Biomedical subjects
Publications and source records attributed to K Ushio.
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The inactive complex of diol dehydrase with hydroxocobalamin was resolved by treatment with SO2-3, followed by dialysis to remove SO2-3, giving the apoenzyme which was reconstitutable into catalytically active holoenzyme upon addition of adenosylcobalamin ("re-activation"). Spectral evidence showed that the enzyme-bound hydroxocobalamin undergoes a Co beta-ligand exchange reaction forming sulfitocobalamin. Sulfitocobalamin was bound to diol dehydrase only loosely, and therefore dissociated from the enzyme. In contrast, neither the enzyme-hydroxocobalamin-5'-deoxyadenosine nor the enzyme-hydroxocobalamin-adenosine complex was resolved and thus re-activated by this procedure. It was shown spectroscopically that the hydroxocobalamin in these complexes does not react with SO2-3, or even with CN-, indicating that the OH group in the Co beta-position was blocked spatially by these enzyme-bound nucleosides. Neither O2-inactivated holoenzyme nor the holoenzyme inactivated suicidally by glycerol or 1,2-ethanediol during dehydration reaction was also re-activated by the same procedure. The complex of the enzyme with cyanocobalamin or methylcobalamin was not resolvable by the SO2-3 treatment. This was because these cobalamins bound to the enzyme were not subject to a ligand exchange reaction with SO2-3.
A fluorescent oxidation product of the molybdenum cofactor was isolated from Escherichia coli nitrate reductase (EC 1.9.6.1) and bovine milk xanthine oxidase (EC 1.2.3.2), which showed a visible absorption band at 395 nm and was dephosphorylated by alkaline phosphatase but not by phosphodiesterase I. The dephosphorylated species was oxidized by periodate to thieno[3,2-g]pterin-2-carbaldehyde which was quantitatively converted to thieno[3,2-g]pterin-2-carboxylic acid by subsequent treatment with Ag2O in 2 N NaOH. These results indicate that the oxidation product of the molybdenum cofactor is a thieno[3,2-g]pterin derivative with an unidentified side chain in the 2 position.
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To induce the inhibition of ALA-D (delta-aminolaevulinic acid dehydratase) activity by lead in vitro, it is necessary to preincubate the enzyme fraction with lead ions and the Hb fraction (factors) together. The combination of two of the three (ALA-D fraction, lead acetate, and Hb fraction) in the preincubation has only a small effect on the activity. Lead preincubated with ALA-D and Hb fractions does not alter the affinity of the enzyme for the substrate, suggesting that the substrate can bind to the enzyme molecules (non-competitive inhibition). The restoration of activity by zinc with dithiothreitol is associated with the removal of lead from ALA-D fraction proteins to which it has bound in vivo and in vitro. The mode of the inhibitory action of tin on ALA-D is similar to that of lead because the inhibition is intensified by the addition of Hb fraction and is restored by heating. The inhibition concentration is, however, higher than that of lead. Of the three methods recovering decreased activity, heating is the most specific to detect inhibition by lead.
CaEDTA 20 mg/kg was administered weekly for 3.5 years after termination of occupational exposure to two lead workers. The diminution half-lives for lead in blood and urine lead mobilized by CaEDTA were 4.8 and 3.3 years respectively for subject 1 following 28 years exposure and 3.3 and 2.0 years respectively for subject 2 following 26 years exposure. The difference in the diminution rate between lead in blood and lead mobilized by CaEDTA was significant in subject 2 (p less than 0.05).
We describe a method of determining coproporphyrin I, III, and I plus III in urine by "high-performance" liquid chromatography. Urine is simply injected after dilution with an equal volume of glacial acetic acid. Some urinary coproporphyrin apparently binds zinc without acetic acid treatment. The working linear range of coproporphyrin concentrations is 10 to 2000 micrograms/L of urine. The sensitivity of the method is sufficient to detect as little as 10 micrograms of coproporphyrins per liter of urine. Analytical recoveries for both coproporphyrins were 96.7-106%. Results by the present method and those by an extraction method (Br J Ind Med 31:72-74, 1974) correlate well (r = 0.975). Mean (and range) coproporphyrin I, III, and I plus III concentrations in urine from normal subjects are 33.7 (7-75), 28.6 (0-130), and 62.2 (7-174) micrograms/L, respectively.
A method is described for the determination of 5'-nucleotidase activity in human erythrocytes and plasma. Using reversed-phase high-performance liquid chromatography; the product (uridine) was separated from the substrate (uridine-5'-monophosphate) in less than 4 min. The activity determined closely agreed with that determined by the conventional method, in which the inorganic phosphate released is measured. The present method eliminates the need for dialysis of enzyme solution prior to the assay, and offers several advantages over other assay methods, including high sensitivity.
Medical consultation rates were compared between a group of lead workers (346 males) and two groups of non-lead workers (317 and 329 males) in a newspaper company where a serious industrial dispute took place over health effects of lead three years before this study. The comparison was also made within the group of lead workers. Maximal blood lead concentrations (PbBs) ranged from 0.1 mumol/kg to 3.6 mumol/kg (74 microgram/100 g) with an average of 1.3 mumol/kg in the past three years. Ages averaged 35 years in lead workers, and 34 and 30 years in non-lead workers, the ranges being 19-55 years in all. The consultation rate of lead workers was significantly higher than that of both the non-lead workers (P less than 0.001), when the number of workers who consulted physicians or dentists once or more for a year per 100 workers (general consultation rate) was compared. Disease-specific consultation rates were also higher in lead workers for seven categories of diseases and injuries such as hypertensive disease and peptic ulcers. When lead workers were divided into three groups by PbB or erythrocytes delta-aminolevulinic acid dehydratase (ALAD) levels, however, no significantly high rate was found in the higher PhB groups not lower ALAD groups in terms of general and disease-specific consultation rates. It was concluded that the industrial dispute might have been a major cause of the high consultation rate of lead workers.
A hypothetical model was introduced to estimate the body burden of chelatable lead from the mobilisation yield of lead by calcium disodium ethylenediamine tetra-acetate (CaEDTA). It was estimated that, on average, 14 and 19% of the body burden was mobilised into the urine during the 24 hours after an injection of 53.4 mumol (20 mg) and 107 mumol (40 mg) CaEDTA per kg bodyweight, respectively. The body burden of chelatable lead ranged from 4 mumol (0.8 mg) to 120 mumol (24.9 mg) (mean 37 mumol (7.7 mg) in lead workers with blood lead concentrations of 0.3-2.9 mumol/kg (6-60 microgram/100 g) (mean 1.4 mumol/kg (29 microgram/100 g)). There were linear relationships between blood lead concentrations and body burden of chelatable lead on a log scale.
Proteins in the ALA-D (delta-aminolaevulinic acid dehydratase) fraction from gel filtration of erythrocyte supernatant (ES) have the highest affinity for lead among erythrocyte constituents in vivo and in vitro. It takes 20-40 hours for erythrocyte components to be equilibrated with lead added in vitro. AT low lead concentrations, under 60 micrograms/100 ml ES, the extent of decrease in ALA-D activity indicates the extent of lead saturation of ALA-D fraction proteins. The saturation is attained at 80-110 micrograms/100 ml ES. Although an appreciable amount of lead is also found in the haemoglobin fraction that contains certain factors concerned in ALA-D inhibition, lead responsible for inducing the inhibition is not bound to haemoglobin fraction proteins but to ALA-D fraction proteins. Of three treatments or agents recovering the enzyme from lead effects, zinc is the only one that can fully restore the inhibition.
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In the previous paper (S. Honda, T. Toraya, and S. Fukui, J. Bacteriol., 143, 1458-1465 (1980)), we reported that the glycerol-inactivated holoenzymes of adenosylcobalamin-dependent glycerol dehydratase and diol dehydratase are rapidly and continually reactivated in toluene-treated cells (in situ) by adenosine 5'-triphosphate (ATP) and divalent metal ions in the presence of free adenosylcobalamin. To elucidate the mechanism of this in situ reactivation, the nature of the binding of various irreversible cobalamin inhibitors to the dehydratases in situ was investigated. In the presence of ATP and Mn2+, enzyme-bound hydroxocobalamin, cyanocobalamin and methylcobalamin were rapidly displaced by added adenosylcobalamin. Without ATP and Mn2+, such displacement did not take place. In contrast, enzyme-bound adeninylbutylcobalamin and adenosylethylcobalamin were essentially not displaceable by the free coenzyme even in the presence of ATP and Mn2+. Inosylcobalamin was a very weak inhibitor irrespective of the presence of ATP and Mn2+. These results indicate that the relative affinity of the enzymes in situ for the cobalamins with simple Co beta ligands was markedly lowered in the presence of ATP and Mn2+, whereas that for the cobalamins with adenine-containing ligands was not. When the glycerol-inactivated holoenzymes in situ were dialyzed against a buffer containing ATP and Mg2+, the inactivated coenzyme moiety dissociated from the enzymes leaving apoproteins. Kinetic evidence was also obtained with the dehydratases in situ that continual displacement of the inactivated coenzyme moiety by adenosylcobalamin takes place during the glycerol dehydration reaction in the presence of ATP and Mn2+. Since the adenosyl group of the bound coenzyme is irreversibly removed from the cobalamin moiety during inactivation by glycerol, all of these data constitute clear evidence that the inactivated holo-dehydratases are reactivated in situ in the presence of ATP and Mn2+ by displacement of the modified coenzyme moiety by free intact adenosylcobalamin (i.e. selective B12-exchange mechanism).
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