Inhibitory effect of aluminum on dopamine beta-hydroxylase from bovine adrenal gland.

Aluminum is a well known neurotoxic agent that is overaccumulated in the substantia nigra of patients affected by Parkinson's disease as well as in certain cerebral areas of other neurodegenerative pathologies such as Alzheimer's disease. Although the role of aluminum in neurodegenerative diseases is yet to be clearly understood, the metal ion is known to substantially alter the activity of several key enzymes in the central nervous system. The present paper reports data on the effect of aluminum on the activity of dopamine-beta-hydroxylase from bovine adrenaL gland utiLized as a model study. The metal ion inhibited the activity of this enzyme with a mixed type mechanism following the Michaelis-Menten equation. In the absence of Al, the enzyme exhibited a Km and Vmax of 2.56 mM of 4.12 pmol/min respectively, while in the presence of Al its Km and Vmax were 3.85 mM and 2.86 pmol/min respectively. The potential implications of aluminum in the etiopathogenesis of neurological disorders are discussed.     

Regulation of dopamine beta-hydroxylase in rat adrenal glands.

Rat adrenal gland levels of dopamine beta-hydroxylase are subject to dual control. Activation of the splanchnic nerves to the adrenal medulla by reserpine induces the synthesis of dopamine beta-hydroxylase without altering the rate of enzyme degradation. In contrast, hypophysectomy causes a decline in steady state dopamine beta-hydroxylase levels by first accelerating the rate of degradation, then by slowing the rate of enzyme synthesis as well. Adrenocorticotropic hormone administration partially reversed the effect of hypophysectomy on dopamin beta-hydroxylase degradation. These findings suggest that the trans-synaptic factors controlling dopamine beta-hydroxylase induction act by a different mechanism (enzyme synthesis) than the hormonal controls regulating steady state levels (enzyme degradation). Thus, active inhibition of enzyme degradation may be an important control in maintenance of steady state enzyme levels.     

Blood dopamine beta-hydroxylase activity and noradrenaline levels in the developing white rat.

Dopamine beta-hydroxylase (DBH) (3,4-dihydroxyphenylethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating) (EC 1.14.17.1) activity in serum of blood obtained by decapitation of white rats at 19, 20, and 21 days in utero, immediately after birth, and postnatally to 70 days, was measured. Noradrenaline (NA) and DBH in plasma from undisturbed, cannulated, postweaning rats were also assayed. During the last few days in utero and the first 2 postnatal days serum DBH activity tripled and then remained elevated during the suckling period. Upon weaning, serum DBH activity declined at first precipitously and then more slowly, until the adult level was reached around 70 days of age. This postweaning decrease in DBH activity was also observed with the cannulated animals. In contrast, plasma NA levels remained low and constant throughout the postweaning period. In suckling rats treated with 6-hydroxydopamine from 2 to 12 days of age, serum DBH activity decreased to less than half its initial value by day 8. It is suggested that the observed changes in serum DBH activity in fetal and postnatal rats reflect ontogenetic changes in sympathetic nerve terminals and that they are probably not correlated with release of NA.     

Insulin-like growth factor I enhances proenkephalin synthesis and dopamine beta-hydroxylase activity in adrenal chromaffin cells

Insulin-like growth factor I (IGF-I) increased both the contents of proenkephalin-derived enkephalin-containing peptides and the activity of dopamine beta-hydroxylase in bovine adrenal chromaffin cells. These increases in dopamine beta-hydroxylase and enkephalin-containing peptides continued for at least 8 days. The half-maximal IGF-I concentration for these effects was approximately 1 nM, with maximal effects observed at 10-30 nM. In contrast, insulin was 1000-fold less potent. Pretreatment of chromaffin cells with IGF-I increased the rate of [35S]proenkephalin synthesis 4-fold compared to untreated cells. Total protein synthesis increased only 1.5-fold under these conditions. These results suggest that IGF-I may be a normal regulator of chromaffin cell function.     

Purification and characterization of dopamine beta-hydroxylase from bovine adrenal medulla.

A new purification procedure that permits large-scale purification of dopamine beta-hydroxylase from bovine adrenal medulla was developed. Whole adrenal medullas were extracted with 0.1% Triton X-100, and the enzyme was purified by precipitation with polyethylene glycol, chromatography on DEAE-cellulose, and adsorption to concanavalin A linked to agarose. The yield of protein and the specific activity were high compared with previously published methods. The enzyme appeared essentially homogenous by the criteria of polyacrylamide gel electrophoresis in the presence or absence of dodecylsulfate, and sedimentation velocity analysis. The purified protein was subjected to amino acid and carbohydrate analyses, and the results were compared with previously published data. We found about 3 mol of copper per mol of protein (tetramer of 290000 daltons). No free sulfhydryl groups could be found. Analysis for NH2-terminal amino acids with [14C]dansyl chloride revealed 2 residues of alanine and 2 residues of serine per tetramer. We found the NH2-terminal amino acid of chromogranin A to be leucine. The results of our analysis for amino acid composition and NH2-terminal amino acids do not support the suggestion that dopamine beta-hydroxylase and chromogranin A contain identical peptide chains.     

Regional differences in microsomal lipid peroxidation and antioxidant levels in the guinea pig adrenal cortex

Lipid peroxidation (LP) and antioxidant levels were studied in the chromatically distinct inner (zona reticularis) and outer (zona fasciculata + zona glomerulosa) zones of the guinea pig adrenal cortex. Ferrous ion (Fe2+) produced a concentration-dependent (10(-5) to 10(-3) M) stimulation of microsomal LP in both zones, but LP, as estimated by malonaldehyde production, was far greater in the inner zone. Although cytosolic ascorbic acid content was similar in the two zones, microsomal tocopherol levels were approx 4 times greater in the outer than inner zone. Subphysiological concentrations of ascorbic acid, like Fe2+, initiated LP to a greater extent in inner than outer zone microsomes; optimal stimulation of LP by ascorbic acid occurred at concentrations of 100-200 microM in both zones. Physiological concentrations of ascorbic acid (1-5 mM), by contrast, did not initiate LP and, in fact, markedly inhibited Fe2+-induced LP in both inner and outer zone microsomal preparations. Outer zone microsomes were more sensitive to the antioxidant effects of ascorbic acid than were inner zone preparations. Addition of alpha-tocopherol to inner zone microsomal suspensions inhibited Fe2+-induced LP. The results indicate that there are regional differences in adrenocortical LP which may be caused by differences in tocopherol content. alpha-Tocopherol may serve important antioxidant functions within the adrenal cortex, thereby contributing to the functional zonation of the gland.     

Purification and some characteristics of an oestrogen sulphotransferase from guinea pig adrenal gland and its non-identity with adrenal pregnenolone sulphotransferase.

An oestrogen sulphotransferase, active towards both oestrone and oestradiol, and of high specific activity, is present in cytosol prepared from adrenal glands of both sexes of English Shorthair and Hartley guinea pigs. The ovarian and testicular cytosolic activities of this enzyme are markedly low in comparison with the adrenal activity. The adrenal enzyme is distinct from an accompanying pregnenolone sulphotransferase as judged by f.p.l.c. gel filtration, chromatofocusing, and differences in activation brought about by the addition of thiol groups. The oestrogen sulphotransferase behaved as a 67 kDa protein on a Sephadex G100 column and as a 48 kDa protein on f.p.l.c. gel-filtration columns. Two forms of the enzyme with apparent pI values of 6.1 and 5.5 were eluted during f.p.l.c. chromatofocusing. Sequential salt fractionation, f.p.l.c. gel filtration and elution from an agarose-hexane-adenosine-3',5'-diphosphate affinity gel has resulted in a preparation which, when resubmitted to f.p.l.c. gel filtration, yields a considerably purified oestrogen sulphotransferase. When submitted to SDS/polyacrylamide-gel electrophoresis under reducing conditions, a main protein band of 34-36 kDa is observed. It is suggested that the enzyme may exist as a dimer in the cytosol.     

Immunocytological localization of dopamine in the guinea pig retina

We examined dopaminergic neurons in the guinea pig retina; antisera against tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT) and an antiserum against gamma-aminobutyric acid (GABA) were used. In the present study, two types of amacrine cells were labeled with an anti-TH antiserum. However, no DBH and PNMT immunoreactivities were seen. The type 1 cell had a larger-sized soma located in the inner nuclear layer with processes ramifying mainly in stratum 1 of the inner plexiform layer (IPL). The type 2 cell had a smaller-sized soma and processes branching in stratum 3 of the IPL. The mean densities were 56.4 +/- 11.5/mm2 for the type 1 cell and 166.6 +/- 30.3/mm2 for the type 2 cell. Double immunocytochemistry using an antiserum against GABA revealed that while none of the type 1 cells showed GABA immunoreactivity, all of the type 2 cells displayed GABA immunoreactivity. Our results suggest that, in the guinea pig retina, the type 1 amacrine cells are pure dopaminergic and the type 2 cells are dopaminergic elements that use GABA as their second transmitter.     

Serum dopamine beta-hydroxylase activity in non-human primates: phylogenetic and genetic implications

1. Serum dopamine beta-hydroxylase (DBH) activity is one to two orders of magnitude higher in man than in any other species previously studied. The high levels of human serum DBH are associated with the inherited allele DBHH. 2. DBH activity was measured in serum from gorillas, chimpanzees, orangutans, gibbons, rhesus monkeys and squirrel monkeys in an attempt to determine how recently in the course of evolution the allele DBHH had originated. 3. Of the non-human primates tested, only gorillas had high levels of serum DBH activity comparable to those found in man. 4. The genetic polymorphism responsible for evaluated serum DBH in man is probably of very recent evolutionary origin.     

The molecular shape of dopamine beta-hydroxylase from chromaffin granules of bovine adrenal medulla

The structural features of the soluble dopamine beta-hydroxylase from chromaffin granules of bovine adrenal medulla were studied using negative staining and platinum shadowing electron microscopic methods. The enzyme was shown to be highly asymmetric as suggested in earlier hydrodynamic studies. The tetramer of the enzyme appeared as four subunits arranged in the shape of a planar rose with an estimated width of 15 nm. A minimum thickness of 3.0 nm for the enzyme monomer was calculated from the shadow length of unidirectionally shadowed molecules. A model composed of four oblate ellipsoid monomers in a tetrameric rose arrangement is proposed for the shape of the dopamine beta-hydroxylase molecule. Two monomers associate edge to edge to form an in-plane dimer and two dimers associate side-by-side with their respective long axes at a slight angle to form a tetramer. Theoretical calculations based on the model are consistent with previous hydrodynamic studies.     

The pH-dependent subunit dissociation and catalytic activity of bovine dopamine beta-hydroxylase

The soluble form of dopamine beta-hydroxylase from bovine adrenal medulla has previously been shown to exist as a tetrameric species of Mr = 290,000 composed of two disulfide-linked dimers. Here we report that this enzyme can also undergo a reversible tetramerdimer dissociation which is dependent on pH. Gel permeation chromatography of dopamine beta-hydroxylase at pH 5.0 demonstrates a Stokes radius of 5.8 nm. When the pH is shifted to 5.7, the Stokes radius changes to 6.9 nm. Sedimentation equilibrium analysis of the purified enzyme demonstrates that this change in molecular size is due to a change in molecular weight. At low protein concentration, the estimated Mr of the enzyme is 145,000 at pH 5.0 and at high protein concentration approaches 290,000 at pH 5.7. This change in Mr is consistent with the existence of a tetramer-dimer dissociation and a change in the equilibrium constant from 1.8 X 10(-6) M to 1.16 X 10(-9) M when the pH is increased from 5.0 to 5.7. This pH-dependent subunit dissociation is correlated with pH-dependent changes in enzyme activity. Purified bovine-soluble dopamine beta-hydroxylase activity is a hyperbolic function of tyramine concentration at pH 5.0. However, the hydroxylase activity displays non-hyperbolic kinetics at pH 6.0. The kinetic data obtained at pH 6.0 can be accounted for by fitting to a model containing two nonidentical catalytic forms of enzyme generated by the pH-dependent partial dissociation of tetrameric enzyme to dimeric subunits. The two catalytic forms have apparently identical maximal velocities; however, they differ in their Michaelis constants for the substrate; the dimeric form having a low Km and the tetrameric form having a high Km. Since the pH inside bovine adrenal medullary chromaffin granules is approximately 5.5, we conclude that the subunits of dopamine beta-hydroxylase are in dynamic dissociation in a physiologically important pH range.