A pyrimidine-guanine sequence-specific ribonuclease from Rana catesbeiana (bullfrog) oocytes.

A pyrimidine-guanine sequence-specific ribonuclease (RC-RNase) was purified from Rana catesbeiana (bullfrog) oocytes by sequential phosphocellulose, Sephadex G75, heparin Sepharose CL 6B and CM-Sepharose CL 6B column chromatography. The purified enzyme with molecular weight of 13,000 daltons gave a single band on SDS-polyacrylamide gel. One CNBr-cleaved fragment has a sequence of NVLSTTRFQLNT/TRTSITPR, which is identical to residues 59-79 of a sialic acid binding lectin from R. catesbeiana eggs, and is 71% homologous to residues 60-80 of an RNase from R. catesbeaina liver. The RC-RNase preferentially cleaved RNA at pyrimidine residues with a 3' flanking guanine under various conditions. The sequence specificity of RC-RNase was further confirmed with dinucleotide as substrates, which were analyzed by thin layer chromatography after enzyme digestion. The values of kcat/km for pCpG, pUpG and pUpU were 2.66 x 10(7) M-1s-1, 2.50 x 10(7) M-1s-1 and 2.44 x 10(6) M-1s-1 respectively, however, those for other phosphorylated dinucleotides were less than 2% of pCpG and pUpG. As compared to single strand RNA, double strand RNA was relatively resistant to RC-RNase. Besides poly (A) and poly (G), most of synthetic homo- and heteropolynucleotides were also susceptible to RC-RNase. The RC-RNase was stable in the acidic (pH 2) and alkaline (pH 12) condition, but could be inactivated by heating to 80 degrees C for 15 min. No divalent cation was required for its activity. Furthermore, the enzyme activity could be enhanced by 2 M urea, and inhibited to 50% by 0.12 M NaCl or 0.02% SDS.     

Purification and molecular characteristics of mitochondrial phosphoenolpyruvate carboxykinase from bullfrog (Rana catesbeiana) liver

Phosphoenolpyruvate carboxykinase from bullfrog liver mitochondria has been purified to electrophoretical and immunological homogeneity by an improved method using hydrophobic chromatography on Sepharose-hexane-GMP and affinity chromatography on phosphocellulose. The molecular weight was determined to be 70,000 by SDS-gel electrophoresis, 65,000 by Sephadex G-100 gel filtration and 72,000 by glycerol gradient centrifugation. The isoelectric point was determined to be 6.2, differing from that of the cytosol enzyme. The rabbit IgG fraction against the mitochondrial PEP carboxykinase precipitated not only the mitochondrial but also the cytosol enzyme. The dissociation constant of the nucleotide-enzyme complex was determined to be 3 microM for GTP, 8.5 microM for GDP, and 171 microM for GMP. The affinity of GTP for the enzyme was reduced in the presence of phosphoenolpyruvate or Mn2+, whereas that of GDP was not changed. GMP inhibited the enzyme competitively with GDP for the phosphoenolpyruvate carboxylation and competitively with GTP for the exchange reaction between [14C]HCO3- and oxaloacetate. The purified enzyme was found to have a cysteine residue which reacted with iodoacetamide to form inactive enzyme. Guanine nucleotides or IDP and Mn2+ at a lower concentration prevented the inactivation by iodoacetamide of the enzyme in a competitive manner. Binding of guanine nucleotide to the enzyme and the relation of the sulfhydryl group to the nucleotide binding are discussed.     

Purification and characterization of cytosol phosphoenolpyruvate carboxykinase from bullfrog (Rana catesbeiana) liver.

Cytosol PEP carboxykinase has been purified to electrophoretic homogeneity from bullfrog liver homogenate. The enzyme is a single polypeptide chain with a molecular weight of approximately 72,000-75,000. The purified enzyme catalyzed oxaloacetate decarboxylation (nucleoside triphosphate-supported), phosphoenolpyruvate carboxylation, and an exchange reaction between oxaloacetate and [14C]HCO3-in the presence of ITP or CTP. Manganese is absolutely required for the enzyme-catalyzed phosphoenolpyruvate carboxylation, whereas it can be replaced by Mg2+ for the oxaloacetate decarboxylation and the exchange reaction. The optimal pH of each reaction is dependent on the divalent metal ion used. The dependence of the enzyme activity on Mn2+ is markedly different in the phosphoenolpyuvate carboxylation and the oxaloacetate decarboxylation reactions.     

Purification and cloning of cytotoxic ribonucleases from Rana catesbeiana (bullfrog)

Ribonucleases with antitumor activity are mainly found in the oocytes and embryos of frogs, but the role of these ribonucleases in frog development is not clear. Moreover, most frog ribonuclease genes have not been cloned and characterized. In the present study, a group of ribonucleases were isolated from Rana catesbeiana (bullfrog). These ribonucleases in mature oocytes, namely RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNase 6, as well as liver-specific ribonuclease RC-RNase L1, were purified by column chromatographs and detected by zymogram assay and western blotting. Characterization of these purified ribonucleases revealed that they were highly conserved in amino acid sequence and had a pyroglutamate residue at their N-termini, but possessed different specific activities, base specificities and optimal pH values for their activities. These ribonucleases were cytotoxic to cervical carcinoma HeLa cells, but their cytotoxicities were not closely correlated to their enzymatic specific activities. Some other amino acid residues in addition to their catalytic residues were implicated to be involved in the cytotoxicity of the frog ribonucleases to tumor cells. Because the coding regions lack introns, the ribonuclease genes were cloned by PCR using genomic DNA as template. Their DNA sequences and amino acid sequences are homologous to those of mammalian ribonuclease superfamily, ~50 and ~25%, respectively.     

Solution structure of the cytotoxic RNase 4 from oocytes of bullfrog Rana catesbeiana

Cytotoxic ribonucleases with antitumor activity are mainly found in the oocytes and early embryos of frogs. Native RC-RNase 4 (RNase 4), consisting of 106 residues linked with four disulfide bridges, is a cytotoxic ribonuclease isolated from oocytes of bullfrog Rana catesbeiana. RNase 4 belongs to the bovine pancreatic ribonuclease (RNase A) superfamily. Recombinant RC-RNase 4 (rRNase 4), which contains an additional Met residue and glutamine instead of pyroglutamate at the N terminus, was found to possess less catalytic and cytotoxic activities than RNase 4. Equilibrium thermal and guanidine-HCl denaturation CD measurements revealed that RNase 4 is more thermally and chemically stable than rRNase 4. However, CD and NMR data showed that there is no gross conformational change between native and recombinant RNase 4. The NMR solution structure of rRNase 4 was determined to comprise three alpha-helices and two sets of antiparallel beta-sheets. Superimposition of each structure with the mean structure yielded an average root mean square deviation (RMSD) of 0.72(+/-0.14)A for the backbone atoms, and 1.42(+/-0.19)A for the heavy atoms in residues 3-105. A comparison of the 3D structure of rRNase 4 with the structurally and functionally related cytotoxic ribonuclease, onconase (ONC), showed that the two H-bonds in the N-terminal pyroglutamate of ONC were not present at the corresponding glutamine residue of rRNase 4. We suggest that the loss of these two H-bonds is one of the key factors responsible for the reductions of the conformational stability, catalytic and cytotoxic activities in rRNase 4. Furthermore, the differences of side-chain conformations of subsite residues among RNase A, ONC and rRNase 4 are related to their distinct catalytic activities and base preferences.     

Glomerular permeability in the bullfrog Rana catesbeiana

Filtration studies suggest similar size pores in the glomerular filters of mammals and amphibians. However, the glomerular wall in the bullfrog exhibits several structural features not found in mammals. The subendothelial space of the basement membrane is often greatly enlarged and infiltrated by cellular elements. The lamina densa of the basement membrane shows extensive variation in thickness and packing of its filaments. On the other hand, the epithelial slits in the bullfrog are closed by a slit diaphragm which appears similar in size and structure to the slit diaphragm in mammals. Horse spleen ferritin, a protein with a hydrodynamic radius of 61 A, was used as an ultrastructural tracer to determine whether the highly variable structure of the basement membrane renders this layer more permeable than its mammalian counterpart. Within 10 min after intravenous injection, ferritin was found throughout the basement membrane and often in clusters within the subepithelial layer adjacent to the slit diaphragm. Virtually no ferritin was found within the urinary space, podocytes, or cells of the proximal tubule. Ferritin distribution was the same in both superficial glomeruli and more deeply lying glomeruli regardless of the method of fixation. These results indicate that in the bullfrog the slit diaphragm is a principal filtration barrier to ferritin and thus to smaller plasma proteins.     

Purification and properties of an alkaline ribonuclease from the hepatic cytosol fraction of bullfrog, Rana catesbeiana.

In the hepatic cytosol fraction of bullfrog, Rana catesbeiana, an alkaline RNase [EC 3.1.4.22] exists in two forms. One is the free form of RNase, which elutes from a carboxymethyl-cellulose column at a concentration of 0.2 M NaC1. The other is a masked or latent form (RNase-RNase inhibitor complex) which is not adsorbed on the carboxymethyl-cellulose column and which can be converted to the free form of RNase by the addition of p-chloromercuribenzoate. Electrophoretically pure RNase was obtained by the following procedure. The unadsorbed fraction of hepatic cytosol on a column of carboxymethyl-cellulose was treated with p-chloromercuribenzoate and then applied to a second carboxymethyl-cellulose column. The molar weight of RNase was determined to be approximately 12,000 by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. From the results of gel filtration, the molecular weight of the RNase-RNase inhibitor complex was 130,000. The RNase hydrolyzed poly C, poly U, and poly I, but not poly A or poly G. When poly C was used as a substrate, 2',3'-cyclic CMP as an intermediate and 3'-CMP as a final product were identified. The results of amino acid analysis indicated the presence of an unusual component. The general properties of the RNase and the RNase-RNase inhibitor complex are also reported.     

Fine structure of the rhombencephalic tela of the bullfrog,Rana catesbeiana

Summary The posterior rhombencephalic tela choroidea of the bullfrog was examined by electron microscopy. This membrane, the pia-ependymal roof of the caudal hindbrain, contains a large central region characterized by cuboidal ependymal cells which surround sizable microscopic apertures — the interependymal pores.Ultrastructurally ependymal cells of this area are characterized by infrequent apical microvilli and cilia. They contain irregularly shaped nuclei and few cytoplasmic organelles that are largely apical in position. The most striking feature is an abundance of cytoplasmic filaments forming an extensive cytoskeleton. Laterally these cells are joined by numerous elaborate desmosomes. The majority of the ependymal cells have a basal lamina consisting of single, double, or triple laminae lying parallel to the basal plasma membrane.Several unusual specializations are seen at the margins of the interependymal pores. The ependymal cells have lateral cytoplasmic processes that form the actual border of each pore. These processes originate from the apical surface of the cell and partially enclose an elaborate network of basal lamina associated with the interependymal pores.These findings demonstrate microscopic apertures in the roof of the fourth ventricle in the bullfrog that are associated with an unusual form of supportive ependyma.     

Cloning of hibernation-related genes of bullfrog (Rana catesbeiana) by cDNA subtraction

Seven genes specifically expressed during hibernation in the bullfrog (Rana catesbeiana) were cloned from a subtracted cDNA library constructed from livers of winter bullfrogs. Those genes were fibrinogen alpha-subunit, fibrinogen gamma-subunit, complement component C3, alpha-1-microglobulin/bikunin precursor (AMBP), transferrin, apoferritin middle subunit and one novel gene. Northern hybridization has indicated that these seven genes were specifically induced or enhanced in winter. Above all, expression of the novel gene was specifically induced in winter in liver, though the expression of that was neither induced in bullfrog nor Xenopus laevis by cold treatment. The novel gene, which was designated as rc-hirp (Rana catesbeiana hibernation-related protein), encoded 420 base pairs length and a putative protein of 139 amino acid residues. Annual analyses of the expression of these genes have suggested that the seven winter-specific genes are playing an important role in hibernation processes.     

Characterization of cholecystokinin receptors in bullfrog (Rana catesbeiana) brain and pancreas

The binding of biologically active 125I-Bolton-Hunter-CCK-33 to bullfrog brain and pancreatic membrane particles was characterized. Both tissues exhibited time-dependent, saturable, reversible, and high affinity binding without evidence for cooperative interaction. Both bullfrog CCK receptors resembled their mammalian counterparts in having acidic pH optima for tracer binding and a Kd of about 0.5 nM. However, the receptors differed from their mammalian counterparts in that (1) the bullfrog brain membranes bound more tracer per mg protein than did the pancreatic membranes, (2) both bullfrog CCK receptors were relatively insensitive to dibutyryl cGMP, and (3) both bullfrog brain and pancreatic CCK receptors exhibited the same general specificity toward a variety of CCK and gastrin peptides. For both tissues, the relative order of receptor binding potency was CCK-8 greater than caerulein = CCK-33 greater than gastrin-17-II greater than CCK-8-ns = gastrin-17-I greater than caerulein-ns greater than gastrin-4 with the sulfated CCK peptides being 1000-fold more potent than their nonsulfated analogs. Sulfated gastrin was also relatively potent, being only 10-fold weaker than CCK-8. Gastrin-4 was 20 000-fold weaker than CCK-8 in interacting with the brain CCK receptor. The latter finding is in sharp contrast to the mammalian brain CCK receptor. We conclude that the bullfrog brain and pancreas contain similar CCK receptors of probable physiological significance and may represent an ancestral condition from which the two distinct CCK receptors present in mammalian brain and pancreas have evolved.     

Arachnoid mater of the bullfrog,Rana catesbeiana

Summary In the bullfrog, the meninges surrounding the central nervous system include an arachnoid mater that contains layers of cells with abundant intermediate filaments (IFs) having unique organizational characteristics. This membrane contains an inner lamina of cells that resemble fibroblasts and an outer lamina of flattened cells that are almost filled with IFs. The IFs of the outer arachnoid are arranged in compact, arching bundles that lie parallel to the outer surface of the central nervous system. Thus, sections cut tangentially to the membrane reveal bending of filament bundles, whereas transverse sections do not. In some cells bordering the subdural space, bundles of filaments are organized into highly-ordered spiral arrays. Attachments to the numerous desmosomes and, apparently, to the nuclear envelope suggest anchoring of cytoplasmic structures by the IF system. Microtubules occur primarily near the plasma membrane and the nucleus. Numerous caveolae also are associated with the plasma membrane.The unusual abundance, organization, and cytoplasmic relations of IFs in the bullfrog arachnoid suggest that this membrane may serve as an important model for study of fundamental cytoskeletal relations and function.     

Tympanic sound radiation in the bullfrog Rana catesbeiana

Members of the Rana catesbeiana clade display sexually dimorphic eardrums. In this species assemblage the eardrum of males can be 50% larger than in females of the same body size. There has been, however, no apparent functional explanation for this dimorphism. Measurements of the acoustical coupling (transfer function) of internally generated sound to the enlarged eardrum of male bullfrogs (R. catesbeiana) show distinct energy peaks coincident with those observed in the spectral envelopes of the release and mating calls. Moreover, when the tympanic membranes are artificially damped the spectrum of the release call is drastically altered and the total amount of power radiated decreases substantially. These observations point to a previously unsuspected role for the ears in the sound broadcasting process of the bullfrog and possibly other anurans with similarly modified tympanic membranes. Accepted: 19 July 1997     

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