Cloning and expression of human placental L-Dopa decarboxylase

L-Dopa decarboxylase (DDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme that catalyses the decarboxylation of L-Dopa to dopamine. In this study we show the expression of DDC in human placental tissue and present data on the molecular cloning and in vitro expression of the active recombinant enzyme. Our analyses indicated the presence of both alternative DDC mRNA splice variants (neuronal and nonneuronal) in human placenta. Cloning of the coding region of the DDC cDNA into the pTrcHisA expression vector led to the production of the enzymatically active recombinant protein. The obtained recombinant enzyme specific activity values were in good agreement with the results obtained for the purified enzyme from human kidney. The availability of active recombinant human DDC could provide information leading to the better understanding of the enzyme's structure and substrate specificity, as well as its regulation and involvement in pathological conditions.     

Purification and characterization of L-DOPA decarboxylase from human kidney

L-DOPA decarboxylase has been purified to homogeneity from post mortem removed human kidneys. Homogeneity was examined by polyacrylamide gel electrophoresis (PAGE) analysis both in the presence and absence of SDS. The enzyme has a molecular weight of 100,000 daltons estimated by gel filtration and 50,000 daltons determined after SDS-PAGE. Human L-DOPA decarboxylase therefore is a dimer. Polyclonal antibodies produced against human L-DOPA decarboxylase react with the 50,000 daltons enzyme subunit after immuno-blotting and also precipitates enzyme activity. Activity against L-DOPA is partially inhibited by 5-hydroxytryptophan (5-HTP). The effect of various cations on L-DOPA decarboxylase activity has also been tested.     

Purification of L-dopa decarboxylase from rat liver and production of polyclonal and monoclonal antibodies against it

L-DOPA decarboxylase [DDC, aromatic-L-amino acid carboxyl-lyase, EC 4.1.1.28] was purified 800-fold from rat liver by several column chromatographic steps. The enzyme (specific activity, about 6 mumol/min X mg protein) had a molecular weight of 100,000 and gave a single band with a molecular weight of 50,000 on SDS-polyacrylamide gel electrophoresis. Its isoelectric point was pH 5.7. The absorption spectrum in the visible region of the purified DDC showed maxima at 330 and 420 nm. Polyclonal and monoclonal antibodies against DDC were produced by using this purified protein as an antigen. Polyclonal anti-DDC serum immunoprecipitated the DDC activities of rat, guinea-pig and rabbit livers (about 1, 10, and more than 100 microliter of antiserum, respectively, were required for 50% precipitation of 2 nmol/min of activity of these enzymes). The monoclonal antibody, named MA-1, belonged to the IgG1 subclass and immunoprecipitated the DDC activities of rat and guinea-pig livers to the same extent (about 0.5 micrograms of IgG was required to immunoprecipitate 2 nmol/min activity of each enzyme), but it did not affect the rabbit enzyme. The antibody MA-1 detected DDC molecules of both the purified enzyme and crude homogenate of rat liver blotted onto a nitrocellulose sheet. Immunohistochemically this antibody also stained specific neurons in the substantia nigra, raphe nucleus and locus coeruleus of rat brain.     

L-DOPA decarboxylase association with membranes in mouse brain

This work presents evidence on the association of active DDC molecules with membranes in mammalian brain. L-DOPA decarboxylase (DDC) is generally considered to be a cytosolic enzyme. Membrane-associated DDC was detected by immunoblotting and enzymatic assay experiments. DDC activity and immunoreactivity could be partially extracted from mammalian brain membranes by detergent. Fractionation of membranes by temperature-induced phase separation in Triton X-114, resulted in the recovery of membrane-associated DDC in separation phases where integral and hydrophobic membrane proteins separate. Treatment of membranes with phosphatidylinositol-specific phospholipase C or proteinase K, did not elute membrane-associated DDC activity, suggesting that a population of DDC molecules exist embedded within membranes. The elucidation of the functional significance of the enzyme's association with membranes could provide us with new information leading to the better understanding of the biological pathways that DDC is involved in.     

Demonstration of immunohistochemical and immunochemical cross-reactivity of L-histidine and L-dopa decarboxylases using antibodies against the two enzymes

Both rat L-histidine decarboxylase (HDC) and guinea-pig L-DOPA decarboxylase (DDC) were shown immunohistochemically and immunochemically to react with anti-rat HDC antibody. No cross-reaction was observed in immunoprecipitation experiments, but both anti-rat HDC antibody and anti-rat DDC antibody immunostained neurons in the substantia nigra, raphe nucleus and locus coeruleus of guinea-pig brain. Moreover, on immunoblotting, anti-rat HDC antibody recognized not only rat HDC but also guinea-pig DDC, but not rat DDC. However, anti-rat DDC antibody showed no immunohistochemical or immunochemical cross-reactivity with rat HDC.     

Structural insight into Parkinson's disease treatment from drug-inhibited DOPA decarboxylase.

DOPA decarboxylase (DDC) is responsible for the synthesis of the key neurotransmitters dopamine and serotonin via decarboxylation of L-3,4-dihydroxyphenylalanine (L-DOPA) and L-5-hydroxytryptophan, respectively. DDC has been implicated in a number of clinic disorders, including Parkinson's disease and hypertension. Peripheral inhibitors of DDC are currently used to treat these diseases. We present the crystal structures of ligand-free DDC and its complex with the anti-Parkinson drug carbiDOPA. The inhibitor is bound to the enzyme by forming a hydrazone linkage with the cofactor, and its catechol ring is deeply buried in the active site cleft. The structures provide the molecular basis for the development of new inhibitors of DDC with better pharmacological characteristics.     

Use of radiolabeled monofluoromethyl-Dopa to define the subunit structure of human L-Dopa decarboxylase

Human L-Dopa decarboxylase (L-aromatic amino acid decarboxylase, DDC) has been purified from pheochromocytoma tissue, a benign tumor of the catecholamine-synthesizing cells of the adrenal medulla. The binding characteristics of a new radiolabeled enzyme-activated suicide inhibitor of DDC ( [3H]monofluoromethyl-Dopa, [3H]MFMD) have been established, and the covalent linkage of the inhibitor to the enzyme has been used to identify that human DDC exists as a dimer of a 50-kDa subunit. An antibody to human DDC identically precipitates the enzyme activity from different human, rat, and mouse tissues. Our data demonstrate the value of [3H]MFMD for probing the structure of DDC and facilitating the purification of this enzyme, and further emphasize the high degree of conservation of the DDC molecule over a wide variety of species.     

Presence of endogenous inhibitor of aromatic L-amino acid decarboxylase in monkey serum

Summary In the course of our studies on the developmental changes of aromatic L-amino acid decarboxylase (AADC) in the serum of Japanese monkeys (Macaca fuscata fuscata), we found the presence of an endogenous inhibitor of AADC in all stages of monkey life. This inhibitor inhibited the serum enzyme activity completely with L-5-hydroxytryptophan (L-5-HTP) as substrate, while the activity was partially inhibited with L-DOPA as substrate. The inhibitor was non-dialyzable, but it could be removed from the monkey serum by DEAE-Sephacel chromatography. After this treatment AADC activities could be detected in the monkey serum by using both L-DOPA and L-5-HTP as substrates. Moreover, the total activity for L-DOPA was augmented by 3-fold in the serum after the removal of the inhibitor. Serum AADC was partially purified from monkey and compared with that of rat using both L-DOPA and L-5-HTP as substrates, but the ratio of the activities for the two substrates did not change significantly in each fraction during purification from either monkey or rat serum.On leave from the University of Rajshahi, Rajshahi, Bangladesh.     

Immunological cross-reactivity of gamma-glutamyltranspeptidases from human and rat kidney, liver, and bile

Antisera to purified gamma-glutamyltranspeptidase (gamma GTP) from human and rat kidney were prepared, and their reactivities toward purified gamma GTP from kidney, liver, and bile were tested. The following results were obtained: 1. On double immunodiffusion, Triton-solubilized gamma GTP, and papain-solubilized gamma GTP from rat kidney gave single precipitin lines which fused completely against antiserum to the purified enzyme from rat kidney. 2. An antigen-antibody complex of human kidney gamma GTP retained about 50% of the catalytic activity of the antigen. 3. Double immunodiffusion showed that the enzymes from human liver, kidney, and bile were immunologically identical. 4. Antiserum to rat kidney gamma GTP partially cross reacted with human gamma GTP, but antiserum to human gamma GTP reacted only very weakly with rat gamma GTP. It is concluded that gamma GTP of human liver, kidney, and bile are immunologically identical and that rat gamma GTP and human gamma GTP have certain antigenic determinants in common.     

Developmental expression and spatial distribution of dopa decarboxylase in Drosophila

Regulation of the dopa decarboxylase gene of Drosophila has been studied at the genetic and molecular levels. Here we report a direct assay for the tissue and temporal regulation of Ddc. A dopa decarboxylase (DDC) peptide was obtained by bacterial expression of a portion of the DDC gene in a pUC plasmid. Antisera raised against this biologically purified DDC peptide react specifically with Drosophila DDC in histological preparations and protein blots. The levels of DDC cross-reacting material closely parallel the levels of enzyme activity observed during development, indicating that DDC is degraded during periods of declining activity. We find that DDC is expressed in only two tissues, namely, the epidermis and the nervous system of the larva and adult. Epidermal DDC was found within the epidermal cells and was not detected in the overlying cuticle. DDC-containing neurons were observed in the central as well as in the visceral nervous system. Paired and unpaired midline neurons in the ventral ganglia are arranged in a segmental pattern. A subset of the DDC-positive neurons appears to correlate with the serotonin-positive neurons suggesting that the others are producing only dopamine. We find that the DDC activity associated with the proventriculus and ovary is due to the presence of DDC in the stomatogastric and caudal system neurons specifically associated with those structures.     

Decarboxylation of p-Tyrosine: A Potential Source of p-Tyramine in Mammalian Tissues

The question of the existence of a p-tyrosine decarboxylase pathway for the formation of p-tyramine in mammalian tissues remains unresolved. Development of a sensitive and specific assay for p-tyrosine decarboxylase has permitted demonstration of this activity in rat tissues and human kidney. Tyrosine decarboxylase was purified to electrophoretic homogeneity by pH 5.0 precipitation, ammonium sulfate precipitation, gel filtration, phenyl-Sepharose chromatography, DEAE-Sephacel chromatography, and preparative isoelectric focusing. A specific rabbit antiserum to tyrosine decarboxylase was also obtained. Purified tyrosine decarboxylase possessed a narrow pH dependency with an optimum at 8.0. Benzene and certain other organic solvents dramatically stimulated tyrosine decarboxylase activity of purified enzyme. Purified tyrosine decarboxylase activity also decarboxylated L-DOPA, 5-hydroxytryptophan, 3,4-dihydroxyphenylserine, o-tyrosine, m-tyrosine, phenylalanine, histidine, and tryptophan, which suggested that the purified enzyme was aromatic L-amino acid decarboxylase. This conclusion was supported by a constant ratio of 5-hydroxytryptophan decarboxylase to tyrosine decarboxylase throughout the purification scheme and by parallel immunoprecipitation of decarboxylase activities by the specific antityrosine decarboxylase antisera. Thus, we report that p-tyrosine is decarboxylated by aromatic L-amino acid decarboxylase and that this metabolic transformation may be an important source of p-tyramine in mammalian tissues. In conclusion, neuronal tissues that synthesize catecholamines or serotonin should now be considered capable of synthesizing p-tyramine and other biogenic amines.     

A study on renin binding protein (RnBP) in the human kidney

We purified, from human kidney, a protein that reacts with rabbit anti-porcine kidney renin binding protein (RnBP) antiserum by trapping with porcine kidney renin. The purified preparation showed a single protein peak on gel filtration by high performance liquid chromatography (HPLC) and two protein bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The latter two kinds of protein were identified as the porcine renin and human kidney protein from their electrophoretic mobilities and reactivity toward rabbit anti-porcine kidney renin and RnBP antisera. The molecular weights of the purified preparation and the human kidney protein were estimated to be 56,000 by HPLC and 43,000 by SDS-PAGE, respectively. The specific activity of porcine renin in the purified preparation was 8.6 mg angiotensin I per mg of protein per h at 37 degrees C and pH 6.5. This specific activity was about one-fifth that of free porcine renin. Therefore, it is suggested from the reactivity toward the anti-porcine RnBP antiserum and inhibitory action toward porcine renin that the human kidney protein is RnBP and that the human RnBP is purified as a complex with porcine renin.     

An organic solvent resistant tyrosinase from Streptomyces sp. REN-21: purification and characterization

We found a tyrosinase, which has high activity in the presence of organic solvents, in the culture filtrate of Streptomyces sp. REN-21. The organic solvent resistant tyrosinase (OSRT) was purified from the culture filtrate by three column chromatographies. About 1.2 mg of purified OSRT was obtained from 5.6 liters of the culture filtrate with a yield of 26.0%. The purified enzyme had a single polypeptide chain with a molecular mass of about 32,000 Da. The optimum pH and temperature of OSRT were pH 7.0 and 35 degrees C using L-beta-(3,4-dihydroxyphenyl)alanine (L-DOPA) as substrate. OSRT showed stereospecificity toward L-, DL-, and D-enantiomers of DOPA or tyrosine. OSRT had 44% of the activity of the control even in the presence of 50% ethanol, while a mushroom tyrosinase showed only 6% activity under the same conditions. Moreover, OSRT retained its original activity even after 20 h of incubation at 30 degrees C in the presence of 30% ethanol.     

Human TRP-1 Has Tyrosine Hydroxylase but no DOPA Oxidase Activity

Human TRP-1 has been immunopurified from normal human melanocytes cultured from black neonatal subjects and used to investigate the catalytic function of TRP-1 for the two substrates, L-tyrosine and L-DOPA. Immunopurified TRP-1 did not demonstrate DOPA staining on SDS/PAGE nor DOPA oxidase (DO) activity with either routine or modified assays. The purified TRP-1 also demonstrated no tyrosine hydroxylase (TH) activity using the routine Pomerantz assay. However, there was apparent TH activity exhibited by immunopurified TRP-1 under conditions with low tyrosine concentration (≤0.8 μCi/ml of ³H-tyrosine), prolonged incubation time (i.e., overnight) and in the absence of the cofactor L-DOPA. Using these latter specific conditions, TH activity was also detected in cell lysates from a tyrosinase-negative albino melanocyte line which exhibited no TH activity with the routine Pomerantz assay. In addition, TH activity under low substrate assay conditions was not exhibited in a melanocyte line derived from a TRP-1 deficient, Brown albino individual. However, the absence of TH in this Brown albino cell line could be compensated for by the addition of L-DOPA to the assay. These results suggested that TRP-1 has some tyrosine hydroxylase but no DOPA oxidase activity. We propose that one function of TRP-1 is to modulate tyrosinase activity by making DOPA available as a cofactor to perpetuate the initial steps in melanogenesis.     

Purification and comparison of phosphoenolpyruvate carboxykinase from the liver and kidney of the Arabian camel (Camelus dromedarius)

1. Phosphoenolpyruvate carboxykinase was partially purified from camel liver and kidney by ammonium sulphate fractionation, gel filtration and ion-exchange chromatography. 2. The specific activity of the purified preparation from liver was 39.2 mumol/min per mg protein. 3. When isolated from the kidney the specific activity of the enzyme was very much higher 155.5 mumol/min per mg protein. 4. The enzyme from the two sources were similar in their pH optimum which was approx. 7.2 and their relative stability to thermal inactivation at 60 degrees C. 5. The mol. wt of the enzyme from both organs was estimated at 80,000 +/- 5000.     

Human 5-lipoxygenase contains an essential iron

The iron content of human 5-lipoxygenase has been determined by a colorimetric assay using the chromogenic ligand FerroZine. The highly active enzyme was obtained from a baculovirus expression system and purified using an ATP-agarose chromatography column (Denis, D., Falgueyret, J.-P., Riendeau, D., and Abramovitz, M. (1991) J. Biol. Chem. 266, 5072-5079). A linear correlation was observed between the enzyme's specific activity and iron content in six different preparations. Enzyme with the highest specific activity (24 mumol of 5-hydroperoxyeicosatetraenoic acid/mg of protein) contained 1.1 mol of iron/mol of enzyme, whereas inactive enzyme contained no detectable iron. The iron is tightly bound to the enzyme and could only be released after inactivation of the enzyme by exposure to oxygen.     

Isolation and characterization of basic superoxide dismutase consisting of Mr-25,000 subunits in rat liver

1. A basic protein (pI = 9.0) exhibiting superoxide dismutase activity was purified to homogeneity from rat liver by DEAE-cellulose, CM-cellulose and S-hexylglutathione affinity gel chromatography, chromatofocusing and Sephadex G-150 gel filtration. 2. The purified enzyme had specific activity of 4700 units/mg protein. The activity was not affected by 2 mM KCN. Manganese was detected in the enzyme preparation; the content was 0.9 mol/mol subunit. The N-terminal sequence of the first 23 amino acids of the enzyme exhibited a strong homology (except at position 11) with the mature protein of human Mn-superoxide dimutase. It is, therefore, concluded that the purified enzyme is Mn-superoxide dismutase. 3. The N-terminal amino acid sequence showed that about 50% of tyrosine at position 11 was substituted by glutamine, suggesting the existence of microheterogeneity of the superoxide dismutase protein. 4. The superoxide dismutase purified here was found to consist of subunits with an apparent relative molecular mass of 25,000. This larger than the value hitherto reported for rat liver Mn-superoxide dismutase (Mr 2,400); the previous low value is attributed to differences in methods. 5. The enzyme was shown by immuno-blotting to be exclusively localized in the mitochondrial fraction in the liver. The tissue content of Mn-superoxide dismutase is organ-specific, and was the highest in heart. The precursor protein of the Mn-superoxide dismutase was not detectable in the liver cytosolic and mitochondrial fractions as well as in several extrahepatic organs (lung, heart, brain, muscle, kidney and testis), suggesting rapid transport across mitochondrial membranes and processing of the superoxide dismutase protein.     

Immuno-cross-reactivity of histidine and dopa decarboxylases

Immuno-cross-reactivity between histidine decarboxylase (HDC) and dopa decarboxylase (DDC) was investigated. By comparing the cDNA sequences of rat HDC with rat and guinea-pig DDCs, we found a region that may possibly be related to the cross-reactivity of anti-rat HDC antibody with guinea-pig DDC. The peptide encoded by this region was synthesized and anti-peptide antibody was prepared. We also purified HDC and DDC homogeniously from fetal rat liver and guinea-pig liver, respectively. On immunoblotting, anti-peptide antibody recognized both rat HDC and guinea-pig DDC. Anti-HDC polyclonal antibody which also recognizes both enzymes detected only rat HDC when it was absorbed by the peptide. This result indicates that this region is responsible for the immuno-cross-reactivity of anti-rat HDC antibody with guinea-pig DDC.     

Synthesis of 5-methyl phenanthridium derivatives: A new class of human DOPA decarboxylase inhibitors

DOPA decarboxylase (DDC) is responsible for the decarboxylation of l-DOPA and related aromatic amino acids and correlates closely with a number of clinical disorders. Sanguinarine, a natural quaternary benzophenanthridine alkaloid (QBA), was reported to be inhibitor of rat DDC and possessed a different inhibitory mechanism. In this study, several natural QBAs were assayed as human DDC inhibitors for the first time. A series of 5-methyl phenanthridium derivatives that contain the basic core structure of QBAs were also synthesized and evaluated as human DDC inhibitors. The title compounds still possessed DDC inhibitory potential. Among the synthesized compounds, 2-hydroxyl-8-methoxy-5-methylphenanthridinium chloride (11k) showed good inhibitory activity with an IC₅₀ value of 0.12 mM. Preliminary structure–activity relationship indicated that DDC inhibitory potential of 5-methyl phenanthridium derivatives correlated with the π-electro densities on CN double bond of iminium cation. The hydroxyl group on compound 11k possibly contributed to the formation of hydrogen bond between DDC and the inhibitor.