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Regulation of dopa decarboxylase expression during colour pattern formation in wild-type and melanic tiger swallowtail butterflies.

The eastern tiger swallowtail butterfly Papilio glaucus shows a striking example of Batesian mimicry. In this species, females are either wild type (yellow and black) or melanic (where most of the yellow colour is replaced by black). In order to understand how these different colour patterns are regulated, we examined the temporal order of wing pigment synthesis via precursor incorporation studies, enzyme assays, and in situ hybridisation to mRNA encoding a key enzyme, dopa decarboxylase. We show that dopa decarboxylase provides dopamine to both of the two major colour pigments, papiliochrome (yellow) and melanin (black). Interestingly, however, dopa decarboxylase activity is spatially and temporally regulated, being utilised early in presumptive yellow tissues and later in black. Further, in melanic females, both dopa decarboxylase activity and early papiliochrome synthesis are suppressed in the central forewing and this normally yellow area is later melanised. These results show that the regulation of enzyme synthesis observed in the yellow/black pattern of a single wing, is similar to that involved in melanism. We infer that dopa decarboxylase activity must be regulated in concert with downstream enzymes of either the melanin and/or the papiliochrome specific pathways, forming part of a developmental switch between yellow or black. This modification of multiple enzyme activities in concert is consistent with a model of melanisation involving coordinate regulation of the underlying synthetic pathways by a single Y-linked (female) factor.

Animals↗

Genomic organization of the rat aromatic L-amino acid decarboxylase (AADC) locus: partial analysis reveals divergence from the Drosophila dopa decarboxylase (DDC) gene structure.

Aromatic L-amino acid decarboxylase (AADC) is responsible for the conversion of L-3,4-dihydroxyphenylalanine (L-DOPA) and L-5-hydroxytryptophan to dopamine and serotonin, respectively, which are important neurotransmitters. We characterized genomic clones derived from the rat AADC locus by Southern blot and nucleotide sequencing analyses to explore the exonal organization of the gene. Our results suggest that the rat AADC gene is relatively large, containing at least 12 exons and spanning at least 40 kb in the rat genome. In this study, nine exons corresponding to 71% of the published cDNA sequence were identified, the smallest of which was as short as 20 base pairs (bp). In the Drosophila dopa decarboxylase (DDC) gene, the sequences homologous to these nine exons are all present in the fourth exon. This implies that either multiple intron sequences have been added to the vertebrate AADC gene or alternatively, deleted from the invertebrate gene after the divergence of vertebrates and invertebrates during evolution.

Animals↗

Comparative and quantitative study of L-dopa decarboxylase mRNA in rat neuronal and non-neuronal tissues.

The L-DOPA decarboxylase mRNA levels were determined by a sensitive S1 nuclease method in four organs and one tumor of adult rat. S1 mapping analysis, with probes corresponding to the mRNA coding region, showed that this region is conserved in all L-DOPA decarboxylase mRNA of neuronal and non-neuronal tissues. The mRNA was not very abundant; its representation varies approximately from 0.00035% of total RNA in the mid brain to 0.013% of total mRNA in the pheochromocytoma. A strong correlation between mRNA level and enzyme amount was observed (correlation coefficient = 0.99). The results indicate that the level of mRNA is a primary factor determining the L-DOPA decarboxylase level.

Adrenal Gland Neoplasms↗

Congenital hyperinsulinism: pancreatic [18F]fluoro-L-dihydroxyphenylalanine (DOPA) positron emission tomography and immunohistochemistry study of DOPA decarboxylase and insulin secretion.

CONTEXT: Congenital hyperinsulinism (HI) is characterized by hypoglycemia related to inappropriate insulin secretion. Focal and diffuse forms of hyperinsulinism share a similar clinical presentation, but their treatment is dramatically different. Preoperative differential diagnosis was based on pancreatic venous sampling, a technically demanding technique. OBJECTIVE: Positron emission tomography (PET) after injection of [18F]fluoro-L-DOPA (L-dihydroxyphenylalanine) has been evaluated for the preoperative differentiation between focal and diffuse HI, by imaging uptake of radiotracer and the conversion of [18F]fluoro-L-dopa into dopamine by DOPA decarboxylase. We propose to validate this test by immunohistochemical approach. PATIENTS AND METHODS: Pancreatic surgical specimens of four focal and three diffuse HI were studied, using anti-DOPA decarboxylase and proinsulin antibodies. The effect of an inhibitor of DOPA decarboxylase (carbidopa) on insulin secretion was evaluated in vivo and in cultured INS-1 cells. RESULTS: Immunohistochemical detection of DOPA decarboxylase showed diffuse staining of Langerhans islets in the whole pancreas in all diffuse cases, in contrast with dense focal staining in all focal cases. Staining of Langerhans islets outside the focal lesion was diffusely but weakly positive. We correlated the localization of DOPA decarboxylase and proinsulin in normal pancreas and in both diffuse and focal HI tissues. The diffuse PET uptake found before treatment in one child with diffuse HI disappeared completely after carbidopa administration, suggesting in vivo that pancreatic cells can take up amine precursors and contain DOPA decarboxylase. The insulin secretion measured in the supernatant was the same whether INS-1 cells were treated by dopamine or Lodosyn or untreated. CONCLUSION: We validate PET with as a consistent test to differentiate diffuse and focal HI.

Adolescent↗

Different mRNAs code for dopa decarboxylase in tissues of neuronal and nonneuronal origin.

A cDNA clone for dopa decarboxylase (EC 4.1.1.28) has been isolated from a rat pheochromocytoma cDNA library and the cDNA sequence has been determined. It corresponds to an mRNA of 2094 nucleotides. The length of the mRNA was measured by primer-extension of rat pheochromocytoma RNA and the 5' end of the sequence of the mRNA was confirmed by the PCR. A probe spanning the translation initiation site of the mRNA was used to hybridize with mRNAs from various organs of the rat. S1 nuclease digestion of the mRNAs annealed with this probe revealed two classes of mRNAs. The comparison of the cDNA sequence and published sequences for rat liver, human pheochromocytoma, and Drosophila dopa decarboxylase supported the conclusion that two mRNAs are produced: one is specific for tissue of neuronal origin and the other is specific for tissues of nonneuronal (mesodermal or endodermal) origin. The neuronal mRNA contains a 5' untranslated sequence that is highly conserved between human and rat pheochromocytoma including a GA stretch. The coding sequence and the 3' untranslated sequence of mRNAs from rat liver and pheochromocytoma are identical. The rat mRNA differs only in the 5' untranslated region. Thus a unique gene codes for dopa decarboxylase and this gene gives rise to at least two transcripts presumably in response to different signals during development.

Adrenal Gland Neoplasms↗

Evidence for PQQ as cofactor in 3,4-dihydroxyphenylalanine (dopa) decarboxylase of pig kidney.

Pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase (EC 4.1.1.28) was purified to homogeneity. Treatment of the enzyme with phenylhydrazine (PH) according to a procedure developed for analysis of quinoproteins gave products which were identified as the hydrazone of pyridoxal phosphate (PLP) and the C(5)-hydrazone of pyrroloquinoline quinone (PQQ). This method failed, however, in quantifying the amounts of cofactor. Direct hydrolysis of the enzyme by refluxing with hexanol and concentrated HCl led to detachment of PQQ from the protein in a quantity of 1 PQQ per enzyme molecule. In view of the reactivity of PQQ towards amines and amino acids, we postulate that it participates as a covalently bound cofactor in the catalytic cycle of the enzyme, in interplay with PLP. Since several other enzymes have been reported to show the atypical behaviour of dopa decarboxylase, it seems that the PLP-containing group of enzymes can be subdivided into pyridoxoproteins and pyridoxo-quinoproteins.

Animals↗

Reaction and substrate specificity of recombinant pig kidney Dopa decarboxylase under aerobic and anaerobic conditions.

Dopa decarboxylase (DDC) catalyzes as main reaction the stereospecific CO(2) abstraction from L-Dopa and L-5-hydroxytryptophan (5-HTP), generating the corresponding aromatic amines, dopamine and serotonin, respectively. Side reactions with turnover time of minutes are also catalyzed by the enzyme. In particular, DDC exhibits half-transaminase activity toward D-aromatic amino acids and oxidative deaminase activity toward aromatic amines. The latter reaction could represent a new activity for this class of enzymes. Studies on the effect exerted by O(2) on reaction specificity of DDC revealed that under anaerobic conditions decarboxylation of L-aromatic amino acids takes place with a k(cat) approximately half of that measured in the presence of O(2), and is accompanied by a decarboxylation-dependent transamination, whereas oxidative deamination of aromatic amines is replaced by half-transamination. Half-transamination of D-aromatic amino acids is unaffected by the presence or absence of O(2). Some structural elements relevant for the control of reaction and substrate specificity of DDC have been identified by means of limited tryptic digestion and site-directed mutagenesis studies. All together, the data indicate that the chemical nature of the substrate, the presence of O(2), the integrity of a mobile loop, the absence of perturbation in the coenzyme-binding cleft and pH are important requirements for the achievement of a closed conformational state where the highest level of reaction specificity is reached.

Amines↗

Purification of an endogenous inhibitor of L-Dopa decarboxylase activity from human serum.

An endogenous inhibitor of L-Dopa decarboxylase was identified and purified from human serum. In Triton X-114 partitioning experiments, the inhibitor was recovered in the detergent enriched phase, suggesting a hydrophobic nature. Purification was achieved by means of proteinase K digestion, ammonium sulphate precipitation, phenyl sepharose hydrophobic chromatography and subsequent extraction from a nondenaturing polyacrylamide gel. This purification scheme resulted in the isolation of a single 25 kDa band, bearing L-Dopa decarboxylase inhibitory activity. The purified molecule was found to be resistant to heat and digestion by various proteases. Proteolytic digestion of the purified inhibitor by pronase and aminopeptidase M was achieved only following carboxymethylation. The biological importance of the presence of an L-Dopa decarboxylase activity inhibitor in normal biological fluids remains to be elucidated. The better understanding of the regulation of Ddc enzymatic activity could prove valuable in the clarification of the enzyme's role in a series of pathological conditions, as well as, in physiological regulatory mechanisms.

Animals↗

Isolation and characterization of the dopa decarboxylase gene of Drosophila melanogaster.

We have isolated chromosomal deoxyribonucleic acid clones containing the Drosophila dopa decarboxylase gene. We describe an isolation procedure which can be applied to other nonabundantly expressed Drosophila genes. The dopa decarboxylase gene lies within or very near polytene chromosome band 37C1-2. The gene is interrupted by at least one intron, and the primary mode of regulation is pretranslational. At least two additional sequences hybridized by in vivo ribonucleic acid-derived probes are found within a 35-kilobase region surrounding the gene. The developmental profile of ribonucleic acid transcribed from one of these regions differs from that of the dopa decarboxylase transcript.

Animals↗

A novel form of DOPA decarboxylase produced in drosophila cells in response to 20-hydroxyecdysone.

Two cloned derivatives of the Kc cell line of Drosophila were shown to produce DOPA decarboxylase following administration of the steroid moulting hormone 20-hydroxyecdysone. In the continuous presence of the hormone at a concentration of 2 X 10(-7) M, DOPA decarboxylase activity first appeared between 48 and 72 h. Because of this lag, the tissue culture system promises to serve as a useful model for those in vivo situations where increases in the hormone titre precede increases in DOPA decarboxylase activity. In clone 7C4, after maximal enzyme activity was achieved at 144 h, the enzyme activity per cell decreased as the cells resumed division following the hormone-induced division arrest. In clone 7E10, cell division never resumed in the presence of 20-hydroxyecdysone and DOPA decarboxylase activity per cell increased continuously from the time it first appeared. When line 7E10 was exposed to a 6-h pulse of the steroid, enzyme activity appeared about 18 h earlier than in the presence of continuous hormone and, further, the cells were released from division arrest. Enzyme activity per cell then declined from an early 96-h maximum. The enzyme produced by the cell lines was immunologically distinct from the enzyme produced in vivo and ion-exchange column chromatography resolved the enzyme from cells and intact organisms into two species.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗

Dopa decarboxylase gene polymorphisms and attention deficit hyperactivity disorder (ADHD): no evidence for association in the Irish population.

Dopa decarboxylase (DDC) is an enzyme which catalyses the decarboxylation of both dopa to dopamine and L-5 hydroxytryptophan to serotonin. Both catecholamines are major neurotransmitters of the mammalian nervous system. It has been suggested that genes involved in the dopaminergic system play a primary role in predisposing to attention deficit hyperactivity disorder (ADHD). In this study, the 4-bp insertion/deletion variant mapped to the first neuronally expressed exon 1 at the dopa decarboxylase gene and two microsatellite markers flanking the gene were investigated for possible association with ADHD. Using HHRR, we observed an increased transmission (though not significant) of the 4-bp insertion (allele 1) to ADHD cases (chi(2) = 2.72, P = 0.1, RR = 1.25). However marginally significant excess transmission of allele 10 (213 bp) of the 3' microsatellite D7S2422 ( approximately 0.75 cM distal to dopa decarboxylase gene) was found (chi(2) = 4.2, P = 0.04, RR=1.48). Interestingly, a haplotype containing both alleles is transmitted more frequently (chi(2)= 5, P = 0.025). Analysing data by the sex of transmitting parent showed a greater relative risk for paternal transmission of the 4-bp insertion allele and allele 10 of the D7S2422 (RR = 1.48 and 1.63 respectively). This provides preliminary evidence that this locus or a closely mapped DNA variant may be involved in the genetic susceptibility to ADHD. However, further studies are required to either confirm or refute these observations.

Alleles↗

DdcDE1, a mutant differentially affecting both stage and tissue specific expression of dopa decarboxylase in Drosophila.

The isolation and characterization of a unique Dopa decarboxylase (Ddc) mutant in Drosophila melanogaster is reported. This mutant, DdcDE1, exhibits stage- and tissue-specific altered Ddc expression. Homozygous DdcDE1 embryos, central nervous systems (CNSs) at pupariation and newly eclosed adult epidermis all have approximately 5% as much specific dopa decarboxylase (DDC) activity as the pr control stock in which DdcDE1 was induced. In contrast, the DdcDE1 epidermis at pupariation has roughly 50% as much DDC activity as controls, a 10-fold increase over the relative activity detected in other tissues and stages. Although the adult cuticle lacks proper pigmentation as expected in flies with low DDC activity (less than or equal to 5%), the bristles unexpectedly have wild-type black pigmentation. This implies that the bristle forming cells have more DDC activity than the rest of the adult epidermis. This variegated phenotype, black bristles and pale cuticle, plus the fact that DdcDE1 was originally isolated in a reciprocal translocation between proximal X heterochromatin and the euchromatic left arm of the second chromosome, 42 bands from the Ddc locus, suggested that the mutant might be an example of position-effect variegation. All tests for position-effect variegation, including persistence of the mutant phenotype when DdcDE1 was removed from the translocation, were negative. At pupariation DDC cross-reacting material (CRM) levels are similar in DdcDE1 and wild-type controls, but in newly eclosed adults CRM levels are approximately 35% of wild-type controls. This suggests that DDC produced by DdcDE1 adults has less activity per DDC molecule than the DDC produced at pupariation by DdcDE1. If the DDC enzyme produced by DdcDE1 at adult eclosion had full DDC activity (35% DDC CRM = 35% DDC activity) then no mutant phenotype would be exhibited by DdcDE1 since flies with as little as 10% activity have a wild-type phenotype. DDC thermolability assays clearly demonstrate that DDC from DdcDE1 is more thermolabile than control DDC at both pupariation and adult eclosion. Furthermore, DDC from adults in both DdcDE1 and the pr control is more thermolabile than DDC from white prepupae. Mixing experiments indicate the difference in DDC thermolability between pr white prepupae and pr adults is not due to a difference in the white prepupal and adult supernatants. This suggests that in wild-type different isoforms of DDC are produced either by differences in post-translational modification or as a result of a different primary amino acid sequence.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

An analysis of dopa decarboxylase expression during embryogenesis in Drosophila melanogaster.

Dopa decarboxylase (DDC) activity appears near the end of embryogenesis in Drosophila. High titers of 20-OH-ecdysone on the other hand are found at midembryogenesis. Several explanations for this lag were investigated, since this hormone has been shown to induce a rapid increase in DDC activity at pupariation (G. P. Kraminsky, W. C. Clark, M. A. Estelle, R. D. Gietz, B. A. Sage, J. D. O'Connor, and R. B. Hodgetts, 1980, Proc. Natl. Acad. Sci. USA 77, 4175-4179). Using immunological and genetical criteria, it was shown that the same structural gene encodes DDC in embryos, mature larvae, and young adults. This rules out the existence of a distinct embryonic DDC gene unresponsive to 20-OH-ecdysone. Second, no evidence was found to support the hypothesis that a delay in the translation of DDC transcripts, produced in response to the elevated titer of 20-OH-ecdysone at midembryogenesis, caused the lag. Northern analysis of the RNA molecules homologous to cloned genomic sequences revealed that DDC transcripts were present at two different times during embryogenesis. A transcript was found in both ovaries and 0- to 2-hr embryos. However, this species disappeared by 4 hr and DDC transcript levels remained low until late in embryogenesis, when a significant increase occurred. This increase was presumably responsible for the appearance of the enzyme at this time. The northern blotting revealed nine DDC transcript species were present during embryogenesis and hybridization to intron-specific probes indicated that five of these contained at least part of one (or both) of the two introns. Three putative mature mRNA species were identified by their small size, relative abundance, apparent lack of intron sequence, and their presence on polysomes. The two mature species found during the late stages were postulated to differ in the length of their poly(A)+ tails. The third mature species was found only in ovaries and very young embryos and may well be of maternal origin. Data are examined in light of the possibility that this species is derived from a precursor initiated at a novel promotor.

Animals↗

The effects of central aromatic amino acid DOPA decarboxylase inhibition on the motor actions of L-DOPA and dopamine agonists in MPTP-treated primates.

1. Endogenous L-DOPA may act as a neuromodulator contributing to the production of motor activity. We now investigate the effects of the centrally acting aromatic amino acid dopa decarboxylase (AADC) inhibitor NSD-1015 (3-hydroxybenzyl hydrazine) on the motor actions of L-DOPA and dopamine agonist drugs in MPTP treated common marmosets. 2. Pretreatment with NSD-1015 (10 - 50 mg kg(-1); i.p.) worsened baseline motor deficits in MPTP-treated common marmosets. Similarly, it abolished L-DOPA (5 - 18 mg kg(-1) s.c.) induced locomotor activity and reversal of disability. NSD-1015 pretreatment inhibited dopamine formation and elevated L-DOPA levels in plasma. 3. The increase in locomotor activity and improvement in disability produced by the administration of the D-1 agonist A-86929 (0.03 - 0. 04 mg kg(-1) s.c.) or the D-2 agonist quinpirole (0.05 - 0.3 mg kg(-1) i.p.) was abolished by NSD-1015 (25 mg kg(-1) i.p.) pretreatment. While the effects of a low dose combination of A-86929 (0.04 mg kg(-1) s.c.) and quinpirole (0.05 mg kg(-1) i.p.) were inhibited by NSD-1015 (25 mg kg(-1) i.p.), there was little effect on the action of a high dose combination of these drugs (0.08 mg kg(-1) A-86929 and 0.1 mg kg(-1) quinpirole). 4. Following central AADC inhibition with NSD-1015 (25 mg kg(-1) i.p.), locomotor behaviour induced by administration of high dose combinations of A-86929 (0.08 mg kg(-1) s.c.) and quinpirole (0.1 mg kg(-1) i.p.) was unaffected by L-DOPA (5 mg kg(-1) s.c.) pretreatment. 5. These results do not support a role for endogenous L-DOPA in spontaneous or drug induced locomotor activity. Rather, they strengthen the argument for the importance of endogenous dopaminergic tone in the motor actions of dopamine agonists.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

The central aromatic amino acid DOPA decarboxylase inhibitor, NSD-1015, does not inhibit L-DOPA-induced circling in unilateral 6-OHDA-lesioned-rats.

The centrally acting aromatic amino acid dopa decarboxylase (AADC) inhibitor, 3-hydroxybenzyl hydrazine (NSD-1015), is widely used to study the neurotransmitter-like actions of L-DOPA. However, the effects of NSD-1015 on L-DOPA-induced motor activity are unclear as both increases and decreases have been reported. We now investigate the effects of NSD-1015 on L-DOPA-induced contralateral circling behaviour in 6-OHDA-lesioned rats and on striatal levels of L-DOPA, 3-O-methyl-DOPA (3-OMD), dopamine, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) using microdialysis techniques. NSD-1015 (50-200 mg/kg i.p.) inhibited AADC activity both in the liver and striatum of normal rats. Administration of NSD-1015 (50-200 mg/kg i.p.), delayed the onset of circling produced by administration of L-DOPA (25 mg/kg i.p.) and carbidopa (12.5 mg/kg i. p.), suggesting blockade of central AADC activity. However, the duration of the L-DOPA-induced circling was prolonged and overall no inhibition of circling behaviour occurred. L-DOPA (25 mg/kg i.p.) plus carbidopa (12.5 mg/kg i.p.) increased extracellular levels of L-DOPA, 3-OMD, dopamine, DOPAC and HVA in the 6-OHDA-lesioned striatum. Pretreatment of rats with the central AADC inhibitor, NSD-1015 (100 mg/kg i.p.), potentiated the increase in dialysate levels of L-DOPA and 3-OMD. However, it did not reduce striatal dopamine levels in the 6-OHDA-lesioned hemisphere, which were elevated following L-DOPA administration. The increases in DOPAC and HVA levels were abolished by NSD-1015 pretreatment. These results suggest that, while NSD-1015 blocks central AADC activity, it also acts as a monoamine oxidase inhibitor so maintaining striatal dopamine concentration by reducing dopamine metabolism. NSD-1015, therefore, may not be an appropriate tool for the study of brain AADC activity and for assessing the neuromodulatory role of L-DOPA.

3,4-Dihydroxyphenylacetic Acid↗

Haplotype analysis indicates an association between the DOPA decarboxylase (DDC) gene and nicotine dependence.

DOPA decarboxylase (DDC; also known as L-amino acid decarboxylase; AADC) is involved in the synthesis of dopamine, norepinephrine and serotonin. Because the mesolimbic dopaminergic system is implicated in the reinforcing effects of many drugs, including nicotine, the DDC gene is considered a plausible candidate for involvement in the development of vulnerability to nicotine dependence (ND). Further, this gene is located within the 7p11 region that showed a 'suggestive linkage' to ND in our previous genome-wide scan in the Framingham Heart Study population. In the present study, we tested eight single nucleotide polymorphisms (SNPs) within DDC for association with ND, which was assessed by smoking quantity (SQ), the heaviness of smoking index (HSI) and the Fagerstrom test for ND (FTND) score, in a total of 2037 smokers and non-smokers from 602 nuclear families of African- or European-American (AA or EA, respectively) ancestry. Association analysis for individual SNPs using the PBAT-GEE program indicated that SNP rs921451 was significantly associated with two of the three adjusted ND measures in the EA sample (P=0.01-0.04). Haplotype-based association analysis revealed a protective T-G-T-G haplotype for rs921451-rs3735273-rs1451371-rs2060762 in the AA sample, which was significantly associated with all three adjusted ND measures after correction for multiple testing (min Z=-2.78, P=0.006 for HSI). In contrast, we found a high-risk T-G-T-G haplotype for a different SNP combination in the EA sample, rs921451-rs3735273-rs1451371-rs3757472, which showed a significant association after Bonferroni correction with the SQ and FTND score (max Z=2.73, P=0.005 for FTND). In summary, our findings provide the first evidence for the involvement of DDC in the susceptibility to ND and, further, reveal the racial specificity of its impact.

Adult↗

Levels of histaminase and L-DOPA decarboxylase activity in the transition from C-cell hyperplasia to familial medullary thyroid carcinoma.

The distribution of calcitonin, L-dopa decarboxylase, and histaminase is studied in sectioned total thyroid glands from patients with different stages of familial medullary thyroid carcinoma. In 5 glands with gross carcinoma and in 3 with early microscopic carcinoma the distribution of all three parameters positively correlates (p less than .01 for each correlation). In contrast, in 6 glands with C-cell hyperplasia only the distribution of calcitonin and L-dopa decarboxylase correlates (r = 0.64, p less than .01) while those for histaminase vs. calcitonin (r = .17, p = N.S.) and histaminase vs. dopa decarboxylase (r = .03, p = N.S.) do not. In the glands with microscopic carcinoma the peak levels of histaminase occur in the areas of disease as defined by immunohistochemical staining of calcitonin; mean histaminase activity is the only one of the three parameters measured that distinguishes between C-cell hyperplasia and microscopic carcinoma (p less than .005). Immunohistochemical staining of histaminase shows positive cells in glands with gross and microscopic carcinoma, but in none of the glands with hyperplasia alone. Histaminase is thus found in high amounts in some malignant C-cells only and may be a useful marker to distinguish between hyperplasia and malignancy in thyroids with early C-cell proliferative disorders.

Amine Oxidase (Copper-Containing)↗

Mechanism-based inactivation of dopa decarboxylase by serotonin.

Pig kidney dopa decarboxylase (DDC) expressed in Escherichia coli is a homodimeric enzyme containing one catalytically active pyridoxal 5'-phosphate active site per subunit. In addition to catalyzing the decarboxylation of -aromatic amino acids, DDC also reacts with 5-hydroxytryptamine (5-HT), converting it to 5-hydroxyindolacetaldehyde and ammonia. These products have been identified by means of the enzymes alcohol dehydrogenase and glutamate dehydrogenase, together with high performance liquid chromatographic and mass spectroscopic analysis. The Kcat and Km values of this reaction were determined to be 0.48 min-1 and 0.47 mM, respectively. The NaBH4-reduced enzyme does not catalyze this reaction. Concurrent with this reaction, 5-HT inactivates DDC in both a time- and concentration-dependent manner and exhibits saturation of the rate of inactivation at high concentrations, with Ki and Kinact values of 0.40 mM and 0.023 min-1, respectively. Protection from inactivation by 5-HT was observed in the presence of the active site-directed inhibitor 3,4-dihydroxy-D-phenylalanine. Inactivation with [2-14C]5-HT results in the incorporation of 1 mol of label/enzyme subunit. Taken together, these findings indicate that 5-HT is both a substrate and a mechanism-based inactivator with a partition ratio for product formation versus inactivation of 21. The absorbance, CD, and fluorometric features of 5-HT-inactivated DDC have also been characterized. A speculative mechanism for the reaction and inactivation consistent with the experimental findings is presented.

Aldehydes↗