Chlorpromazine binding to Na+, K+-ATPase and photolabeling: involvement of the ouabain site monitored by fluorescence.

This work reports the results of ultraviolet irradiation on the interaction of the phototoxic antipsychotic drug chlorpromazine (CPZ) with the sodium pump Na+, K+-ATPase. The study was performed by monitoring the fluorescence modifications of CPZ itself and of the specific probe anthroylouabain (AO). CPZ association with Na+, K+-ATPase was found to modify the kinetics of CPZ-photodegradation. It was demonstrated that UV irradiation produces a stable fluorescent photoproduct of CPZ covalently bound to Na+, K+-ATPase. The fluorescent probe AO, which specifically binds to the extracellular ouabain site of the pump, was used to localize the CPZ binding site. UV-irradiation of AO-labeled Na+, K+-ATPase treated with CPZ at concentration about 20 microM produced dose-dependent modifications of the AO fluorescence, e.g. increased quantum yield and blue shift. The results demonstrated that CPZ binds near the ouabain site. The photo-induced reaction of CPZ with AO-labeled Na+, K+-ATPase protected the ouabain site from the aqueous environment. It was also found that UV irradiation of CPZ-treated enzyme obstructs the binding of AO, which suggested occlusion of the ouabain site. This effect can be evaluated for a potential use of CPZ in photochemotherapy.

Identification and distribution of chondroitin sulfate in the three electric organs of the electric eel, Electrophorus electricus (L.).

The electrogenic tissue of the electric eel Electrophorus electricus (L.) is distributed in three well-defined electric organs, the Main electric organ, Sach's organ and Hunter's organ. Sulfated glycosaminoglycan (GAG) composition was characterized in the three electric organs of the electric eel. Sulfated GAGs were analyzed in the electric organs using metachromatic staining, biochemical analysis including electrophoresis before and after specific enzymatic or chemical degradations, and immunostaining with an antibody against chondroitin sulfate (CS). Our results showed in the three electric organs that CS was the main sulfated GAG species detected, accompanied by small and diminutive amounts of CS/dermatan sulfate hybrid chains and heparan sulfate (HS), respectively. However, HS was not detected in the Sach's organ. CS was predominantly detected in the innervated membrane face of the electroplaques in the three electric organs. Our findings extend previous observations on the GAG composition in the electric organs of E. electricus and provide new information regarding the tissue distribution and location of CS.

Purification and amino acid sequence of fructose-1,6-bisphosphate aldolase from the electric organ of Electrophorus electricus (L.).

A soluble fructose-1,6-bisphosphate aldolase enzyme has been purified 50.2-fold (2.36%) at the homogeneity from the electric organ of Electrophorus electricus by one step of DEAE-52 anion exchange chromatography followed by Superose-12 gel filtration-FPLC. Like other aldolase enzymes the E. electricus protein is a dimer with two identical subunits of 45 kDa. The N-terminal (20 residues) revealed a high homology with S. aurata (75%, goldfish), R. ratus and M. musculus (mouse, 80%) enzymes.

Influence of nitric oxide donors on the intrinsic fluorescence of Na+,K+-ATPase and effects on the membrane lipids.

Effects of the nitric oxide donors S-nitroso-glutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) on Na+,K+-ATPase-rich membrane fragments purified from pig kidney outer medulla were studied using intrinsic fluorescence and ESR of spin-labeled membranes. These S-nitrosothiols differently affected the intrinsic fluorescence of Na+,K+-ATPase: GSNO induced a partial quenching, whereas SNAP produced no alteration. Quenching can be due to a direct modification of exposed tryptophan residues or to an indirect effect caused by reactions of nitrogen oxide reactive species with other residues or even with the membrane lipids. Pre-incubation of Na+,K+-ATPase with 0.4mM GSNO resulted in a modest inhibition of ATPase activity (about 24%) measured under optimal conditions. Stearic acid spin-labeled at the 14th carbon atom (14-SASL) was used to investigate membrane fluidity and the protein-lipid interface. SNAP slightly increased the mobility of bulk lipids from Na+,K+-ATPase-rich membranes, but did not change the fraction of bulk to protein-interacting lipids. Conversely, treatment with GSNO extinguished the ESR signals from 14-SASL, indicating generation of free radicals with high affinity for the lipid moiety. Our results demonstrated that membranes influence bioavailability of reactive nitrogen species and bias the activity of different S-nitrosothiols.

Toxicity induced by Hg2+ on choline acetyltransferase activity from E. electricus (L.) electrocytes: the protective effect of 2,3 dimercapto-propanol (BAL).

BACKGROUND: The effect of mercury (Hg(2+)) on the activity of choline acetyltransferase (ChAT) from electrocytes of Electrophorus electricus (L.) was studied due to the importance of this enzyme and acetylcholine in many neurochemical functions such as arousal, learning, and memory. MATERIAL/METHODS: Mercury, which has affinity to thiol groups, acted as a potent inhibitor of ChAT, which was obtained by differential centrifugation and ammonium sulfate precipitation, at 80%, from the main electric organ homogenate. RESULTS: Mercury inhibition presents different kinetic behaviors for both enzyme substrates: noncompetitive to choline and of mixed type to AcCoA, with inhibition constants on the order of 0.5 to 1.0 microM. The enzyme activity was recovered using 2,3 dimercapto-propanol (BAL), a well-known chelate for sulphydryl groups and metals, which acted as a protecting agent and was able to revert the Hg(2+) inhibition at a concentration of 10 (-6) M. After treatment with this metal and in the presence of 2,3 dimercapto-propanol, 70% of the enzyme activity was recovered for AcCoA and 80% for choline. CONCLUSIONS: The observed inhibition is likely due to direct protein interaction, because the addition of BAL reversed the effects of HgCl(2) on ChAT activity. The results cast new light on the mechanisms of mercurial neurotoxicity.

Choline acetyltransferase detection in normal and denervated electrocyte from Electrophorus electricus (L.) using a confocal scanning optical microscopy analysis

Acetylcholine is the neurotransmitter responsible for the transmission of impulses from cholinergic neurons to cells of innervated tissues. Its biosynthesis is catalyzed by the enzyme Choline acetyltransferase that is considered to be a phenotypically specific marker for cholinergic system. It is well known that the regulation of Choline acetyltransferase activity under physiological and pathological conditions is important for development and neuronal activities of cholinergic functions. We observed the distribution of Choline acetyltransferase in sections from the normal and denervated main electric organ sections of Electrophorus electricus (L.) by immunofluorescence using a anti-Choline acetyltransferase antibody. The animals were submitted to a surgical procedure to remove about 20 nerves and after 30 and 60 days, they were sacrificed. After 30 days, the results from immunohistochemistry demonstrated an increase on the Choline acetyltransferase distribution at denervated tissue sections when compared with the sections from the normal contralateral organ. A very similar labeling was observed between normal and denervated tissue sections of the animals after 60 days. However, Choline acetyltransferase activity (nmolesACh/ min/ mg of protein) in extracts obtained from electrocyte microsomal preparation, estimated by Fonnun's method (Fonnun 1975), was 70 per cent lower in the denervated extracts.