A. Hassón-Voloch

Inhibition of acetylcholinesterase from Electrophorus electricus (L.) by tricyclic antidepressants

The effects of tricyclic antidepressants drugs (TCA) amitriptyline, imipramine and nortriptyline, on purified Electrophorus electricus (L.) acetylcholinesterase (AChE; acetylcholine hydrolase, EC 3.1.1.7) were studied using kinetic methods and specific fluorescent probe propidium. The antidepressants inhibited AChE activity by a non-competitive mechanism. Inhibition constants range from 200 to 400 microM. Dimethylated amitriptyline and imipramine were more potent inhibitors than the monomethylated nortriptyline. Fluorescence measurements using bis-quaternary ligand propidium were used to monitor ligand-binding properties of these cationic antidepressants to the AChE peripheral anionic site (PAS). This ligand exhibited an eight-fold fluorescence enhancement upon binding to the peripheral anionic site of AChE from E. electricus (L.) with K(D)=7 x 10(-7)M. It was observed that TCA drugs displaced propidium from the enzyme. On the basis of the displacement experiments antidepressant dissociation constants were determined. Similar values for the inhibition constants suggest that these drugs have similar affinity to the peripheral anionic site. The results also indicate that the catalytic active center of AChE does not participate in the interaction of enzyme with tricyclic antidepressants. These studies suggest that the binding site for tricyclic antidepressants is located at the peripheral anionic site of E. electricus (L.) acetylcholinesterase.

Biochemical and cytochemical localization of ATPases on the membranes of the electrocyte of Electrophorus electricus

SummaryThe localization of (Na+-K+) ATPase in the intact electrocyte of the electric organ of Electrophorus electricus (L.) and its subcellular fractions was investigated by biochemical and cytochemical methods. The distribution of AChE activity in the subcellular fractions was also comparatively analysed with this enzyme serving as a marker of the innervated membranes of the electrocyte. After application of cytochemical method of Farquhar and Palade to glutaraldehyde-fixed tissue, reaction was observed only at the membranes of vesicles localized at the periphery of the electrocyte. Previously fixed electrocytes, incubated in Ernsts medium showed reaction only at the vesicles whereas in unfixed tissue reaction also appeared at other membranes (surface and invaginations) of the anterior and posterior faces. This reaction was significantly inhibited in the presence of ouabain or in the absence of K+. Inhibition of Na+-K+-ATPase by glutaraldehyde fixation was also confirmed by biochemical analysis.

Isolation and characterization of a highly purified flagellar membrane fraction from trypanosomatids.

The flagellar membrane of trypanosomatids has certain ultrastructural and antigenic characteristics that make its biochemical analysis very interesting. We have obtained a highly purified flagellar membrane fraction from epimastigotes of Trypanosoma cruzi and promastigotes of Herpetomonas samuelpessoai. The fractions consisted of regular spherical vesicles. Membrane fractions fixed in a glutaraldehyde solution containing filipin and freeze-fractured showed few intramembranous particles, and many protuberances indicative of filipin-sterol complexes. The protein composition of all the subflagellar fractions was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The flagellar membrane fraction showed more than twenty bands. We wish particularly to point out the presence of six bands corresponding to proteins of 160, 135, 41, 31, 26 and 18 kDa which were not present in the axoneme-paraxial structure fraction. The blot of flagellar membrane proteins successively incubated with concanavalin A and horseradish peroxidase showed intense binding of the lectin to proteins of 117 and 87 kDa, and less strong binding to several other minor bands. Our results showed that although the membrane of the flagellum of trypanosomatids had few proteins, it seemed rich in glycosylated elements.

Electrocyte (Na+, K+)ATPase inhibition induced by zinc is reverted by dithiothreitol

The Mg(2+)-dependent (Na(+),K(+))ATPase maintains several cellular processes and is essential for cell excitability. In view of the importance of the enzyme activity, the interaction and binding affinities to substrates and metal ions have been studied. We determined the effect of Zinc ion (Zn(2+)) on the (Na(+),K(+))ATPase activity present in both conducting (non-innervated) and post-synaptic (innervated) membranes of electrocyte from Electrophorus electricus (L.). Zn(2+) is involved in many biological functions and is present in pre-synaptic nerve terminals. This metal, which has affinity for thiol groups, acted as a potent competitive inhibitor of (Na(+),K(+))ATPase of both membrane fractions, which were obtained by differential centrifugation of the E. electricus main electric organ homogenate. We tried to recover the enzyme activity using dithiothreitol, a reducing agent. Kinetic analysis showed that dithiothreitol acted as a non-essential non-competitive activator of (Na(+),K(+))ATPase from both membrane fractions and was able to revert the Zn(2+) inhibition at mM concentrations. In the presence of dithiothreitol, this metal behaved as a competitive inhibitor of (Na(+),K(+))ATPase in the non-innervated membrane fractions and presented a non-competitive inhibition of (Na(+),K(+))ATPase in innervated membrane fractions. This difference may be attributed to formation of a Zn-dithiothreitol complex, as well as the involvement of other binding sites for both agents. The consequences of the enzyme inhibition by Zn(2+) may be considered in regard to its neurotoxic effects.

Effect of Li+ and Ba2+ on the electrocyte membrane-bound (Na+ + K+)-ATPase

Membrane-bound (Na+ + K+)-ATPase activity from the non-innervated and innervated faces of Electrophorus electricus (L.) electric organ, obtained by differential centrifugation, was measured using AChE as an enzyme marker for membranes derived from the post-synaptic area (fraction P3) of the electrocyte. The effect of Li+ and Ba2+ on (Na+ + K+)-ATPase activity of the two membrane fractions (P2 and P3) was analysed with respect to K+ and Mg2+ ions, after the I50 estimation. The kinetics of the reactions with these cations were investigated showing that Li+ inhibits P2 uncompetitively and for P3 presented a mixed type inhibition. Ba2+ behaved as an hyperbolic mixed type inhibitor for P2 and a linear mixed type inhibitor for P3 fraction.

Creatine kinase from the electric organ of Electrophorus electricus (L.)—isozyme analysis

Creatine kinase (CPK) isozymes of extracts from the electric organ, dorsal muscle and brain of Electrophorus electricus (L.) were analysed with Cellogel electrophoresis. A single component corresponding to the MB-form was obtained for both electric organ and the dorsal muscle. The BB-form was present in the brain extract. 2. Upon acetone fractionation of the aqueous of electric organ, the final fraction was submitted to gel filtration and presented a single peak of CPK activity. 3. Characterization of this fraction by thin-layer gel filtration indicated an apparent molecular weight of 80,000 which corresponds to the enzyme dimeric structure. 4. The implications of this finding with the muscular origin of the electric organ are discussed.

The effect of mercury and aluminum on sodium-potassium-Mg2+ dependent-adenosine triphosphatase activity of electrophorus electricus (L.) electrocyte

Abstract 1. 1. Na + ,K + -ATPase, which mediates the active transport of Na + and K + across the plasma membrane, is found in equivalent amounts in both plasma membranes of the electrocyte, the anterior, non-innervated (fraction P 2 ) and the posterior, innervated (fraction P 3 ) obtained by differential centrifu gation of Electrophorus electricus (L.) electric organ. 2. 2. The kinetic effects of Hg 2+ and A1 3+ , described as neurotoxic metals, on the Na + ,K + -ATPase activity of the two membrane fractions (P 2 and P 3 ) were analysed with respect to Na + and K + ions, after the I 50 estimation of each metal. 3. 3. Mercury is a potent Na + ,K + -ATPase inhibitor in the nanomolar range. In all cases, it behaved as a mixed partial hyperbolic inhibitor. 4. 4. Aluminum was shown to be a poor enzyme inhibitor. Changing the K + concentration, it behaved as a mixed linear inhibitor (P 2 fraction) and as a non-essential mixed activator (fraction P 3 ). Aluminum behaved as a partial hyperbolic inhibitor for both P 2 and P 3 fractions with respect to Na + concentration. 5. 5. The observation of the variable kinetic behaviour of P 2 and P 3 led us to attribute these differences to the Na + ,K + -ATPase electrocyte isoenzymes which occur in different proportions in these fractions (Gomes-Quintana et al. , 1992 Comp. biochem. Physiol. 103B/3 623–628).

Lactate dehydrogenase from the electric organ of electrophorus electricus (L.)—isozyme analysis

Abstract 1. 1. Analysis of LDH isozymes from the electric organ of Electrophorus electricus (L.) was performed with Cellogel and disc electrophoresis. 2. 2. Several extracting media were employed for the enzyme analysis and only a single component with LDH activity was obtained, which had the same mobility as the muscle LDH of Electrophorus. 3. 3. Sucrose gradient ultracentrifugation analysis confirmed the presence of only one form of LDH in electric organ extracts. 4. 4. The identity of this form with muscle LDH confirms that the electric organ results from the differentiation of muscular tissue.

Cytochemistry and freeze-fracture of membranes isolated from the electrocyte of Electrophorus electricus (L.).

SummaryMembranes were isolated from the main electric organ of Electrophorus electricus and studied by means of cytochemistry and freezefracture. The membrane fractions consisted of vesicles inside-in as determined by localization of anionic sites using colloidal iron and cationized ferritin particles. The anionic sites were not homogeneously distributed on the surface of the vesicle. Freeze-fracture showed the presence of intramembranous particles associated with either protoplasmic (P) or extracellular (E) faces of the membrane. Regions of the membrane without particles were observed. The results are discussed in relation to the existence of association between intramembranous particles and membrane receptors.

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 μM 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.