Yasuhiro Sagara

Molecular cloning of a cDNA for rat liver monoamine oxidase B

The cDNA for rat monoamine oxidase B mRNA was isolated from liver cDNA library in lambda gt11 using specific antibody and oligonucleotide probes derived from FAD-containing peptide of the enzyme. The primary structure of the protein, deduced from the nucleotide sequence, consisted of 520 amino acid residues and its molecular weight was calculated to be 58.4 kD which is in good agreement with that of the in vitro-synthesized peptide. FAD-binding site is located in the carboxy-terminal region. There is no typical structural feature common to the targeting signals for mitochondria, the periodic distribution of basic amino acids spaced by several uncharged residues, at its amino-terminal region. This region has an uninterrupted stretch of 14 hydrophobic residues.

Effect of three triterpenoids, lupeol, betulin, and betulinic acid on the stimulus-induced superoxide generation and tyrosyl phosphorylation of proteins in human neutrophils.

BACKGROUND The roots of Anemone raddeana are used in Chinese folk medicine for curing rheumatism and neuralgia. METHODS The three triterpenoids lupeol, betulin and betulinic acid were isolated from ethanol extracts of the roots of A. raddeana. The effect of these triterpenoids on superoxide generation and tyrosyl phosphorylation of proteins in human neutrophils was investigated. RESULTS The superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was significantly suppressed by betulin and lupeol depending on the concentration of the triterpenoids. The suppressive effect of betulinic acid was low. The phorbol 12-myristate 13-acetate (PMA)-induced superoxide generation was suppressed by betulin in a concentration-dependent manner, but not by lupeol and betulinic acid. In contrast, the superoxide generation induced by arachidonic acid (AA) was suppressed by lupeol, while betulin and betulinic acid weakly enhanced the AA-induced superoxide generation. Lupeol and betulin suppressed tyrosyl phosphorylation of a 45.0-kDa protein in fMLP-treated human neutrophils in parallel to the suppression of fMLP-induced superoxide generation, but betulinic acid did not. Lupeol, betulin and betulinic acid showed no hemolytic effect even at a concentration of 500 micromol/l. CONCLUSIONS Lupeol and betulin suppress superoxide generation by preventing tyrosyl phosphorylation of a 45.0-kDa protein in human neutrophils, and may have pharmaceutical applications.

Essential role of the Arg112 residue of cytochrome P450cam for electron transfer from reduced putidaredoxin

Cytochrome P450cam (CYP101) of Pseudomonas putida PpGl in which Arg112 is substituted by Cys was isolated by in vitro random mutagenesis of the camC gene DNA coding for P450cam. The absorption spectra of the purified mutant enzyme were similar to those of the wild type enzyme, but its substrate‐dependent NADH oxidation activity in the presence of putidaredoxin (Pd) and putidaredoxin reductase (PdR) was extremely low. The rate constant of electron transfer from reduced Pd to the heme of the mutant P450cam, measured on an anaerobic stopped flow apparatus, was 1/400 of that of the wild type enzyme and the dissociation constant of the mutant P450cam for oxidized Pd was several fold higher than that of the wild type enzyme. A considerable decrease in mid‐point potential of the mutant enzyme was also noted. We conclude that Arg112, which is located on the surface of the P450cam molecule and hydrogen‐bonded to one of the heme propionate chains, plays an essential role in the electron transfer from Pd.

Effect of six flavonoid compounds from Ixeris sonchifolia on stimulus-induced superoxide generation and tyrosyl phosphorylation in human neutrophils

BACKGROUND Ixeris sonchifolia (Bge.) Hance is an herbal medication used in China as an analgesic. METHODS The effect of six flavonoid compounds isolated from Ixeris sonchifolia (Bge.) Hance on stimulus-induced superoxide generation and phosphorylation of tyrosine residues of protein in human neutrophils was investigated. The six compounds examined were luteolin 7-glucuronide methylester (LGME), luteolin 7-glucuronide ethylester (LGEE), luteolin 7-glucoside (LG), luteolin 7-glucopyranosyl-(1-->6)-glucoside (LGG6), luteolin 7-glucopyranosyl-(1-->2)-glucoside (LGG2) and apigenin 7-glucoside (AG). RESULTS When the cells were preincubated with these six flavonoids, the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was significantly suppressed in a concentration-dependent manner. These flavonoids also suppressed the superoxide generation induced by arachidonic acid (AA). The rate of suppression by these flavonoids was AG>LG, LGG6, LGEE, LGG2>LGME. In case of the superoxide generation induced by phorbol 12-myristate 13-acetate (PMA), LG, LGG6 and AG suppressed the superoxide generation but LGME, LGEE and LGG2 gave no effect. When the cells were incubated with fMLP in the presence of LGME, LGEE and AG, fMLP-induced tyrosyl phosphorylation of 45-kDa proteins of the cells was dose-dependently suppressed in parallel to the suppression of fMLP-induced superoxide generation. CONCLUSION Flavonoids suppress tyrosine phosphorylase in a dose-dependent manner, and may have pharmacoceutical applications.

Putative functions of phenylalanine-350 of Pseudomonas putida cytochrome P-450cam

Cytochrome P-450cam hydroxylates d-camphor, using molecular oxygen and reducing equivalents transferred via putidaredoxin. We constructed mutant genes in which Phe-350 of P-450cam was replaced by Leu, Tyr, or His by site-directed mutagenesis, expressed them in Escherichia coli, purified the mutant proteins, and compared their enzymic properties with those of the wild type P-450cam. NADH oxidation rate of the Tyr mutant in the reconstituted system with putidaredoxin and putidaredoxin reductase was similar to that of the wild type enzyme, while the Leu mutant and the His mutant showed 67% and 17% activity of that of the wild type, respectively. The affinities of these mutant proteins for camphor and the oxidized form of putidaredoxin were much the same as those of the wild type protein. Rate constants for the reduction reaction of P-450cam by reduced putidaredoxin, a physiological electron donor for P-450cam, of Tyr and His mutants were much the same as that of the wild type enzyme, whereas the Leu mutant showed approx. half that of the wild type. Thus, the aromatic ring of Phe-350 of P-450cam probably contributes to enhancing efficiency of the electron transfer yet does not seem to be essential for the reaction.

Significant contribution of arginine-112 and its positive charge of Pseudomonas putida cytochrome P-450cam in the electron transport from putidaredoxin

Cytochrome P-450cam of Pseudomonas putida is a prototype of various eukaryotic cytochrome P-450 molecules. Arg-112 located on the surface of this protein is highly conserved among various other cytochromes P-450. In this study, we constructed mutant genes for P-450cam in which Arg-112 was replaced by Gln or Glu, expressed them in Escherichia coli and purified the mutant proteins. Their enzymic activities were analyzed in the reconstituted system to determine the function of Arg-112. Kd values for d-camphor of Arg112-Gln and Arg112-Glu were much the same as those of the wild-type enzyme, whereas Kd values for the oxidized form of putidaredoxin, which is an acidic protein and is the redox partner of P-450cam, were 240 and 530 microM, respectively. These values are 8 and 19 times larger than that of the wild-type enzyme (28 microM), thereby indicating lower affinities of the mutant enzymes for the oxidized putidaredoxin. Reaction rate constants for reduction by the reduced form of putidaredoxin, measured using the stopped flow method, were 45.5, 9.0 x 10(-3) and 9.0 x 10(-4) s-1 for the wild type, Arg112-Gln and Arg112-Glu, respectively. Thus, Arg-112 of P-450cam plays an important role in the interaction with putidaredoxin and in the high efficiency of the electron transfer; the positive charge of the residue seeming to contribute to the process. The yields in Escherichia coli, the heme contents in the purified fractions and heat stability of the mutant proteins were lower than those of the wild type enzyme, suggesting that Arg-112 of P-450cam is also important for stability of P-450cam.

In vitro synthesis of monoamine oxidase of rat liver outer mitochondrial membrane

Abstract Monoamine oxidase, an intrinsic protein of outer mitochondrial membrane, was purified from bovine liver and rabbit antibody against the enzyme was prepared. The antibody could react with the monoamine oxidase of rat liver mitochondria. When rat liver RNA was translated in vitro using rabbit reticulocyte lysate and monoamine oxidase peptide in the translation products was immunoprecipitated by the antibody, the peptide was detected in the products programmed by the messenger RNAs from total and free polysomes but not that from bound polysomes. The enzyme synthesized in vitro had the same apparent molecular size as the mature protein in outer mitochondrial membrane.

Effect of three triterpenoid compounds isolated from root bark of Aralia elata on stimulus-induced superoxide generation and tyrosyl phosphorylation and translocation of p47phox and p67phox to cell membrane in human neutrophil

BACKGROUND Root bark of Aralia elata is used as a folk medicine for neurasthenia, rheumatism, diabetes, hepatitis virus and spasm of the stomach in China, Japan and Russia. METHODS The effect of three triterpenoid compounds isolated from root bark of A. elata on stimulus-induced superoxide generation and tyrosyl phosphorylation and translocation of p47(phox) and p67(phox) to cell membrane was investigated. The three compounds examined were Elatoside A, Elatoside C, and Tarasaponin V. RESULTS When the cells were preincubated with these compounds, the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was suppressed in a low concentration range. However, the superoxide generation was significantly enhanced by 40 micromol/l triterpenoid, and was again suppressed in the higher concentration range. In the case of superoxide generation induced by phorbol 12-myristate 13-acetate (PMA), the compounds had no obvious effect on the superoxide generation in low concentration but suppressed that at 40 micromol/l. These compounds also efficiently suppressed the superoxide generation induced by arachidonic acid (AA) at 10 micromol/l. In parallel to the effect on the fMLP-induced superoxide generation, these compounds suppressed fMLP-induced tyrosyl phosphorylation and the translocation to membrane of cytosolic compounds, p47(phox) and p67(phox) at 10 and 80 micromol/l but not at 40 micromol/l. CONCLUSIONS Triterpenoid saponins examined in this study effect stimulus-induced superoxide generation and tyrosyl phosphorylation and translocation to membrane of p47(phox) and p67(phox) in a concentration-dependent manner, and may have some pharmaceutical applications.

Effect of sarsasapogenin and its derivatives on the stimulus coupled responses of human neutrophils

METHODS The effects of three sapogenins (sarsasapogenin, tigogenin and hecogenin) on the stimulus-induced superoxide generation and protein tyrosyl phosphorylation in human neutrophils were investigated. RESULTS When the cells were preincubated with sapogenin, three sapogenins dose-dependently suppressed the superoxide generations induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA), respectively. In both cases, their effects were in the order: sarsasapogenin>tigogenin>hecogenin. While sarsasapogenin suppressed the superoxide generation induced by arachidonic acid (AA) as well, the superoxide generation was scarcely suppressed by tigogenin and significantly enhanced by hecogenin. In parallel to their effects on the superoxide generation, the three sapogenins dose-dependently suppressed the fMLP-induced and PMA-induced tyrosyl phosphorylations of 45 kDa protein in neutrophils, respectively. CONCLUSIONS Of the sapogenins tested, sarsasapogenin may have the most clinical use as it suppresses superoxide generation.

The effects of serum iminodipeptides and prednisolone on superoxide generation and tyrosyl phosphorylation of proteins in neutrophils from a patient with prolidase deficiency.

The aim of this study was to examine the effects of serum iminodipeptides and prednisolone on superoxide generation and tyrosyl phosphorylation of proteins in neutrophils from a patient with prolidase deficiency, and also to find the causative effects of superoxide on inflammatory skin lesions. When the neutrophils from a patient with prolidase deficiency (PDPPMN) were preincubated with prolyl-proline (Pro-Pro), which is one of the iminodipeptides found at high concentration in the serum of patients with prolidase deficiency, the N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide generation was enhanced in a concentration-dependent manner, although the extent of enhancing effect was lower than that in neutrophils from healthy humans (HPPMN). Pro-Pro also enhanced superoxide generation induced by opsonized zymosan (OZ) in PDPPMN but not that induced by arachidonic acid or phorbol 12-myristate 13-acetate. Herbimycin A and genistein decreased the fMLP- and OZ-induced superoxide generations after priming by Pro-Pro. 1-(5-isoquinoline-sulfonyl)-2-methyl-piperazine (H-7) and staurosporine did not decrease, but rather enhanced, the superoxide generation in a low concentration range. When PDPPMN were prepared, tyrosyl phosphorylation of 45 kDa protein in PDPPMN had already occurred. The phosphorylation was scarcely increased by incubation of the cells with Pro-Pro, in contrast to that in HPPMN. Genistein decreased the phosphorylation of 45 kDa protein in both PDPPMN and HPPMN. These results suggest that the priming effect of iminodipeptides on superoxide generation in PDPPMN is coupled with phosphorylation of 45 kDa protein by protein tyrosine kinase. Protein tyrosine kinase may play a critical role(s) in the regulatory mechanism of priming by iminodipeptides and activation of NADPH oxidase in the patients neutrophils. In prolidase deficiency, the characteristic skin manifestations are inflammatory indurations and chronic leg ulcers. Prednisolone improves the ulcers, and this compound decreased the fMLP- and OZ-induced superoxide generation and tyrosyl phosphorylation of 45 kDa protein in the patients neutrophils after priming by Pro-Pro. When inflammatory skin lesions were present, the levels of iminodipeptides in the patients serum were elevated and the superoxide generation by neutrophils was up-regulated. When skin lesions were healing or absent, the levels of iminodipeptides in the patients serum and superoxide generation by neutrophils were higher than those of healthy controls but lower than those in the inflammatory stages. Thus, the enhancement of superoxide generation by neutrophils via serum iminodipeptides would be one of the inducers of inflammatory skin lesions. Corticosteroid administration might be a therapeutic modality of choice for skin lesions.