Arno Rudi Schwantes

Adaptative features of ectothermic enzymes--I. Temperature effects on the malate dehydrogenase from a temperate fish Leiostomus xanthurus.

1. Following electrophoresis the s-MDH activity of Leiostomus xanthurus and many other species of fish and amphibian appears in three sharp, equally-spaced, anodal bands. Each band is a dimer (AA, AB and BB) and two loci are active. 2. In Leiostomus tissue extracts A and B subunits are present are differing quantitative levels and their activities can be modified by changes in environment temperature. 3. Thermostability and thermal dependency tests show that, similar to what occurs during acclimatization, the AA isozyme is more stable to heat than is the BB isozyme. The BB isozyme is activated by low temperatures and is rapidly inactivated by high temperatures. 4. Extracts from a variety of fishes, amphibians, reptiles and birds suggest that when only one or two s-MDH bands are present, they behave as dose the AA homodimer in Leiostomus Xanthurus, i.e., are stable at elevated temperatures.

Loci that encode the lactate dehydrogenase in 23 species of fish belonging to the orders Cypriniformes, Siluriformes and Perciformes: Adaptative features

Abstract 1. 1. Electrophoretic analyses of the lactate dehydrogenase isozyme patterns of 23 species of tropical fishes belonging to the orders Cypriniformes, Siluriformes and Perciformes indicates that at least two LDH loci—Ldh-A and Ldh-B are active. In the Perciformes species, in addition to these loci, the Ldh-C is also present, and restricted in its expression to the eye and brain. 2. 2. Genetic variants were detected in the Ldh-A and Ldh-B loci of Leporinus friderici and in the Ldh-B locus of Schizodon borelli Cypriniformes. 3. 3. An increase in the occurrence of the Ldh-BB′ and Ldh-B′B′ allotypes of Leporinus friderici was observed along the successive collects that started in 1980. This change in phenotypic frequency suggests that this polymorphism could be related to environmental conditions.

Adaptative features of ectothermic enzymes—IV. Studies on malate dehydrogenase of Astyanax fasciatus (Characidae) from lobo reservoir (São Carlos, São Paulo, Brasil)

1. Skeletal muscle and heart supernatant malate dehydrogenase (s-MDH) from a subtropical fish, Astyanax fasciatus consists of three electrophoretically anodal bands. Each band is a dimer (AA, AB and BB) and two loci are active. 2. In A. fasciatus tissue extracts, A and B subunits are present at differing quantitative levels and their activities are almost season-independent. However, the relative activity of each homodimer in relation to total s-MDH estimated by densitometry of gels or of each homodimer purified by chromatography varies with temperature. The more anodic homodimer is thermolabile and the less anodic one is thermostable. 3. The pH optimum of s-MDH is 7.5, of AA is 6.5 and of BB is 7.8. 4. The BB isozyme is more sensitive to high concentrations of substrate and has a Km temperature-independent. The AA isozyme is not inhibited by high concentrations of oxaloacetate and shows a Km temperature-dependent with a fourteenfold increase between 20 degrees and 40 degrees C.

Adaptative features of ectothermic enzymes—II. The effects of acclimation temperature on the malate dehydrogenase of the spot, Leiostomus xanthurus

1. Isozyme patterns and thermostability of the skeletal muscle and heart malate dehydrogenase (s-MDH) from the spot acclimated to different temperatures were examined. 2. No changes in isozyme patterns were seen for MDH in any of the tissues examined in response to 15 degrees and 20 degrees C acclimation. 3. The A-homodimer, which was more thermostable showed an increase in its relative activity, whereas the B-homodimer, which was more heat sensitive, showed a decrease during warm acclimation. However, after different periods of low temperature incubation, these samples showed a decrease in their subunit ratios. 4. Concerning the effect to thermal acclimation on the thermostability of MDH, it was found that skeletal muscle samples of the 30 degrees C-acclimated spot were more stable to heat than the 20 degrees and 15 degrees C-acclimated fishes.

Adaptative features of enzymes from family sciaenidae (perciformes)—I. studies on soluble malate dehydrogenase (s-MDH) and creatine kinase (CK) of fishes from the south coast of uruguay

1. Electrophoretic analysis of the soluble malate dehydrogenase (s-MDH) and creatine kinase (CK) isozyme patterns of three species of temperate fishes (Scianidae, Perciformes) indicates at least two loci for s-MDH, Mdh-A and Mdh-B, and four CK, Ck-A, Ck-B, Ck-C and Ck-D. 2. The subunits encoded by these loci occurred at different levels in different tissues and organs analyzed. 3. Through electrophoretic analysis the products of these loci showed different behaviour to changes in temperature. 4. Relative activities of s-MDH and CK isozymes were compared by Klebes (1975) method to determine pattern of divergence of duplicated gene expression in the three studied species.

Biological aspects of Amazonian fishes--VI. Hemoglobins and whole blood properties of Semaprochilodus species (Prochilodontidae) at two phases of migration.

Electrophoretic patterns of hemoglobins, red blood cell phosphates, Bohr effect of blood and stripped hemoglobin, ATP and GTP modulation of Hb-O2 affinity of two species belonging to the genus Semaprochilodus (S. insignis and S. taeniurus--jaraquis), captured at two different locations in Rio Negro, near Manaus (Amazonas), are described. High Bohr effect values were found in these two species and in jaraqui-açu (a possible hybrid between them). In S. insignis a higher Bohr effect for migrating individuals was observed, while S. taeniurus showed the reverse. The main erythrocytic phosphates found in these two species are ATP and GTP. They have a large influence on the O2-Hb binding properties in both species. The GTP/ATP ratio showed large differences between catching places. The two species have different electrophoretic patterns. Semaprochilodus insignis showed four anodal components which showed quantitative variation between catching places; S. taeniurus had three anodal components and a similar quantitative variation.

Electrophoretic studies on polyploid amphibians—III. Lack of locus duplication evidences through tetraploidization

Abstract 1. 1. An investigation of 9 enzymatic systems by starch-gel and polyacrylamide electrophoreses in the tetraploid and diploid species Odontophrynus americanus and O. cultripes is presented. The electrophoretic patterns of five enzymes in the glucose-metabolizing system (critical enzymes) and 4 enzymes (peripheral enzymes) which were not directly involved in the above-mentioned system are compared which resulted in the conclusion that the diploid and tetraploid populations of O. americanus are identical in mobility and number of bands. 2. 2. Comparison between the tetrapoloid O. americanus and a closely related diploid species O. cultripes shows that the critical enzymes, ODH and ADH, exhibit equal number of bands. Most enzymes studied differ in the relative mobility of their bands, with the exception of HBDH, ADH and ODH. 3. 3. Polymorphism is detected for the tetrazolium oxidase (TO), due to two alleles F and S, both in the diploid and tetraploid species of O. americanus and in O. cultripes . The tetraploid O. americanus differs from the diploid species in having two extra phenotypes, suggesting a tetrasomic mode of inheritance. Electrophoreses of individual oocytes show that, in the tetraploid O. americanus , the four genes for TO are active in each cell. 4. 4. Our present results support the assumption that O. americanus 4n is a recent autotetraploid. Gene duplications detected in the tetraploid are prior to polyploidy.

Patterns of gene expression during Prochilodus scrofa (Characiformes: Prochilodontidae) embryogenesis—II. Soluble malate dehydrogenase

Abstract 1. 1. The patterns of expression of the lactate dehydrogenase loci in the differentiated adult tissues and in the early embryonic stages (0.8–180 hr post-fertilization) of curimbata, Prochilodus scrofa, were investigated. 2. 2. As in other Characiformes species, only two bidirectionally divergent LDH loci—LDH-A∗ and B ∗ were differentially expressed among the tissues and sequentially during the embryogenesis of this species. The LDH-A∗ locus was expressed predominantly in white skeletal muscle, and the B ∗ locus in all other tissues examined. 3. 3. LDH-B4 was present throughout embryogenesis and was the unique LDH isozyme until 20 hr post-fertilization (post-hatching). Ldh-A subunit activity, however, was detected only 34 hr post-fertilization (stage 7) and then, only as heterotetramers containing Ldh-B subunits. This later development detection of A subunits and the appearance of both subunits under heteropolymeric forms suggest the concomitant synthesis of these two subunits at this time of development. 4. 4. The A subunits, which predominate in skeletal muscle where they presumably play an important biochemical role in anaerobic energy production, are first detected when curimbata larvae show more accentuate swimming motions. 5. 5. Three allelic variants for LDH-A∗ locus, A ∗ 300 , A ∗ 1000 and A ∗ -400 were detected in adult curimbata. In heterozygous embryos (offspring from matings type II, III and IV), heteropolymers with maternal and/or paternal A100 and A1000 subunits are synchronously expressed 34 hr post-fertilization.

Electrophoretic studies on polyploid amphibians—I. 6-phosphogluconate dehydrogenase (6-PGD)

Abstract 1. 1. Mobility, activity and number of 6-PGD electrophoretic bands of the diploid and the tetraploid Odontophrynus americanus frogs are the same. This confirms previous cytological findings that, although genes are duplicated in number in the tetraploid, diploidization did not yet occur. Thus, locus duplication cannot be detected in O. americanus (4n). 2. 2. The 6-PGD of the triploid hybrids follows a trisomic mendelian pattern with two codominant alleles. Three bands were found: maternal (AA), hybrid intermediate (AC) and paternal (CC) with an intensity ratio of 4:4:1, respectively. 3. 3. The hypotheses on gene regulation in diploid, triploid and tetraploid Odontophrynus are discussed.

Biochemical and physiological properties of the lactate dehydrogenase allozymes of the Brazilian teleost, Leporinus friderici, Anostomidae, Cypriniformes

Abstract 1. 1. Lactate dehydrogenase polymorphism of Leporinus friderici is determined by two loci, each one encoded by two alleles—Ldh-A, Ldh-A′ and Ldh-B and Ldh-B′. 2. 2. Biochemical characterization of the different LDH phenotypes was carried out. Substrate saturation curves show that extracts, where the LDH-B predominates, are much more sensitive to substrate inhibition and have higher thermostability than skeletal muscle extracts where the LDH-A predominates. 3. 3. Pyruvate affinity parameters of the different LDH-B phenotypes show differential values in all temperatures assayed. LDH-BB phenotypes presented always greater affinity values than the LDH-BB′ and B′B′ ones. 4. 4. The different LDH-B phenotypes show differential thermostability when tested at 65°C, so, the LDH-B′B′ phenotype had higher stability than the LDH-BB′ and LDH-BB phenotypes. This was corroborated with the isozymes partially purified. 5. 5. A variation was detected in the LDH-B phenotype frequencies since the experiment was started in 1980. 6. 6. All these features suggest that this polymorphism may have an adaptive significance in relation to environmental variability, i.e. temperature fluctuations and water pollution due to industrial waste that notably increased in these last years.