, low affinity system. Homocystine inhibited the uptake of cystine, lysine and arginine by isolated rat renal cortical tubules. The inhibition of homocystine on cystine uptake appeared to occur on both the high and low Km system for tubule cell entry of cystine. The data suggest that the low Km system for homocystine transport is shared with cystine and the dibasic amino acids. These data extend the knowledge of homocystine metabolism and provide a rational basis for new approaches to the treatment of homocystinuria.
Assuntos
Homocistina/metabolismo , Túbulos Renais/metabolismo , Aminoácidos/farmacologia , Animais , Arginina/metabolismo , Transporte Biológico Ativo , Cistina/metabolismo , Cinética , Lisina/metabolismo , Masculino , Ratos , Ratos Endogâmicos , Fatores de Tempo , Distribuição TecidualRESUMO
Uptake of L-cystine by brush-border membrane vesicles isolated from rat renal-cortical tissue was time-dependent and occurred in the absence of cystine reduction. A significant capacity for vesicular binding of cystine was observed. The amount bound increased with time of incubation and could be displaced by thiol reagents. At early time points, cystine uptake measured the transport of cystine into the intravesicular space. Total cystine uptake was mediated by multiple transport systems, including a low-Km high-affinity component which was shared by lysine, arginine, ornithine and glutamine and on which hetero-exchange diffusion of lysine and cystine was demonstrated.
Assuntos
Membrana Celular/metabolismo , Cistina/metabolismo , Córtex Renal/metabolismo , Microvilosidades/metabolismo , Aminoácidos/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Técnicas In Vitro , Córtex Renal/efeitos dos fármacos , Cinética , Masculino , Microvilosidades/efeitos dos fármacos , Ratos , Sódio/metabolismo , Compostos de Sulfidrila/farmacologiaRESUMO
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, Km1 = 0.067 mM and Km2 = 5.26 mM. The uptake of glycine was also Na+ gradient dependent, and exhibited a two Km system, Km1 = 0.22 mM and Km2 = 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.
Assuntos
Glicina/metabolismo , Túbulos Renais/metabolismo , Prolina/metabolismo , Animais , Arginina/metabolismo , Transporte Biológico , Sistema Livre de Células , Concentração de Íons de Hidrogênio , Túbulos Renais/ultraestrutura , Cinética , Lisina/metabolismo , Masculino , Membranas/metabolismo , Metilglucosídeos/metabolismo , Fenilalanina/metabolismo , Ratos , Sódio/fisiologiaRESUMO
Glutamine uptake by rat renal brushborder vesicles occurred via two distinct saturable processes with Km values of 0.145 and 8.5 mM which were stimulated by both ionic and sodium gradients with a pH optimum of 6.8--7.1. Glutamic acid uptake also occurred by a two-component system with Km values of 0.016 and 3.60 mM. Both components were stimulated specifically by a sodium gradient. The low Km system for glutamic acid had a pH optimum of 7.2--7.4. Glutamine entry at 0.06 mM was inhibited by a variety of amino acids at 3 mM, including dibasic amino acids, glycine, valine, and phenylalanine. Glutamic acid entry at 0.06 mM was inhibited 20--30% by 3 mM phenylalanine, valine, alpha-aminoisobutyric acid, and glutamine. No metabolic alteration of glutamic acid was observed on incubation with membrane vesicles, but glutamine was significantly hydrolyzed to glutamic acid upon prolonged incubation. Hydrolysis of glutamine was negligible at 15 sec incubation which was employed for measurement of initial rate of entry. These studies provide support for the existence of an uptake system in the brushborder of the renal proximal tubule cell capable of handling the reabsorption of glutamine normally present in glomerular filtrate.
Assuntos
Glutamatos/metabolismo , Glutamina/metabolismo , Rim/metabolismo , Aminoácidos/metabolismo , Animais , Transporte Biológico Ativo , Glutamatos/administração & dosagem , Glutamina/administração & dosagem , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Masculino , Microvilosidades/metabolismo , Ratos , Sódio/administração & dosagemRESUMO
Proline transport into renal brushborder membrane vesicles isolated from human kidney is mediated by two uptake systems. The high-affinity system is stimulated by a Na gradient and appears to be shared with glycine while the low-affinity system is not. Uptake curves of low concentrations of proline exhibit a Na-gradient-dependent overshoot indicative of electrogenic transport. The proline transport systems observed in isolated human renal brushborder membrane vesicles appear to have characteristics similar to those in rat kidney membranes.
Assuntos
Córtex Renal/metabolismo , Microvilosidades/metabolismo , Prolina/metabolismo , Alanina/metabolismo , Glicina/metabolismo , Humanos , Hidroxiprolina/metabolismo , Técnicas In Vitro , Túbulos Renais Proximais/metabolismo , CinéticaRESUMO
The effects of freezing renal tissue from rat, dog, and man on the time course of uptake of proline and alpha-methyl-D-glucoside by subsequently isolated brush border membrane vesicles was examined and compared with uptake patterns by membranes isolated from tissue that had never been frozen. The overshoot phenomenon was used as the critical criterion for viability of the transport systems. Membranes isolated from frozen rat and dog kidney possessed intact transport systems for proline and alpha-methyl-D-glucoside capable of producing normal or even enhanced overshoot patterns. Freezing human kidney prior to membrane isolation resulted in severe impairment of the vesicle transport capabilities. Freezing a crude membrane suspension, however, allowed the subsequent purification of only partially intact systems.
Assuntos
Membrana Celular/metabolismo , Rim/metabolismo , Metilglucosídeos/metabolismo , Metilglicosídeos/metabolismo , Microvilosidades/metabolismo , Prolina/metabolismo , Ratos Endogâmicos , Preservação de Tecido/métodos , Animais , Transporte Biológico , Pré-Escolar , Cães , Congelamento , Humanos , Rim/ultraestrutura , Masculino , Microscopia Eletrônica de Varredura , Microvilosidades/enzimologia , RatosRESUMO
The ability of eight stripping agents to solubilize five marker enzymes from rat renal brush border membranes isolated by three different preparative methods was examined. Protein and enzyme activities - alkaline phosphatase (APase), L-leucine aminopeptidase (LAPase), gamma-glutamyl transpeptidase (GGTase), gamma-glutamyl hydrolase (GGHase) and maltase - solubilized by the treatments were expressed as percent of total activity recovered in excess of control values. The relative enzyme activity and the solubilization factor were determined for each marker enzyme in every treated sample and the treatments with the eight agents compared. Trypsin treatment released > 80% of LAPase and < 10% of total membrane protein. Papain treatment released only 16--23% of total membrane protein but most of the enzyme activities except APase. Neuraminidase had no solubilizing effect. 4--10% of total membrane protein was solubilized by LiCl treatment but no marker enzyme activities were released. Less total membrane protein was released by treatment with proteolytic enzymes or LiCl than with the detergents Triton X-100, hexadecyltrimethylammonium bromide, sodium deoxycholate, and sodium dodecylsulfate. APase activity was the least readily solubilized. Correlating the degree of solubilization for five marker enzymes with the types of stripping agents used and with the appearance of the membrane surface when examined by electron microscopy led to the suggestion that LAPase, GGTase, GGHase and maltase molecules are part of an interwoven surface layer of membrane proteins which can be disrupted by transamidation and transesterification reactions. APase appears to be more strongly associated with the intact lipid matrix than the bulk of the membrane protein.