Identification and optimization of tyrosine hydroxylase activity in Mucuna pruriens DC. var. utilis

Tyrosine hydroxylase, an iron containing tetrahydrobiopterin dependent monooxygenase (tyrosine 3-monooxygenase; EC 1.14.16.2), catalyzes the rate-limiting step in which l-dopa is formed from the substrate l-tyrosine. l-Dopa concentration and activity of l-tyrosine hydroxylase enzyme were measured in roots, stem, leaves, pods, and immature seeds of Mucuna pruriens. Immature seeds contained maximum l-dopa content and mature leaves possessed maximum catalytic activity of tyrosine hydroxylase. Tyrosine hydroxylase from leaf homogenate was characterized as a 55 kDa protein by SDS-PAGE and Western-blot analysis with monoclonal mouse IgG2a tyrosine hydroxylase antibody. The conditions for maximum tyrosine hydroxylase activity from the leaf extract were optimized with respect to temperature, pH, cofactor 6-MPH₄, and divalent metal ions. The tyrosine hydroxylase from leaf extract possessed a K _m value of 808.63 μM for l-tyrosine at 37°C and pH 6.0. The activity of the enzyme was slightly inhibited at 2,000 μM l-tyrosine. Higher concentrations of the cofactor 6-MPH₄, however, completely inhibited the synthesis of l-dopa. Tyrosine hydroxylase converted specific monophenols such as l-tyrosine (808.63 μM) and tyramine (K _m 1.1 mM) to diphenols l-dopa and dopamine, respectively. Fe(II) activated the enzyme while higher concentration of other divalent metals reduced its activity. For the first time, tyrosine hydroxylase from M. pruriens is being reported in this study.     

Tolerance to acid soil conditions of the velvet beans Mucuna pruriens var. utilis and M. deeringiana

Velvet beans, fast growing leguminous cover crops used in the humid tropics, are shallow rooted on acid soils. This might be due to an inherent branching pattern, to an intrinsic toxicity of the acid subsoil or to a relative preference for root development in the topsoil. Such preference could be based on soil chemical factors in the subsoil or on physical factors such as penetration resistance or aeration. In a field experiment with two species of velvet bean (Mucuna pruriens var. utilis and M. deeringiana) all topsoil was removed and plants were sown directly into the acid subsoil. Root development was neither affected by this treatment nor by P fertilization or liming. In the absence of topsoil good root development in the exposed upper layer of subsoil was possible, so the hypothesis of a toxicity per se of the subsoil could be rejected. To test whether poor root development in the subsoil in the presence of topsoil is due to an inherent branching pattern of the plant or to a relative preference for topsoil, a modified in-growth core technique was used. Local topsoil and subsoil and an acid soil with a higher exchangeable Al content were placed in mesh bags at different depths and at different bulk densities, with and without lime and/or P fertilizer. A comparison of root development in mesh bags placed in the topsoil or subsoil showed that position and thus inherent branching pattern is not important. Root development in the subsoil was poor when this soil was placed in a mesh bag in the topsoil, but in an acid soil of much higher exchangeable Al content and higher percentage Al saturation more roots developed. In a second experiment in mesh bags, bulk density of the repacked soil in the range 1.0–1.5 g cm^-3 had no significant effect on root development. P fertilization and a high rate of liming of the soil placed in the mesh bag had a positive effect on root length density. It is concluded that poor root development in the acid subsoil under field conditions is due to a relative preference for topsoil. Al saturation and bulk density of the soil are not directly involved in this preference, but differences in availability of P and Mg or in Ca/Al ratios might play a role.     

Tolerance to acid soil conditions of the velvet beans Mucuna pruriens var. utilis and M. deeringiana

Fast growing, climbing leguminous cover crops such as the velvet beans can be used to reclaim weed-infested, degraded soils in the humid tropics, especially land covered by the grass Imperata cylindrica; they climb over the grass leaves and shade the grass out if their cover lasts long enough. Tolerance of two species of velvet bean to eroded soils was investigated by removing topsoil and directly sowing into the subsoil; plots where topsoil was not removed were used as a control. The response to small amounts of P fertilizer and lime was also tested. Removal of the topsoil resulted in retarded growth of both species, in increased dry matter content of the shoot, in decreased specific leaf area and in increased leaf weight ratio, due to shorter internodes. Six weeks after planting the leaf area index (LAI) was about 1.2 where topsoil was retained, sufficient for a shading effect on Imperata. Where topsoil had been removed, the LAI was only 0.6. Mucuna pruriens var. utilis showed a faster aboveground growth than M. deeringiana; the species did not differ in tolerance to eroded soil. Small amounts of P fertilizer had no significant effect on the growth of both Mucuna species. Shoot: root ratios, on a dry weight basis, were much lower when topsoil had been removed, about 3.7 and 2.4 for M. p. utilis and M. deeringiana respectively, compared to 6.2 and 3.3 where topsoil was retained. Removal of topsoil led to reduced Mg and to increased Al concentrations in roots, and to increased levels of Mn and Al in shoots. In the second year no effect of lime or residual effect of P application was found on growth of Mucuna or Imperata. Removal of the topsoil had little effect on the growth of weeds after the cover crop had been harvested. Due to the high Al tolerance of Imperata, reclamation by Mucuna will be less effective if the topsoil has been lost by erosion.     

Limited uptake, translocation and enhanced metabolic degradation contribute to glyphosate tolerance in Mucuna pruriens var. utilis plants

Velvet bean (Mucuna pruriens, Fabaceae) plants exhibits an innate, very high resistance (i.e., tolerance) to glyphosate similar to that of plants which have acquired resistance to this herbicide as a trait. We analyzed the uptake of [(14)C]-glyphosate by leaves and its translocation to meristematic tissues, and used scanning electron micrographs to further analyze the cuticle and 3D capillary electrophoresis to investigate a putative metabolism capable of degrading the herbicide. Velvet bean exhibited limited uptake of glyphosate and impaired translocation of the compound to meristematic tissues. Also, for the first time in a higher plant, two concurrent pathways capable of degrading glyphosate to AMPA, Pi, glyoxylate, sarcosine and formaldehyde as end products were identified. Based on the results, the innate tolerance of velvet bean to glyphosate is possibly a result of the combined action of the previous three traits, namely: limited uptake, impaired translocation and enhanced degradation.     

Study on the Chemical Constituens of Mucuna pruriens var. utilis (With Black seed Coat)

Five compounds were first isolated from the legumes of Mucuna pruriens (L.) DC. var. utilis (Wall. ex Wight) Baker ex Burck (M. atrocarpum Metcalf) with black seed coat. They were identified as L-dopa, stizolamine, D-erythro-neopterin, 6-hydroxymethylpterin and isoxanthopterin.     

Tolerance and avoidance of Al toxicity by Mucuna pruriens var. utilis at different levels of P supply

Previous laboratory experiments showed that velvet bean Mucuna pruriens is moderately tolerant to the presence of Al (up to 185 µM) in the root environment, but that it only develops a shallow root system in acid soils. Field experiments showed that Mucuna can tolerate acid subsoil conditions in a homogeneous root environment, but avoids subsoil if topsoil is present. Subsequent split-root experiments with a recirculating nutrient solution showed that this subsoil avoidance may be based on an Al avoidance mechanism in the root system. This Al avoidance mechanism, however, was not evident when phosphorus (P) supply to the whole plant was adequate. We thus hypothesized that surface application of P may help to overcome Al avoidance in the subsoil.In a field experiment on an ultisol in Lampung (Indonesia), only a moderate increase in aboveground biomass production was found for a wide range of P application rates, although the soil was low in available P, and the P adsorption isotherm was very steep. An increased P status of the topsoil and an increased P concentration in the aboveground biomass (from 50 to 75 mmol kg^-1) had no effect on root development in the subsoil.     

Biotic elicitation mediated in vitro production of L-DOPA from Mucuna pruriens (L.) DC. cell cultures

In Vitro Cellular & Developmental Biology - Plant - With the emerging rise in the need for drugs extracted from various plant sources, there also arises the need for the optimum production of...     

Nutritional characterization of Mucuna pruriens: 4. Does replacing soybean meal with Mucuna pruriens in lamb diets affect ruminal,blood and tissue l-dopa concentrations?

The harmful effects of the 3,4-dihydroxy-l-phenylalanine (l-dopa) in Mucuna pruriens (Velvet bean) seeds have limited its use as a protein supplement for monogastrics and humans. Little is known about the extent of metabolism of Mucuna l-dopa in ruminants or its accumulation in ruminant tissues consumed as food by humans. This study aimed to determine if replacing soybean meal (SB) with Mucuna increases concentrations of l-dopa in rumen fluid, blood and muscle tissue of lambs. Twenty-seven RM lambs (RM; initial body weight, BW = 33.8 ± 5.44 kg) and 12 Florida Native (FN; initial BW = 24.9 ± 8.63 kg) lambs were assigned to four treatments and fed a basal diet of coastal bermudagrass hay, corn grain, and liquid molasses for 42 (FN) or 49 days (RM) and then slaughtered. Dietary supplements were formulated by substituting 0 (SB), 330 (Lo), 670 (Med) or at least 1000 (Hi) g/kg of SB with rolled Mucuna seeds (l-dopa, 24 g/kg dry matter, DM). Body weight was measured weekly and carcass characteristics and concentrations of l-dopa in rumen fluid, blood and the sterno-mandibularis muscle were measured at slaughter when concentrations of blood urea N, glucose, haptoglobin and cerruloplasmin were also measured. Lambs fed SB had higher (P<0.05) average daily gain than those fed Mucuna (0.20 versus 0.15 kg/day for RM; 0.21 versus 0.14 kg/day for FN). However, concentrate protein source did not affect dressing and concentrations of blood urea N, or blood glucose. Feeding the Hi diet versus the SB diet did not increase concentrations of blood cerruloplasmin, or l-dopa or concentrations of l-dopa metabolites in blood. No l-dopa was found in the ruminal fluid of the lambs and l-dopa concentrations in the sterno-mandibularis muscle were low (i.e., <5 ng l-dopa/g), and unaffected (P>0.05) by diet. Ingested Mucuna l-dopa was extensively metabolized in lambs, and did not accumulate to toxic levels in muscle tissues.     

Effect of temperature on aflatoxin production in Mucuna pruriens seeds.

This paper describes the effect of temperature on the level of aflatoxin production in Mucuna pruriens seeds. The highest level of aflatoxin B1 (1.75 micrograms/g) was detected in the samples incubated at 25 degrees C for three weeks. At 20, 30, and 35 degrees C, aflatoxin levels were 0.30 to 0.56, 0.37 to 1.20, and 0.26 to 0.65 micrograms/g, respectively. The lowest concentration of aflatoxin B1 (0.10 to 0.29 microgram/g) was produced at 15 degrees C.     

Ultrastructural and biochemical investigations of protein mobilization of Mucuna pruriens (L.) DC. cotyledons and embryo axis

The mobilization of storage reserves, with particular emphasis on storage proteins of Mucuna pruriens (L.) DC., cotyledons, and embryo was investigated from the ultrastructural and biochemical points of view. Proteins and starch were the two main storage substances in cotyledons, and proteins and lipids were the main ones in the embryo. Embryo protein bodies were smaller and fewer in number than those of cotyledons. Structural and ultrastructural data determined between 24 and 48 h after imbibition and between 48 and 72 h after imbibition, the end of significant embryo and cotyledon protein mobilization, respectively, indicating more precocious storage protein mobilization in the axis than cotyledons. Moreover, storage protein mobilization in embryo and cotyledons occurred before the end of germination. Water soluble proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, producing 29 bands with molecular weights from 14 to 90 KDa. Embryo extract contained more proteins than cotyledon extract, contained seven characteristic bands, and showed a higher variability of the optical density trend than cotyledon.     

Induction of phenoloxidase in cell suspension cultures of Mucuna pruriens L.

The effects of limitating nitrogen-containing compounds in the medium and of adding the amino-acid analogues p-fluorophenylalanine and ethionine on both phenoloxidase activity and the accumulation of L-3,4-dihydroxyphenylalanine (L-DOPA) are reported for cell suspension cultures of Mucuna pruriens. Nitrogen limitation of the cultures, or the addition of p-fluorophenylalanine or ethionine to the culture medium resulted in an increased phenoloxidase activity. There appeared to be an inverse relationship between phenoloxidase activity and the acccumulation of L-tyrosine into L-DOPA by alginate-entrapped cells occurred at a higher rate when phenoloxidase activity was increased.Abbreviations pFPA p-fluorophenylalanine - L-DOPA L-3,4-dihydroxyphenylalanine     

Inhibition of phenylalanine hydroxylase activity by alpha-methyl tyrosine, a potent inhibitor of tyrosine hydroxylase.

Inhibitors of phenylalanine hydroxylase and tyrosine hydroxylase were used in the assay of phenylalanine hydroxylase in liver and kidney of rats and mice. Parachlorophenylalanine (PCPA), methyl tyrosine methyl ester and dimethyl tyrosine methyl ester showed 5–15% inhibition while α-methyl tyrosine seemed to inhibit phenylalanine hydroxylase to the extent of 95–98% at concentrations of 5 × 10 ⁻⁵M –1 × 10 ⁻⁴M. After a phenylketonuric diet (0.12% PCPA + 3% excess phenylalanine), the liver showed 60% phenylalanine hydroxylase activity and kidney 82% that present in pair-fed normals. Hepatic activity was normal after 8 days refeeding normal diet whereas kidney showed 63% of normal activity. The PCPA-fed animals showed 34% in liver and 38% in kidney as compared to normals; in both cases normal activity was noticed after refeeding. The phenylalanine-fed animals showed activity similar to that seen in phenylketonuric animals. The temporary inducement of phenylketonuria in these animals may be due to a slight change in conformation of the phenylalanine hydroxylase molecule; once the normal diet is resumed, the enzyme reverts back to its active form. This paper also suggests that α-methyl tyrosine when fed in conjunction with the phenylketonuric diet may suppress phenylalanine hydroxylase activity completely in the experimental animals thus yielding normal tyrosine levels as seen in human phenylketonurics.     

Al avoidance and Al tolerance ofMucuna pruriens var.utilis: Effects of a heterogeneous root environment and the nitrogen form in the root environment

InMucuna pruriens var.utilis, grown with nitrate-N in a hydroponic split-root system, an Al avoidance reaction of root growth was observed, which was ascribed to local P stress in the Al containing compartment. The Al avoidance reaction was similar to the avoidance ofMucuna roots of acid subsoil in the field where roots grew preferentially in the topsoil. In the present paper the effect of different N forms (NO₃ ^– and NH₄ ⁺) on the reactions ofMucuna to Al were studied, since in acid soils N is present as a mixture of NO₃ ^– and NH₄ ⁺. No interaction between the N form and Al toxicity was found. A hydroponic split-root experiment with NH₄NO₃ nutrition, which is comparable to the situation in the field, showed that under these conditions Al avoidance did not occur. It is concluded that a relation between the Al avoidance reaction ofMucuna and P stress is still likely.Abbreviations D_r root diameter - L_pr total root length per plant - L_rw specific root length - NRA nitrate reductase activity - S/R shoot: root ratio     

Direct inhibition of tyrosine hydroxylase activity by catechol estrogens.

Catechol estrogens, the 2-hydroxylated metabolites of estrogens, recently shown to be formed in brain, inhibit tyrosine hydroxylase, the enzyme that catalyzes the pivotal step in the biosynthesis of the neurotransmitters dopamine and norepinephrine. The nature of the inhibition is by competition with the pterin cofactor and thus resembles feedback inhibition of the enzyme by catecholamines.     

Allosteric effect of tetrahydrobiopterin cofactors on tyrosine hydroxylase activity.

Allostery of tyrosine hydroxylase was found by kinetical studies of partially purified tyrosine hydroxylase from clonal rat pheochromocytoma PC12h cells. Positive cooperativity toward the cofactors, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin [(6R)BH4] and (6S)-L-erythro-5,6,7,8-tetrahydrobiopterin [(6S)BH4], was observed. It is indicated that biopterin might be the regulatory factor of the enzyme polymers, which changes the affinity for the cofactor itself. Moreover, the stereochemical structure of (6R)BH4, the naturally-occurring cofactor, took an important role on the kinetical properties of the enzyme in concern with L-tyrosine.     

A simple and sensitive fluorescence assay for tyrosine hydroxylase activity.

A simple and sensitive fluorescence assay for tyrosine hydroxylase (TH) activity wasdevised based on rapid isolation of enzymatically formed dopa by a double-column procedure fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminum oxide). Interfering substances were removed by the first Amberlite CG-50 column. Dopa was adsorbed on the second aluminum oxide column, then eluted with 0.5 m acetic acid, and assayed by the highly sensitive hydroxyindole method of Johnson et al. (1973, Anal. Biochem.54, 129–136). The standard incubation mixture (total volume, 0.5 ml) contained 0.3 mm l-tyrosine, 1.0 mm 6-methyl-5,6,7,8-tetrahydropterin, 100 mm mercaptoethanol, and an optimal concentration of ferrous ion. d-Tyrosine was used for the blank incubation. Recovery of dopa added to the standard incubation mixture as internal standard was about 70% and was reproducible. The fluorescence characteristics of the product were the same as those of authentic dopa. Blank fluorescence was very low even with crude enzyme preparations. The limit of sensitivity was 100 pmol of dopa formed, which is close to the sensitivity of radioassays. TH activity in homogenates of rat brain stem or human putamen could be assayed in the standard incubation system containing ferrous ion. The validity of this fluorescence assay has been shown by the agreement between the values obtained by this method and by radioassay using l-[U-¹⁴C]tyrosine as substrate. In the rapid assay procedure dopa in the eluate from aluminum oxide was assayed directly by native fluorescence. Although the sensitivity was about 1 nmol, this rapid assay procedure was found to be particularly useful for the purification of TH.     

Stimulatory effect of copper and zinc sulphate on plant regeneration,glutathione-S-transferase analysis and assessment of antioxidant activities in Mucuna pruriens L. (DC)

Plant Cell, Tissue and Organ Culture (PCTOC) - The effect of heavy metals, CuSO4 and ZnSO4 on morphogenic response of cotyledonary node explants of Mucuna pruriens (L.), a fast growing tropical...     

Reduction in brain tyrosine hydroxylase activity following acetylcholinesterase blockade in rats.

Activation of cholinergic neurons in the brain is produced by administration of the acetylcholinesterase inhibitors physostigmine and diisopropylfluorophosphate (DFP). This activation has a biphasic effect on tyrosine hydroxylase (EC 4.14.3-) activity. The acute effect of DFP, 1 mg/kg, intraperitoneally, or physostigmine, 0.2 mg/kg, intravenously, or 10 mug, intraventricularly, was a rapid reduction in tyrosine hydroxylase activity in the hypothalamus. The activities of DOPA decarboxylase (EC 4.1.1.28) and dopamine-beta-hydroxylase (EC 1.14.17.1) were not changed. In contrast to the acute effect, chronic administration of physostigmine, 0.2 mg/kg, intravenously, twice daily for 7 days produced an increase in tyrosine hydroxylase activity in the hypothalamus. The rapid acute effects may be due to an allosteric inactivation of tyrosine hydroxylase, while the chronic effects may reflect enzyme induction.