A new continuous spectrophotometric assay method for DOPA oxidase activity of tyrosinase

Sensitive assay methods for tyrosinase are essential not only for the understanding the process of pigment production but also for the development of effective inhibitors of tyrosinase. To develop an efficient assay method, we applied thymol blue to reaction mixtures. The enzyme kinetic study revealed that DOPA oxidase activity of tyrosinase in thymol blue-applied reaction system was more sensitively measured, even under lower enzyme units compared with the previous report with significant enhancement of Vmax while affinity change on substrate was not observed. To test whether this method could be applicable to the inhibition and the inactivation kinetic study of tyrosinase, the effect of kojic acid, a well-known tyrosinase inhibitor, and sodium chloride respectively, have been studied. Conclusively, thymol blue method can assay tyrosinase activity with sensitivity and is applicable to the inhibition and the inactivation study of tyrosinase.     

Reactions of DOPA (3,4-dihydroxyphenylalanine) decarboxylase with DOPA.

The study of DOPA (3,4-dihydroxyphenylalanine) decarboxylase by steady-state methods is difficult because multiple reactions occur. The reaction with DOPA was studied at enzyme concentrations between 20 and 50 micrometer by direct observation of the bound coenzyme by using stopped-flow and conventional spectrophotometry. Four processes were observed on different time scales and three of these were attributed to stages in the decarboxylation. The fourth was attributed to an accompanying transamination that renders the enzyme inactive. It was clear that much, if not all, of the 330 nm-absorbing coenzyme present in the free enzyme plays an active part in the decarboxylation, since it is converted into 420 nm-absorbing material in the first observable step. An intermediate absorbing maximally at 390 nm is formed in a slower step. Rate and equilibrium constants have been determined and the ratio of decarboxylation to transamination was estimated to be 1200:1.     

A new assay for collagenolytic activity.

A new assay procedure for the determination of collagenolytic activity is presented. The substrate can be prepared by simple reduction of the purified acidsoluble rat tail tendon collagen with NaB³H₄. Collagenase activity is determined by measurement of soluble tritiated collagen peptides released. It has proven to be a method with a high degree of sensitivity and reproducibility.     

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.     

A simple and continuous assay for decarboxylase activity using a carbon dioxide-selective electrode

A carbon dioxide electrode has been used for kinetic studies of decarboxylase activity. The methodology used requires only a sensitive pH meter operating in the millivolt mode with the electrode. The CO₂ release may be followed continuously. Compared to other assay procedures the method outlined offers the advantages of rapidity, continuity, specificity, and sufficient precision. Techniques for the calibration of the electrode response are discussed. The applicability of this assay was appraised by determining the K_m of lysine decarboxylase. The value obtained showed good agreement with that reported in literature. The procedure might be useful for the study of enzyme-inhibitor interactions.     

Purification of DOPA decarboxylase from bovine striatum

Pyridoxal phosphate-dependent DOPA decarboxylase has been purified from bovine striatum to a specific activity of 1.6 U/mg protein. After ammonium sulfate precipitation (30–60%) it was purified by DEAE-Sephacel, Sephacryl S-200, and TSK Phenyl 5 PW chromatography. The purified enzyme showed a single silver staining band with polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions. The bovine striatal DOPA decarboxylase is a dimer (subunit M_r = 56000 by SDS-PAGE) with a native M_r of 106000 as judged by chromatography on Sephacryl S-200 and by sedimentation analysis. Similar to the DOPA decarboxylase purified from non-CNS tissues, the bovine striatal enzyme requires free sulfhydryl groups for activity, is strongly inhibited by heavy metal ions, and can decarboxylate 5-hydroxytryptophan as well. It should be noted, however, that the final enzyme preparation is enriched in DOPA decarboxylase activity. The distribution of the DOPA decarboxylase and 5-HTP decarboxylase activities also varies among several bovine brain regions. In addition, heat treatment of the enzyme preparation inactivated the two decarboxylation activities at different rates.Abbreviations AADC Aromatic L-amino Acid Decarboxylase - CNS Central Nervous System - DOPA 3,4-dihydroxyphenylalanine - DTT Dithiothreitol, 5-HTP - 5-hydroxytryptophan - Mr relative molecular weight - PLP pyridoxal 5-phosphate - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Part of this paper was presented at the 1987 Annual Pharmacology and Toxicology Conferences held at University of North Dakota School of Medicine, North Dakota, USA Res Commun Psychol Psychiat Behav 12: 227–228, 1987 (Abstr).     

A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase

A pH-sensitive colorimetric assay has been established to quantitatively measure glutamate decarboxylase (GAD) activity in bacterial cell extracts using a microplate format. GAD catalyzes the irreversible α-decarboxylation of l-glutamate to γ-aminobutyrate. The assay is based on the color change of bromocresol green due to an increase in pH as protons are consumed during the enzyme-catalyzed reaction. Bromocresol green was chosen as the indicator because it has a similar pK_a to the acetate buffer used. The corresponding absorbance change at 620 nm was recorded with a microplate reader as the reaction proceeded. A difference in the enzyme preparation pH and optimal pH for GAD activity of 2.5 did not prevent this method from successfully allowing the determination of reaction kinetic parameters and the detection of improvements in enzymatic activity with a low coefficient of variance. Our assay is simple, rapid, requires minimal sample concentration and can be carried out in robotic high-throughput devices used as standard in directed evolution experiments. In addition, it is also applicable to other reactions that involve a change in pH.     

A rapid histidine decarboxylase assay

Histidine decarboxylase (EC 4.1.1.22) catalyzes the conversion of histidine to histamine. Because current assays for enzyme activity are time consuming and require additional enzymes or large amounts of tissue, a rapid radioisotopic assay was devised. Using commercially available radioactive histidine (without additional purification), the enzyme mediates the formation of histamine. The product is resolved from precursor by paper electrophoresis in a formic acid-acetic acid solution for 15 min. After drying and ninhydrin staining, radioactive histamine is measured by liquid seintillation spectrometry. This assay procedure is sensitive enough to measure decarboxylase activity in milligram quantities of rat brain.     

A new robust kinetic assay for DAP epimerase activity

DAP epimerase is the penultimate enzyme in the lysine biosynthesis pathway. The most versatile assay for DAP epimerase catalytic activity employs a coupled DAP epimerase–DAP dehydrogenase enzyme system with a commercial mixture of DAP isomers as substrate. DAP dehydrogenase converts meso-DAP to THDP with concomitant reduction of NADP⁺ to NADPH. We show that at high concentrations, accumulation of NADPH results in inhibition of DAPDH, resulting in spurious kinetic data. A new assay has been developed employing DAP decarboxylase that allows the reliable characterisation of DAP epimerase enzyme kinetics.     

Characterization of DOPA decarboxylase mRNA in rat pheochromocytoma

Total poly (A+) RNA has been extracted from rat pheochromocytoma and translated in vitro by means of a reticulocyte lysate system. We show that two antisera, prepared against pig kidney DOPA decarboxylase (DDC) or rat pheochromocytoma DDC, immunoprecipitate an in vitro synthetized 50 kDa polypeptide identified as DDC by competition experiments with pure DDC. The proportion of specific mRNA has been calculated and represents 0.05% of total poly A+ mRNA. Its size has been established by electrophoresis in methylmercuric hydroxide containing agarose gel, corresponding to a 2.2 kb length mRNA.     

Spectrophotometric assay for ornithine decarboxylase

A rapid and sensitive spectrophotometric assay for ornithine decarboxylase is described. It is based on the observation that the product of ornithine decarboxylase, putrescine, reacts with 2,4,6-trinitrobenzenesulfonic acid to give a colored product soluble in 1-pentanol whereas ornithine does not. The amount of putrescine produced by the enzyme was determined by measuring the absorbance of the 1-pentanol extract of the reaction mixture at 420 nm, and by comparing the results to those obtained by the trapping of 14CO2 and by HPLC assays. The three assays were found to be equivalent in sensitivity, with the spectrophotometric assay having the advantages of being relatively rapid, requiring only common laboratory equipment, and not requiring the use of radioactive isotopes.     

A highly sensitive high-throughput luminescence assay for malonyl-CoA decarboxylase

Malonyl-CoA decarboxylase (MCD) catalyzes the conversion of malonyl-CoA to acetyl-CoA and thereby regulates malonyl-CoA levels in cells. Malonyl-CoA is a potent inhibitor of mitochondrial carnitine palmitoyltransferase-1, a key enzyme involved in the mitochondrial uptake of fatty acids for oxidation. Abnormally high rates of fatty acid oxidation contribute to ischemic damage. Inhibition of MCD leads to increased malonyl-CoA and therefore decreases fatty acid oxidation, representing a novel approach for the treatment of ischemic heart injury. The commonly used MCD assay monitors the production of NADH fluorometrically, which is not ideal for library screening due to potential fluorescent interference by certain compounds. Here we report a luminescence assay for MCD activity. This assay is less susceptible to fluorescent interference by compounds. Furthermore, it is 150-fold more sensitive, with a detection limit of 20 nM acetyl-CoA, compared to 3 μM in the fluorescence assay. This assay is also amenable to automation for high-throughput screening and yields excellent assay statistics (Z′ > 0.8). In addition, it can be applied to the screening for inhibitors of any other enzymes that generate acetyl-CoA.     

A spectrophotometric assay for meso-diaminopimelate decarboxylase and L-alpha-amino-epsilon-caprolactam hydrolase

A spectrophotometric assay for the activities of mesodiaminopimelate decarboxylase and L-alpha-amino-epsilon-caprolactam hydrolase is described. With the commercially available enzyme saccharopine dehydrogenase lysine formed either by decarboxylation of meso-diaminopimelate or by hydrolysis of L-alpha-amino-epsilon-caprolactam is converted to saccharopine with the concomitant oxidation of NADH, which is monitored by the decrease in absorbance at 340 nm. For meso-diaminopimelate decarboxylase this assay can be performed either as an endpoint determination, when working with crude extracts, or as a continuous spectrophotometric assay of partially purified enzyme preparations. The activity of L-alpha-amino-epsilon-caprolactam hydrolase can only be assayed by the endpoint method because of the great differences in the pH optima of the hydrolase and the saccharopine dehydrogenase.