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PubMed1984

Adrenocortical cytochrome P-450 responsible for cholesterol side chain cleavage (P-450scc) is phosphorylated by the calcium-activated, phospholipid-sensitive protein kinase (protein kinase C).

Purified bovine adrenocortical cytochrome P-450scc (specific for cholesterol side chain cleavage in the inner mitochondrial membrane) was selectively phosphorylated in vitro by a Ca2+-activated, phospholipid-sensitive protein kinase (protein kinase C) preparation, whereas cyclic AMP dependent and two cyclic nucleotide independent kinases were ineffective. Cytochrome P-450scc incorporated a maximum of 4 mol of phosphate in the presence of protein kinase C within 15 min at 30 degrees C, with apparent Km and Vmax of 0.14 mumol and 0.76 pmol/min, respectively. Serine and threonine were the two target aminoacids phosphorylated in a ratio of about 1:1. In the presence of 1 microM Ca2+, a mixture of phosphatidylserine and diolein (or a potent tumor promoter phorbol ester) was required for optimal cytochrome P-450scc phosphorylation. In addition, purified inner mitochondrial membrane preparations from adrenocortical mitochondria were found to contain protein kinase C activity. These findings, together with the previous demonstration that activators of protein kinase C such as a potent phorbol ester activates steroidogenesis of intact adrenocortical cells, suggest that phosphorylation of P-450scc should be examined for its possible role in the regulation of adrenocortical functions.

Adrenal Cortex↗

PubMed2004

A deficiency at the gene coding for zeta-carotene desaturase characterizes the sunflower non dormant-1 mutant.

The non dormant-1 (nd-1) mutant of sunflower (Helianthus annuus L.) is characterized by an albino and viviparous phenotype. Pigment analysis by spectrophotometer and HPLC demonstrated in nd-1 cotyledons the absence of beta-carotene, lutein and violaxanthin. Additionally, we found a strong accumulation of zeta-carotene and, to a lesser extent, of phytofluene and cis-phytoene in nd-1 seedlings grown in very dim light (1 micro mol m(-2) s(-1)). These results suggested that zeta-carotene desaturation was impaired in the mutant plants. To understand the molecular basis of the nd-1 mutation, we cloned and characterized the zeta-carotene desaturase (Zds) gene from sunflower. A reconstructed full-length sequence (1,916 bp) of the Zds cDNA was obtained from homozygous Nd-1/Nd-1 wild-type plants. It contains a 1,761-bp CDS, 62 nucleotides of 5'-untranslated region (UTR), and 77 nucleotides of 3'-UTR. The predicted protein (64.9 kDa) consists of 587 amino acid residues with a putative transit sequence for plastid targeting in the N-terminal region and a typical amino oxidase domain that includes the flavin adenosine dinucleotide (FAD) binding motif. The phylogenetic analysis demonstrated that the sunflower Zds was clustered to marigold (Tagetes) Zds gene, for which it showed an overall aminoacidic identity of 96.6% and resulted strictly correlated with other Zds sequences of higher plants. Interestingly, RT-PCR analyses showed that nd-1 plants were unable to accumulate Zds transcripts. Sequence information from the Zds cDNA was used to design specific primers and to isolate the full-length exons/introns region of the gene. The sunflower Zds gene (HaZds) comprises 14 exons and 13 introns scattered in a ca. 5.0-kb region. Also, HaZds showed a high conservation of the distribution and size of the exons with rice Zds gene. Based on genomic Southern analysis, the nd-1 genome disclosed a large deficiency at the Zds locus.

Amino Acid Sequence↗

PubMedDate not supplied

Oxidative deamination of thialysine by snake venom L-aminoacid oxidase.

Thialysine is oxidatively deaminated by snake venom L-aminoacid oxidase at alkaline pH. The oxygen consumption curves show a characteristic diphasic course: the quick uptake of half a mole of oxygen per mole of substrate, in aggreement with a typical oxidative deamination, is followed by a slow extra oxygen consumption. The first product of the reaction is the corresponding alpha-oxo-epsilon-amino acid, which spontaneously cyclizes to the internal Schiff base 5-6-dihydro-delta 3,1,4-thiazin-3-carboxylic acid (TZCA). This latter has been identified by its UV absorption spectrum, by some chemical reactions, by paper chromatography, and by the production of cystamine and glyoxylic acid after prolonged oxidation of thialysine followed by acid hydrolysis. The possibility of an alpha-beta elimination reaction giving rise to cysteamine from thialysine, coupled to the oxidative deamination, has been excluded.

Amino Acid Oxidoreductases↗

PubMed1975

Synthesis and release of histamine studied on slices from rat hypothalamus.

In slices from rat hypothalamus, incubated in the presence of 3H-L-histidine (3H-L-His), the aminoacid was rapidly taken up by a saturable process, and partially converted into 3H-histamine (3H-HA). The overall conversion was prevented either by inhibitors of L-histidine decarboxylase or by aromatic aminoacids competing with L-His uptake. The synthesis process exhibited Michaelis--Menten kinetics with an affinity of the aminoacid not different from that for the decarboxylase in homogenates; however the Vmax in homogenates was more than 10 times higher than in slices. Depolarization of the slices by 50 mM potassium resulted in: (a) a calcium-dependent release of 3H-HA which was more marked than that previously reported for endogenous HA, (b) a significant acceleration in the rate of 3H-HA synthesis, which was characterized by an unchanged Km but a significantly elevated Vmax. The regulation of HA synthesis did not appear to depend on end-product inhibition since it was not midified by the addition of exogenous HA. The release of 3H-HA was followed by the accumulation of 3H-methylhistamine, which was enhanced by a monoamine oxidase inhibitor. Aminoguanidine, a diamine oxidase inhibitor, had no effect on catabolism. The involvement of mast-cells in the storage of a fraction of endogenous HA in hypothalamic slices was assessed by the significant releasing effect of compound 48/80. Hence, the data support the existence of two distinct HA stores in the brain: depolarization relases the amine and increases its synthesis, probably in neurones, whereas compound 48/80 releases it from a slowly turning-over store, probably in mast-cells.

Amino Acid Oxidoreductases↗

PubMed2003

Oxidase enzyme immobilisation through electropolymerised films to assemble biosensors for batch and flow injection analysis.

Glucose oxidase, lactate oxidase, L-aminoacid oxidase and alcohol oxidase were immobilised on new films based on 2,6-dihydroxynaphthalene (2,6-DHN) copolymerised with 2-(4-aminophenyl)-ethylamine (AP-EA) onto the Pt electrodes. The electropolymerisation was performed by cyclic voltammetry. Different scan rates and scan potential ranges were investigated and selected according to the monomers used. These sensors were tested for hydrogen peroxide, ascorbic acid and acetaminophen by cyclic voltammetry and amperometry. The amperometric studies were carried out in batch as well as in a flow injection analysis (FIA) system. Analytical parameters such as reproducibility, interference rejection, response time, buffer, storage and operational time of the sensors have been studied. These films were also characterised by X-ray photoelectron spectroscopy (XPS). Different strategies for enzyme immobilisation were performed and discussed: enzyme entrapment in the film during the electropolymerisation and covalent attachment of the enzyme to the film via a carbodiimide (1-ethtl-3-(3-dimethylaminopropyl)carbodiimide, EDC) or glutaraldehyde. Different parameters were considered in order to optimise the immobilisation procedures. Results provide a guide to design high sensitive, stable and interference-free biosensors. In addition, studies were performed using these probes in an original FIA based on solenoidal valves. Sensor stability, life time and dynamic range were also optimised in these conditions.

Biosensing Techniques↗

PubMed2000

Mammalian class theta GST and differential susceptibility to carcinogens: a review.

Glutathione S-transferases (GSTs) are an important part of the cellular detoxification system and, perhaps, evolved to protect cells against reactive oxygen metabolites. Theta is considered the most ancient among the GSTs and theta-like GSTs are found in mammals, fish, insects, plants, unicellular algae, and bacteria. It is thought that an ancestral theta-gene underwent an early duplication before the divergence of fungi and animals and further duplications generated the variety of the other classes of GSTs (alpha, mu, phi, etc.). The comparison of the aminoacidic homologies among mammals suggests that a duplication of an ancient GST theta occurred before the speciation of mammals and resulted in the subunits GSTT1 and GSTT2. The ancestral GST theta has a dehalogenase activity towards several halogenated compounds, such as the dichloromethane. In fact, some aerobic and anaerobic methylotrophic bacteria can use these molecules as the sole carbon and energy source. The mammalian GST theta cannot sustain the growth of bacteria but still retains the dehalogenating activity. Therefore, although mammalian GST theta behaves as a scavenger towards electrophiles, such as epoxides, it acts also as metabolic activator for halogenated compounds, producing a variety of intermediates potentially dangerous for DNA and cells. For example, mice exposed to dichloromethane show a dose-dependent incidence of cancer via the GSTT1-1 pathway. Because GSTT1-1 is polymorphic in humans, with about 20% of Caucasians and 80% of Asians lacking the enzyme, the relationship between the phenotype and the incidence of cancer has been investigated extensively in order to detect GSTT1-1-associated differential susceptibility towards endogenous or exogenous carcinogens. The lack of the enzyme is related to a slightly increased risk of cancer of the bladder, gastro-intestinal tract, and for tobacco-related tumors (lung or oral cavity). More pronounced risks were found in males with the GSTT1-null genotype for brain diseases and skin basal cell carcinomas not related to sunlight exposures. Moreover, there was an increased risk of kidney and liver tumors in humans with the GSTT1-1 positive genotype following exposures to halogenated solvents. Interestingly, the liver and kidney are two organs that express the highest level of GST theta in the human body. Thus, the GSTT1-1 genotype is suspected to confer decreased or increased risk of cancer in relation to the source of exposure; in vitro studies, mostly conducted on metabolites of butadiene, confirm the protective action of GSTT1-1, whereas, thus far, experimental studies prove that the increasing risk is limited.

Amino Acid Sequence↗

PubMed2001

Inhibitory effect of naringin on the micronuclei induced by ifosfamide in mouse, and evaluation of its modulatory effect on the Cyp3a subfamily.

Naringin (Nar) is a flavonone found in high amount in grapefruit. In in vitro studies to determine its antimutagenicity results have been both positive and negative. On the other hand, an increase in the bioavailability of some medicaments have been observed when these are ingested together with grapefruit. It has been suggested that the effect may be related to the inhibition of the human enzyme Cytochrome P450 (CYP) 3A4 by Nar, an enzyme with a high aminoacid sequence homology with the Cyp3a in mouse. The present study was designed for three main purposes: (1) to determine whether Nar has a genotoxic effect in mouse in vivo. This was evaluated by measuring the rate of micronucleated polychromatic erythrocytes (MNPE); (2) to determine its antigenotoxic and its anticytotoxic potential on the damage produced by ifosfamide (Ifos). The first study was done by scoring the rate of MNPE, and the second one by establishing the index polychromatic erythrocytes/normochromatic erythrocytes (PE/NE); and (3) to explore whether its antigenotoxic mechanism of action is related to an inhibitory effect of Nar on the expression of the Cyp3a enzyme, an effect which could avoid the biotransformation of Ifos. A single oral administration was used for all groups in the experiment: three groups were given different doses of Nar (50, 250, and 500 mg/kg), other groups received the same doses of Nar plus an administration of Ifos (60 mg/kg), another group treated with distilled water and another with Ifos (60 mg/kg) were used as negative and positive controls, respectively. The micronuclei and the cell scoring were made in blood samples taken from the tail of the animals at 0, 24, 48, 72, and 96 h. The results showed that Nar was neither genotoxic nor cytotoxic with the doses tested, but Ifos produced an increase in the rate of MNPE at 24 and 48 h. The highest value was 24+/-1.57 MNPE per thousand cells at 48 h. The index PE/NE was significantly reduced by Ifos at 24 and 48 h. Concerning the antigenotoxic capacity of Nar, a significant decrease was observed in the MNPE produced by Ifos at the three tested doses. This effect was dose-dependent, showing the highest reduction in MNPE frequency (54.2%) at 48 h with 500 mg/kg of Nar. However, no protection on the cytotoxicity produced by Ifos was observed. Immunoblot analysis was used to assess the Cyp3a expression in liver and intestinal microsomes from mouse exposed orally to Nar. An induction in the Cyp3a protein was observed in both intestinal and hepatic microsomes from treated mice. This induction correlated with an increase in erythromycin N-demethylase activity. These data suggest that other mechanism(s) are involved in the antigenotoxic action of naringin.

Animals↗