Low molecular weight immunoglobulins in Rana catesbeiana tadpoles.

Rana catesbeiana tadpoles formed high and low m.w. antibodies in response to immunization with a bacteriophage. Although the neutralizing activity associated with the low m.w. immunoglobulins was relatively weak, the existence of antibodies in this class was clearly demonstrated by radioimmunoelectrophoresis. Moreover, two antigenically distinct variants of the low m.w. antibodies were detected. These were serologically indistinguishable from the two types of low m.w. immunoglobulin previously isolated from the serum of adult frogs of this species.     

Isolation and preliminary characterization of two varieties of low molecular weight immunoglobulin in the bullfrog, Rana catesbeiana.

Two varieties of low m.w. immunoglobulins have been isolated from the serum of Rana catesbeiana frogs. They are highly cross-reactive, although each also contains unique antigenic determinants. Since both low m.w. immunoglobulins were identified in the serum of 22 individual frogs, it was concluded that they are isotypic variants. The light chains of R. catesbeiana and mammalian high and low m.w. immunoglobulins are similar in electrophoretic mobility on polyacrylamide gels containing sodium dodecyl sulfate. The heavy chains of fropg high m.w. immunoglobulins have the mobility of mammalian mu-chains; the heavy chains of both variants of frog low m.w. immunoglobulins migrate between mammalian mu- and gamma-chains in approximately the position of mammalian alpha-chains. An unusual structural feature of the R. catesbeiana high ald low m.w. immunoglobulins is that the unreduced proteins are partially dissociated in sodium dodecyl sulfate.     

Heavy chain of human high molecular weight and low molecular weight kininogens binds calcium ion

An antibody subpopulation, anti high molecular weight (anti-HMW) kininogen-Ca2+ antibody able to bind specifically to the HMW kininogen-Ca2+ complex, was isolated from anti-HMW kininogen antiserum. Partially purified anti-HMW kininogen antibody was applied to a HMW kininogen-Sepharose column equilibrated with 40 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 7.5, containing 1.0 M NaCl and 1 mM CaCl2, and anti-HMW kininogen-Ca2+ antibody was eluted with 5 mM ethylenediaminetetraacetic acid. As a result of characterization by enzyme-linked immunosorbent assay, this antibody specifically recognized the cyanogen bromide cleaved fragment 1 (CB-1) region (1-160 amino acid sequence) of the heavy chain of kininogen molecules in the presence of Ca2+ or Mg2+. Furthermore, circular dichroism (CD) experiments showed that the conformational changes of HMW kininogen and heavy chain were induced by metal ions such as Ca2+ and Mg2+ and that these changes were due to the conformational change of the CB-1 region of the heavy chain. The dissociation constant (Kd) for the heavy chain-Ca2+ measured by CD analysis at 214 nm was found to be 0.33 +/- 0.09 mM (mean +/- SD). The number of Ca2+-binding sites of heavy chain calculated from the Hill plot was 1.15 +/- 0.04 (mean +/- SD). Then, a possible Ca2+-binding site was found in the amino-terminal portion of the heavy chain of kininogen molecules.     

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.     

Hexokinase III from Rana catesbeiana.

1. Hexokinase III was partially purified from the liver of the American bullfrong, Rana catesbeiana, using DEAE-cellulose column chromatography. 2. It was inhibited by glucose concentrations above 5 x 10(-5) M (pH 5.9), 10(-4) M (pH 6.7) or 10(-3) M (pH 7.5). 3. There was virtually no inhibition by excess glucose at pH 8.7. 4. The maximum velocity of the reaction increased with increasing pH. 5. Galactose could not be utilized as a substrate. 6. Classical Michaelis-Menten kinetics were obtained with respect to ATP, with no evidence of allostery. 7. The apparent Michaelis constant for ATP was 0.23 +/- 0.013 mM in the presence of 0.2 mM glucose at pH 7.5.     

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     

A functionally active heavy chain derived from human high molecular weight urokinase

Human high molecular weight urokinase, a plasminogen activator, when minimally reduced with 0.01 M 2-mercaptoethanol for 10 h at pH 8.0 and 25 degrees C and then carboxymethylated with sodium iodoacetate, gave two chains, a functionally active heavy chain with about 80% of the original activity and a light chain. These two chains were found to be linked by a single interchain disulfide bond. The functionally active heavy chain can be isolated by an affinity chromatography method with [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester]-Sepharose. The light chain, which has no enzyme activity, is not adsorbed to the affinity matrix, whereas the active heavy chain was adsorbed and subsequently eluted. The active heavy chain was further purified by gel filtration on Sephadex G-100. This preparation was found to be homogeneous by both analytical and sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The molecular weight of the active heavy chain was determined to be 33,000 by Sephadex G-100 gel filtration and 31,000 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Its specific activity, with L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide, was determined to be 208,000 IU/mg of protein. Approximately 87% active sites were found by p-nitrophenyl-p'-guanidino-benzoate titration with a molar activity of 7.41 X 10(9) IU/mmol of active site. The active heavy chain when compared to low molecular weight urokinase has a similar molecular weight, specific activity, and amino acid composition. The NH2-terminal residue found in the active heavy chain was lysine which was the same as that found in low molecular weight urokinase, whereas the NH2-terminal residues found in high molecular weight urokinase were serine and lysine. Serine is the NH2-terminal residue of the light chain of high molecular weight urokinase. The steady state kinetic parameters of activation of human Glu-plasminogen by the active heavy chain were also similar to low molecular weight urokinase, as were the amidase parameters of these enzymes. The Michaelis constants of activation (Kplg) were 2.11 and 2.21 microM, respectively; the catalytic rate constants of activation (kplg) were 51.7 and 44.1 min-1, respectively, with second order rate constants, kplg/Kplg of 24.5 and 20.2 microM-1 min-1, respectively.     

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.     

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.     

牛蛙(Rana catesbeiana)染色体研究

对牛蛙（Rana catesbeiana,Shaw）的染色体组型进行了研究。观察骨髓的C-中期细胞,结果证明牛蛙的体细胞染色体数目2n=26,其中有5对大型染色体和8对小型染色体,可以分成A、B、C3个组,雌性和雄性个体间没有发现异型性染色体。上述结果与前人的研究结果基本一致。但是作者发现牛蛙骨髓细胞的第7、8、10、12号染色体上均有次缢痕。牛蛙的核型为2n：26=22m＋4sm。     

The development of nerve-muscle junctions in Rana catesbeiana tadpoles

The physiological properties of developing nerve-muscle junctions in Rana catesbeiana tadpoles are described. Developing neurons at different stages of ontogeny formed functional synaptic connections with a section of tail muscle implanted in place of the hind limb bud. Transmission is quantal in nature, sensitive in normal ways to calcium and magnesium concentrations, and conforms to a Poisson distribution. The quantal content is initially low and increases with development. Mepp's occur randomly and have low frequencies which increase slightly with development. The size of a single quantum of transmitter does not change during development. The muscle fibers are multiply innervated, resulting in Epp's with distinct peaks and complex skewed mepp amplitude histograms. No significant increases were observed in the level of differentiation of the developing motor neurons as a result of their having innervated a portion of mature tail muscle. The numbers of developing motor neurons increased in the experimental lateral motor column, and a lag in their maturity was observed relative to motor neurons in the control lateral motor column.     

Cathepsins J and K: high molecular weight cysteine proteinases from human tissues

Human tissue extracts contained two high Mr proteinases active in hydrolyzing the fluorogenic substrate Cbz-phe-arg-aminomethylcoumarin. By gel filtration chromatography, cathepsins J and K had apparent molecular weights of 230,000 and 650,000, respectively. Both enzymes were cysteine proteinases with optimum activity at pH 6.2-6.8; neither had aminopeptidase activity. Human kidney, lung and spleen were rich sources of these enzymes, while liver contained moderate amounts. Cathepsins J and K were partially characterized and appeared to differ from the mammalian high Mr cysteine proteinases described in the literature. In rat liver and kidney and in mouse liver, cathepsin J was localized in the particulate fraction, whereas cathepsin K was not detected in these tissues.