ABSTRACT
The mitochondrial outer membrane enzyme kynurenine 3-hydroxylase (K3H) is an NADPH-dependent flavin mono-oxygenase involved in the tryptophan pathway, where it catalyzes the hydroxylation of kynurenine. K3H was transiently expressed in COS-1 cells as a glutathione S-transferase (GST) fusion protein, and the pure recombinant protein (rec-K3H) was obtained with a specific activity of about 2000 nmol.min-1.mg-1. Rec-K3H was shown to have an optimum pH at 7.5, to use NADPH more efficiently than NADH, and to contain one molecule of non-covalently bound FAD per molecule of enzyme. The mechanism of the rec-K3H-catalyzed reaction was investigated by overall initial-rate measurements, and a random mechanism in which combination of the enzyme with one substrate does not influence its affinity for the other is proposed. Further kinetic studies revealed that K3H activity was inhibited by both pyridoxal phosphate and Cl-, and that NADPH-catalyzed oxidation occurred even in the absence of kynurenine if 3-hydroxykynurenine was present, suggesting an uncoupling effect of 3-hydroxykynurenine with peroxide formation. This observation could be of clinical interest, as peroxide formation could explain the neurotoxicity of 3-hydroxykynurenine in vivo.
Subject(s)
Mixed Function Oxygenases/metabolism , Animals , COS Cells , Chlorides/metabolism , Chlorides/pharmacology , Flavin-Adenine Dinucleotide/metabolism , Humans , Hydrogen-Ion Concentration , Ions , Kinetics , Kynurenine/analogs & derivatives , Kynurenine/metabolism , Kynurenine 3-Monooxygenase , Mixed Function Oxygenases/antagonists & inhibitors , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/isolation & purification , Molecular Weight , NAD/metabolism , NADP/metabolism , NADPH Oxidases/metabolism , Oxidation-Reduction , Peroxides/metabolism , Pyridoxal Phosphate/metabolism , Pyridoxal Phosphate/pharmacology , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Substrate Specificity , Thermodynamics , TransfectionABSTRACT
Cerebral ischaemia results in significant brain damage, but the molecular mechanisms associated with ischaemia-induced brain injury are not well defined. We have adopted an improved differential-display method to search for new ischaemia-related genes. Among the different cDNAs isolated following transient forebrain ischaemia in rat, PH3.3 was selected for further studies. The search for homologies revealed that it is the rat homologue to human zinc finger motif 1 (ZFM1), also called mammalian splicing factor 1 (SF1). With Northern blot, PH3.3 hybridized with three mRNA species of 2.3, 2.9 and 3.6 kb, significantly increased at 6 h and 5 days after the ischaemic insult. These findings were extended also to another animal model. In situ hybridization in ischaemic gerbils showed that PH3.3 mRNA was induced in the dentate gyrus as early as 4 h post-ischaemia. Expression peaked at 2 days in the whole hippocampus and cortex, and then progressively decreased towards sham levels. By day 4, expression had disappeared almost entirely from the cells in the CA1 region of the hippocampus, concomitant with the degeneration of pyramidal neurons. Interestingly, ZFM1/SF1 has been recently identified as activated following p53-induced apoptosis. Several lines of evidence suggest that p53 may play two roles in the post-ischaemic brain. The primary role of p53 is to activate DNA repair processes, but if repair fails, apoptosis will be initiated. Thus, ZFM1/SF1 may represent a relevant link between p53 and the neuroprotective/neurodegenerative processes which follow cerebral ischaemia.
Subject(s)
Brain Chemistry/physiology , Brain Ischemia/metabolism , Carrier Proteins/genetics , DNA-Binding Proteins , Dentate Gyrus/blood supply , Nuclear Proteins/genetics , Transcription Factors , Amino Acid Sequence , Animals , Blotting, Northern , Cloning, Molecular , DNA Probes , Dentate Gyrus/chemistry , Gene Expression/physiology , Gerbillinae , In Situ Hybridization , Male , Molecular Sequence Data , Polymerase Chain Reaction , RNA Splicing/physiology , RNA Splicing Factors , RNA, Messenger/analysis , Rats , Rats, Wistar , Tumor Suppressor Protein p53/genetics , Zinc Fingers/geneticsABSTRACT
Protein tyrosine phosphatases (PTPases) have been implicated in the control of cell proliferation and differentiation. To isolate new members of this family potentially involved in cell growth regulation, we looked for PTPase sequences differently expressed in proliferating or quiescent NIH 3T3 fibroblasts. The full-length cDNA of one of these growth-regulated genes, named PTP35, was isolated from a 3T3 library and found to encode the murine IA-2 PTPase-related sequence. Endogenous PTP35 mRNA steady-state levels were found to be strictly regulated during cell growth in 3T3 fibroblasts, being high in actively cycling cells and barely detectable in density-arrested cells. Both PTP35 mRNA and protein levels could be induced in quiescent cells by mitogenic stimulation. The growth factor specificity and kinetics of this induction were analyzed in detail.
