Esterase-6 allozymes: Biochemical studies of two common and one rare variant inDrosophila melanogaster

The biochemical properties of three allozymes coded by theEst-6 locus, two common forms (EST-6^S and EST-6^F) and one rare form (EST-6^VF), were studied. The results show the existence of differences in isoelectric point, activity, activation energy, K_m, and temperature coefficient among the three variants, especially between the two common forms and the one rare form. The specific activity of the rare enzymatic variant seems to be less affected by temperature variation. The possible significance of these findings in relation to the mechanism of reproduction is briefly discussed.     

Evolutionary genetics of Metridium senile. I. Kinetic differences in phosphoglucose isomerase allozymes

Populations of the sea anemone Metridium senile from the northeast coast of the United States exhibit a one-locus, two-allele polymorphism for phosphoglucose isomerase. No additional hidden variation is exposed by changes in pH, gel pore size, or heat denaturation. The allozymes are similar in pH optimum, sensitivity of K _m to pH, and sensitivity of K _m and V _max to temperature. In other respects they are functionally different, with the fast allozyme having a 3.5-fold higher specific activity and a slightly higher K _m of fructose-6-phosphate than the slow form. In these respects, heterozygotes produce a mixture of enzymes that appears to function roughly as the sum of its component parts. Comparisons of V _max/K _m ratios reveal significant differences among genotypes, with the fast form having higher values at all temperatures than the slow form and heterozygotes falling intermediate. In addition, there is a significant difference among genotypes in sensitivity of this parameter to temperature, with the fast homozygote and heterozygote displaying greater sensitivity than the slow homozygote. Temperature is probably an important selective agent in maintaining this polymorphism.Supported by Grant T-4 from the Health Research and Services Foundation, NSF DEB77-14442, NIH GM25809, and NIH GM28024.     

Evolutionary relationships of the Lake Malawi Oreochromis species: evidence from allozymes

Three tilapiine species belonging to the endemic Lake Malawi species flock known as' chambo' Oreochromis ( Nyasalapia ) karongae , O. (N.) lidole and O. (N.) squamipinnis and the species Oreochromis (Oreochromis) shiranus were collected from the wild. These four species were analysed at 43 enzyme loci using starch gel electrophoresis. No significant deviations from Hardy-Weinberg equilibrium were observed after correcting for multiple simultaneous testing. The expected heterozygosity ( H _e) was lowest in O. (O.) shiranus ( H _e=0.082). The three chambo species had higher levels: H _e=0.103 to H _e=0.116. The three chambo species as a whole were polymorphic (99%) at 16 different loci sharing 10 of these in common. No fixed differences between the three chambo species were observed but highly significant allele frequency differences existed between all the chambo species. The F _ST calculated for the three chambo species was 0051, closer to intra-specific than to inter-specific levels. O. (O.) shiranus could be clearly separated from all chambo species at five fixed loci. Comparison of the allozyme data from these species with five species from the same sub genera support the hypothesis that the chambo form a monophyletic group and that O. (N.) macrochir or a related species represents the sister taxon.     

Inhibition of phosphoglucose isomerase allozymes from the wing polymorphic waterstrider,Limnoporus canaliculatus,by pentose shunt metabolites

Inhibition of phosphoglucose isomerase (PGI) allozymes from the wing-polymorphic waterstrider, Limnoporus canaliculatus, by three pentose-shunt metabolites was studied at several different temperatures. This was done to determine if the allozymes exhibited a differential ability to participate in lipid biosynthesis via differential partitioning of carbon flux through the pentose shunt versus glycolysis. 6-Phosphogluconate and erythrose-4-phosphate proved to be strong competitive inhibitors of PGI, while sedoheptulose-7-phosphate was a very weak inhibitor. The PGI allozymes from L. canalicualtus were differentially inhibited by 6-phosphogluconate at two of the three temperatures studied. However, this property does not appear to be an adaptive difference between the allozymes but, rather, a correlated effect resulting from variation in substrate binding. Estimates of reaction rates for the allozymes indicate that the differences in inhibition result in no detectable differences in reaction velocities. Thus, no evidence in support of the hypothesis that PGI allozymes from Limnoporus canaliculatus were adapted to function in different metabolic capacities via differential inhibition was obtained in this study. However, the importance of this characteristic in allozymic adaptation in natural populations remains an open question.Supported by NSF Grant DEB 7908802 and UPHS Grant GM 21133 to R. K. Koehn and an NSF dissertation improvement grant to A. J. Zera.     

Polymorphism of erythrocyte glucosephosphate isomerase in sheep

An electrophoretic analysis of glucosephosphate isomerase (GPI) in seven Italian sheep populations suggests that this locus is more polymorphic than previously supposed. The observed phenotype distributions are in agreement with the hypothesis of the existence of three codominant alleles, GPI*F, GPI*S and GPI*N, GPI*S being the most frequent (0.935 ÷ 1.000).     

Genetic control of two electrophoretic variants of glucosephosphate isomerase in the mouse (Mus musculus)

The autosomal variation and the genetic control of GPI has been determined by a comparison of electrophoretic patterns of F₁ and backcross progeny of three inbred strains of mice. The locus controlling the production of GPI in the mouse has been designated Gpi-1. Two alleles at this locus have been described and designated Gpi-1 ^a and Gpi-1 ^b, which represent, respectively, the slow and fast electrophoretic forms. Twenty-seven inbred strains of mice have been classified for these two alleles. The absence of close linkage of Gpi-1 to seven other genetic loci has been determined. It has been demonstrated that the polymorphism of Gpi-1 is widely distributed in feral mice. GPI was expressed in vitro and in four types of malignant tumors.Supported by U.S. Public Health Service Grants GM-09966, from General Medical Sciences, and GY 4193.     

Genetic control of glucosephosphate isomerase (GPI) in common carp (Cyprinus carpio L.)

In common carp, a freshwater fish species of tetraploid origin, GPI enzymes are present in two variants: GPI-A and GPI-B. GPI-A is coded by two loci segregating for two (GPI-A 1*) and six (GPI-A2*) alleles. Experimental crosses of the ornamental (Koi) variety of common carp revealed that GPI-B is coded by only one locus (GPI-B*). Another GPI-B* locus must have been silenced in the process of functional diploidization. It was also shown that the GPI-A2* locus segregated independently from the GPI-B* locus, demonstrating that the loci are located on different chromosomes.     

Population structure of seabass in Portugal: evidence from allozymes

Starch-gel electrophoresis was used to assess the level and distribution of genetic variation in juvenile European seabass sampled from five different nursery grounds along the coast of Portugal between November 1992 and February 1994. Thirty-eight loci were examined, of which six ( AAT-3*, ADA*, GPI-1*, GPI-2*, G3PDH-2*, SOD* ) were polymorphic at the 99% level and were used in population surveys. Low but statistically significant multilocus F _ST values suggest that population structuring exists along the Portuguese coast. There is some restriction in gene flow between the population at Faro and all other sites to the north.     

Electrophoretic studies on glucosephosphate isomerase and phosphoglucomutase in two types of Anisakis larvae

Agatsuma T. 1982. Electrophoretie studies on glucosephosphate isornerase and phosphoglucomutase in two types of Anisakis larvae. International Journal for Parasitology12: 35–39. Enzyme electrophoresis was carried out between the larval forms. Type I and II, of Anisakis using starch gel. In glucose-phosphate isomerase, considerable polymorphisms were found in each type. At least 5 alleles appear to occur at this enzyme locus in natural populations of both types. Out of 5 alleles, 3 were common to both types. No significant difference was found in their frequencies. However, each larval form can be easily distinguished by the electrophoretic mobility of phosphoglucomutase. It was concluded that enzyme electrophoresis is an alternative useful tool for the identification of larval forms of Anisakis.     

Structural and functional differentiation of two clinally distributed glucosephosphate isomerase allelic isozymes from the teleost Fundulus heteroclitus

The teleost Fundulus heteroclitus (L.) possesses two loci, Gpi-A and Gpi-B, for the glycolytic enzyme, glucose-phosphate isomerase (GPI; D- glucose-6-phosphate ketol-isomerase; E.C. 5.3.1.9). The Gpi-B locus is polymorphic in Fundulus, with two common alleles, Gpi-Bb and Gpi-Bc, distributed in a clinal manner in populations along the east coast of North America. Since this clinal distribution is strongly correlated with a temperature gradient, we asked whether the GPI-B2 allozymes were functionally adapted to the thermal environment in which a given phenotype predominated. The two major GPI-B2 allozymes were purified to homogeneity and were characterized as to molecular weight, isoelectric pH, thermal denaturation, and kinetic parameters. Both GPI-Bb2 and GPI- Bc2 allozymes have molecular masses of 110 kD, and they have isoelectric pHs of 6.4 and 6.6, respectively. The GPI-Bb2 allozyme was more stable to thermal denaturation than was the GPI-Bc2 enzyme. Kinetic properties of the allelic isozymes were investigated both as a function of pH and as a function of temperature. At 25 degrees C, over the pH range considered, there were no significant differences between allozymes, either in Km for fructose-6-phosphate or in Ki for 6- phosphogluconate, but apparent Vmax values differed between pH 7.5 and pH 8.5. All steady-state kinetic parameters showed strong temperature dependence, but the allozymes differed only in the Ki for 6- phosphogluconate at temperatures greater than 30 degrees C. On the basis of the observed structural and functional differences alluded to above, the hypothesis that the major allelic isozymes of the Gpi-B locus were functionally equivalent was rejected. However, it is not yet known whether these structural and functional differences have any significance at higher levels of biological organization.      

Schistosoma mansoni: Inhibition of glucosephosphate isomerase and glycolysis by sugar phosphates

Glucosephosphate isomerase (EC 5.3.1.9) of Schistosoma mansoni is inhibited competitively by a number of tetrose, pentose, and hexose phosphates with inhibitor constant (K_i) values in the range of 0.5 to 400 μM. The most potent inhibitor is 5-phospho-d-arabinonate which resembles the cis-enediolate transition state intermediate of the reaction. These analogs were also found to be effective inhibitors of the production of lactate from glucose by suitably supplemented worm homogenates. The rank order of potency of inhibition of glycolysis was inversely related to the magnitudes of the K_i values for glucosephosphate isomerase. These K_i values were similar to those previously reported for mammalian glucosephosphate isomerase, suggesting similarities in the steric and electronic characteristics of the active sites of these isofunctional enzymes. This conclusion was further supported by the observed pH dependence of the inhibition by 5-phospho-d-arabinonate. Although glucosephosphate isomerase is not a rate-limiting enzyme of glycolysis, in the conventional sense, its selective inhibition could be of chemotherapeutic importance, in part because of the accumulation in glycolyzing systems of glucose 6-phosphate which is a potent feedback inhibitor of hexokinase.     

Contrasting population genetic structures using allozymes and the inversion polymorphism in Drosophila buzzatii

Second chromosome inversion and genotypic frequencies at seven allozyme loci, differentially associated with inversions, were determined in seven natural populations of Drosophila buzzatii. The patterns of variation of allozymes and the inversion polymorphisms were significantly different, indicating the role of adaptive differentiation for the latter. Moreover, the patterns of population structure varied among allozyme loci, suggesting the operation of diversifying selection for certain loci. Differentiation was negligible for Leucyl‐amino peptidase (Lap) and Peptidase‐2 (Pep‐2), low to moderate for Aldehyde oxidase (Aldox), Peptidase‐1 (Pep‐1) and Esterase‐1 (Est‐1) and high for Esterase‐2 (Est‐2) and Xanthine dehydrogenase (Xdh). Significant linkage disequilibria were detected between inversions and Aldox, Est‐1, Est‐2 and Xdh. Multiple regression analyses of inversion and allele frequencies on environmental variables revealed the existence of clines for inversions, Est‐1, Est‐2, Xdh and Aldox along altitudinal, latitudinal and/or climatic gradients. Tests using conditional allele frequencies showed that Est‐1 and Aldox clines could be accounted for by hitchhiking with inversions, whereas natural selection should be invoked to explain the clines observed for Est‐2 and Xdh.     

Crystal structure of glucose-6-phosphate isomerase from Thermus thermophilus HB8 showing a snapshot of active dimeric state

Glucose-6-phosphate isomerase (GPI) is a glycolytic enzyme with ill-defined oligomeric state. In order to obtain insight into the correlation between oligomerization and the catalytic function of this enzyme, the crystal structure of GPI from the extreme thermophile Thermus thermophilus HB8 (TtGPI) has been determined at 1.95 Å resolution. The crystallographic asymmetric unit contains an apparent dimer. The core fold of protomer and the interprotomer spatial arrangement of the dimer are similar to those of already reported crystal structures of other GPIs. The active site is located on the dimer interface, and putative catalytic residues are well conserved among the GPIs. These results suggest that the observed dimeric state of TtGPI in the crystal is biologically relevant and that this enzyme uses a common catalytic mechanism for the isomerase reaction. Gel-filtration chromatography, chemical cross-linking, sedimentation equilibrium by analytical ultracentrifugation, and dynamic light-scattering experiments indicate that TtGPI exists in a dynamic equilibrium between monomeric and dimeric states in solution. Several factors potentially contributing to the thermal stability of TtGPI protomer were identified: (i) a decrease in denaturation entropy by the shorter polypeptide length and by amino acid composition, including the increased number of proline residues and a higher arginine-to-lysine ratio; (ii) a larger number of ion pairs; and (iii) a reduction in cavity volume. From these results, it is suggested that transient dimer formation is sufficient for the catalytic function and that the TtGPI protomer itself has intrinsically higher thermal stability.     

Comparison of purified acid phosphatase allozymes inDrosophila virilis: Differences in carbohydrate content and composition of the allozymes

Three acid phosphatase (EC 3.1.3.2) allozymes (ACPH¹, ACPH², and ACPH⁴) ofDrosophila virilis show different activities as measured by electrophoretic techniques. Recently, it was suggested that these differences are attributable to the variable ability of the allozymes to be incorporated into lysosomes (Narise, S.,Genet. Res. Cambr., 45:143, 1985). Immunoelectrophoresis demonstrated that the activity differences between these electrophoretic variants coincided with differences in the amount of the enzyme protein in soluble fractions but not in whole cell-free extracts. These results support the idea that acid phosphatase allozymes inD. virilis are cell-localization variants. We examined the problem by structural analysis of both the protein and the carbohydrate moieties of these allozyme glycoproteins, since lysosomal enzymes are known to become localized in lysosomes through their carbohydrate moieties. The three ACPH allozymes were purified to homogeneity from their respective homozygotes and compared with respect to amino acid composition and carbohydrate content and composition. Amino acid compositions were similar, while content and compositions of neutral sugars were significantly different. The neutral sugar content of ACPH¹ was 9.2%; that of ACPH², 21.0%; and that of ACPH⁴, 7.3%. A trace of hexosamines, but noN-acetylneuraminic acid, was found in the ACPH allozymes. Isoelectric points varied corresponding to their electrophoretic mobilities, which were not changed by treatment with alkaline phosphatase and neuraminidase.     

Contribution to the phylogeny of pangasiid catfishes based on allozymes and mitochondrial DNA

Phylogenetic inferences based on molecular data provide support for the recognition of some Pangasius subgenera and, or species groups as distinct genera. It is suggested that Pangasiidae and Schilbeidae have diverged from a common ancestor probably since late Miocene. Moreover, the data provide evidence for the existence of two distinct episodes of speciation in Pangasiidae. The most ancient occurred in the middle Miocene and concerned emergence of the different genera, while the second referred to an intense speciation event in the genus Pangasius , probably between the early Miocene and the late Pliocene. Finally, this study revealed the existence of two new species of Pangasius .     

Distribution of erythrocytic allozymes in two sibling species of greater galago [Galago crassicaudatus E. Geoffroy 1812 and G. garnettii (Ogilby 1838)]

This study investigated the use of erythrocyte enzymes as indicators of the presence or absence of gene flow between the sibling species G. crassicaudatus and G. garnettii. Fifty-five animals deriving from 14 different source populations were included in the analyses. In addition to hemoglobin, eight enzyme systems were examined: acid phosphatase, adenylate kinase, carbonic anhydrase II, esterase D, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, peptidase A, and peptidase B. of these, adenylate kinase, glucose-6-phosphate dehydrogenase, hemoglobin, peptidase A, and peptidase B showed no interspecific or intraspecific variation. Esterase D was polymorphic in certain populations of G. crassicaudatus but not in others or in G. garnettii. Acid phosphatase and 6-phosphogluconate dehydrogenase were polymorphic in G. garnettii but monomorphic in all G. crassicaudatus populations. The taxa showed fixation for different alleles at the carbonic anhydrase II locus, indicating a lack of gene exchange between the taxa. We suggest that acid phosphatase, 6-phosphogluconate dehydrogenase, and carbonic anhydrase II may be used as genetic markers in the identification of these two taxa.     

Isozymes of glucosephosphate isomerase (PGI) in fishes of the subclass actinopterygii

A compilation of the species of fishes of the subclass Actinopterygii for the study of the PGI isozyme system is given. PGI appears to be codified by more than one locus in fishes; 65% of the species analysed here have two loci for PGI. PGI duplication in fishes and the relationship of isozymes of PGI with temperature and metabolism are discussed.     

Mitochondrial DNA and allozymes reveal high dispersal abilities and historical movement across drainage boundaries in two species of freshwater fishes from inland rivers in Queensland, Australia

This study used allozymes and mtDNA variation to test that: 1) populations of two fish species, Nematolosa erebi and Retropinna semoni , in lowland rivers in central Australia were highly connected within drainages, 2) populations in different drainages were highly differentiated and 3) there was evidence of historical connections between two major lowland drainages in inland Australia. Levels of genetic differentiation among populations within drainages were low, but still statistically significant, indicating that populations were not as highly connected as had been predicted. Populations from the Murray–Darling and the Lake Eyre drainages were highly differentiated, indicating no contemporary dispersal across drainage boundaries. Both species showed evidence of historical connections between the two drainage basins, although estimates of the time that these last occurred differed between the two species. Nematolosa erebi populations from the two drainages were estimated to have been separated c . 150 000 years ago, whereas populations of R. semoni , were estimated to have been separated c . 1.5 million years ago.