Frog Liver Research Articles

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

1. 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. 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. 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.

Comparative studies on AMP-deaminase—VI. Antigenic properties of the enzymes from frog liver, frog muscle and hen white muscle

1. 1. AMP-deaminases from frog muscle, frog liver and hen white muscle were weak antigens, however specific rabbit anti-serum against each enzyme could be obtained. The most effective as an antigen was frog liver AMP-deaminase. 2. 2. Anti-frog liver deaminase serum was the most specific when tested with different deaminases as antigens.

Developmental changes in non-histone chromosomal proteins of bullfrog ( Rana catesbeiana)

1. 1. Chromatins were isolated from liver, brain, kidney, spleen, heart muscle and erythrocytes of the bullfrog and its tadpole, Rana catesbeiana and their non-histone chromosomal proteins (NHC proteins) were compared by a simple electrophoretic method. 2. 2. No qualitative but quantitative variations in the composition of NHC proteins were found among the tissues examined. 3. 3. The tissues of tadpole and frog were classified into three groups according to their electrophoretic pattern of NHC proteins; brain, heart muscle and spleen showing almost the same pattern, liver and kidney showing different pattern in some components, and erythrocytes showing markedly different pattern. 4. 4. Component D3 occurring in the frog liver, not in the tadpole, was found to increase in the tadpole liver during natural and thyroxine-induced metamorphosis.

Comparative study of Dieldrin effects on amphibian liver and early embryo phospholipids

1. The effect of Dieldrin on phospholipid content and composition was compared in adult frog liver and in early embryos. 2. Male frogs are more resistant to acute Dieldrin treatment than females. Total phospholipids content increases in the liver of male animals while in females decreases. 3. Early embryo development was not affected morphologically by the addition of 0.02–10.0 mg of Dieldrin/1; however phospholipid content and composition were altered. 4. Adaptative changes in the embryo due to chronically administered Dieldrin is suggested.

On 4-hydroxyphenylpyruvate dioxygenase of adult frog liver

1. 1. It has been reported that 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27) activity in the liver from Rana esculenta is present only after autolysis or trypsin digestion, which releases a heat- and acid-stable inhibitor of low molecular mass. 2. 2. Attempts to demonstrate similar effects with the liver enzyme from adult Rana pipiens were unsuccessful. Trypsin had only an inhibitory effect on the enzyme activity in crude extracts. 3. 3. Both untreated and trypsin-treated enzyme had a molecular mass of about 100,000 daltons as determined by gel filtration. The pI was around pH 4.6. One pH-optimum between pH 7 and 8 was observed. 4. 4. At pH 7.5 and 37°C the basal enzyme activity was 1.3 μmol/min per g of protein. It was increased six-fold by a reductant in the presence of catalase. Fe 2+ (50 μM) increased the activity further 1.6-fold when the reaction was carried out in Tris-HCl buffer, but not in potassium phosphate buffer. 5. 5. The K m for 4-hydroxyphenylpyruvate was 50 μM and the V max was around 10 μmol/min per g of soluble protein with reductively activated enzyme. 6. 6. Substrate inhibition was observed above 20 μM concentrations of 4-hydroxyphenylpyruvate.

Tyrosine aminotransferase activity of frog ( Rana esculenta) liver

1. 1. The presence of tyrosine aminotransferase is reported both in particulate and soluble fractions of frog liver. 2. 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. 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.

Microperoxisomes in the epithelial cells of the amphibian urinary bladder: an electron microscopic demonstration of catalase and malate synthase.

All cells that comprise the epithelium of the toad urinary bladder were found to contain small ovoid to tubular membrane-bound bodies with a finely granular matrix. Such organelles were devoid of dense cores (nucleoids). These microperoxisomes reacted positively when incubated for the demonstration of catalase or malate synthase activity. In the toad liver, peroxisomes as well as microperoxisomes were seen. Histochemically, both demonstrated catalase activity; neither showed malate synthase activity. The presence of malate synthase, a glyoxylate cycle enzyme, in toad urinary bladder microperoxisomes may make these latter organelles unique among vertebrates, since malate synthase has been thought to be absent in higher animals.

Omega- and (omega-1)-hydroxylation of 1-dodecanol by frog liver microsomes.

Frog liver microsomes catalyzed the hydroxylation of 1-dodecanol into the corresponding omega- and (omega-1)-hydroxy derivatives. The hydroxylation rate for 1-dodecanol was much lower than that for lauric acid. Both NADPH and O2 were required for hydroxylation activity. NADH had no effect on the hydroxylation. The hydroxylating system was inhibited 49% by CO at a CO:O2 ratio of 4.0. The formation of omega-hydroxydodecanol was more sharply inhibited by CO than was the formation of (omega-1)-hydroxydodecanol, implying that more than one cytochrome P-450 was involved in the hydroxylation of 1-dodecanol and that CO has a higher affinity for the P-450 catalyzing the omega-hydroxylation. The formation of laurate during the incubation of 1-dodecanol with frog liver microsomes suggests that a fatty alcohol oxidation system is also present in the microsomes. NAD+ was the most effective cofactor for the oxidation of 1-dodecanol and NADP+ had a little effect. Pyrazole (an inhibitor of alcohol dehydrogenase) had a slight inhibitory effect on the oxidation and sodium azide (an inhibitor of catalase) had no effect.

Isolation and characterization of xenopus laevis albumin mRNA.

The messenger RNA for albumin was isolated from the liver of male frogs. Purification was achieved using oligo(dT)-cellulose chromatography and sucrose gradient centrifugation under denaturing conditions. As judged by translational activity in a cell-free protein-synthesizing system derived from rabbit reticulocytes, albumin mRNA was enriched 259-fold as compared to the total ribonucleic acid of the liver cells. Purified albumin mRNA migrated after sucrose gradient centrifugation as a single symmetrical peak of approximately 17 S and also moved as a single band following denaturing agarose gel electrophoresis. Albumin mRNA possess properties compatible with the presence of a poly(adenylic acid) sequence. Translation in vitro yielded a product which is immunoprecipitable with anti-(frog albumin) and which showed a single radioactive peak having a molecular weight of about 74000 in sodium dodecylsulfate/polyacrylamide gel electrophoresis. Complementary DNA was synthesized using reverse transcriptase, and, as a template, purified albumin mRNA. Following hybridization under conditions of excess RNA, the rot1/2 of albumin mRNA was found to be 1.8 X 10-3 mol . s . 1-1. This result also confirmed that albumin nRNA had been isolated in a highly purified form.

Cytosolic and mitochondrial phospho enolpyruvate carboxykinase of the bullfrog, Rana catesbeiana, liver

The mitochondrial and cytosolic phospho enolpyruvate 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 phospho enolpyruvate 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 phospho enolpyruvate 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 phospho enolpyruvate carboxykinase localized in the mitochondria is of central importance in phospho enolpyruvate synthesis from oxaloacetate

Monoamine oxidase in frog liver and brain

1. 1. Studies with clorgyline and deprynyl as inhibitors showed that there are two types of mitochondrial flavoprotein monoamine oxidase (MAO) in frog brain and liver that resemble type A and type B MAO in many mammalian organs. 2. 2. The enzymic reactions occured by a double-displacement (ping-pong) mechanism. 3. 3. In frog liver, there is also another type of mitochondrial MAO, which contains copper and pyridoxal phosphate as cofactor, but its activity is very low with benzylamine as substrate.

Comparative studies on AMP-deaminase—IV. Amino acids composition of the enzymes from rat, rabbit, hen, frog, pikeperch skeletal muscle and frog liver

1. 1. Amino acids analysis showed a great degree of homology between vertebrate muscle AMP-deaminases. 2. 2. Significant differences were found in the amino acid composition of frog liver AMP-deaminase and skeletal muscle enzymes. 3. 3. A significant amount of glucosamine was found only in the rabbit red muscle enzyme. 4. 4. Frog liver AMP-deaminase had an isoelectric point distinct from the isoelectric points of muscle enzymes.

Comparative studies on AMP-deaminase—V. Change of enzyme activity during storage

1. 1. The specific activities of homogenous AMP-deaminases isolated from rat, rabbit, hen, frog and perch skeletal muscle increased during storage at 4°C up to 2 weeks. A longer storage caused gradual decrease of activity. 2. 2. No change of specific activity was observed during storage of the enzymes from human and pikeperch skeletal muscle and from frog liver. 3. 3. The absorbancy ratio A 280/A 260 was in the range 1–2 for each enzyme studied, being stable during storage of a given enzyme preparation. 4. 4. Limited proteolysis with trypsin resulted in a significant increase of activity of AMP-deaminase in crude extracts of rat muscle and hen white as well as red muscles. 5. 5. Upon trypsin treatment the specific activity of AMP-deaminase purified from rat muscle increased while negligible activity increase with the enzymes purified from hen white and red muscles was observed.

Hepatic microsomal mixed-function oxidase in the frog, Xenopus laevis

1. 1. Hepatic microsomal mixed-function oxidase of the frog, Xenopus laevis appears to be similar to that of fish and mammals. 2. 2. However, the levels of protein in liver microsomes, and specific contents and activity of cytochrome P450, cytochrome c reductase, aldrin and chlordane epoxidase, and naphthalene and chlordene hydroxylase of frog liver are about 30–40% of these of male rats or mice, and about 65% of that of the adult bluegill. 3. 3. The levels are extremely low in frog larvae but keep increasing after metamorphosis reaching the adult levels between 9 and 12 weeks after tail absorption. 4. 4. The levels of enzyme activity correspond to the P450 concentration and not to the level of cytochrome c reductase activity. 5. 5. In adult frogs the enzyme activity can be affected only slightly by diets. 6. 6. Inducers of P450 (hexobarbital) and P448 ( β-naphthoflavone) cause about 50% increase in cytochrome P450 concentration but did not increase P450 catalyzed reactions.

Effect of detergents on the microsomal cytochrome P-450-dependent monooxygenases from frog and rat liver

Abstract 1. 1. A frog liver microsomal hydroxylase system was more susceptible to inhibition by five detergents (sodium cholate, sodium deoxycholate, Triton X-100, Emulgen 913 and Tween 20) in the assay for laurate hydroxylation than the rat liver microsomal hydroxylase system. This suggests that liver microsomal cytochrome P-450-dependent monooxygenases from different sources may not have the same response to detergents. 2. 2. Emulgen 913 was the most effective detergent for the solubilization of cytochrome P-450 from frog and rat liver microsomes among the five detergents. 3. 3. The average level of cytochrome P-450 as determined in the frog ( 0.401 ± 0.023 nmol/mg protein ) is lower than that in the rat (0.60 or 1.19 ± 0.10 ), rabbit ( 1.45 ± 0.35 ), swine ( 0.59 ± 0.16 ), and approximately the same as in the human ( 0.39 ± 0.14 ) and the cat ( 0.380 ± 0.085 ).

Occurrence of avidin-like biotin-binding capacity in various vertebrate tissues and its induction by tissue injury

1. 1.|Avidin-like biotin-binding capacity was found in the liver and ovary of the common frog and in the kidney or lung of various avian species. 2. 2. The induction of biotin-binding capacity by tissue injury was marked in various avian species, while in the lizard and in the skin of the common frog it was slight. 3. 3. Avidin-like biotin-binding capacity or its induction was not found in the tissues of rainbow trout and three mammalian species studied.

Stimulation of septation in mitochondria by diamide, a thiol oxidising agent

Diamide (10-(4)M), a thiol oxidizing agent, rapidly promotes septation in isolated frog liver mitochondria an also in situ in liver slices. The effect is partially inhibited by dithioerythritol. DNP does not have this effect, and it is concluded that diamide does not promote septation via an uncoupling action. The septate mitochondria have a different appearance from typical dividing mitochondria previously described; it is suggested that (1) diamide may act by favouring the fusion of the internal membranes, and (2) -SH oxidation is of importance in mitochondria in ageing and in various pathological conditions. The ways in which septa may develop in mitochondria in the orthodox and condensed configuration are discussed.

Cell-free translation and thyroxine induction of carbamyl phosphate synthetase I messenger RNA in tadpole liver.

Total RNA of tadpole and frog (Rana catesbeiana) liver was isolated by either 7 or 8 M guanidine . HCl extraction and translated in a cell-free protein-synthesizing system derived from rabbit reticulocytes. The identity of carbamyl phosphate synthetase I[carbamoyl-phosphate synthase (ammonia); ATP:carbamate phosphotransferase (dephosphorylating), EC 2.7.2.5] synthesized in vitro with the purified enzyme was established as follows: (i) immunoprecipitation by a specific antibody; (i) comigration with purified carrier enzyme on sodium dodecyl sulfate/polyacrylamide gel electrophoresis; (iii) copurification with carrier enzyme by affinity chromatography on Cibacron Blue F3GA-coupled agarose; and (iv) formation of identical proteolytic cleavage products. Inclusion of protease inhibitors in the system resulted in no apparent change in the polypeptide molecular weight. These results indicate that carbamyl phosphate synthetase I is synthesized as a polypeptide that is indistinguishable from the mature enzyme by the analytical methods used and that it is not grossly modified during its transport into mitochondria. The level of translatable mRNA for carbamyl phosphate synthetase-I in tadpole liver was increased about 2-fold 1 day after thyroxine treatment and did not change significantly through 4 subsequent days of treatment. Thus the thyroxine-induced synthesis of carbamyl phosphate synthetase I in tadpole liver is at least partly due to an increase of translatable mRNA for this enzyme.

Effect of the venom of the scorpion,Heterometrus fulvipes, on certain aspects of nitrogen metabolism in different tissues in the frog,Rana hexadactyla

Proteolytic activity increased in brain, gastrocnemius muscle and liver of frog due toHeterometrus fulvipes envenomation. Structural protein content decreased and soluble protein content increased in experimental muscle (EM) and the change of these in contralateral muscle (CM), brain and liver was insignificant. Total free amino acid level decreased in EM and increased in CM, brain and liver. Ammonia level increased significantly in all the three tissues while urea content decreased. All these changes were maximum around 24 hr after envenomation and thereafter tended towards normalcy except in EM where there was no tendency towards normal level.

Improved Synthesis of CMP-Sialates Using Enzymes from Frog Liver and Equine Submandibular Gland

An efficient method for the preparation of CMP-N-acetylneuraminic acid using crude or partially purified CTP:N-acylneuraminate cytidylyltransferase from equine submandibular gland or frog liver is described. The yield of the sugar nucleotide after purification by ion-exchange chromatography and gel filtration was 95%. The compound was studied by 360 MHz 1H NMR spectroscopy in addition to the usual chemical and physical analyses. The preparation of radioactive or unlabelled CMP 4-O-methyl-N-acetylneuraminic acid, which is not known to occur in nature, was achieved in 17% yield with the aid of the equine enzyme.