Fructose 1,6-bisphosphate aldolase of Drosophila melanogaster: comparative sequence analyses around the substrate-binding lysyl residue

The amino acid sequence of a 103 residue segment encompassing the substrate-binding active site lysyl residue of fructose 1,6-bisphosphate aldolase from Drosophila melanogaster is determined. The sequence is identical to more than 70% with the structure of rabbit muscle aldolase and with the known partial sequences of the sturgeon muscle, trout muscle, and ox liver enzymes. The homology of the insect enzyme with the vertebrate aldolases strongly implies a similar tertiary structure folding.     

Extended amino acid sequences around the active-site lysine residue of class-I fructose 1,6-bisphosphate aldolases from rabbit muscle, sturgeon muscle, trout muscle and ox liver.

1. Amino acid sequences covering the region between residues 173 and 248 [adopting the numbering system proposed by Lai, Nakai & Chang (1974) Science 183, 1204-1206] were derived for trout (Salmo trutta) muscle aldolase and for ox liver aldolase. A comparable sequence was derived for residues 180-248 of sturgeon (Acipenser transmontanus) muscle aldolase. The close homology with the rabbit muscle enzyme was used to align the peptides of the other aldolases from which the sequences were derived. The results also allowed a partial sequence for the N-terminal 39 residues for the ox liver enzyme to be deduced. 2. In the light of the strong homology evinced for these enzymes, a re-investigation of the amino acid sequence of rabbit muscle aldolase between residues 181 and 185 was undertaken. This indicated the presence of a hitherto unsuspected -Ile-Val-sequence between residues 181 and 182 and the need to invert the sequence -Glu-Val- to -Val-Glx- at positions 184 and 185. 3. Comparison of the available amino acid sequences of these enzymes suggested an early evolutionary divergence of the genes for muscle and liver aldolases. It was also consistent with other evidence that the central region of the primary structure of these enzymes (which includes the active-site lysine-227) forms part of a conserved folding domain in the protein subunit. 4. Detailed evidence for the amino acid sequences proposed has been deposited as Suy Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Purification and properties of fructose diphosphate aldolase from Ascaris suum muscle

A fructose diphosphate aldolase has been isolated from ascarid muscle and crystallized by simple column chromatography and an ammonium sulfate fractionation procedure. It was found to be homogeneous on electrophoresis and Sephadex G-200 gel filtration. This enzyme has a fructose diphosphate/fructose 1-phosphate activity ratio close to 40 and specific activity for fructose diphosphate cleavage close to 11. K_m values of ascarid aldolase are 1 × 10⁻⁶m and 2 × 10⁻³m for fructose diphosphate and fructose 1-phosphate, respectively. The enzyme reveals a number of catalytic and molecular properties similar to those found for class I fructose diphosphate aldolases. It has C-terminal functional tyrosine residues, a molecular weight of 155,000, and is inactivated by NaBH₄ in presence of substrate. Data show the presence of two types of subunits in ascarid aldolase; the subunits have different electrophoretic mobilities but similar molecular weights of 40,000. Immunological studies indicate that the antibody-binding sites of the molecules of the rabbit muscle aldolase A or rabbit liver aldolase B are structurally different from those of ascarid aldolase. Hybridization studies show the formation of one middle hybrid form from a binary mixture of the subunits of ascarid and rabbit muscle aldolases. Hybridization between rabbit liver aldolase and ascarid aldolase was not observed. The results indicate that ascarid aldolase is structurally more related to the mammalian aldolase A than to the aldolase B.     

Proliferative zones in the brain of the Amur sturgeon fry. Interactions with neuromeres and migration of secondary matrix zones

Neurogenesis in the forebrain region was studied in the Amur sturgeon Acipenser schrenki fry using immunocytochemical marking of the proliferative nuclear antigen. The brain zones with high proliferative activities were located at the brain ventricle surface facing the periventricular cavity. In addition to the periventricular zone of primary proliferation, several secondary proliferative zones were found in the forebrain region of the Amur sturgeon Acipenser schrenki.     

Polymorphism of the mitochondrial DNA control region in eight sturgeon species and development of a system for DNA-based species identification

Intraspecific and interspecific nucleotide sequence variations of the mtDNA control region (D-loop) were studied with mtDNAs isolated from tissue specimens of more than 1400 sturgeons of nine species: Russian sturgeon Acipenser gueldenstaedtii, Persian sturgeon A. persicus, Siberian sturgeon A. baerii, Amur sturgeon A. schrenkii, Fringebarbel sturgeon A. nudiventris, sterlet A. ruthenus, stellate sturgeon A. stellatus, beluga Huso huso, and kaluga H. dauricus. The results were used to analyze the interspecific variation of the mtDNA control region in the given set of species and to develop a test system of ten species-specific primers, which allowed species identification from noninvasive tissue samples, spawn, and food products of eight species. The system proved suitable for multiplex PCR. A method was developed for the first time to reliably differentiate the A. baerii mitotype and the baerii-like mitotype of A. gueldenstaedtii. It was found that, although genetically separate, A. gueldenstaedtii and A. persicus are relatively young species and have common mitochondrial haplotypes, precluding their identification via mtDNA analysis alone. To develop a system for species identification of A. gueldenstaedtii and A. persicus, it is necessary to study the polymorphism of nuclear markers.     

Fructose 1,6-diphosphate aldolase of Drosophila melanogaster: comparative sequence analyses of the cysteine-containing peptides.

Following tryptic digestion four cysteine-containing peptides per monomer have been isolated from fructose 1,6-diphosphate aldolase of Drosophila melanogaster. Sequence analyses of the peptides showed that three of the four cysteinyl residues appear to occur in homologous positions to three of the eight cysteines of rabbit muscle aldolase. Moreover they seem to be homologous also to three of the six sulfhydryl groups in sturgeon aldolase. The fourth cysteine-containing peptide of Drosophila aldolase has no homologous SH peptide either in the rabbit or in the sturgeon enzyme, but corresponds to another tryptic peptide in the rabbit aldolase. As deduced from homology all four SH peptides are localized in the buried region of the molecule. This conclusion is confirmed by the fact that all four cysteine-containing peptides have been isolated from the central cyanogen bromide fragment. Drosophila aldolase has no exposed thiol groups, thus demonstrating that these residues are not essential either in catalytic activity or for the stabilization of the three-dimensional structure.     

A comparative study of aldolase from human muscle and liver

Aldolase was purified from human skeletal muscle and human liver by techniques capable of processing large quantities (10-20kg) of tissue. The methods used also proved convenient for isolating aldolase on a large scale from other mammalian and avian sources. Aldolase from both human liver and muscle was crystallized; each gave two crystalline forms, depending on the conditions of crystallization. X-ray studies on the muscle aldolase crystals suggest a close structural similarity between human and rabbit muscle aldolase. Aldolases from human muscle and liver have similar pH optima and pH stability but their stability to heat treatment differs. The effect of heat on the enzymes may therefore provide an easy means of distinguishing them. The kinetic constants K(m) and k(cat.) for these aldolases are similar to other mammalian aldolases. Amino acid analyses and tryptic peptide ;mapping' show that the primary structures of the two aldolases differ greatly.     

Alteration of thermoregulation behavior in juvenile fish in relation to satiation level

Thermoregulation behavior of satiated and hungry juvenile sterlet Acipenser ruthenus, the Siberian sturgeon Acipenser baeri, and the goldfish Carassius auratus was studied. Significant alterations of thermoregulation behavior appear in juvenile fish one day after food deprivation. Hungry fish preferred, on average, lower temperatures, their temperature range was broader, and the locomotor activity was higher than in satiated fish.     

Limited proteolysis of liver and muscle aldolases: Effects of subtilisin,cathepsin B,and Staphylococcus aureus protease

Limited proteolysis of rabbit liver and muscle aldolases by subtilisin and cathepsin B results in decreased catalytic activity, associated with the release of acid-soluble peptides from the COOH terminus. Analysis of the sequence of these peptides confirms the COOH-terminal sequence of the muscle enzyme and provides new information on the COOH-terminal sequence of the liver enzyme. As previously reported for muscle aldolase, cathepsin B releases mainly dipeptides from the COOH terminus of liver aldolase. The COOH-terminal sequence of rabbit liver aldolase is SerThrGlnSerLeuPheThrAla SerTyrThrTyr. The Gln-Ser bond is resistant to Staphylococcus aureus protease, which hydrolyzes a GluSer bond at the corresponding positions in the muscle enzyme.     

The low level of differences between mitogenomes of the Sakhalin sturgeon <Emphasis Type="Italic">Acipenser mikadoi</Emphasis> Hilgendorf, 1892 and the green sturgeon <Emphasis Type="Italic">A. medirostris</Emphasis> Ayeres, 1854 (Acipenseridae) indicates their recent divergence

The complete mitochondrial genome of the Sakhalin sturgeon Acipenser mikadoi and two mitogenomes of the Amur sturgeon A. schrenckii were sequenced using Roche 454 technology. The mitogenomes of the green sturgeon A. medirostris (obtained from GenBank) and the Sakhalin sturgeon differ as much as the mitogenomes of two mtDNA haplogroups (SM and BG) found in the same population of the Amur sturgeon: 0.0042 ± 0.0006 and 0.0036 ± 0.0005 substitutions per site (Tamura–Nei distance, TrN), respectively. The differences of these mitogenome pairs from mitogenomes of sister species (kaluga A. dauricus and white sturgeon A. transmontanus) are 3–6 times larger: 0.0260 ± 0.0015 and 0.0102 ± 0.0008, respectively. Thus, the differences between the mitogenomes of the Sakhalin and green sturgeons can be attributed to the variability at the intraspecific level. The time that has passed since the divergence of the Sakhalin and green sturgeons is considered to be much shorter than was previously believed: approximately 0.16 rather than 9.60 million years.     

Development of mesonephros in prolarvae of acipenserids (acipenseridae)

Development of the excretory system in early ontogenesis of acipenserids—the Russian sturgeon Acipenser gueldenstaedtii, the starred sturgeon A. stellatus, the sterlet A. ruthenus, and the great sturgeon Huso huso—is investigated histologically. In acipenserids, the mesonephros develops after hatching. At the 36th stage of development (after Detlaf et al., 1981), the mesonephros is a consecutively arranged group of spherical rudiments of placodes situated along the Wolffian duct. Morphological differentiation of mesonephros as the first generations of renal corpuscles, vascular glomeruli, and convoluted tubules is completed up to the 45th stage, i.e., to the moment of transition of prolarvae to active feeding.     

TWO CLASS I ALDOLASES IN KLEBSORMIDIUM FLACCIDUM (CHAROPHYCEAE): AN EVOLUTIONARY LINK FROM CHLOROPHYTES TO HIGHER PLANTS1

Two fructose-bisphosphate aldolases(EC 4.1.2.13) from Klebsormidium flaccidum Silver, Mattox and Black-well were purified by affinity elution from phosphocellulose. The two enzymes were subsequently separated by HPLC on an anion-exchange column (QAE-silica). The aldolase eluting first represented 5% of the total activity; the other aldolase represented the remaining activity. The activity of the enzymes was not reduced by the presence of 1 mM EDTA or increased by 0.1 mM Zn²⁺, establishing their character as class I type (Me²⁺ independent) aldolases. The K_m(fructose-1,6-bisphosphate) values were 1.7 and 34.7 μM for the enzyme eluting first and second, respectively, from the QAE-silica column. The subunit molecular masses, as determined by SDS-PACE, were 40.5 and 37 kD; the specific activities of the purified enzymes were 7.9 and 24.7 · mg^?1 protein, respectively. The two aldolases of K. flaccidum are homologous to the cytosol and chloroplast specific isoenzymes of higher plants by several criteria and are therefore probably located in the same cellular compartments in K. flaccidum. The K_m and specific activity for the chloroplast aldolase of K. flaccidum are three times higher than for the chloroplast aldolase of higher plants, a remarkable difference. Immunotitration with specific antisera against the chloroplast aldolase of Chlamydomonas reinhardtii Dangeard and spinach showed that the chloroplast aldolase of K. flaccidum was immunochemically intermediate in structure to the respective aldolases of C. reinhardtii and higher plants. K. flaccidum is the second species of Charophyceae (besides Chara foetida Braun) with two class I aldolases as in higher plants whereas two species of Chlorophyceae have only one class I aldolase and, under some conditions, an additional class II (Me²⁺ dependent) aldolase. Thus, aldolases may turn out, in addition to the known enzymes of glycolate conversion and urea degradation, be a novel enzyme system to evaluate algal evolution along with cytological features.     

Amino acid sequence of an invertebrate FBP aldolase (from Drosophila melanogaster)

The complete amino acid sequence of FBP aldolase from Drosophila melanogaster has been determined. The enzyme contains four identical subunits of 360 amino acid residues. The primary structure of the monomer was established using automated Edman degradation on fragments prepared by CNBr-cleavage, by partial acid cleavage at the unique Asp-Pro bond and by oxidative cleavage at the three tryptophan residues. Manual Edman-Chang degradation was used on smaller peptides obtained by digestion with Staphylococcus aureus V8 protease, trypsin or chymotrypsin. The primary structure of Drosophila aldolase exhibits very extensive homology with the sequence of rabbit muscle aldolase (71% identity), thus explaining the early observation that Drosophila and mammalian aldolases form active interspecies hybrid quaternary structures (Brenner-Holzach, O. and Leuthardt, F., Eur. J. Biochem. (1972) 31, 423-426).     

Genetic characterization of Amur sturgeon <Emphasis Type="Italic">Acipenser schrenckii</Emphasis> and its hybrid caught around Hokkaido

Genetic characterization was performed in five individuals of wild Amur sturgeon Acipenser schrenckii, and/or its presumed hybrid caught around Hokkaido, using a mitochondrial DNA (mtDNA) marker and two markers of nuclear DNA (nDNA). Genetic analyses indicated that two of the five fish had the mtDNA haplotype of Kaluga, Huso dauricus, whereas the nDNA markers indicated signs for both A. schrenckii and H. dauricus genotypes, referring to a hybrid origin. The other three fish were plausibly pure A. schrenckii. The results indicated the importance of combined usage of mtDNA and nDNA markers for correct species identification in sturgeon.     

Organization of diencephalon of the sturgeons. External nuclei of the pretectal area

Cytoarchitectonics of the external nuclei of the pretectal area was studied in four species of the sturgeons: great sturgeon, Huso huso L., Kura sturgeon, Acipenser gueldenstaedtii persicus n. Kurensis Belyaeff, stellate sturgeon, Ac. stellatus Pall. and barbel sturgeon, Ac. nudiventris Lov. Study of morphological organization of the structures was carried out using routine Nissl staining and Bilschowski impregnation technique in Viktorov’s modification. Similar structure of this pretectal area was found in the species. Five nuclear formations were described in this pretectal area: parvocellular and magnocellular external pretectal nuclei, laminiform nucleus of pretectum, dorsal pretectal nucleus, and the accessory optic nucleus. Comparison of the obtained data with the literature revealed a high level of differentiation of the area of pretectum in the sturgeons. Also, a general conclusion is given on the organization of the whole pretectal area in the sturgeons.     

Distribution of the enzyme activity in the intestine of beluga <Emphasis Type="Italic">Huso huso</Emphasis> and Russian sturgeon <Emphasis Type="Italic">Acipenser gueldenstaedtii</Emphasis> (Acipenseridae)

Data on distribution of digestive enzymes along the intestinal tract of mature specimens of the beluga Huso huso and Russian sturgeon Acipenser gueldenstaedtii are presented. The absence of a distinct proximo-distal gradient of the enzyme activity can be explained by short length of the intestine of sturgeons. Considerable differences are found in the distribution pattern of enzymes along the intestinal tract of beluga and Russian sturgeon. The effect of the method of calculation of the enzyme activity on the data on the proximo- distal gradient is considered.     

Selective predation by reintroduced juvenile Lake sturgeon (<Emphasis Type="Italic">Acipenser fulvescens</Emphasis>) in Ft. Loudoun reservoir,Tennessee (USA)

Following water quality and minimum flow improvements to the impounded Tennessee and Cumberland Rivers, juvenile lake sturgeon (Acipenser fulvescens) have been restocked annually since 2000. Our goal was to seasonally assess foraging mode of this recovering population in Ft. Loudoun Reservoir in the Upper Tennessee River. During 2014–15, individuals were captured using trot-lines in a 13-km reach that supports the greatest density of lake sturgeon. We used colonic flushing and gastric lavage techniques to obtain diet. We took systematic benthic sediment grabs along multiple transects throughout the reach and opportunistically deployed rock cages filled with hard substrates to assess potential prey that colonize hard surfaces. Foraging modes of lake sturgeon were determined by comparing relative abundances of invertebrate taxa in the gut contents (6581 invertebrates) of 28 fish to the relative abundances of the same invertebrate taxa collected from the resource base (1667 invertebrates). Proportional similarity, Levin’s niche breadth, and Manly’s index were used to assess the degree of prey selectivity. Lake sturgeon fed selectively on a narrow range of available prey consisting mostly of larval chironomids (93% composition by number during warm season, 96% during cool season), some genera of which they prey upon selectively, primarily Chironomus sp., but to a lesser extent Procladius, Ablabesmyia, Coelotanypus, and Cryptochironomus spp. Meanwhile, other abundant taxa in the resource base were avoided, such as Oligochaetes, Hexagenia mayflies, and the chironomid Glyptotendipes. Our results illustrate that assessing seasonally available prey from habitat utilized by lake sturgeon is important when investigating diet preference.     

Periventricular and central nuclei of the pretectal area of diencephalon of the sturgeons

Cytoarchitectonics of periventricular and central nuclei of the pretectal area was studied in four species of the sturgeons: the great sturgeon Huso huso, L., the Russian sturgeon Acipenser gъldenst?dti persicus n. kurensis, Belyaeff, the starred sturgeon Ac. stellatus, Pall., and the barbel sturgeon Ac. nudiventris, Lov.; this pretectum part has a similar structure. Study of these parts of the pretectal area was carried out by methods of Nissl and Bielshowskii modified by Viktorov. In this part of the pretectal area, nine nuclear structures were described, eight of them—nuclear; these are ventral periventricular pretectal nucleus and its dorsal component, dorsal periventricular pretectal nucleus, nucleus of medial longitudinal bundle, subcomissural organ, medial and lateral intercalate nuclei, and central and posterior pretectal nuclei. The main attention has been paid to the issue of the evolutional progression of this part of the pretectal area in the sturgeons as compared with other Actinopterygii.     

Comparison of the subunit and primary structures of the pyruvate kinases from rabbit and sturgeon muscles.

The structures of the pyruvate kinases isolated from rabbit and sturgeon muscles were compared. Both enzymes are composed of subunits of 56000 mol.wt. Amino acid compositions of the two enzymes are similar, but not identical. Examination of the peptides produced by CNBr cleavage demonstrated that there are at least some highly homologous regions in the two proteins. There are only two replacements between an 18-residue portion of the polypeptide chain of rabbit muscle pyruvate kinase and a portion of the polypeptide chain of the enzyme isolated from sturgeon muscle.     

Primary structure at the active sites of beef and rabbit liver aldolases

The sequence of the tryptic heptaeicosapeptide isolated from the active site of beef liver aldolase has been determined and shown to be identical with that of the corresponding peptide from rabbit liver aldolase. The sequence is Ala-Leu-Asn-Asp-His-His-Val-Tyr-Leu-Glu-Gly-Thr-Leu-Leu-βGlys-Pro-Asn-Met-Val-Thr-Ala-Gly-His-Ala-Cys-Thr-Lys. There is extensive homology with peptides isolated from the same region of a variety of muscle aldolases.