Catharacta maccormicki blood samples were collected in the winter (October) and in the summer (February) in order to study the intraerythrocytic organic phosphates, hemoglobin (Hb) electrophoretic patterns, oxygen blood equilibrium and stripped Hbs, as well as the effect of 2,3-biphosphoglycerate (BPG) and inositol hexaphosphate (IHP) on oxygen affinity. All the samples (five from the winter and five from the summer) showed the same electrophoretic pattern: one minor fast component and one major slow one. No differences in oxygen affinity and Bohr effect in the samples collected in the winter and in the summer were found. Oxygen affinity was higher in the stripped Hb than in the blood. BPG seemed to have no effect on the functional properties of skua Hb while IHP does. No BPG was found in any sample. Both inositol pentaphosphate (IP5) and IHP were found in all the samples. The IP5/IHP ratio in the winter samples was 3.0 while in summer 3.5. Adenosine diphosphate (ADP) was found in samples from both the seasons. Adenosine monophosphate (AMP) and adenosine triphosphate (ATP) were present only in the summer samples while guanosine triphosphate (GTP) was found in the winter samples. Since IP5 and IHP are very powerful HB allosteric effectors, ATP and GTP might function as other protein modulators.

Kinetic properties and thermal stabilities of Geophagus brasiliensis skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37) and its isolated isoforms were analyzed to examine a possible sMDH-B* locus duplication in a fixation process influenced by genetic drift. Two optimal pHs were detected: 7.5 for AB1 unfractionated muscle phenotype and its B1 isoform, and 8.0 for AB1B2 unfractionated muscle phenotype, A and B2 isoforms. While G. brasiliensis A isoform could be characterized as thermostable, the duplicated B isoform cannot be assumed as thermolabile. Km values for isolated B2 isoforms were 1.6 times lower than for B1. A duplication event in progress best explains the electrophoretic six-band pattern detected in G. brasiliensis, which would be caused by genetic drift.

O número de escamas em séries laterais (SNS) de exemplares de Mugil sp. (Mugilidae, Perciformes) coletados na região estuarino-lagunar de Cananéia, Estado de São Paulo, varia de 33 a 39. A fim de tentar determinar se exemplares com diferentes SNS corresponderiam a diferentes espécies ou populações de tainhas, foram analisadas as propriedades eletrocinéticas, cinéticas e de termoestabilidade da sua lactato desidrogenase (LDH). A exemplo de muitos teleósteos, a LDH de Mugil sp. mostrou-se codificada por 3 locos gênicos: LDH-A*, LDH-B* e LDH-C*. Método de diluições seriadas aplicado a 10 diferentes tecidos dessa espécie sugeriu um padrão bidirecionalmente divergente de expressão desses locos da LDH. Não foi observada nenhuma associação entre padrão eletroforético, inativação térmica, respostas cinéticas e diferentes SNS. Os valores de Km (pyr) aparente aqui obtidos mostraram-se similares aos obtidos por outros autores para músculo e coração, bem como aos de suas isoformas purificadas. O efeito do NaCl nos valores de Km e Vmax de Mugil sp. (SNS de 35 e 39) indica que esse sal se comporta como um inibidor competitivo, já que decresce a afinidade enzima-substrato. Assim, os comportamentos eletrocinéticos e de termoestabilidade, valores de Km e Vmax e os efeitos do NaCl sobre esses não nos permitem caracterizar as tainhas capturadas na região estuarino-lagunar de Cananéia, com amplitudes de SNS de 33 a 39, como diferentes espécies ou populações.

The scale number in lateral sets (SNS) of Mugil sp. (Mugilidae, Perciformes) collected in the lagoon-estuarine region of Cananéia, State of São Paulo ranges from 33 to 39. Electrokinetic, kinetic and thermostability properties of lactate dehydrogenase (LDH) were tested to determine if individuals with different SNS correspond to different species or populations of mullet. As in many other teleosts, LDH-A*, LDH-B*, and LDH-C* loci were detected. Through a two-fold serial dilution method applied to 10 different tissues of Mugil sp., a bidirectionally divergent expression of these loci was suggested. No association among LDH electrophoretic pattern, thermal inactivation, kinetic responses and different SNS was observed. The apparent Km (pyr) values obtained here were similar to Km values obtained by other authors for muscle and heart LDH or their purified isoforms. The effect of NaCl on Km and Vmax values of Mugil sp. (35 and 39 SNS individuals) indicates that this salt behaves as a competitive inhibitor, since it decreases enzyme-substrate affinity. Thus, electrokinetic and thermostability behavior, Km and Vmax values and the effect of NaCl do not permit us to consider these mullets, with SNS ranging from 33 to 39, as belonging to different populations or species.

A fim de explicar o padrão eletroforético de seis componentes detectado para a malato desidrogenase solúvel (MDH, EC 1.1.1.37) em 84% dos exemplares de G. brasiliensis analisados (Cichlidae, Perciformes), uma duplicação recente no loco sMDH-B* é sugerida. Diluições seriadas de Klebe realizadas com extratos de músculo esquelético mostraram para as subunidades B1 e B2 o mesmo ponto final visual sugerindo um padrão de expressão não divergente para esses genes duplicados. Uma vez que não existe evidência de poliploidia na família Cichlidae, é sugerido que a duplicação no loco sMDH-B* seja resultante de uma duplicação regional. Especificidade tissular, termoestabilidade e propriedades cinéticas mostraram-se similares para as isoformas B, em ambos os fenótipos detectados, sugerindo estarem esses sob a ação do mesmo gene regulador. Os resultados similares obtidos para os fenótipos de três (AB1) e seis (AB1B2) componentes aqui analisados não mostraram nenhum indicativo de vantagem adaptativa deste último sobre o primeiro, em região subtropical.

A recent locus duplication hypothesis for sMDH-B* was proposed to explain the complex electrophoretic pattern of six bands detected for the soluble form of malate dehydrogenase (MDH, EC 1.1.1.37) in 84% of the Geophagus brasiliensis (Cichlidae, Perciformes) analyzed (AB1B2 individuals). Klebe's serial dilutions were carried out in skeletal muscle extracts. B1 and B2 subunits had the same visual end-points, reflecting a nondivergent pattern for these B-duplicated genes. Since there is no evidence of polyploidy in the Cichlidae family, MDH-B* loci must have evolved from regional gene duplication. Tissue specificities, thermostability and kinetic tests resulted in similar responses from both B-isoforms, in both sMDH phenotypes, suggesting that these more recently duplicated loci underwent the same regulatory gene action. Similar results obtained with the two sMDH phenotypes did not show any indication of a six-banded specimen adaptive advantage in subtropical regions.

No presente trabalho, mostramos em ortólogos da sMDH de 21 espécies de peixes subtropicais das ordens Characiformes, Siluriformes e Perciformes, três diferentes estabilidades térmicas. A primeira, caracterizada pela termoestabilidade do componente menos anódico ou isoforma-A, foi detectada em 52% de todas as espécies. A segunda, exibida por 29% das espécies aqui analisadas, caracterizou-se por um padrão não-divergente de termoestabilidade dos locos sMDH-A* e sMDH-B*. Na terceira resposta, obtida em 19% das espécies analisadas (as 4 espécies Siluriformes), foi observada a termoestabilidade da banda mais anódica ou isoforma-B. O efeito da temperatura ambiental na atividade relativa de isoformas parálogas e ortólogas de duas dessas espécies com diferentes respostas térmicas (Leporinus friderici - sMDH-A*, termoestável, e Pimelodus maculatus, termoestabilidade reversa ou padrão eletroforético reverso), coletadas em meses de inverno e de verão, mostrou que as subunidades A e B estão presentes em seus extratos de tecidos em diferentes níveis quantitativos e suas atividades relativas são, praticamente, independentes da época de coleta. Na incubação de extratos de tecidos dessas 2 espécies a 50°C, diferentes respostas de inativação térmica foram dadas pelos locos da sMDH. Em P. maculatus, a resposta obtida em músculo esquelético ajudou a escolher a hipótese mais provável - a da termoestabilidade reversa, onde a banda mais anódica ou isoforma-B é a sMDH termoestável. Assim, diferenças na susceptibilidade à temperatura parecem ter ocorrido entre homólogos ortólogos da sMDH, nessas espécies, onde a banda mais rápida, normalmente músculo-específica e termolábil na maioria dos teleósteos, aparece em P. maculatus como a isoforma termoestável.

Electrophoretic thermostability tests of soluble malate dehydrogenases (sMDH) isozymes in tissue extracts of 21 subtropical fish belonging to the orders Characiformes, Siluriformes and Perciformes showed three distinct results. The first, characterized by thermal stability of the slowest-migrating band or A-isoform, was detected in 52% of all species. The second, exhibited in 29% of the species analyzed, had a bidirectionally divergent pattern of their sMDH locus expression, and was characterized by a nondivergent thermostability pattern of both sMDH-A* and B*. In the third category, obtained in 19% of the species studied (the four Siluriformes species), thermostability of the fastest-migrating bands, or B-isoforms, was observed. Comparison of the effects of habitat temperature on the activity of paralogous and orthologous isoforms in tissue extracts of two of these species with different thermostability properties (Leporinus friderici - thermostable sMDH-A*, and Pimelodus maculatus - reverse thermostability properties or reverse electrophoretic pattern), collected during winter and summer months, showed that A and B subunits were present at different quantitative levels and their activities were nearly season independent. Differences in susceptibility to temperature (50°C) of both sMDH loci from tissue extracts of these species were found. In P. maculatus, these susceptibilities helped strengthen one of the hypotheses: the reverse thermostability pattern, where the fastest-migrating band or the B-isoform was the thermostable sMDH. Thus, temperature differences among orthologous homologues of sMDH seem to have occurred in these acclimatized species, where the fastest-migrating band, usually muscle specific and thermolabile in most teleosts, appeared in P. maculatus as the thermostable isoform.