Immunohistochemical localization of 5-oxo-L-prolinase, an enzyme of the gamma-glutamyl cycle, in porcine brain microvessels.
The immunohistochemical analysis of the distribution of 5-oxo-L-prolinase in porcine brain at the light microscopic level was performed with an antibody raised against the enzyme purified from pig kidney. The present study reveals the specific expression of 5-oxo-L-prolinase in brain capillaries with an average diameter of 4.1+/-0.9 microm, while larger blood vessels remain unstained. Porcine kidney and skeletal muscle show no endothelial-specific staining with the antibody. In some cases, the asymmetrical staining pattern in cross and longitudinal sections of brain microvessels indicate endothelial- but also pericyte-specific expression. (+info)
Molecular cloning, sequencing, and expression in Escherichia coli of the gene encoding a novel 5-oxoprolinase without ATP-hydrolyzing activity from Alcaligenes faecalis N-38A.
The gene encoding a novel 5-oxoprolinase without ATP-hydrolyzing activity from Alcaligenes faecalis N-38A was cloned and characterized. The coding region of this gene is 1,299 bp long. The predicted primary protein is composed of 433 amino acid residues, with a 31-amino-acid signal peptide. The mature protein is composed of 402 amino acid residues with a molecular mass of 46,163 Da. The derived amino acid sequence of the enzyme showed no significant sequence similarity to any other proteins reported so far. The 5-oxoprolinase gene was expressed in Escherichia coli by using a regulatory expression system with an isopropyl-beta-D-thiogalactopyranoside-inducible tac promoter, and its expression level was approximately 16 mg per liter. The purified enzyme has the same characteristics as the authentic enzyme, except for the amino terminus, which has three additional amino acids. The enzyme was markedly inhibited by p-chloromercuribenzoic acid, EDTA, o-phenanthroline, HgCl(2), and CuSO(4). The EDTA-inactivated enzyme was completely restored by the addition of Zn(2+) or Co(2+). In addition, the enzyme was found to contain 1 g-atom of zinc per mol of protein. These results suggest that the 5-oxoprolinase produced by A. faecalis N-38A is a zinc metalloenzyme. (+info)
A simultaneous assay method for L-glutamate and L-pyroglutamate contents in soy sauce using a 5-oxoprolinase (without ATP hydrolyzing activity).
L-Glutamine and L-glutamate, which are important flavor components in soy sauce, are converted to L-pyroglutamate during brewing. Therefore, it is necessary that the L-glutamate and L-pyroglutamate contents can be measured accurately. We developed a simultaneous assay method for L-glutamate and L-pyroglutamate by using 5-oxoprolinase (without ATP hydrolyzing activity) and glutamate oxidase. By this method, the L-pyroglutamate could be measured accurately in a range of 0.05 to 1.0 mM in the presence of 1.0 mM L-glutamate. This system is effective for process and quality controls. (+info)
Protective role of L-2-oxothiazolidine-4-carboxylic acid in cisplatin-induced renal injury.
BACKGROUND: Oxidative stress and inflammation are implicated in the pathogenesis of cisplatin-induced nephrotoxicity. l-2-oxothiazolidine-4-carboxylic acid (OTC) is a cysteine prodrug, and increases cellular glutathione (GSH). OTC is converted to cysteine by the intracellular enzyme, oxoprolinase. To date, the protective role of OTC on cisplatin-induced renal injury has not been investigated. The purpose of the present study was to examine the protective effect of OTC on cisplatin-induced renal injury and to examine the mechanism of its protection. METHODS: Mice were treated with cisplatin with or without administration of OTC. The generation of reactive oxygen species (ROS), expression of intercellular adhesion molecule (ICAM)-1 and monocyte chemoattractant protein (MCP)-1 were determined in the kidney using 2',7'-dichlorofluorescein diacetate, immunostaining or western blot analysis. Nuclear factor (NF)-kappaB activity, infiltration of F4/80-positive cells and apoptosis were also investigated in addition to renal function and histology using electrophoretic mobility shift assay, immunostaining, western blot analysis, uridine triphosphate (dUTP) nick-end labelling or periodic acid-Schiff staining. The effect of OTC on superoxide dismutase activity and GSH level in cisplatin-treated normal adult human kidney (HK-2) cells were measured using assay kits. RESULTS: The administration of OTC resulted in a significant reduction of cisplatin-induced ROS production, the p65 subunit of NF-kappaB translocation into nucleus, expression of ICAM-1, caspase 3 activity, expression of MCP-1 and the infiltration of macrophages into renal tissue. OTC markedly ameliorated renal damage induced by cisplatin through antioxidant and anti-inflammatory effect. CONCLUSIONS: These results suggest that OTC can be a potential therapeutic agent in cisplatin-induced renal injury through decreasing the ROS levels and activation of NF-kappaB. (+info)
Inborn errors in the metabolism of glutathione.
Glutathione is a tripeptide composed of glutamate, cysteine and glycine. Glutathione is present in millimolar concentrations in most mammalian cells and it is involved in several fundamental biological functions, including free radical scavenging, detoxification of xenobiotics and carcinogens, redox reactions, biosynthesis of DNA, proteins and leukotrienes, as well as neurotransmission/neuromodulation. Glutathione is metabolised via the gamma-glutamyl cycle, which is catalyzed by six enzymes. In man, hereditary deficiencies have been found in five of the six enzymes. Glutathione synthetase deficiency is the most frequently recognized disorder and, in its severe form, it is associated with hemolytic anemia, metabolic acidosis, 5-oxoprolinuria, central nervous system (CNS) damage and recurrent bacterial infections. Gamma-glutamylcysteine synthetase deficiency is also associated with hemolytic anemia, and some patients with this disorder show defects of neuromuscular function and generalized aminoaciduria. Gamma-glutamyl transpeptidase deficiency has been found in patients with CNS involvement and glutathionuria. 5-Oxoprolinase deficiency is associated with 5-oxoprolinuria but without a clear association with other symptoms. Dipeptidase deficiency has been described in one patient. All disorders are very rare and inherited in an autosomal recessive manner. Most of the mutations are leaky so that many patients have residual enzyme activity. Diagnosis is made by measuring the concentration of different metabolites in the gamma-glutamyl cycle, enzyme activity and in glutathione synthetase and gamma-glutamylcysteine synthetase deficiency, also by mutation analysis. Prenatal diagnosis has been preformed in glutathione synthetase deficiency. The prognosis is difficult to predict, as few patients are known, but seems to vary significantly between different patients. The aims of the treatment of glutathione synthesis defects are to avoid hemolytic crises and to increase the defense against reactive oxygen species. No treatment has been recommended for gamma-glutamyl transpeptidase, 5-oxoprolinase and dipeptidase deficiency. (+info)
Metabolomic analysis via reversed-phase ion-pairing liquid chromatography coupled to a stand alone orbitrap mass spectrometer.
A futile cycle, formed between two ATP-dependant gamma-glutamyl cycle enzymes, gamma-glutamyl cysteine synthetase and 5-oxoprolinase: the cause of cellular ATP depletion in nephrotic cystinosis?
Cystinosis, an inherited disease caused by a defect in the lysosomal cystine transporter (CTNS), is characterized by renal proximal tubular dysfunction. Adenosine triphosphate (ATP) depletion appears to be a key event in the pathophysiology of the disease, even though the manner in which ATP depletion occurs is still a puzzle. We present a model that explains how a futile cycle that is generated between two ATP-utilizing enzymes of the gamma-glutamyl cycle leads to ATP depletion. The enzyme gamma-glutamyl cysteine synthetase (gamma-GCS), in the absence of cysteine, forms 5-oxoproline (instead of the normal substrate, gamma-glutamyl cysteine) and the 5-oxoproline is converted into glutamate by the ATP-dependant enzyme, 5-oxoprolinase. Thus, in cysteine-limiting conditions, glutamate is cycled back into glutamate via 5-oxoproline at the cost of two ATP molecules without production of glutathione and is the cause of the decreased levels of glutathione synthesis, as well as the ATP depletion observed in these cells. The model is also compatible with the differences seen in the human patients and the mouse model of cystinosis, where renal failure is not observed. (+info)
Selective inhibition of gamma-glutamyl-cycle enzymes by substrate analogs.
Substrate analogs have been obtained that selectively inhibit the reactions of the gamma-glutamyl cycle or that are susceptible to only limited metabolism by the cycle. Thus, glutathione synthesis may be inhibited and analogs of glutathione may be synthesized that do not participate in transpeptidation. Specific inhibitors of gamma-glutamylcyclotransferase and 5-oxoprolinase have been obtained. The findings offer new approaches to the in vivo study of the cycle and also to the design of more specifically directed analogs of inhibitors such as methionine sulfoximine and 6-diazo-5-oxonorleucine. (+info)