Cytosolic and mitochondrial phosphoenolpyruvate carboxykinase of the bullfrog,Rana catesbeiana,liver

The mitochondrial and cytosolic phosphoenolpyruvate carboxykinase (GTP: oxaloacetate carboxy-lyase(transphosphorylating), EC 4.1.1.32) occurring in the bullfrog (Rana catesbeiana) liver were studied. The enzymes in the two intracellular compartments of both tadpole and adult frog liver were immunologically identical. Both radioactively-labelled forms of the mitochondrial and cytosolic phosphoenolpyruvate carboxykinase from bullfrog liver were imported at the same rate into intact mitochondria in vitro. The mitochondrial and cytosolic enzyme activities did not respond to the administration of glucagon, glucocorticoid, quinolinate and d-mannoheptulose which are known as enhancers of phosphoenolpyruvate carboxykinase, but were found to increase during natural metamorphosis. The former activity was markedly increased in the tadpoles treated with 3,5,3′-triiodothyronine. It was supposed that in the bullfrog liver the phosphoenolpyruvate carboxykinase localized in the mitochondria is of central importance in phosphoenolpyruvate synthesis from oxaloacetate     

Glycogen degradation and inhibition of enzyme activity in the metamorphosing tadpole hepatocyte

Glycogen concentration in the frog tadpole hepatocyte was quantitatively measured and found to decrease markedly during metamorphosis. It is postulated that this disappearance of glycogen may result in increased glucose concentration. Tryptophan oxygenase activity, which normally decreases during metamorphosis, was further depressed by glucose injection. Glycogen breakdown into glucose, followed by glucose inhibition of the enzyme, may, therefore, be a normally occurring mechanism in the intact liver.     

Changes in Polyamine Contents Development of the Frog, Microhyla ornata

Putrescine, spermidine and spermine levels were measured during development, metamorphosis and adult life of the frog, Microhyla ornata . Development of Microhyla was accompanied by high fluctuating levels of putrescine and spermidine with low and steady levels of spermine. Putrescine was the major polyamine during development from egg to mature tadpole. During metamorphosis both putrescine and spermidine decreased significantly; but the decrease in putrescine content was more rapid than that of spermidine. Thus, in the freshly metamorphosed frog, the concentration of spermidine exceeded that of putrescine. In most of the adult tissues also spermidine concentration was higher than putrescine and spermine. While the free form of putrescine and spermidine increased during early development of the fertilized egg to tadpole, the levels of protein conjugated polyamines decreased. In the free form, putrescine was the major polyamine while in the protein conjugated form spermidine concentration was higher than putrescine and spermine. Thus polyamine pattern is different in early development, during metamorphosis and in differentiated adult tissues of this frog. ∞-Difluoromethylornithine treatment at early blastula stage did not interfere with the normal development of Microhyla embryos.     

Biochemical and Immunological Characterization of Collagenase in Tissues of Metamorphosing Bullfrog Tadpoles

Tissue inhibitor of metalloproteinases (TIMP, a specific inhibitor of collagenase) was found to inhibit thyroid hormone-induced tail regression, suggesting the important role of collagenase in this process. Collagenase was purified from culture media of back skin of tadpole of bullfrog, Rana catesbeiana . Anti-tadpole collagenase polyclonal antisera were obtained against the purified enzyme. The antibody inhibited the activity of tadpole collagenase. The antisera reacted to tissues of adult bullfrogs, tadpoles of african clawed frog, Xenopus laevis , and adult newts, Cynopus pyrrhogaster , and also reacted to human fibroblast collagenase. Immunoblot analyses suggested that tadpole collagenase lacks the procollagenase which is generally found in mammalian collagenases. Intense immunological stains were observed for the tissues of thyroid hormone-treated tadpoles as compared to those of untreated animals. Thyroid hormone increased amounts of collagenase not only in epidermal layer but also in mesenchymal tissues including fibroblastic cells.     

Cytochrome c oxidase from the liver of bullfrog, Rana catesbeina and change in its turnover rate during metamorphosis

1. Cytochrome c oxidase was purified from the liver mitochodria of bullfrog (Rana catesbeina). The heme a content of the purified enzyme was 13.5 nmol per mg protein. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the enzyme protein was composed of nine polypeptide subunits having molecular weights of 42 000, 27 000, 25 000, 20 000, 15 000, 13 000, 8 600, 5 400 and 3 600. The purified enzyme from the adult frog was immunologically identical with that from the tadpole. 2. The rates of synthesis and degradation of cytochrome c oxidase were 5.2- and 2.0-times higher at metamorphic climax than at premetamorphic stage, respectively. The amount of the enzyme in the liver was highest at metamorphic climax.     

ERYTHROPOIESIS DURING AMPHIBIAN METAMORPHOSIS : II. Immunochemical Study of Larval and Adult Hemoglobins of Rana catesbeiana

Rabbit antibodies were prepared against the major hemoglobin components of the larval and adult stages of R. catesbeiana. The properties of the antisera were studied by double immunodiffusion, precipitation, and complement fixation. The antisera to tadpole and frog hemoglobins did not cross-react with either hemoglobin or apohemoglobin. The anti-serum against frog hemoglobin was used for the detection of frog hemoglobin in tadpoles undergoing either natural or thyroxine-induced metamorphosis. It was shown that frog hemoglobin is detectable first in the liver, indicating that the liver is the site of erythrocyte maturation during metamorphosis.     

Thyroxine elicits divergent changes in mRNA levels for two urea cycle enzymes and one gluconeogenic enzyme in tadpole liver

Thyroxine-induced metamorphosis of the tadpole to the frog (Rana catesbeiana) is marked by increased activities of the urea cycle enzymes in liver. Cloned cDNAs for two mammalian urea cycle enzymes--carbamyl-phosphate synthetase I and argininosuccinate synthetase--were shown to cross-hybridize with the corresponding mRNAs in tadpole liver. Thyroxine treatment produced nearly 10-fold, coordinate increases in hybridizable mRNA levels for these two enzymes in tadpole liver. This increase is sufficient to account for reported increases in enzyme levels and synthesis rates, demonstrating that thyroxine largely regulates concentrations of these enzymes at a pretranslational step(s). In contrast, levels of phosphoenolpyruvate carboxykinase mRNA in tadpole liver decreased by more than 90% following thyroxine treatment. This differs from the thyroxine-induced increases in synthesis rates of enzyme and mRNA reported for phosphoenolpyruvate carboxykinase in rat liver. However, the decreased levels of this mRNA in tadpole liver may represent a secondary response due to thyroxine-stimulated release of insulin.     

Gene switching at Xenopus laevis metamorphosis

During the climax of amphibian metamorphosis many tadpole organs remodel. The different remodeling strategies are controlled by thyroid hormone (TH). The liver, skin, and tail fibroblasts shut off tadpole genes and activate frog genes in the same cell without DNA replication. We refer to this as “gene switching”. In contrast, the exocrine pancreas and the intestinal epithelium dedifferentiate to a progenitor state and then redifferentiate to the adult cell type. Tadpole and adult globin are not present in the same cell. Switching from red cells containing tadpole-specific globin to those with frog globin in the liver occurs at a progenitor cell stage of development and is preceded by DNA replication. Red cell switching is the only one of these remodeling strategies that resembles a stem cell mechanism.     

Purification and properties of the manganese superoxide dismutase from the liver of bullfrog, Rana catesbeiana

A manganese superoxide dismutase (Mn-SOD) from the liver of bullfrog, Rana catesbeiana, was purified to electrophoretic homogeneity. The enzyme has a molecular weight of about 84,000 and is composed of four identical subunits, each containing one manganese atom. The amino acid composition of the enzyme is similar to that of Mn-SODs isolated from human and chicken livers, but differs considerably from that of the Escherichia coli enzyme (D. Barra et al. (1984) J. Biol. Chem. 259, 12595-12601; R. A. Weisiger and I. Fridovich (1973) J. Biol. Chem. 248, 3582-3592; H. M. Steinman (1978) J. Biol. Chem. 253, 8708-8720). The N-terminal amino acid is lysine. The sequence of 23 amino acid residues in the N-terminal region was determined. It shows excellent homologies with those of the human and chicken enzymes (H. M. Steinmam and R. L. Hill (1973) Proc. Natl. Acad. Sci. USA 70, 3725-3729; C. Ditlow et al. (1982) Carlsberg Res. Commun. 47, 81-91). The frog liver enzyme is also located exclusively in the mitochondrial matrix. Immunologically the same enzyme is also found in the tadpole liver, in an amount of about one-half of that in the adult bullfrog.     

ERYTHROPOIESIS DURING AMPHIBIAN METAMORPHOSIS : III. Immunochemical Detection of Tadpole and Frog Hemoglobins (Rana catesbeiana) in Single Erythrocytes

Rabbit antibodies specific for the major tadpole and frog hemoglobin components of R. catesbeiana were used for the detection of the two hemoglobins inside single cells. The antisera, after fractionation by ammonium sulfate precipitation and diethylaminoethyl (DEAE)-cellulose chromatography, were conjugated with fluorescein isothiocyanate for the antifrog hemoglobin serum and tetramethylrhodamine isothiocyanate for the antitadpole hemoglobin serum. The conjugated fractions, refractionated by stepwise elution from a DEAE-cellulose column, were used for the fluorescent staining of blood smears, liver tissue imprints, and smears of liver cell suspensions. Both simultaneous and sequential staining with the two fluorescent preparations indicated that larval and adult hemoglobins were not present within the same erythrocyte during metamorphosis. In other experiments, erythroid cells from animals in metamorphosis were spread on agar containing specific antiserum. Precipitates were formed around the cells which contain the particular hemoglobin. The percentages of cells containing either tadpole or frog hemoglobin were estimated within the experimental error of the method. The data showed that the two hemoglobins are in different cells. It is concluded that the hemoglobin change observed during the metamorphosis of R. catesbeiana is due to the appearance of a new population of erythroid cells containing exclusively frog hemoglobin.     

Female-offspring communication in a Taiwanese tree frog, Chirixalus eiffingeri (Anura: Rhacophoridae)

We assessed the roles of visual and olfactory cues in female-tadpole communication inChirixalus eiffingeri . The mean cumulative time that at least one tadpole was active or begged for food was significantly longer when a female C. eiffingeri was present than when a plastic frog was introduced and when no frog was present. Tadpoles did not respond visually to a female frog physically separated from them by transparent Plexiglas. However, tadpoles were more active in water conditioned by female frogs than in unconditioned water. Tadpole activity was further elevated by water conditioned by a female frog and tadpoles. Tadpoles were more active in water conditioned by male frogs than in unconditioned water, but water conditioned by a male frog and tadpoles did not further elevate tadpole activity. Thus, water conditioned by adults of either sex contains substances that increase tadpole activity, but only females show a synergistic effect with conditioning by tadpoles. Copyright 2002 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour     

Nuclear ribonucleoprotein complexes of amphibian liver. I. Characterization of the complex and its small molecular weight RNA moiety.

Nuclear RNA-protein complexes containing small molecular weight RNAs were isolated from hepatic nuclei of Rana catesbeiana tadpoles and frogs according to a procedure normally used for the isolation of heterogeneous nuclear ribonucleoprotein complexes from other eukaryotic tissues. Preliminary characterization of the tadpole nuclear RNP indicated a particle size of 50--70 S in sucrose density gradients and a buoyant density of 1.40 gm/ml in CsCl gradients. When analyzed on SDS-polyacrylamide gels, this complex was observed to contain at least 40 polypeptides ranging in molecular weight from 15,000 to 200,000. Nuclear RNA-protein complexes were also isolated from adult frog hepatic nuclei by the same protocol and the RNA moiety which had been purified from the frog complex was compared with the nuclear RNA isolated from the tadpole particles. Electrophoretic analysis of the nuclear RNA-protein-associated RNA revealed minor qualitative and quantitive differences in the more than 25 discrete bands (4--9 S) associated with each particle. Base analysis of tadpole and frog nuclear RNA revealed a nucleotide composition of approximately 50% adenosine plus uridine nucleotides, with an unusually high content of cytosine residues (approximately 30%). Comparison of the two RNA samples demonstrated a large increase in the adenosine content of frog unclear RNA, and the presence of a minor base in frog nuclear RNA which was absent in the tadpole sample. These results indicated that changes in the RNA content of the amphibian nuclear RNP complex had occurred during bullfrog development.     

Rhodanese activity and total sulfur content in frog and mouse liver

The activity of rhodanese was histochemically tested in cryostat sections of the frog (Rana temporaria) and mouse liver. The activity levels were evaluated in sections, and the results were expressed as the ratio of the area of all granules, products of the enzymatic test, to the total analyzed area (area fraction). The present study confirmed the biochemically detected activity of rhodanese, and showed a large pool of endogenous sulfane sulfur donors, substrates for rhodanese, in the frog liver. The area of a single granule corresponded to the size of the mitochondrium, what suggests enzyme localization in this organelle. In view of this it should be considered whether the rhodanese activity, biochemically detected in the cytosolic fraction of the frog liver, results from the enzyme action. The total content of sulfur in cryostat sections of the mouse and frog liver was calculated and compared on the basis of the Energy Dispersion Spectrum (EDS) obtained by a scanning microscope. These studies showed higher total sulfur content in the frog liver than in the mouse liver. The high total content of sulfur in the frog liver in autumn might be associated with sulfur storing for protein biosynthesis during the period of hibernation.     

Metamorphic Changes in Glycolipids and Myelin Proteins and 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase in Bullfrog and Axolotl Brains

Abstract: The metamorphic changes in levels of glycolipids and myelin proteins and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) in the brains of bullfrog tadpoles, adult frogs, and axolotls were investigated, with particular emphasis on myelin maturation. The concentrations of cerebroside. sulfatide, and galactosyldiacylglycerol gradually increased from the onset of prometamorphosis throughout the active metamorphic period and then greatly increased after metamorphosis was completed. The ratio of glucocerebroside to galactocerebroside increased greatly in the prometamorphic period and then rapidly decreased to the frog level during the climax period. The fatty acid compositions of cerebroside and sulfatide showed a developmental change, with 24:1 being more predominant in the later metamorphic stage. The proportion of hydroxy fatty acids increased up to the onset of the prometamorphic stage and thereafter remained constant at ∼ 50% of the total. The CNP activity remained unchanged throughout metamorphosis at 60% that in frog myelin and increased in the adult frog. The composition of tadpole myelin proteins remained constant during metamorphosis, with large basic protein being the most abundant, and in the frog, proteolipid protein and large basic protein were present in comparable amounts. The two adult forms of axolotl, i.e., the neotenous and metamorphosed forms, exhibited almost identical myelin constituents, and CNP activity in the neotenous form amounted to one-fifth that in the bullfrog. These results indicate that active biosynthesis of myelin marker components occurs as metamorphosis proceeds, but more pronounced changes of myelin components occur after metamorphosis is completed.     

The life span of red blood cells in the amphibian larvae, Rana catesbeiana

Nearly one hundred amphibian tadpoles were made anemic by phenylhydrazine injection. During the recovery period radioactive thymidine was incorporated into the DNA of the nucleated amphibian red blood cells. Over the next 135 days blood was drawn and smears of circulating red blood cells were prepared. The blood smears from each tadpole were autoradiographed and percentage of labeled nuclei (PLN) determined. The average life span of tadpole red blood cells was calculated from the change in PLN, and is about 100 days. It is concluded that during the transition from tadpole to frog, the tadpole red blood cell life span must be drastically shortened to account for the hemoglobin transition observed during amphibian metamorphosis.     

Comparative study on vertebrate liver AMP deaminases

Similar activity of AMP deaminase was found in rat, hen, turtle and flounder liver when estimated at high AMP concentration. The enzyme activity was of an order of magnitude higher in frog liver. Simple step by step phosphocellulose column chromatography revealed two forms of AMP deaminase in chicken and flounder liver and one form in the liver of rat and turtle. All enzymes (except for frog liver AMP deaminase) were activated by ATP. The enzymes from rat, frog and both forms from flounder were also activated by ADP. GTP exhibited a variety of effects. The enzyme from rat and turtle was inhibited, both forms from hen and flounder were activated and frog liver enzyme was not influenced.     

Characterization of γ-glutamyltranspeptidase in the liver of the frog: 2. Response to season,temperature and thyroid hormone in Rana pipiens

The impact of season and temperature on frog liver γ-glutamyltranspeptidase was assessed by measuring the activity of this enzyme in plasma membranes isolated from the livers of Rana pipiens obtained as summer and winter frogs; subjected to short-term (3 weeks) temperature acclimation; and subjected to multiple-temperature shifts. Plasma levels of T₃ were determined. γ-Glutamyltranspeptidase was found to be 2·2-fold higher in the summer frog relative to the winter frog; decreased by 44 percent in the summer frog by cold acclimation and increased by 1·7-fold in the winter frog by warm acclimation; and increased by 1·9-fold in the summer frog and 2·8-fold in the winter frog subjected to multiple-temperature shifts. Plasma T₃ levels were found to be 42-fold higher in the summer frog relative to the winter frog; decreased by 42 percent by cold acclimation and increased by 2·9-fold by warm acclimation; and decreased by 39 percent and 38 percent in the summer and winter frogs subjected to multiple temperature shifts. T₃ replacement during the last phase of the multiple-temperature shift protocol, restored the plasma T₃ levels to 75 percent of the control levels and prevented the increase evoked by the multiple-temperature shifts in γ-glutamyl-transpeptidase activity. Indeed, enzyme activity in the T₃ replaced state was 19 percent lower than in the control state. The involvement of thyroid hormone as a negative regulator of enzyme activity is discussed.     

Tyrosine aminotransferase activity of frog (Rana esculenta) liver

1. The presence of tyrosine aminotransferase is reported both in particulate and soluble fractions of frog liver. 2. The activity of the soluble enzyme of frog liver was investigated with regard to its dose and time dependence, its substrate specificity and concentration dependence, its thermal sensitivity as well as pH and temperature dependence. 3. It appears that the properties of the soluble tyrosine aminotransferase of frog liver are in close agreement with those reported for the mammalian liver enzyme.     

Sequential up-regulation of thyroid hormone beta receptor, ornithine transcarbamylase, and carbamyl phosphate synthetase mRNAs in the liver of Rana catesbeiana tadpoles during spontaneous and thyroid hormone-induced metamorphosis.

During both spontaneous and thyroid hormone (TH)-induced metamorphosis, the Rana catesbeiana tadpole undergoes postembryonic developmental changes in its liver which are necessary for its transition from an ammonotelic larva to a ureotelic adult. Although this transition ultimately results from marked increases in the activities and/or de novo synthesis of the urea cycle enzymes, the precise molecular means by which TH exerts this tissue-specific response are presently unknown. Recent reports, using RNA from whole Xenopus laevis tadpole homogenates and indirect means of measuring TH receptor (TR) mRNAs, suggest a correlation between the up-regulation of TR beta-mRNAs and the general morphological changes occurring during amphibian metamorphosis. To assess whether or not this same relationship exists in a TH-responsive tissue, such as liver, we isolated and characterized a cDNA clone containing the complete nucleotide sequence for a R. catesbeiana urea cycle enzyme, ornithine transcarbamylase (OTC), as well as a genomic clone containing a portion of the hormone-binding domain of a R. catesbeiana TR beta gene. Through use of these homologous sequences and a heterologous cDNA fragment encoding rat carbamyl phosphate synthetase (CPS), we directly determined the relative levels of the TR beta, OTC, and CPS mRNAs in liver from spontaneous and TH-induced tadpoles. Our results establish that TH affects an up-regulation of mRNAs for its own receptor prior to up-regulating CPS and OTC mRNAs. Moreover, results with cultured tadpole liver demonstrate that TH, in the absence of any other hormonal influence, can affect an up-regulation of both the TR beta and OTC mRNAs.     

Tyrosine aminotransferase activity of frog (Rana esculenta) liver--II. Comparative aspects of intracellular distribution

1. A subcellular fractionation procedure for frog liver is reported and validated by the distribution pattern of several marker enzymes, also in comparison with rat liver. 2. The subcellular distribution of tyrosine aminotransferase was investigated in frog liver as compared to rat liver: a different distribution of the enzyme was observed, being the activity mostly recovered in mitochondrial and cytosolic compartments. 3. Results indicate that mitochondrial tyrosine aminotransferase of both frog and rat liver is a matrix enzyme, even if differences are observed concerning its release from the organelles upon detergent treatment.