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Partial purification and characterization of the enzyme trimethylamine oxide demethylase from whiting (Merlangius merlangus)
Havemeister, W. (2000). Partial purification and characterization of the enzyme trimethylamine oxide demethylase from whiting (Merlangius merlangus). Schriften der Bundesforschungsanstalt für Fischerei, 25. Bundesforschungsanstalt für Fischerei: Hamburg. 160 pp.
Part of: Schriften der Bundesforschungsanstalt für Fischerei. Bundesforschungsanstalt für Fischerei: Hamburg. ISSN 0438-4547, more

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    Marine

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  • Havemeister, W.

Abstract
    The development ofa discontinuous assay system (standard trimethylamine oxide demethylase-TMAO-ase-assay) composed of two steps, the TMAO-ase reaction and the measurement of FA, enabled a fast, reliable, and sensitive measurement of the enzyme TMAO-ase (E.C. 4.1.2.32). TMAO-ase can be considered as a lysosomal enzyme, bound to or within Iysosomes, but existing as well in the soluble fraction. A pH-optimum of 4.5- 5.0 and the simultaneous presence of TMAO-ase activity and activity of acid phosphatase and a-glucosidase indicated the lysosomal origin of TMAO-ase. Determination of the distribution of whiting TMAO-ase indicated high activity in kidney. Therefore optimization of the extraction procedure was performed with kidney. Extraction was optimized using several homogenization techniques, varying temperature during homogenization and extraction, addition of salt, and application of detergents. Extraction studies as well as localization of TMAO-ase demonstrated that TMAO-ase exists in two forms: a soluble and an insoluble, membrane bound part. Several chromatographic techniques (gel filtration, IEC, HlC, IMAC) were applied for purification of TMAO-ase. Gel filtration with Sepharose 4B and 6B resulted in separation of two main TMAO-ase rich fractions: a high MW , membrane bound fraction (approx. 2000 kDa) and a lower MW, soluble fraction (150-600 kDa) which was enriched up to 5-fold. Soluble and insoluble TMAO-ase both were heat stable up to 30°C -40°C for 20 min. Partially purified TMAO-ase showed maximum activity at 25°C and pH 4.5 -5.0; its activation energy was 5.2 kcal/mol°K to 6.0 kcal/mol°K depending on the buffer used for measurement. Isoelectric focusing of crude and partially purified TMAO-ase showed bands in the weak acidic pI range. For partially purified, soluble TMAO-ase four distinct bands in the pI range between 6.2 -6.6 were identified by the enzyme specific stain. These bands were assessed by silver staining and TMAO-ase standard assay. The four bands might indicate that soluble TMAO-ase is composed of four isoenzymes. SDS-electrophoresis of high and low MW TMAO-ase fractions under reduced and non-reduced conditions demonstrated that TMAO-ase was composed of several sub-units in the MW range between 4x10³ -6.6x104 Da. The band pattern of soluble and insoluble fractions were identical and sub-units therefore probably linked by SH-groups. Investigations of substrate specificity were performed with synthesized and well characterized TMAO-analogues (EDMAO, DEMAO, TEAO). These amine oxides were also cleaved by TMAO-ase but with decreasing affinity due to stereochemical reasons. The apparent Km values found for TMAO and EDMAO were 5-7-11.5 mM and 11.2-16.8 mM demonstrating similar affinity of TMAO-ase for these substrates.

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