E. J. M. Pennings

Serotonin metabolism in migraine

To investigate systemic serotonin (5-HT) metabolism in migraine, we determined platelet and platelet-free plasma concentrations of 5-HT, its precursors tryptophan and 5-hydroxytryptophan, and its main metabolite 5-hydroxy-indoleacetic acid (5-HIAA), as well as the activities of the platelet enzymes monoamine oxidase and phenolsulfotransferase in classic and common migraineurs. Between attacks, migraineurs had lower plasma 5-HT and higher 5-HIAA levels than did healthy controls and patients with tension headache. During migraine attacks, plasma 5-HT levels were substantially higher than during attack-free periods, while 5-HIAA concentrations and platelet enzyme activities were lower. Platelet 5-HT was reduced only during common, but not classic, migraine attacks. We hypothesize that systemic 5-HT metabolism is enhanced in migraineurs during headache-free periods and transiently decreases during attacks, presumably due to a fall in enzymatic degradation. Furthermore, platelet behavior differs during migraine attacks with and without aura, and release of platelet 5-HT cannot (exclusively) be held accountable for the rise of plasma 5-HT during migraine attacks.

Overexpression of a tryptophan decarboxylase cDNA in Catharanthus roseus crown gall calluses results in increased tryptamine levels but not in increased terpenoid indole alkaloid production

The enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28) catalyses a key step in the biosynthesis of terpenoid indole alkaloids inC. roseus by converting tryptophan into tryptamine. Hardly anytdc mRNA could be detected in hormone-independent callus and cell suspension cultures transformed by the oncogenic T-DNA ofAgrobacterium tumefaciens. Supply of tryptamine may therefore represent a limiting factor in the biosynthesis of alkaloids by such cultures. To investigate this possibility, chimaeric gene constructs, in which atdc cDNA is linked in the sense or antisense orientation to the cauliflower mosaic virus 35S promoter and terminator, were introduced inC. roseus cells by infecting seedlings with an oncogenicA. tumefaciens strain. In the resulting crown gall tumour calluses harbouring thetdc sense construct, an increased TDC protein level, TDC activity and tryptamine content but no significant increase in terpenoid indole alkaloid production were observed compared to empty-vector-transformed tumour calluses. In tumour calluses containing thetdc antisense construct, decreased levels of TDC activity were measured. Factors which might be responsible for the lack in increased terpenoid indole alkaloid production in thetdc cDNA overexpressing crown gall calluses are discussed.

Melanin content of cultured human melanocytes and UV-induced cytotoxicity.

Cultured melanocytes originating from persons with different skin phototypes were utilized for measurement of endonuclease sensitive sites induced by UVB and the determination of cell survival after UVA or UVB irradiation. During culture, the melanocytes largely maintained their phenotypic characteristics according to their original skin phototype. Total melanin concentrations were 4.9 times higher in the darker skin phototype (IV-VI) melanocytes when compared to the cells from lighter skin phototypes (I-III). Also phaeomelanin contents were higher (2.5 times) in the skin phototype (IV-VI) melanocytes which implies that the cells from light skin types contain less melanin, but a relatively high proportion of phaeomelanin. After UVB irradiation a stronger induction of endonuclease sensitive sites was found for melanocytes with a lower level of total melanin and a high content of pheomelanin. By measuring the clone forming ability in different melanocyte cultures after UVB irradiation, significant better survival was found in case of the cells with the higher melanin content. Despite the large variations in melanin content, no significant difference in survival after UVA irradiation could be demonstrated in this way. Our results suggest a protective effect of melanin for UVB and indicate the importance of the measurements of melanin content and composition when different parameters of UV-induced damage are studied in melanin producing cells.

Assay of strictosidine synthase from plant cell cultures by high-performance liquid chromatography

An HPLC assay is described for the enzyme strictosidine synthase in which the formation of strictosidine and the decrease of tryptamine can be followed at the same time. In cell cultures of Catharanthus roseus significant amounts of strictosidine glucosidase activity were detected. In crude preparations, the strictosidine synthase reaction is therefore best measured by the secologanin-dependent decrease of tryptamine. In this way, the specific synthase activity in a cell free extract was found to be 56 pkat/mg of protein. Inclusion of 100 mM D(+)-gluconic acid-delta-lactone in the incubation mixture inhibited 75% of the glucosidase activity, without inhibiting the synthase activity. The synthase activity was readily separated from the glucosidase activity by gel filtration on Sephadex G-75 or Ultrogel AcA-44. Cell cultures of Tabernaemontana orientalis did not contain measurable amounts of strictosidine glucosidine activity. The specific strictosidine synthase activity was 130-200 pkat/mg of protein during the growth of this cell culture. Strictosidine synthase is stable at -20 degrees C for at least 2 months.

Assay of tryptophan decarboxylase from Catharanthus roseus plant cell cultures by high-performance liquid chromatography.

An assay is described for the enzyme tryptophan decarboxylase from plant cell suspension cultures. It is based on the fluorometric detection of tryptamine by HPLC on a LiChrosorb RP-8 Select B column. Tryptophan decarboxylase from Catharanthus roseus was induced by transferring 14-day-old cells into an induction medium. Optimum activity was found 2 days after transfer, the increase being 5- to 10-fold. When kept at -15 degrees C the crude enzyme lost half its activity in about 7 days. The rate of the decarboxylation reaction was linear for at least 3 h at 35 degrees C.

Purification and characterization of strictosidine synthase from a suspension culture of Cinchona robusta

Abstract Four isoforms of strictosidine synthase (EC 4.3.3.2), which catalyses the stereospecific condensation of secologanin and tryptamine, were purified to h

Assay of platelet monoamine oxidase in whole blood

Monoamine oxidase (MAO; EC 1.4.3.4.) was measured in whole blood with kynuramine as the substrate. Optimal circumstances were determined. By use of selective inhibitors, gradient centrifugation, dilution of samples and removal of thrombocytes from whole blood it was shown that this assay of MAO in whole blood is in fact a determination of platelet MAO. No reversible endogenous inhibitors are present in the blood. Because preparation of platelet-rich plasma may lead to considerable losses of specific subpopulations with relatively low or high enzyme activities, the advantage of using whole blood is that the MAO activity is determined in the whole platelet population.

Tryptophan decarboxylase from Catharanthus roseus is a pyridoxoquinoprotein

Tryptophan decarboxylase (EC 4.1.1.28) from Catharanthus roseus was purified to homogeneity. The native enzyme has an M r of about 96000 as estimated from native PAGE. After SDS‐PAGE, three protein bands were visible corresponding with M r 49000, 33000 and 17000. The N‐termini of the 49 kDa protein and the 33 kDa protein were identical. Antibodies against the 49 kDA protein also reacted strongly with the two smaller proteins. It is concluded that the native enzyme consists of two subunits of M r 49000. Tryptophan decarboxylase appears to be a pyridoxo‐quinoprotein, since two molecules of pyridoxal phosphate and two molecules of covalently‐bound pyrroloquinoline quinone were found per enzyme molecule.

Assay of catechol O-methyltransferase by determination of the m- and p-O-methylated products using high-performance liquid chromatography

A new assay of catechol O-methyltransferase (EC 2.1.1.6) is described by determination of the m- and p-O-methylated products of 3,4-dihydroxybenzoic acid. The method involves DEAE-Sephadex A-25 chromatography and reversed-phase high-performance liquid chromatography on a LiChrosorb 5 RP 18 column. The liquid chromatographic solvent system consisted of 0.05 m acetic acid in methanol:water (1:4, vv), pH 3,2. meta/para ratios of O-methylation are easily obtained by this method. Dimethylation has not been observed with a partially purified enzyme preparation from rat liver.

Tyrosine and cysteine are substrates for blackspot synthesis in potato

Partially purified blackspot pigments from potato tubers (Solanum tuberosum L.) of two commercial cultivars were subjected to a microassay for melanin, which consisted of specific chemical degradation and subsequent HPLC analysis. Permanganate oxidation yielded pyrrole-2,3,5-tricarboxylic acid, whereas hydrolysis in hydriodic acid liberated aminohydroxyphenylalanine isomers. These results indicate that the polymeric pigments, which have previously been found to contain a protein matrix, carry crosslinked 5,6-dihydroxyindole-2-carboxylic acid and benzothiazine units. This leads to the conclusion that free tyrosine and free cysteine are incorporated in the proteinaceous pigments via the polyphenol oxidase catalysed pathway of melanogenesis in the process of blackspot formation. The findings are in accordance with the hypothesis that the process of blackspot formation is a non-regulated cascade of reactions in disintegrated tuber cells, rather than a finely tuned biosynthesis.