Immunocytochemical localization of a non-vitellogenin, estrogen-induced plasma protein in the hepatocyte of the American bullfrog, Rana catesbeiana

Recently, a non-vitellogenin, estrogen-induced frog plasma protein of unknown function and site of synthesis, which has been given the temporary name, protein-RcX, was isolated and partially characterized by Mitchell et al. In the present study, the protein A-gold immunocytochemical technique was applied to investigate its site of secretion; this was found to be the hepatocyte of the estradiol-17 beta-treated adult, male American bullfrog, Rana catesbeiana. Specific immunolabeling for protein-RcX was present over those intracellular compartments involved in protein secretion, i.e., the rough endoplasmic reticulum, Golgi apparatus and secretory granules, in addition, lysosomes were also labeled. No specific labeling for this protein was observed on hepatocytes of normal, non-estrogen treated, adult, male bullfrogs. Further, the labeling was abolished when plasma containing protein-RcX was added to the antibody prior to incubation but remained when purified vitellogenin was added. These observations support the hypothesis that protein-RcX is a non-vitellogenin, estrogen-induced plasma protein which is synthesized and secreted in parallel with vitellogenin by the hepatocyte of the estrogen-treated frog.     

Purification and characterization of cathepsin D-like proteinase from the tadpole tail of bullfrog, Rana catesbeiana

1. An acid aspartic proteinase in the regressing tadpole tail was purified about 800-fold with a 36% recovery. 2. The mol. wt of the enzyme was found to be 42,000 on gel filtration and 38,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively. 3. The purified enzyme had a maximum activity at pH 3.5 and an apparent Km of 0.084% with acid-denatured hemoglobin as substrate. 4. The enzyme activity was strongly inhibited by pepstatin. In addition, diazoacetylnorleucine methyl ester inactivated the enzyme in the presence of cupric ions. 5. The enzyme was identified as a cathepsin D (EC. 3.4.23.5)-like proteinase.     

Purification and characterization of NADPH-dependent methemoglobin reductase from the nucleated erythrocytes of bullfrog, Rana catesbeiana

Two protein components having a NADPH-dependent methemoglobin reductase activity were purified to electrophoretic homogeneity from the erythrocytes of the bullfrog, Rana catesbeiana. Their molecular properties were investigated. The components were separated by isoelectric focusing, having discrete bands of pI 5.0 and 7.5, respectively. The pI 5.0 component, designated F-5.0, was faint yellow, with a broad absorption in the range of 400-450 nm, while the pI 7.5 component, designated F-7.5, was colorless and did not absorb in that range. The molecular weight was estimated to be 22,000 for both components by gel filtration and SDS-PAGE. When F-5.0 was subjected to isoelectric focusing repeatedly, the protein part of that component gradually moved to and refocused at pH 7.5, leaving a yellow color at acidic pH. Both F-5.0 and F-7.5 were highly specific for NADPH and had the same kinetic properties in catalyzing the reduction of MB, DCPIP, FMN, or FAD, and that of methemoglobin or cytochrome c in the presence of a certain dye. They were also indistinguishable from one another in their amino acid compositions and were completely identical in the N-terminal sequence of 24 amino acid residues. These findings strongly suggest that the two components can be attributed to the same enzyme molecule, carrying an identical protein moiety but interacting differently with some unidentified biological pigments, and that they are equivalent in their molecular and kinetic properties to the NADPH-dependent enzyme(s) occurring in human erythrocytes.     

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.     

Developmental and diel profiles of plasma corticosteroids in the bullfrog,Rana catesbeiana

Corticosteroids synergize with the thyroid hormone (TH) at late metamorphic stages and might have a role in the hormonal regulation of amphibian metamorphosis. This role could be influenced by diel fluctuations, particularly if the peak of the plasma corticoids changed in relation to the TH peaks. Diel variation in plasma corticosteroids was studied in Rana catesbeiana prometamorphic and climax tadpoles on 18:6, 12:12 and 6:18 light:dark (LD) cycles. Cortisol (hydrocortisone; HC) and aldosterone (ALDO) exhibited different, but LD cycle-specific, circadian fluctuations at prometamorphosis, whereas corticosterone (CORT) was undetectable (less than 1.18 ng/ml). HC, ALDO and CORT rhythms became synchronous at early metamorphic climax on all LD cycles, although the cosinor-derived acrophases, which occurred around the time of the dark:light transition, shifted approximately 6 h earlier from 18L:6D to 6L:18D. On both 18L:6D and 12L:12D, the acrophase of HC changed little from prometamorphosis to climax, whereas that of ALDO underwent a major phase shift. On 6L:18D, both the ALDO and the HC acrophases shifted at climax. These LD cycle-specific phase shifts of the diel rhythms placed the acrophases of the corticoids in different phase relationships to that of the previously determined thyroxine (T(4)) acrophase at climax, and may partially explain the influence of the light regimen on metamorphic timing. The pronounced diel variations in the corticoid concentrations from the troughs to the peaks show that hormone levels are a function of the time of day and the environmental lighting regimen, which need to be taken into account in measuring the level of plasma hormones in amphibians. The 24-h means calculated from the data of all the sampling times showed that only plasma ALDO and CORT, but not HC, rose markedly at climax, although there were significant LD cycle-related differences in the mean levels of both HC and ALDO at prometamorphosis, and in HC at climax. Additional work sampling at mid-light showed that plasma CORT peaked at Stage XXIII, decreased at the end of climax, and remained low in the postmetamorphic froglet at 2.1 ng/ml. In the adult bullfrog, CORT was clearly the predominant corticosteroid at 34.3 ng/ml, whereas HC and ALDO levels were only approximately 1.3 ng/ml.     

Purification, characterization, and amino acid sequences of pepsinogens and pepsins from the esophageal mucosa of bullfrog (Rana catesbeiana)

Two pepsinogens (pepsinogens 1 and 2) were purified from the esophageal mucosa of the bullfrog (Rana catesbeiana), and their molecular weights were determined to be 40,100 and 39,200, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal 70-residue sequences of both pepsinogens are the same, including the 36-residue activation segment. Furthermore, a cDNA clone encoding frog pepsinogen was obtained and sequenced, which permitted deduction of the complete amino acid sequence (368 residues) of one of the pepsinogen isozymogens. The calculated molecular weight of the protein (40,034) coincided well with the values obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results are incompatible with the previous report (Shugerman R. P., Hirschowitz, B. I., Bhown, A. S., Schrohenloher, R. E., and Spenney, J. G. (1982) J. Biol. Chem. 257, 795-798) that the major pepsinogen isolated from the bullfrog esophageal gland is a unique "mini" pepsinogen with a molecular weight of approximately 32,000-34,000. The two pepsinogens were immunologically indistinguishable from each other and related to human pepsinogen C. The deduced amino acid sequence was also more homologous with those of pepsinogens C than those of pepsinogens A and prochymosin. These results indicate that the frog pepsinogens belong to the pepsinogen C group. They were both glycoproteins, and therefore, this is the first finding of carbohydrate-containing pepsinogens C. Both pepsinogens were activated to pepsins in the same manner by an apparent one-step mechanism. The resulting pepsins were enzymatically indistinguishable from each other, and their properties resembled those of tuna pepsins.     

Pharmacokinetics of kanamycin in the bullfrog, Rana catesbeiana

1. Kanamycin disposition was studied in bullfrogs (Rana catesbeiana) following single doses IP. Both plasma t1/2 and Vd of the drug increased with increasing time after drug indicating redistribution and tight binding of kanamycin to deep tissue compartments. 2. Kanamycin was eliminated unchanged with a t1/2 plasma = 27 hr; perilymph = 89 hr; endolymph = 183 hr; aqueous humor = 54 hr; and CSF = 58 hr. 3. Kanamycin was absorbed by frogs from environmental water. 4. Environmental conditions must be carefully specified and monitored, as well as the physiological state of the animals when studying the effects of drugs on Amphibia.     

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.     

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.     

Biomechanics of vibration reception in the bullfrog,Rana catesbeiana

The opercularis system (OPS) of amphibians consists of an opercularis muscle that connects the shoulder girdle skeleton to the operculum, a movable element in the oval window of the otic capsule. The role of the OPS in reception of vibrations was examined in bullfrogs (Rana catesbeiana) tested in various postures that manipulated differential motion between the shoulder girdle (the origin of the opercularis muscle) and skull (including the inner ear). Amplitude and phase relationship of motions of the suprascapular cartilage of the shoulder girdle and the posterior skull were also measured during these tests. 1. Microphonic responses to vertical vibrations from 25-200 Hz were typically highest when frogs were in a normal, sitting posture with the head held off the vibrating platform. Responses from animals in which the head directly contacted the platform were often less (by up to 10 dB at certain frequencies). Responses from all test positions were highest at lower frequencies, especially between 50-100 Hz. 2. Suprascapular accelerations were typically highest in the normal, sitting posture, and at lower frequencies (50-75 Hz) were often greater than that of the vibrating platform by up to 8 dB. The shoulder girdle skeleton of the bullfrog is therefore readily affected by vertical substrate motion. 3. The amplitude of microphonic responses in the different test postures did not correspond well with head acceleration. Rather, response amplitude corresponded best with the absolute difference between shoulder and head motion. For example, in the normal posture, suprascapular motion was much greater than head motion, and responses were relatively high. If only the head was vibrated, head motion was high and shoulder motion low, and responses also were relatively high. If the head and body were vibrated together, their motions were similar, and responses to the same platform accelerations were often reduced. Phase differences between shoulder and head motions were small at the frequencies examined and may be of little functional significance. The importance of differences in shoulder and head motion suggests that the resulting differential motion of the operculum and inner ear fluids can produce waves that stimulate appropriate end organs (such as the saccule). 4. Removal of the opercularis muscle reduced responses up to 18 dB at certain frequencies in some of the test postures. The most significant reductions were observed in those postures with a significant difference between shoulder and head motion (such as the normal posture).(ABSTRACT TRUNCATED AT 400 WORDS)     

Primary structure of a ribonuclease from bullfrog (Rana catesbeiana) liver

A pyrimidine base-specific ribonuclease was purified from bullfrog (Rana catesbeiana) liver by means of CM-cellulose column chromatography and affinity chromatography on heparin-Sepharose CL-6B, which gave single band on SDS-slab electrophoresis. The primary structure of the bullfrog liver RNase was determined. It consisted of 111 amino acid residues, including 8 half-cystine residues. From the sequence, it was concluded that three disulfide bridges in RNase A were conserved in the bullfrog RNase, that a disulfide bridge in RNase A [Cys65-Cys126 (RNase A numbering)] was deleted, and that a new disulfide bridge was created in the C-terminal part of the enzyme. In this frog RNase, the amino acid residues thought to be essential for catalysis in bovine pancreatic RNase A were conserved except for Asp121 (RNase A numbering). The sequence homology of the bullfrog liver RNase with bovine pancreatic RNase A was 30.6%. The sequence of bullfrog liver RNase was very similar to those of lectins obtained from bullfrog egg by Titani et al. [Biochemistry (1988) 26, 2189-2194] and R. japonica egg by Kamiya et al. [Seikagaku (in Japanese) (1989) 60, 733; and personal communication from Kamiya, Y., Oyama, F., Oyama, R., Sakakibara, F., Nitta, K., Kawauchi, H., and Titani, K.]. The sequence homology between the bullfrog liver RNase and the two lectins was 70.2 and 64.8%, respectively.     

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     

Central respiratory pattern generation in the bullfrog, Rana catesbeiana

There are two components to breathing pattern generation the production of the pattern of neural discharge associated with individual breaths, and the pattern in which breaths are produced to effect ventilation. Bullfrogs typically breathe with randomly distributed breaths. When respiratory drive is elevated, breathing becomes more regular and often episodic. Studies on in vitro brainstem-spinal cord preparations of the adult bullfrog and in situ preparations of decerebrate, paralyzed, unidirectionally ventilated animals suggest that output from the central rhythm generator in frogs is conditional on receiving some input and that a host of central inputs remain even in the most reduced preparations. There appear to be descending inputs from sites in the dorsal brainstem just caudal to the optic chiasma that cluster breaths into episodes, a strong excitatory input caudal to this site but rostral to the origin of the Vth cranial nerve and, possibly, segmental rhythm generators throughout the medulla that are normally entrained to produce the normal breathing pattern. The data also suggest that the shape of the discharge pattern (augmenting, decrementing) and timing of outputs (alternating vs synchronous) associated with motor outflow during each breath are also dependent on the interconnections between these various sites.     

Characterization of the cDNA encoding bullfrog, Rana catesbeiana, osteocalcin and two forms of the protein isolated from bone

A full-length cDNA clone encoding osteocalcin from the bullfrog, Rana catesbeiana (bone Gla-protein, BGP) has been isolated, and the complete coding sequence for the 100-amino-acid pre-pro-osteocalcin protein was determined. The amino acid sequence of Rana catesbeiana osteocalcin, especially the mature 49-amino acid sequence, is closer to the mammalian than to the fish, Sparus osteocalcin. Rana mature osteocalcin has a similarity of 67% with human or 59% with rat osteocalcin, and only 42% with fish mature osteocalcin. The 51-amino-acid pre-pro-peptide contains the expected hydrophobic leader sequence and the dibasic Arg-Arg sequence preceding the NH2-terminal Ser of the mature 49-amino-acid Rana osteocalcin. The pro-peptide sequence also contains the expected motif of polar and hydrophobic residues, which targets vitamin K-dependent gamma-carboxylation of three specific Glu residues at positions 17, 21, and 24 in the mature protein. At the native protein expression levels, extraction from Rana cortical bone in the presence of protease inhibitor cocktail resulted in the isolation of two distinct forms of osteocalcin, P-1 and P-2, with a 3:2 distribution. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and amino acid sequence analysis of the N-terminal domain, we confirmed that P-1 is the intact 49-residue osteocalcin with N-terminal SNLRNAVFG., and that P-2 lacks four amino acids from the N-terminus, (NAVFG.). These results demonstrate the existence of a form of osteocalcin lacking four N-terminal amino acids in Rana bone, and that mature Rana osteocalcins remained highly conserved in their molecular evolution, especially with respect to the conservation of the C-terminal domain (residues 14-49).     

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.     

Purification and characterization of inhibitor against the cysteine proteinase of Rana catesbeiana

1. Inhibitors of cysteine proteinase were found in tadpole tail of metamorphosing bullfrog. 2. One of the inhibitors was purified by affinity chromatography with CM-papain agarose, gel filtration with Superose 12 and ion exchange chromatography with Mono S. 3. The molecular weight of the inhibitor was 130,000-140,000 and the isoelectric point was pH 9.6. 4. The inhibitor had inhibitory effects on ficin, papain and tadpole tail cysteine proteinase. 5. The inhibitor is possibly involved in the regulation of muscle degradation in tail regression of metamorphosing tadpole.