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Renal transport of neutral amino acids. Demonstration of Na+-independent and Na+-dependent electrogenic uptake of L-proline, hydroxy-L-proline and 5-oxo-L-proline by luminal-membrane vesicles.

ABSTRACT: Uptake of L-proline, hydroxy-L-proline and 5-oxo-L-proline by luminal-membrane vesicles isolated either from whole cortex or from pars convoluta or pars recta of proximal tubules was studied by a spectrophotometric method. Uptake of L-proline and hydroxy-L-proline by vesicles from whole cortex was mediated by both Na+-dependent and Na+-independent, but electrogenic, processes, whereas transport of 5-oxo-L-proline in these vesicles was strictly Na+-dependent. Eadie-Hofstee analysis of saturation-kinetic data suggested the presence of multiple transport systems in luminal-membrane vesicles from whole renal cortex for the uptake of all these amino acids. Tubular localization of the transport systems was studied by the use of vesicles derived from pars convoluta and from pars recta. In pars recta transport of all three amino acids was strictly dependent on Na+ and occurred via a high-affinity system (half-saturation: 0.1-0.3 mM). Cation-dependent but Na+-unspecific transport of low affinity for L-proline and hydroxy-L-proline was exclusively localized to the pars convoluta, which also contained a Na+-preferring system of intermediate affinity (half-saturation: L-proline, 0.75 mM; hydroxy-L-proline, 1.3 mM). 5-Oxo-L-proline was transported by low-affinity and Na+-dependent systems in both pars convoluta and pars recta. Competition experiments revealed that transport systems for L-proline and hydroxy-L-proline are common, but indicated separate high-affinity transport systems for 5-oxo-L-proline and L-proline in luminal-membrane vesicles from pars recta. The physiological importance of the presence of various neutral amino acid-transport systems in different segments of the proximal tubule is discussed.

SUBMITTER: Roigaard-Petersen H

PROVIDER: S-EPMC1153590 | biostudies-other | 1984 May

REPOSITORIES: biostudies-other

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Project description:Hydroxyprolines, such as trans-4-hydroxy-l-proline (T4LHyp), trans-3-hydroxy-l-proline (T3LHyp), and cis-3-hydroxy-l-proline (C3LHyp), are present in some proteins including collagen, plant cell wall, and several peptide antibiotics. In bacteria, genes involved in the degradation of hydroxyproline are often clustered on the genome (l-Hyp gene cluster). We recently reported that an aconitase X (AcnX)-like hypI gene from an l-Hyp gene cluster functions as a monomeric C3LHyp dehydratase (AcnXType I). However, the physiological role of C3LHyp dehydratase remained unclear. We here demonstrate that Azospirillum brasilense NBRC 102289, an aerobic nitrogen-fixing bacterium, robustly grows using not only T4LHyp and T3LHyp but also C3LHyp as the sole carbon source. The small and large subunits of the hypI gene (hypIS and hypIL, respectively) from A. brasilense NBRC 102289 are located separately from the l-Hyp gene cluster and encode a C3LHyp dehydratase with a novel heterodimeric structure (AcnXType IIa). A strain disrupted in the hypIS gene did not grow on C3LHyp, suggesting its involvement in C3LHyp metabolism. Furthermore, C3LHyp induced transcription of not only the hypI genes but also the hypK gene encoding Δ1-pyrroline-2-carboxylate reductase, which is involved in T3LHyp, d-proline, and d-lysine metabolism. On the other hand, the l-Hyp gene cluster of some other bacteria contained not only the AcnXType IIa gene but also two putative proline racemase-like genes (hypA1 and hypA2). Despite having the same active sites (a pair of Cys/Cys) as hydroxyproline 2-epimerase, which is involved in the metabolism of T4LHyp, the dominant reaction by HypA2 was clearly the dehydration of T3LHyp, a novel type of T3LHyp dehydratase that differed from the known enzyme (Cys/Thr).IMPORTANCE More than 50 years after the discovery of trans-4-hydroxy-l-proline (generally called l-hydroxyproline) degradation in aerobic bacteria, its genetic and molecular information has only recently been elucidated. l-Hydroxyproline metabolic genes are often clustered on bacterial genomes. These loci frequently contain a hypothetical gene(s), whose novel enzyme functions are related to the metabolism of trans-3-hydroxyl-proline and/or cis-3-hydroxyl-proline, a relatively rare l-hydroxyproline in nature. Several l-hydroxyproline metabolic enzymes show no sequential similarities, suggesting their emergence by convergent evolution. Furthermore, transcriptional regulation by trans-4-hydroxy-l-proline, trans-3-hydroxy-l-proline, and/or cis-3-hydroxy-l-proline significantly differs between bacteria. The results of the present study show that several l-hydroxyprolines are available for bacteria as carbon and energy sources and may contribute to the discovery of potential metabolic pathways of another hydroxyproline(s).

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Renal transport of neutral amino acids. Demonstration of Na+-independent and Na+-dependent electrogenic uptake of L-proline, hydroxy-L-proline and 5-oxo-L-proline by luminal-membrane vesicles.

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