[New enzyme activity of leukotriene A4 hydrolase].
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Amino Acid Sequence↗
Biomedical subjects
N Ohishi
Publications and source records attributed to N Ohishi.
Leukotriene A4 hydrolase, a bifunctional enzyme. Distinction of leukotriene A4 hydrolase and aminopeptidase activities by site-directed mutagenesis at Glu-297.
We previously obtained evidence for intrinsic aminopeptidase activity for leukotriene (LT)A4 hydrolase, an enzyme characterized to specifically catalyse the hydrolysis of LTA4 to LTB4, a chemotactic compound. From a sequence homology search between LTA4 hydrolase and several aminopeptidases, it became clear that they share a putative active site for known aminopeptidases and a zinc binding domain. Thus, Glu-297 of LTA4 hydrolase is a candidate for the active site of its aminopeptidase activity, while His-296, His-300 and Glu-319 appear to constitute a zinc binding site. To determine whether or not this putative active site is also essential to LTA4 hydrolase activity, site-directed mutagenesis experiments were carried out. Glu-297 was mutated into 4 different amino acids. The mutant E297Q (Glu changed to Gln) conserved LTA4 hydrolase activity but showed little aminopeptidase activity. Other mutants at Glu-297 (E297A, E297D and E297K) showed markedly reduced amounts of both activities. It is thus proposed that either a glutamic or glutamine moiety at 297 is required for full LTA4 hydrolase activity, while the free carboxylic acid of glutamic acid is essential for aminopeptidase.
Amino Acid Sequence↗
Generation of hydroxyl radical from linoleic acid hydroperoxide in the presence of epinephrine and iron.
When linoleic acid hydroperoxide was reacted with ferrous iron, the electron spin resonance signals characteristic of the spin adduct of 5,5-dimethyl-1-pyrroline-N-oxide and the hydroxyl radical were detected. Although the signals were not detected with the hydroperoxide and ferric iron, they were actually found if epinephrine was added, indicating that the hydroxyl radical could be generated from the hydroperoxide upon reduction of the latter with ferrous iron formed by epinephrine. The possible generation of the hydroxyl radical from lipid peroxides in vivo was discussed from the viewpoint of pathogenesis of lipid peroxide-related diseases.
Cyclic N-Oxides↗
[Therapeutic effects of cefprozil in the treatment of various infectious diseases in children].
Therapeutic effects of cefprozil (CFPZ, BMY-28100), a new cephalosporin, were examined in various infectious diseases in children. Clinical efficacy rates were 50% (2/4) in acute bronchitis, 80% (4/5) in pharyngitis, 0% in laryngitis, 100% (7/7) in tonsillitis, 100% (8/8) in impetigo contagiosa, furuncle and posthitis. Hence, the overall efficacy rate was 84% (21/25). Adverse effects were observed in 1 case with slightly elevated serum GOT and GPT. Changes in serum concentrations and urinary excretion of CFPZ were examined in 4 and 2 children without infection, respectively. T 1/2 values obtained were between 1 hour to 2 hours (bioassay). Six hour recovery rates in urine were 51.8% and 77.8% (bioassay). CFPZ was considered to be a safe and useful drug in treating various infectious diseases in children.
Administration, Oral↗
Pharmacokinetics and distribution of recombinant human erythropoietin in rats with renal dysfunction.
Pharmacokinetics and distribution were studied following intravenous administration of recombinant human erythropoietin (rh-EPO, CAS 11096-26-7) to 5/6-nephrectomized rats. 5/6-Nephrectomy caused a significant decrease of total body clearance of rh-EPO. Renal function, therefore, is thought to affect the metabolism in extrarenal tissues. The metabolism of EPO might be indirectly associated with renal function. There were no differences of the concentration in the bone marrow and spleen between 5/6-nephrectomized rats and non-nephrectomized rats, suggesting that 5/6-nephrectomy does not cause a change in the affinity of rh-EPO for target tissues. Repeated administration of rh-EPO caused a slight reduction of the biological half-life in 5/6-nephrectomized rats as well as in non-nephrectomized rats, suggesting stimulation of a compensatory metabolism in extrarenal tissues.
Animals↗
Pharmacokinetics and distribution of recombinant erythropoietin in rats.
Pharmacokinetics and distribution of recombinant human erythropoietin (rh-EPO, Epoch, CAS 11096-26-7) were studied in rats. After intravenous administration, concentrations of rh-EPO in the plasma declined biexponentially. AUC increased proportionally with the dose. The half-life of a-phase was prolonged with increasing doses. The low volume (Vss) of the distribution of rh-EPO was 65-79 ml/kg, which indicate a mainly extracellular distribution of this drug. The half-life of beta-phase was 1.98-2.7 h. The total body clearance was 18-21 ml/h/kg, and was independent of the dose. The renal clearance was much lower than total body clearance, suggesting that extrarenal clearance, presumably metabolism, plays a role in elimination. rh-EPO was eliminated by metabolism. The profiles of times vs concentration of radioactivity in the bone marrow and spleen, which are considered to be target tissues, were different from that of the plasma; namely bone marrow and spleen showed Cmax at 30 min after dosing, and this time, radioactivity in these tissues showed almost intact rh-EPO. At the dose of 0.1 micrograms/kg, the concentration in the bone marrow was about 2-fold higher than that of plasma, the concentration in the spleen was lower than that in the plasma 30 min after dosing of rh-EPO. The distributions of rh-EPO in both tissues were saturable.
Animals↗
Distribution of recombinant human erythropoietin following multiple intravenous administration and effects of age on the distribution in rats.
Multiple administration of 125I-recombinant human erythropoietin (125I-rh-EPO) did not cause an accumulation of intact EPO in tissue. The gel filtration profile of tissue from rats given multiple doses of 125I-rh-EPO was very similar to that for rats given single doses of 125I-rh-EPO, suggesting that intact rh-EPO (CAS 11096-26-7) does not accumulate. Pretreatment of rats with non-labeled rh-EPO did not cause a change in the ratio of metabolites to intact rh-EPO, as judged by the gel filtration data. This indicates that multiple administrations do not cause a change in metabolic pathways in the tissues. The tissue to plasma concentration ratio (t/p ratio) of rh-EPO in the target organ, i.e., the bone marrow and spleen, decreased with increase of age; the spleen showed a marked decrease in t/p ratio as compared to the bone marrow. On the other hand, t/p ratios in the kidney and liver did not change for periods of 4 to 31 weeks.
Aging↗
Structurally different rat liver medium-chain acyl CoA dehydrogenases directed by complementary DNAs differing in their 5'-region.
Different forms of rat liver medium-chain acyl CoA dehydrogenase (MCAD) (EC 1.3.99.3) were produced in Escherichia coli carrying expression plasmids (pRMCADm-1 approximately 9) differing at the 5'-region of the cDNA. The proteins expressed could be readily extracted from the cells. The protein (approximately 44 kDa) directed by pRMCADm-3 showed the highest activity and was readily purified to homogeneity. The purified enzyme contained non-covalently bound FAD and was similar to rat liver mitochondrial enzyme in all respects examined. The purified protein (approximately 45 kDa) directed by pRMCADm-1 did not contain FAD and showed no enzymatic activity. Therefore, the leader peptide disturbs the binding of FAD to the apoprotein. The purified protein (approximately 40 kDa) directed by pRMCADm-6 did not contain FAD. Thus, the deletion of the NH2-terminal portion of the apoprotein to some extent results in its inability to combine with FAD.
Acyl-CoA Dehydrogenase↗
Micro-determination of L-gulono-gamma-lactone oxidase activity.
Highly sensitive assay method of L-gulono-gamma-lactone oxidase (GLO) was constructed. In this method, L-ascorbic acid formed in the enzymatic reaction was converted to its bis(dinitrophenyl)hydrazone derivative, and the amount of the latter was determined by high-performance liquid chromatography. Twenty picomoles of ascorbic acid was detected, which makes this method 25 times more sensitive than the previously used dipyridyl one. By the present method, a minute activity of GLO in liver microsomes prepared from rats of the Osteogenic Disorder Shionogi strain (ODS-od/od) could be measured.
Animals↗
Comparative pharmacokinetics and distribution of human urinary erythropoietin and recombinant human erythropoietin in rats.
Comparative pharmacokinetic and distribution studies of human recombinant erythropoietin (rh-EPO, Epoch, CAS 11096-26-7) and human urinary erythropoietin (u-EPO) were performed in rats. The pharmacokinetic parameters following intravenous administration of rh-EPO were similar to those of u-EPO. With respect to the concentrations of radioactivity in the liver reflecting uptakes of asialoglycoprotein, there were no differences in the concentrations between 125I-rh-EPO and 125I-u-EPO. The concentrations of radioactivity in the target organs, i.e., bone marrow and spleen in rats given 125I-rh-EPO were very similar to those of rats given 125I-u-EPO. These facts are most likely to reflect the similarity in pharmacological action.
Animals↗
Biosynthesis and function of leukotriene B4. Immunochemical study of leukotriene A4 hydrolase and identification of putative leukotriene B4 receptor.
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Animals↗
Preparation of iodine labeled recombinant human erythropoietin.
125I-labelled recombinant human erythropoietin (125I-rh-EPO; CAS for EPO: 11096-26-7) was prepared by using iodo-gen (1,3,4,6-tetrachloro-3,6-diphenylglycouril), which showed a similar immunoreactivity to anti rh-EPO anti-rabbit serum and a similar pharmacokinetics with that of non-labeled rh-EPO.
Animals↗
[Clinical efficacy of cefpirome against various infectious diseases in children].
The clinical efficacy and the pharmacokinetics of cefpirome (CPR, HR 810), a new semisynthetic cephalosporin derivative, were investigated in children with various infectious diseases. CPR showed high blood peak levels, relatively long half-life and high levels in urine. Excellent clinical efficacy was obtained in 2/3 cases with pneumonia, 3/4 cases with upper respiratory infection, 2/2 cases with cutaneous and soft tissue infection and 1/1 case with urinary tract infection. The overall efficacy rate was 80%. No clinical adverse effects were observed while slightly elevated GPT and GOT, decreased platelet were detected in 4 cases without clinical problems. CPR should be an useful and safe drug in treating infectious diseases in children.
Adolescent↗
Leukotriene A4 hydrolase is a zinc-containing aminopeptidase.
A comparison of amino acid sequences revealed that leukotriene A4 (LTA4) hydrolase is homologous to various types of aminopeptidases. Consistently with the finding, the purified LTA4 hydrolases from both human and guinea pig sources contained equimolar zinc ion, as determined by atomic absorption spectrometry. The enzyme had a significant amount of aminopeptidase activity toward synthetic peptide substrates. Both LTA4 hydrolase and aminopeptidase activities were inhibited by o-phenanthroline, p-chloromercuribenzoic acid, and Leu-thiol with similar IC50 values. Co-purification as well as co-immunoprecipitation of both enzyme activities with an affinity-purified antibody against LTA4 hydrolase strongly suggest that the two enzyme activities reside in a single protein.
Aminopeptidases↗
Immunological quantitation and immunohistochemical localization of leukotriene A4 hydrolase in guinea pig tissues.
We prepared a highly specific polyclonal antibody against leukotriene (LT) A4 hydrolase using a recombinant human enzyme. Using this antibody, we quantified LTA4 hydrolase protein content in the cytosols of guinea pig tissues. The enzyme protein content correlated well with the enzyme activity with a correlation coefficient of 0.87. However, the enzyme activity per mg of the enzyme in the cytosols was low, particularly in the liver and adrenal gland, compared with the specific activity of the purified enzyme. These observations suggest the presence of inhibitory substances and/or inactive enzymes in the cytosols of these tissues. To determine the cellular localization of LTA4 hydrolase in tissues other than blood cells, we carried out immunohistochemical examinations of guinea pig tissues. We identified epithelial cells in the tracheobronchial system and gastrointestinal tract, smooth muscle cells in the bronchi and aorta, vascular endothelial cells, and the intestinal plexus as novel cellular sources of the enzyme in the parenchyme of the tissue. Thus, LTA4 hydrolase was widely distributed in various types of parenchymal cells in the tissues, and this observation warrants further investigations on the biological activities of LTB4 in these cells and tissues.
Animals↗
Purification and characterization of human lung leukotriene A4 hydrolase.
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Chromatography↗
Fluid flow in a strongly curved pipe.
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Arteriosclerosis↗
Leukotriene A4 hydrolase from guinea pig lung: the presence of two catalytically active forms.
Leukotriene A4 hydrolase was purified to apparent homogeneity from the guinea pig lung. The molecular weight was determined to be 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited two active forms with different pI values (5.7 and 5.4) depending on the presence or absence of SH-reducing reagents during purification procedures. No significant differences were observed between both forms of the enzyme as regards the catalytic properties. The N-terminal 20 amino acid sequence (PEVVDTXSLASPATVXRTKH) showed a 90% identity to the human enzyme with a constitutive substitution of Ile-3 and Ser-14 (human) by Val-3 and Thr-14 (guinea pig), respectively.
Amino Acids↗