Immunocytochemical study of tyrosine hydroxylase and dopamine β-hydroxylase immunoreactivities in the rat pancreas

An immunohistochemical and immunoelectron microscopic study was used to demonstrate tyrosine hydroxylase (TH) and dopamine -hydroxylase (DBH) immunoreactivities in the rat pancreas. Small TH immunoreactive cells were found in close contact with large TH immunonegative ganglion cells among the exocrine glands and were occasionally found in some islets. Some of these TH immunoreactive cells were also DBH immunopositive. The immunoreaction product was seen diffusely in the cytoplasm and in the granule cores of TH immunoreactive cells. All intra-pancreatic ganglion cells were immunoreactive for DBH, but not for TH. The TH immunoreactive cells were identified as small intensely fluorescent (SIF) cells due to their localization and morphological characteristics and showed no insulin, glucagon, somatostatin or pancreatic polypeptide immunoreactivities. These results indicate that SIF cells may release dopamine or noradrenaline to adequate stimuli while the intra-pancreatic ganglion cells with only DBH may not synthesize catecholamines in a normal biosynthetic pathway. TH immunoreactive nerve bundles without varicosities and fibers with varicosities, associated or unassociated with blood vessels, were found in both the exocrine and endocrine pancreas. Close apposition of TH immunoreactive nerve fibers to the smooth muscle and endothelial cells of the blood vessels was observed. A close apposition between TH immunoreactive nerve fibers and exocrine acinar cells and islet endocrine cells was sometimes found in the pancreas. The immunoreaction product was seen diffusely in the axoplasm and in the granular vesicles of the immunoreactive nerve fibers. Since no TH immunoreactive ganglion cells were present in the rat pancreas, the present study suggests that noradrenergic nerve fibers in the pancreas may be extrinsic in origin, and may exert an effect on the regulation of blood flow and on the secretory acitivity of the acinar cells, duct cells and endocrine cells.     

Polymorphism in the 3′ untranslated region of the phenylalanine hydroxylase gene detected by enzyme mismatch cleavage: evolution of haplotypes

A polymorphism was identified in 3 untranslated region of the phenylalanine hydroxylase gene using the newly described mutation detection method, enzyme mismatch cleavage. This polymorphism, 1546 G A, was linked to three mutations on several haplotype backgrounds. A group of haplotypes was identified as evolving from the one ancestral haplotype on which this base substitution occurred. The possible Celtic or Viking origin of this polymorphism is discussed.     

Role of the HIF-1α/Nur77 axis in the regulation of the tyrosine hydroxylase expression by insulin in PC12 cells

Tyrosine hydroxylase (TH), catalyzing the conversion of tyrosine into l -DOPA, is the rate-limiting enzyme in dopamine synthesis. Defects in insulin action contribute to alterations of TH expression and/or activity in the brain and insulin increases TH levels in 1-methyl-4-phenylpyridinium (MPP+)-treated neuronal cells. However, the molecular mechanisms underlying the regulation of TH by insulin have not been elucidated yet. Using PC12 cells, we show for the first time that insulin increases TH expression in a biphasic manner, with a transient peak at 2 hr and a delayed response at 16 hr, which persists for up to 24 hr. The use of a dominant negative hypoxia-inducible factor 1-alpha (HIF-1α) and its pharmacological inhibitor chetomin, together with chromatin immunoprecipitation (ChIP) experiments for the specific binding to TH promoter, demonstrate the direct role of HIF-1α in the early phase. Moreover, ChIP experiments and transfection of a dominant negative of the nerve growth factor IB (Nur77) indicate the involvement of Nur77 in the late phase insulin response, which is mediated by HIF-1α. In conclusion, the present study shows that insulin regulates TH expression through HIF-1α and Nur77 in PC12 cells, supporting the critical role of insulin signaling in maintaining an appropriate dopaminergic tone by regulating TH expression in the central nervous system.     

Further studies on the substrate spectrum of phytanoyl-CoA hydroxylase: implications for Refsum disease?

Refsum disease is a peroxisomal disorder characterized by adult-onset retinitis pigmentosa, anosmia, sensory neuropathy, ataxia, and an accumulation of phytanic acid in plasma and tissues. Approximately 45% of cases are caused by mutations in phytanoyl-CoA hydroxylase (PAHX), the enzyme catalyzing the second step in the peroxisomal alpha-oxidation of 3-methyl-branched fatty acids. To study the substrate specificity of human PAHX, different 3-alkyl-branched substrates were synthesized and incubated with a recombinant polyhistidine-tagged protein. The enzyme showed activity not only toward racemic phytanoyl-CoA and the isomers of 3-methylhexadecanoyl-CoA, but also toward a variety of other mono-branched 3-methylacyl-CoA esters with a chain length down to seven carbon atoms. Furthermore, PAHX hydroxylated a 3-ethylacyl-CoA quite well, whereas a 3-propylacyl-CoA was a poor substrate. Hydroxylation of neither 2- or 4-methyl-branched acyl-CoA esters, nor long or very long straight-chain acyl-CoA esters could be detected. The results presented in this paper show that the substrate specificity of PAHX, with regard to the length of both the acyl-chain and the branch at position 3, is broader than expected. Hence, Refsum disease might be characterized by an accumulation of not only phytanic acid but also other 3-alkyl-branched fatty acids.     

Reconstruction of the astaxanthin biosynthesis pathway in rice endosperm reveals a metabolic bottleneck at the level of endogenous β-carotene hydroxylase activity

Astaxanthin is a high-value ketocarotenoid rarely found in plants. It is derived from β-carotene by the 3-hydroxylation and 4-ketolation of both ionone end groups, in reactions catalyzed by β-carotene hydroxylase and β-carotene ketolase, respectively. We investigated the feasibility of introducing an extended carotenoid biosynthesis pathway into rice endosperm to achieve the production of astaxanthin. This allowed us to identify potential metabolic bottlenecks that have thus far prevented the accumulation of this valuable compound in storage tissues such as cereal grains. Rice endosperm does not usually accumulate carotenoids because phytoene synthase, the enzyme responsible for the first committed step in the pathway, is not present in this tissue. We therefore expressed maize phytoene synthase 1 (ZmPSY1), Pantoea ananatis phytoene desaturase (PaCRTI) and a synthetic Chlamydomonas reinhardtii β-carotene ketolase (sCrBKT) in transgenic rice plants under the control of endosperm-specific promoters. The resulting grains predominantly accumulated the diketocarotenoids canthaxanthin, adonirubin and astaxanthin as well as low levels of monoketocarotenoids. The predominance of canthaxanthin and adonirubin indicated the presence of a hydroxylation bottleneck in the ketocarotenoid pathway. This final rate-limiting step must therefore be overcome to maximize the accumulation of astaxanthin, the end product of the pathway.     

Electron microscopic analysis of tyrosine hydroxylase,dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase immunoreactive innervation of the hypothalamic paraventricular nucleus in the rat

Summary The catecholaminergic innervation of the hypothalamic paraventricular nucleus (PVN) of the rat was studred by preembedding immunocytochemical methods utilizing specific antibodies which were generated against catecholamine synthesizing enzymes. Phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive terminals contained 80–120 nm dense core granules and 30–50 nm clear synaptic vesicles. The labeled boutons terminated on cell bodies and dendrites of both parvo- and magnocellular neurons of PVN via asymmetric synapses. The parvocellular subnuclei received a more intense adrenergic innervation than did the magnocellular regions of the nucleus. Dopamine--hydroxylase (DBH)-immunopositive axons were most numerous in the periventricular zone and the medial paryocellular subnucleus of PVN. Labeled terminal boutens contained 70–100 nm dense granules and clusters of spherical, electron lucent vesicles. Dendrites, perikarya and spinous structures of paraventricular neurons were observed to be the postsynaptic targets of DBH axon terminals. These asymmetric synapses frequently exhibited subsynaptic dense bodies. Paraventricular neurons did not demonstrate either PNMT or DBH immunoreactivity. The fibers present within the nucleus which contained these enzymes are considered to represent extrinsic afferent connections to neurons of the PVN.Tyrosine hydroxylase (TH)-immunoreactivity was found both in neurons and neuronal processes within the PVN In TH-cells, the immunolabel was associated with rough endoplasmic reticulum, free ribosomes and 70–120 nm dense granules. Occasionally, nematosome-like bodies and cilia were observed in the TH-perikarya. Unlabeled axons established en passant and bouton terminaux type synapses with these TH-immunopositive cells. TH-immunoreactive axons terminated on cell bodies as well as somatic and dendritic spines of paraventricular parvocellular neurons. TH-containing axons were observed to deeply invaginate into both dendrites and perikarya of magnocellular neurons.These observations provide ultrastructural evidence for the participation of central catecholaminergic neuronal systems in the regulation of the different neuronal and neuroendocrine functions which have been related to hypothalamic paraventricular neurons.Supported by NIH Grant NS 19266 to W.K. Paull     

Ejection of structural zinc leads to inhibition of γ-butyrobetaine hydroxylase

γ-Butyrobetaine hydroxylase (BBOX) is a 2-oxoglutarate and Fe(II) dependent oxygenase that catalyses an essential step during carnitine biosynthesis in animals. BBOX is inhibited by ejection of structural zinc by a set of selenium containing analogues. Previous structural analyses indicated that an undisrupted N-terminal zinc binding domain of BBOX is required for catalysis. Ebselen is a relatively potent BBOX inhibitor, an observation which may in part reflect its cardioprotective properties.     

Appearance of tyrosine hydroxylase,aromatic amino-acid decarboxylase,dopamine β-hydroxylase and phenylethanolamine N-methyltransferase during the ontogenesis of the adrenal medulla

Summary The cellular localization of the enzymes tyrosine hydroxylase (TH), aromatic amino-acid decarboxylase (or dopa decarboxylase, DDC), dopamine -hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in the adrenal medulla of adult rats and rat fetuses (14th, 17th, 18th, 19th and 21st day) was examined. In the prenatal stages the medullary blastema and an adjacent part of the primitive sympathetic trunk were also investigated. Tissues were fixed in ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). Cryostat sections (10 m in thickness) were stained by the indirect immunofluorescence technique. Rabbit antibodies to TH (isolated from human pheochromocytoma), DDC, DBH and PNMT (the latter three isolated from bovine adrenal medulla) were used. Sections incubated with serum of non-immunized rabbits were used as controls.In the adult adrenal medulla, two cell types can be distinguished. One cell type contains only TH, DDC and DBH. The other cell type contains PNMT in addition. It is concluded that these cells correspond to the noradrenaline-(NA-) and adrenaline-(A-)storing cells respectively. In all prenatal stages TH, DDC and DBH are found in the primitive sympathetic trunk, in the medullary blastema, and in the medullary cells which have migrated into the cortical anlage. PNMT is observed for the first time on the 18th day. Moreover, PNMT could only be demonstrated inside the adrenal gland. From these observations it is concluded that the capacity to synthesize NA is developed even before the medullary cells have reached the cortical anlage. On the contrary, the capacity to synthesize A seems to be acquired only after this contact is established. The hypothesis is put forward that this phenomenon might indicate the induction of PNMT by glucocorticoids secreted by the fetal cortex.This study was supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.) and by the Swedish Medical Research Council (04X-2887-10C). Its results have in part been reported at the 105th Meeting of the Dutch Anatomical Society (Abstract: Acta morphologica neerlando-scandinavica, 14, 251, 1976)     

Purification and properties of calf liver γ-butyrobetaine hydroxylase

Calf liver γ-butyrobetaine hydroxylase has been purified some 400-fold by DEAE, gel permeation, and hydroxylapatite chromatography. The homogeneous enzyme is a dimer of 46,000-dalton subunits. The K_m values for substrates and cofactors and the apparent activation constants for ascorbate and catalase have been determined. Inhibition of the enzyme by a number of divalent metals supports the function of sulfhydryl groups in metal binding. An antibody to the enzyme has been obtained; this does not cross-react with homogeneous γ-butyrobetaine hydroxylase from a Pseudomonas strain. The antibody, coupled to Sepharose 4B, has been used to purify the calf liver hydroxylase 350-fold in one step.     

Protein kinase C-dependent dephosphorylation of tyrosine hydroxylase requires the B56δ heterotrimeric form of protein phosphatase 2A

Tyrosine hydroxylase, which plays a critical role in regulation of dopamine synthesis, is known to be controlled by phosphorylation at several critical sites. One of these sites, Ser40, is phosphorylated by a number of protein kinases, including protein kinase A. The major protein phosphatase that dephosphorylates Ser40 is protein phosphatase-2A (PP2A). A recent study has also linked protein kinase C to the dephosphorylation of Ser40 [1], but the mechanism is unclear. PP2A isoforms are comprised of catalytic, scaffold, and regulatory subunits, the regulatory B subunits being able to influence cellular localization and substrate selection. In the current study, we find that protein kinase C is able to phosphorylate a key regulatory site in the B56δ subunit leading to activation of PP2A. In turn, activation of the B56δ-containing heterotrimeric form of PP2A is responsible for enhanced dephosphorylation of Ser40 of tyrosine hydroylase in response to stimulation of PKC. In support of this mechanism, down-regulation of B56δ expression in N27 cells using RNAi was found to increase dopamine synthesis. Together these studies reveal molecular details of how protein kinase C is linked to reduced tyrosine hydroxylase activity via control of PP2A, and also add to the complexity of protein kinase/protein phosphatase interactions.     

Immunofluorescent and biochemical studies on tyrosine hydroxylase and dopamine-β-hydroxylase of the bullfrog sciatic nerves

Summary Immunofluorescence specific for tyrosine hydroxylase (TH) or dopamine--hydroxylase (DBH) was accentuated in both proximal and distal segments of the sciatic nerve after ligation. Estimations of the enzyme activities confirmed the above results. Mean axoplasmic flow rates of TH and DBH in bullfrog sciatic nerve were found to be 8 and 123 mm/day, respectively. They were decreased by colchicine or by cold temperatures (4° C).     

γ-Butyrobetaine hydroxylase: A unique protective effect of catalase

Catalase stimulates the activity of homogeneous γ-butyrobetaine hydroxylase by approximately 300-fold. The stimulation of the hydroxylation reaction elicited by catalase is saturable, and although a number of proteins may be substituted for catalase, none is as effective. γ-Butyrobetaine hydroxylase is also irreversibly inactivated in the presence of one of its substrates, oxygen, and its cofactor, ascorbate. This inactivation of the hydroxylase activity may be prevented by (i) the presence of high concentrations (2 mg/ml) of various proteins, (ii) the presence of catalytic concentrations (20 μg/ml) of catalase, or (iii) the presence of 10 mm histidine or dithiothreitol. Oxidized species of ascorbate do not appear to be responsible for the inactivation process. Time-dependent inactivation is also observed when γ-butyrobetaine hydroxylase is preincubated with hydrogen peroxide generated by the glucose oxidase-catalyzed oxidation of glucose. At low concentrations, superoxide dismutase was not as effective as an equivalent protein concentration of catalase in protecting against inactivation, and hydroxyl radical scavengers were completely ineffective. In measurements of γ-butyrobetaine hydroxylase activity, the presence of catalase both stimulates the catalytic activity of the hydroxylase and protects the enzyme from inactivation by a product of the interaction of components in the assay mixture, presumably hydrogen peroxide.     

The distribution of neurones immunoreactive for β-tyrosine hydroxylase,dopamine and serotonin in the ventral nerve cord of the cricket,Gryllus bimaculatus

The cellular localization of the biogenic amines dopamine and serotonin was investigated in the ventral nerve cord of the cricket, Gryllus bimaculatus, using antisera raised against dopamine, -tyrosine hydroxylase and serotonin. Dopamine-(n<-70) and serotonin-immunoreactive (n<-120) neurones showed a segmental arrangement in the ventral nerve cord. Some neuromeres, however, did not contain dopamine-immunoreactive cell bodies. The small number of stained cells allowed complete identification of brain and thoracic cells, including intersegmentally projecting axons and terminal arborizations. Dopamine-like immunostaining was found primarily in plurisegmental interneurones with axons descending to the soma-ipsilateral hemispheres of the thoracic and abdominal ganglia. In contrast, serotonin-immunostaining occurred predominantly in interneurones projecting via soma-contralaterally ascending axons to the thorax and brain. In addition, serotonin-immunoreactivity was also present in efferent cells and afferent elements. Serotonin-immunoreactive, but no dopamine-immunoreactive, varicose fibres were observed on the surface of some peripheral nerves. Varicose endings of both dopamine-and serotonin-immunoreactive neurones occurred in each neuromere and showed overlapping neuropilar projections in dorsal and medial regions of the thoracic ganglia. Ventral associative neuropiles lacked dopamine-like immunostaining but were innervated by serotonin-immunoreactive elements. A colocalization of the two amines was not observed. The topographic representation of neurone types immunoreactive for serotonin and dopamine is discussed with respect to possible modulatory functions of these biogenic amines in the central nervous system of the cricket.     

Induction of microsomal aryl hydrocarbon (3,4-benzo(α)pyrene) hydroxylase and cytochrome P-450k in rat kidney cortex: I. Characteristics of the hydroxylase system

Both the rat kidney cortex aryl hydrocarbon hydroxylase activity and cytochrome P-450_K are induced by benzo(α)pyrene treatment. Following a single injection of benzo(α)pyrene, maximal hydroxylase activity and cytochrome P-450_K content occur at 24 hr, returning to control levels within 72–96 hr. Induction of both the enzyme activity and hemoprotein is inhibited by cycloheximide. The enzyme system is localized in the microsomal fraction, has an absolute requirement for NADPH and molecular oxygen, and a pH optimum at 7.4; the induced activity is linear with microsomal protein concentration up to 0.8 mg and with time up to 20 min. Both the hydroxylase activity and cytochrome P-450_K follow the same pattern of inactivation with increasing temperature. The apparent K_m for the induced hydroxylase was 7.7 μm and V was increased fourfold above control value. In the presence of laurate, a substrate for the kidney microsomal cytochrome P-450_K-dependent monooxygenase system, the amount of inhibition of hydroxylase activity corresponded to the level of activity present in untreated kidney cortex microsomes. α-Naphthoflavone (10^?5m), a type I inducer (36) produced a greater inhibitory effect on the induced hydroxylase activity than on the control (55% vs 20%). The presence of cytochrome c or carbon monoxide markedly decreased hydroxylase activity. This evidence in addition to aforementioned characteristics of the enzyme suggests a cytochrome P-450_K-dependent aryl hydroxylase activity which differs from that present in the control rat.     

Immunocytochemical localization of tyrosine hydroxylase,dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase in the adrenal glands of the frog and rat by a peroxidase-antiperoxidase method

Summary The subcellular locilazations of tryrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) in the adrenal glands of the frog and rat have been examined by a peroxidase-antiperoxidase (PAP) method. TH was localized in the ground substance of the adrenaline-containing cells and noradrenaline-containing cells, but not in the nucleus or in the mitochondria. TH was also located on the outside of the membrane of the chromaffin granules. DBH was observed only inside the granules. PNMT was found not only in the ground substance but also on the membrane of some adrenaline-containing granules. Cortical lipid cells of the frog adrenals did not show TH-, DBH-, and PNMT-reactions. The negative reactions to TH-, DBH-, and PNMT-antiserum exhibited by the summer cells of the frog adrenals prove that they belong to the cortical cells.     

Genetic expression of aryl hydrocarbon hydroxylase activity in the mouse: Distinction between the “responsive” homozygote and heterozygote at the Ah locus

β-Napththoflavone administration induces certain monooxygenase activities, such as aryl hydrocarbon (benzo[a]pyrene) hydroxylase, and cytochrome P₁-450 formation in the “responsive” C57BL/6 and C3H/He inbred mouse strains, whereas these changes are absent or relatively small in the so-called nonresponsive $\text{DBA}\text{2}$ inbred strain. Dose-response curves—with the use of large numbers of animals of the same age and sex and with either β-naphthoflavone or the much more potent 2,3,7,8-tetrachlorodibenzo-p-dioxin as inducer—reveal a small, but statistically significant, difference in the hydroxylase induction between the $\text{C}\text{57}\text{BL}\text{6}\text{J}$ homozygote and the ($\text{C}\text{57}\text{BL}\text{6}\text{J}$)($\text{DBA}\text{2}\text{J}$)F₁ heterozygote in liver, kidney, bowel, and lung. The (C3H/HeJ)($\text{DBA}\text{2}\text{J}$)F₁ heterozygote displays additive inheritance in each of these same tissues.     

Up-regulation of IL-1 receptor type I and tyrosine hydroxylase in the rat carotid body following intraperitoneal injection of IL-1β

It is well established that reciprocal modulation exists between the central nervous system and immune system. Interleukin (IL)-1β, a proinflammatory cytokine secreted at early stage of immune challenge, has been recognized as one of the informational molecules in immune-to-brain communication. However, how this large molecule is transmitted to the brain is still unknown. In recent years it has been reported that the cranial nerves, especially the vagus, may play a pivotal role in this regard. It is proposed that IL-1β may bind to its corresponding receptors located in the glomus cells of the vagal paraganglia and then elicit action potentials in the nerve. The existence of IL-1 receptor type I (IL-1RI) in the vagal paraganglia has been shown. The carotid body, which is the largest peripheral chemoreceptive organ, is also a paraganglion. We hypothesize that the carotid body might play a role similar to the vagal paraganglia because they are architectonically similar. Recently we verified the presence of IL-1RI in the rat carotid body and observed increase firing in the carotid sinus nerve following IL-1β stimulation. The aim of this study was to observe the changes in expression of IL-1RI and tyrosine hydroxylase (TH), a rate-limiting enzyme for catecholamine synthesis, in the glomus cells of the rat carotid body following intraperitoneal injection of IL-1β. The radioimmunoassay result showed that the blood IL-1β level was increased after the intraperitoneal injection of rmIL-1β (750 ng/kg) from 0.48 ± 0.08 to 0.78 ± 0.07 ng/ml (P < 0.05). Immunofluorescence and Western blot analysis showed that the expression of IL-1RI and TH in the rat carotid body was increased significantly following peritoneal IL-1β stimulation. In addition, double immunofluorescence labeling for TH and PGP9.5, a marker for glomus cells, or TH immunofluoresence with hematoxylin-eosin (HE) counterstaining revealed that a considerable number of glomus cells did not display TH immunoreactivity. These data provide morphological evidence for the response of the carotid body to proinflammatory cytokine stimulation. The results also indicate that not all of the glomus cells express detectable TH levels either in normal or in some abnormal conditions. Xi-Jing Zhang and Xi Wang are co-first authors.     

Phenylalanine hydroxylase deficiency in the Slovak population: Genotype–phenotype correlations and genotype-based predictions of BH4-responsiveness

We investigated the mutation spectrum of the phenylalanine hydroxylase gene (PAH) in a cohort of patients from 135 Slovak PKU families. Mutational screening of the known coding region, including conventional intron splice sites, was performed using high-resolution melting analysis, with subsequent sequencing analysis of the samples showing deviated melting profiles compared to control samples. The PAH gene was also screened for deletions and duplications using MLPA analysis. Forty-eight different disease causing mutations were identified in our patient group, including 30 missense, 8 splicing, 7 nonsense, 2 large deletions and 1 small deletion with frameshift; giving a detection rate of 97.6%. The most prevalent mutation was the p.R408W, occurring in 47% of all alleles, which concurs with results from neighboring and other Slavic countries. Other frequent mutations were: p.R158Q (5.3%), IVS12 + 1G>A (5.3%), p.R252W (5.1%), p.R261Q (3.9%) and p.A403V (3.6%). We also identified three novel missense mutations: p.F233I, p.R270I, p.F331S and one novel variant: c.− 30A>T in the proximal part of the PAH gene promoter. A spectrum of 84 different genotypes was observed and a genotype based predictions of BH4-responsiveness were assessed. Among all genotypes, 36 were predicted to be BH4-responsive represented by 51 PKU families. In addition, genotype–phenotype correlations were performed.     

Agaricus bisporus tyrosinase—II. Characterization of hydroxylase and dehydrogenase activities

• 1.1. Preparative Isoelectric focusing (PIEF) was used to isolate hydroxylasic and dehydrogenasic activities, at different pI.
• 2.2. The fraction at pI 4.7 and 4.9 displays a pure dehydrogenase activity (substrate l-DOPA).
• 3.3. This fraction did not react with tyrosine, either in the spot-test or in absorption spectra (200–620 nm), and did not exhibit any oxygen consumption.
• 4.4. The fraction at pI 4.1 and 4.3 reacted with both l-DOPA and tyrosine as substrate, showing dehydrogenase and hydroxylase activity.
• 5.5. The latter activity was confirmed by the oxygen consumption test, showing that molecular oxygen is used to ortho-hydroxylate tyrosine.