We studied whether lead exposure increased the serum lipid peroxide (LPO) level and inhibited blood superoxide dismutase (SOD) activity in workers with occupational exposure to lead and rats injected with lead. We examined the following subjects: (1) manual workers (712 males) from 18 to 59-years-old in steel production with occupational exposure to lead, (2) office workers (155 males) without exposure to lead, (3) rats subcutaneously injected with lead in concentrations of 10 or 20 mg/kg as lead acetate. The nutritional intakes of manual workers and office workers were approximately equal. Serum LPO and high-density lipoprotein cholesterol (HDL-CL) levels in manual workers (LPO: 4.4 +/- 1.9 nmol/ml, HDL-CL: 55.6 +/- 14.2 mg/dl) were significantly higher than those in office workers (LPO: 4.0 +/- 1.4 nmol/ml, HDL-CL: 53.0 +/- 13.9 mg/dl). Serum LPO level in the manual workers increased with an increase of the lead concentration in the blood, while blood SOD activity decreased. Similar phenomena were observed in rats subcutaneously injected with lead acetate. Furthermore, the addition of lead at higher than 20-microM concentrations to non-treated rats liver microsomes increased NADPH-dependent liquid peroxidation, and these lead concentrations inhibited bovine erythrocyte SOD activity in vitro assay system. In conclusion, the present results seem to indicate that the increase of serum LPO level in workers with occupational exposure to lead is due not only to the stimulation of lipid peroxidation, but also to the inhibition of SOD activity by exposure to lead in the manufacturing processes.

Tyrosine hydroxylase (TH), an iron-containing enzyme, catalyzes the first and rate-limiting step of catecholamine biosynthesis, and requires tetrahydrobiopterin (BH4) as a cofactor. We found that preincubation of recombinant human TH with BH4 results in the irreversible inactivation of the enzyme at a concentration far less than the Km value toward BH4 in spite of its cofactor role, whereas oxidized biopterin, which has no cofactor activity, does not affect the enzyme activity. We show that TH is inactivated by BH4 in competition with the binding of dopamine. The sequential addition of BH4 to TH results in a gradual decrease in the intensity of the fluorescence and CD spectra without changing their overall profiles. Sedimentation velocity analysis demonstrated an association of TH molecules with each other in the presence of BH4, and studies using gel-permeation chromatography, turbidity measurements, and transmission electron microscopy demonstrated the formation of amorphous aggregates with large molecular weights following the association of the TH proteins. These results suggest that BH4 not only acts as a cofactor, but also accelerates the aggregation of TH. We propose a novel mechanism for regulating the amount of TH protein, and discuss its physiological significance.

Abstract:The relationship between airborne concentration of beryllium in the working environment and workers' beryllium lymphocyte transformation test (Be-LTT) values was examined based on data obtained from a four-year survey (1992)(1993)(1994)(1995) conducted at beryllium-copper alloy manufacturing factories. This study showed that the T cells of workers continuously exposed to beryllium of more than 0.01 /Im3 could be activated and that the cell-mediated immune response of workers could be promoted. On the other hand, the Be-LTT of workers exposed to beryllium levels of less than 0.01 pglm3 was shown to be unaffected by beryllium. These findings suggest that beryllium sensitization is not manifested when level of beryllium in working environment are less than 0.01 pglm3. Therefore, in such cases workers do not develop Chronic beryllium disease (CBD). We concluded that the Be-LTT can be applied as a medical indicator to detect the development of CBD.

Tyrosine hydroxylase (TH), which converts L-tyrosine to L-DOPA, is a rate-limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by feedback inhibition by catecholamine products including dopamine. To investigate the specific portion of the N-terminus of TH that determines the efficiency of dopamine inhibition, wild-type and N-terminal 3 5 , 38-, and 44-amino acid-deleted mutants (del-35, del-38, and del-44, respectively) of human TH type l were expressed as a maltose binding protein fusion in Escherichia coli and purified as a tetrameric form by affinity and size-exclusion chromatography. The fused-form wild-type enzyme possessed almost the same specific enzymatic activity as the previously reported recombinant nonfused form. Although maximum velocities of all N-terminus-deleted forms were about one-fourth of the wild-type value, there was no difference in Michaelis constants for L-tyrosine or (6R)-(~-erythro-l',2'-dihydroxypropyl)-2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine (GRBPH,) among the four enzymes. The iron contents incorporated into the three N-terminus-deleted mutants were significantly lower than that of wild type. However, there was no substantial difference in incorporated iron contents among the three mutants. The deletion of up to no less than 38 amino acid residues in the N-terminus made the enzyme more resistant to dopamine inhibition than the wild-type or del-35 TH form. Dopamine bound to the del-38 more than to the del-35 TH form. However, when incubation with dopamine was followed by further inhibition with the cofactor GRBPH, dopamine was expelled more readily from the del-38 than from the del-35 TH form. These observations suggest that the amino acid sequence G l~~~-A r g~~-A r g~~ plays a key role in determining the competition between dopamine and GRBPH, and affects the efficiency of dopamine inhibition of the catalytic activity. Key Words: Tyrosine hydroxylase [TH; tyrosine 3-monooxygenase; L-tyrosine, tetrahydr0pterin:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.21, which catalyzes the conversion of L-DOPA from L-tyrosine (Nagatsu et al., 1964), is a rate-limiting enzyme in the biosynthesis of catecholamines (Levitt et al., 1965). The enzyme requires a reduced pterin (Nagatsu et al., 1964), and (6R)-(~-erythro-1 ,2'-dihydroxypropyl)-2-amino-4-hydroxy-5,6,7,8-tebxhydropteridine (6R-tebxhydrobiopteri~ 6RBPH4) is the natural cofactor (Kaufman, 1963;Brenneman and Kaufman, 1964;Matsuura et al., 1985). TH also requires ferrous iron (Nagatsu et al., 1964;Shiman et al., 1971). TH consists of a catalytic domain (C-domain) and a regulatory domain (R-domain) (Abate and Joh, 1991). The C-domain is located at the C-terminal two-thirds of the molecule and binds the substrates (L-tyrosine and molecular oxygen) and the cofactor (6RBPH,). In contrast, the R-domain has been assigned to the N-terminal end (Hoeldtke and Kaufman, 1977;Abate et al., 1988;Abate and Joh, 1991) and has an important role in substrate specificity and in control of the catalytic activ...

GTP cyclohydrolase I (GCH) is the rate-limiting enzyme for the synthesis of tetrahydrobiopterin and its activity is important in the regulation of monoamine neurotransmitters such as dopamine, norepinephrine and serotonin. We have studied the action of divalent cations on the enzyme activity of purified recombinant human GCH expressed in Escherichia coli. First, we showed that the enzyme activity is dependent on the concentration of Mg-free GTP. Inhibition of the enzyme activity by Mg 2+ , as well as by Mn 2+ , Co 2+ or Zn 2+ , was due to the reduction of the availability of metal-free GTP substrate for the enzyme, when a divalent cation was present at a relatively high concentration with respect to GTP. We next examined the requirement of Zn 2+for enzyme activity by the use of a protein refolding assay, because the recombinant enzyme contained approximately one zinc atom per subunit of the decameric protein. Only when Zn 2+ was present was the activity of the denatured enzyme effectively recovered by incubation with a chaperone protein. These are the first data demonstrating that GCH recognizes Mg-free GTP and requires Zn 2+ for its catalytic activity. We suggest that the cellular concentration of divalent cations can modulate GCH activity, and thus tetrahydrobiopterin biosynthesis as well.Keywords: GTP cyclohydrolase I; magnesium; recombinant protein; tetrahydrobiopterin; zinc.Metal ions are essential for many physiological functions of the brain. They may also induce or aggravate numerous neurodegenerative processes. Thus, it is important to understand the roles of metal ions in normal and pathological brain functions.GTP cyclohydrolase I (GCH) is the rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin (BH 4 ), and the cellular BH 4 content is regulated mainly by the activity of this enzyme. BH 4 is an essential cofactor for three aromatic amino-acid monooxygenases ) phenylalanine, tyrosine, and tryptophan hydroxylases -and for nitric oxide synthase [1]. BH 4 deficiency caused not only a decrease in the activity of these enzymes but also a decrease in the protein levels of tyrosine hydroxylase and nitric oxide synthase [2,3]. Therefore, the availability of BH 4 affects the amounts of neurotransmitters such as catecholamines, serotonin, melatonin and nitric oxide. The role of BH 4 in the activity of nitric oxide synthase also makes BH 4 an important factor for the immune system and endothelial cell function.Various hormones and cytokines are known to induce the expression of the GCH gene in neural, lymphocytic and endothelial cells, and in different cell lines, resulting in an increased BH 4 content [4][5][6][7][8]. At the post-transcriptional level, BH 4 was shown to inhibit, and phenylalanine to stimulate, GCH activity through interaction with GFRP, a GTP cyclohydrolase I feedback regulatory protein [9]. GCH, which is a homodecameric protein, shows positive cooperativity against the GTP substrate [10] and phenylalanine changes the substrate velocity curve from sigmoidal to hyperbolic [11].Recent...