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.     

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.     

Purification and characterization of cytosol-specific phosphoenolpyruvate carboxykinase from chicken liver

Phosphoenolpyruvate carboxykinase of chicken liver cytosol was purified to homogeneity by procedures including affinity chromatography with GTP as a ligand. The purified enzyme showed a molecular weight of 68,000 on gel electrophoresis in the presence of dodecyl sulfate. Comparative studies on this enzyme and its isozyme purified from chicken liver mitochondria were performed. As regards amino acid composition, the cytosolic enzyme was quite different from the mitochondrial enzyme, but was rather similar to rat liver cytosolic phosphoenolpyruvate carboxykinase. Specific activities of the cytosolic enzyme were 30-100% higher than those of the mitochondrial enzyme for oxaloacetate-CO2 exchange, oxaloacetate decarboxylation, and phosphoenolpyruvate carboxylation reactions, though the relative rates of the activities were similar, decreasing in the order given. Apparent Michaelis constants for oxaloacetate in the oxaloacetate decarboxylation reaction were 11.6 and 17.9 microM for the cytosolic and the mitochondrial enzyme, respectively, but the values for GTP, GDP, phosphoenolpyruvate, and CO2 in the oxaloacetate decarboxylation and phosphoenolpyruvate carboxylation reactions were 1.3-2.2 times higher for the cytosolic enzyme than for the mitochondrial enzyme. Thus, the fundamental catalytic properties of the chicken liver phosphoenolpyruvate carboxykinase isozymes were rather similar, despite the marked difference in amino acid compositions.     

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 characterization of the isozymes of phosphoenolpyruvate carboxykinase from rabbit liver

Procedures are described for the purification of the mitochondrial and cytosolic isozymes of phosphoenolpyruvate carboxykinase from rabbit liver. Examination of the purified isozymes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated apparent homogeneity and identical molecular weights of approximately 65,000. Gel filtration chromatography of the native isozymes, however, yielded apparent molecular weights of 68,000 and 56,000 for the cytosolic and mitochondrial isozymes, respectively. The isoelectric points as determined by chromatofocusing were 5.8 for the mitochondrial isozyme and 5.0 for the cytosolic isozyme. The purified isozymes were readily separable on ion-exchange columns, with the cytosolic isozyme showing the greater affinity. A minor amount of cross-reactivity was apparent when each isozyme was immunotitrated with polyclonal antibodies raised in goat against the opposite isozyme. Peptide maps obtained by high pressure liquid chromatography of both tryptic digests and cyanogen bromide digests of the isozymes showed that many of the peaks were not coincident, suggesting that differences in the sequences are found throughout the primary structures of the isozymes.     

A mitochondrial phosphoenolpyruvate carboxykinase from rat brain

Phosphoenolpyruvate carboxykinase from the rat brain has been purified approximately 6000-fold. This purified enzyme was stable at −20 °C for several months.     

Purification and characterization of a non-vitellogenin, estrogen-induced plasma protein from the American bullfrog Rana catesbeiana

A non-vitellogenin, estrogen-induced protein has been detected for the first time in the plasma of male Rana catesbeiana. A greater than 90% purification of this plasma protein was achieved by salt fractionation with Mg(II) followed by ion-exchange chromatography on DEAE- and CM-cellulose. Immunoelectrophoretic analysis with various antisera showed no immunological cross-reactivity between this protein and vitellogenin. The molecular mass of the purified protein was determined to be 116 000 daltons by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and 105 000 daltons by analytical ultracentrifugation. Sedimentation studies indicate the protein is a nonaggregating spherical monomer with a sedimentation coefficient of 7.5 S. Amino acid analysis demonstrated a composition different from that of vitellogenin and lipovitellin A. Limited proteolysis with trypsin, chymotrypsin, and Bacillus subtilis protease revealed no common peptides on SDS-polyacrylamide gels. Phosphate analysis indicated that, on a molar basis, the non-vitellogen, estrogen-induced protein had less than or equal to 3% of the phosphate found in vitellogenin. Further studies of the structure, function, and metabolism of this protein may reveal information relating to the hormonal control of vitellogenesis.     

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.     

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.     

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.     

Purification and comparison of phosphoenolpyruvate carboxykinase from the liver and kidney of the Arabian camel (Camelus dromedarius)

1. Phosphoenolpyruvate carboxykinase was partially purified from camel liver and kidney by ammonium sulphate fractionation, gel filtration and ion-exchange chromatography. 2. The specific activity of the purified preparation from liver was 39.2 mumol/min per mg protein. 3. When isolated from the kidney the specific activity of the enzyme was very much higher 155.5 mumol/min per mg protein. 4. The enzyme from the two sources were similar in their pH optimum which was approx. 7.2 and their relative stability to thermal inactivation at 60 degrees C. 5. The mol. wt of the enzyme from both organs was estimated at 80,000 +/- 5000.     

An alkaline thiol proteinase in the liver mitochondria of bullfrog, Rana catesbeiana

The mitoplasts were prepared from bullfrog (Rana catesbeiana) liver mitochondria by treatment with digitonin and were then separated into the matrix and inner membrane fractions. The matrix fraction thus obtained was free of lysosomal contaminations and exhibited a distinct proteinase activity. pH dependency of the matrix proteinase activity measured in the presence and absence of iodoacetamide revealed that the matrix contained at least two kinds of proteinase, a major alkaline thiol proteinase having an optimal pH at 8.5 and a minor neutral proteinase having an optimal pH at 7.5. The major matrix proteinase activity was strongly inhibited by leupeptin, chymostatin, antipain and E64-C, an inhibitor of Ca2+-dependent thiol proteinase, while it was scarcely affected by diethylpyrocarbonate. The activity was also inhibited by DTNB and p-chloromercuribenzoate. Addition of hydrocarbon compounds such as ethylene glycol, glycerol, Triton X-100 and poly (ethylene glycol) to the reaction mixture was found to decrease the matrix proteinase activity. Neither cytochrome c nor glutamate dehydrogenase was hydrolyzed when subjected to the matrix proteinase activity in vitro. On the other hand, cytochrome c oxidase was effectively hydrolyzed, and the enzyme associated with the mitochondrial innermembrane fragments was partially hydrolyzed by the major matrix proteinase activity.