Mitochondrial and supernatant glutamate-oxalacetate transaminases (EC 2.6.1.1) and supernatant glutamate-pyruvate transaminase (EC 2.6.1.2) were purified 89, 204 and 240-fold respectively, from dolphin muscle. Starch gel electrophoresis of crude and purified perparations revealed that all three enzymes exist as single forms. Km values of a-ketoglutarate, alanine, pyruvate and glutamate for the glutamate-pyruvate transaminase were 0.45, 8.2, 0.87 and 15 mM, respectively. For the glutamate-oxalacetate transaminases, the Km values of a-ketoglutarate, aspartate, oxalacetate and glutamate were 0.76, 0.50, 0.10 and 9.4 mM, respectively, for the mitochondrial form and 0.13, 2.4, 0.06 and 3.2 mM, respectively, for the supernatant form. In all cases, as the assay pH was decreased from pH 7.3, the Km values of the a-keto acids decreased while those of the amino acids increased. This caused the apparent equilibrium constants for the glutamate-oxalacetate transaminases to remain independent of pH. These values were 9.2 and 6.8 for the mitochondrial and supernatant forms, respectively where K'eq = [asPartate][α-ketoglutarate]/[glutamate][oxalacetate]. Studies of the inhibition of the glutamate-oxalacetate transaminases by dicarboxylic acids indicated that these enzymes may be controlled by pools of metabolic intermediates. Three key roles are suggested for the transminases in the energy metabolism of the diving mammal. First, it is believed that a combined action of the transaminases could enhance energy production during hypoxia by providing (1) fumarate from aspartate for the ATP producing reversal of succinate dehydrogenase and (2) α-ketoglutarate from glutamate for the GTP producing succinyl thiokinase reaction. Next, diving mammals probably accumulate more NADH than other mammals during hypoxia. The glutamate-oxalacetate transaminases seem particularly well suited for restoring redox balance via the malate-aspartate cycle after aerobic metabolism is resumed. Finally, since migrating divers oxidize large amounts of stored fats, the combined reactions of the transaminases could be instrumental in providing increased supplies of oxalacetate to condense with the fat derived acetyl CoA in the citrate synthase reaction. / Science, Faculty of / Zoology, Department of / Graduate

The concerns of the present study were to determine 1) the conditions necessary to measure displaceable [³H] L-aspartate binding to membrane fractions of the rat brain, 2) whether the binding demonstrated the charcteristics of the site which is active in vivo, and 3) whether the acidic amino acid neurotransmitters aspartate and glutamate bind to identical or different sites by comparing the pharmacological specificities of the [³H] L-aspartate binding with that of [³H] L-glutamate. The conditions of the [³H] L-aspartate binding assay were determined in synaptosomal and total particulate fractions of whole rat brain. The reaction mixture which included the membrane fraction suspended in Tris-HCl buffer (pH 7.4) in the presence or absence of the compound under test, was incubated at 37°C for 30 minutes. The reaction was stopped by centrifugation and the radioactivity in the pellet counted by liquid scintillation spectrometry. The [³H] L-aspartate binding was characterized in total particulate fractions of rat cerebellum. The apparent dissociation constant (K[sub=D]) and maximum binding (Bmax), as determined by Scatchard analysis, are 1.64 ± 0.34 μM and 7711 ± fmol/mg protein respectively. The displaceable binding is reversible, saturable, independent of the presence of NA⁺, has an affinity in the range where the neurotransmitter is active in vivo, and demonstrates a pharmacological specificity which includes stereospecificity. The compounds tested to demonstrate the pharmacological specificity were L-aspartate (IC[sub=50] = 1.81 μM), D-aspartate (IC[sub=50] = 46.6 μM), L-glutamate (IC[sub=50] = 1.24 μM), N-methyl-DL-aspartate (inactive), kainate (inactive), D-alpha-aminoadipate (inactive), and L-alpha-aminoadipate (IC[sub=50] =7.12 μM). The pharmacological specificity of [³H] L-aspartate binding was different from that of [³H] L-glutamate. When the binding data only are considered, therefore, separate receptors for aspartate and glutamate are indicated. The pharmacological specificity of the [³H] L-aspartate binding, that is the affinity of the binding site for N-methyl-DL-aspartate, D- and L-alpha-aminoadipate, however, does not correlate with the potency of these compounds derived from iontophoretic studies. L-alpha-aminoadipate is very effective while N-methyl-DL-aspartate and D-alpha-aminoadipate do not displace the [³H] L-aspartate binding. In iontophoretic studies, N-methyl-D-aspartate and D-alpha-aminoadipate are very potent as compared to aspartate while L-alpha-aminoadipate Is inactive. The [³H] L-aspartate binding then may not represent the site which is active in vivo. The characteristics of the aspartate site in vivo, however, may not be truely represented in iontophoretic studies because of, for example, uptake of the compounds. The aspartate binding site, therefore, must be identified as that which is activated in vivo. The question of separate receptors for aspartate and glutamate then must still be resolved. / Science, Faculty of / Zoology, Department of / Graduate

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / In the present work, behavioral, neurochemical (determination of monoamines and metabolites levels rat in striatum), histopatological and biochemical (lipoproteins and transaminases) alterations produced by cocaine, ethanol and the association of theses were analyzed. Females Wistar rats (180-200 g) were treated during 7 days with cocaine (Coc 10 and 20 mg/kg, i.p.), ethanol (Et 2 and 4g/kg, p.o.) and the association of theses (Coc 10 mg + Et 2g - low interaction doses; Coc 20 mg + Ethanol 4g - high interaction doses). The results demonstrated that the spontaneous locomotor activity (SLA) was increased after cocaine administration and decreased after ethanol in both doses. It was not observed alterations in the SLA in the association cocaine + ethanol. The treatment with cocaine and ethanol caused an increase in dopamine level. The association cocaine + ethanol in higher doses caused an increase of dopamine and serotonin and decrease of DOPAC levels, suggesting that those drugs would can actuate directly in those systems or, indirectly, across a process of modulation. Cocaine, ethanol and the association of theses, after subcronic administration and in both doses, caused a donwregulation of D2-like receptors, not by recurring alterations in the values of Kd. The values of Bmax and Kd of the M1 + M2-like receptors have not already suffered alterations. In the biochemical study, the administration of cocaine induced an increase the concentrations of TGO and triglycerides, and decrease of the concentrations of TGP, total cholesterol and HDL. The treatment with ethanol decreases the levels of HDL, total cholesterol and triglycerides. The association cocaine + ethanol caused in both doses decrease of triglycerides, HDL, TGP and total cholesterol. All treatments did promote histopatological alterations in cardiac and hepatic woven. Ours results suggest that the association cocaine + ethanol appears to interfere more intense in the systems of neurotransmitters and in the biochemical parameters than the use of the isolated drugs. / No presente trabalho foram estudadas as alteraÃÃes comportamentais, neuroquÃmicas (determinaÃÃes dos nÃveis de monoaminas e metabÃlitos), histopatolÃgicas e bioquÃmicas (lipoproteÃnas e transaminases) em corpo estriado de ratos tratados com cocaÃna e etanol isoladamente ou em associaÃÃo. Foram utilizadas ratas Wistar (180-200 g), que foram tratadas durante 7 dias com cocaÃna (Coc 10 e 20 mg/kg, i.p.), etanol (Et 2 e 4g/kg, v.o.) e a associaÃÃo destes (Coc 10 mg + Et 2g - interaÃÃo baixas doses; Coc 20 mg + Etanol 4g - interaÃÃo altas doses). Os resultados demonstraram que a atividade locomotora espontÃnea (ALE) foi aumentada apÃs administraÃÃo de cocaÃna em ambas as doses e diminuÃda apÃs a administraÃÃo de etanol em ambas as doses. NÃo foram observadas alteraÃÃes na ALE na associaÃÃo cocaÃna + etanol. O tratamento com cocaÃna e etanol causou um aumento de dopamina, sem alteraÃÃes nos demais neurotransmissores e metabÃlitos. A associaÃÃo cocaÃna + etanol em altas doses, promoveu aumento dos nÃveis de dopamina, diminuiÃÃo de DOPAC e aumento dos nÃveis de 5-HT, sugerindo que essas drogas poderiam atuar diretamente nesses sistemas ou, indiretamente, atravÃs de um processo de modulaÃÃo. A cocaÃna, etanol e a associaÃÃo destes, apÃs administraÃÃo sub-crÃnica e em ambas as doses, causou uma downregulation em receptores D2-sÃmile, nÃo ocorrendo alteraÃÃes nos valores de Kd. Os valores de Bmax e Kd dos receptores M1 + M2-sÃmile nÃo sofreram alteraÃÃes. No estudo bioquÃmico, a administraÃÃo de cocaÃna induziu um aumento nas concentraÃÃes de TGO e triglicerÃdeos, e diminuiÃÃo das concentraÃÃes de TGP, colesterol total e HDL. O tratamento com etanol diminuiu os nÃveis de HDL, colesterol total e triglicerÃdeos. A associaÃÃo cocaÃna + etanol promoveu em ambas as doses diminuiÃÃo de trigicerÃdeos, HDL, TGP e colesterol total. Todos os tratamentos promoveram alteraÃÃes histopatolÃgicas em tecido cardÃaco e hepÃtico. Nossos resultados sugerem que a associaÃÃo cocaÃna + etanol parece interferir de maneira mais intensa nos sistemas de neurotransmissÃo e nos parÃmetros bioquÃmicos do que o uso das drogas isoladas.