Characterization of trimethylamine-N-oxide (TMAO) demethylase activity from fish muscle microsomes

A crude microsomal fraction isolated from red hake (Urophycis chuss) muscle demethylated trimethylamine-N-oxide (TMAO). Two cofactor systems were capable of stimulating activity; the system of NADH and FMN required anaerobic conditions while the other system, composed of iron and cysteine and/or ascorbate functioned in the presence or absence of oxygen. The components of each cofactor system functioned synergistically and kinetic parameters were established for each. Of several amine compounds common to fish muscle, TMAO was the only substrate demethylated by the microsomes. Activity was inhibited by iodoacetamide, potassium cyanide, and sodium azide under certain conditions, but not by carbon monoxide. An enzymic nature of the reaction was demonstrated by the properties of heat lability, sensitivity to protease treatment, the requirement of microsomes for TMAO demethylation and by the exhibition of typical hyperbolic kinetics with respect to substrate (TMAO). Moreover, TMAO demethylation by the microsomes was 3 to 4 orders of magnitude faster than the non-enzymic reaction and the reaction was specific for dimethylamine (DMA) as product. It appears the two cofactor systems may share a common catalytic unit in the process of TMAO demethylation.     

Partial purification and characterization of trimethylamine-N-oxide demethylase from lizardfish kidney

Trimethylamine-N-oxide demethylase (TMAOase) from lizardfish (Saurida micropectoralis) was partially purified by acidification and diethylaminoethyl (DEAE)-cellulose chromatography. The enzyme was purified 82-fold with a yield of 65.4%. The optimum pH and temperature were 7.0 and 50 degrees C, respectively. TMAOase was stable to heat treatment up to 50 degrees C and the activation energy was calculated to be 30.5 kJ mol(-1) K(-1). Combined cofactors (FeCl(2), ascorbate and cysteine) were required for full activation. FeCl(2) exhibited a higher stimulating effect on TMAOase activity than FeCl(3). At concentration less than 2 mM, ascorbate was more stimulatory to the activity than cysteine. The activity was tolerant of NaCl concentration up to 0.5 M. The enzyme had a K(m) for TMAO of 16.2 mM and V(max) of 0.35 micromol min(-1) and was able to convert TMAO to dimethylamine (DMA) and formaldehyde. The molecular mass of enzyme was estimated to be 128 kDa based on activity staining.     

The metabolic fate of isotopically labeled trimethylamine-N-oxide (TMAO) in humans

Trimethylamine-N-oxide (TMAO) is associated with chronic disease risk. However, little is known about the metabolic fate of dietary TMAO. This study sought to quantitatively elucidate the metabolic fate of orally consumed TMAO in humans. As part of a crossover feeding study, healthy young men (n=40) consumed 50-mg deuterium-labeled methyl d9-TMAO (d9-TMAO), and enrichments of TMAO and its derivatives were measured in blood for 6 h, urine and stool, as well as skeletal muscle in a subset of men (n=6). Plasma d9-TMAO was detected as early as 15 min, increased until 1 h and remained elevated through the 6-h period. TMAO exhibited an estimated turnover time of 5.3 h, and ~96% of the dose was eliminated in urine by 24 h, mainly as d9-TMAO. No d9-TMAO was detected in feces. Notably, d9-TMAO and d9-trimethylamine were detected in skeletal muscle (n=6) at 6 h, and the enrichment ratio of d9-TMAO to d9-trimethylamine was influenced by a genetic variant in flavin-containing monooxygenase isoform 3 (FMO3 G472A). These results suggest that the absorption of orally consumed TMAO is near complete and does not require processing by gut microbes. TMAO exhibits fast turnover in the circulation with the majority being eliminated in urine within 24 h. A small portion of the dose, however, is taken up by extrahepatic tissue in a manner that appears to be under the influence of FMO3 G472A polymorphism. This trial was registered at clinicaltrials.gov as NCT02558673.     

Partial purification of ethanolaminephosphotransferase from rat brain microsomes

Rat brain ethanolaminephosphotransferase (CDPethanolamine : 1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) was solubilized by treating rat brain microsomes with buffered solutions containing octyl glucoside or Triton X-100. The solubilized enzyme was stable both at 4 degrees C and at -18 degrees C. A partial purification was obtained using an ion-exchange chromatographic procedure. The partially purified enzyme showed four major bands in SDS-polyacrylamide gel electrophoresis; its specific activity was increased by a factor of 37 compared to that of the membrane-bound enzyme. Glycerol and diacylglycerol were effective as stabilizers. Phosphatidylcholine, lysophosphatidylcholine and phosphatidylserine increased both the specific activity and the stability of the partially purified enzyme.     

Effects of urea and trimethylamine-N-oxide (TMAO) on the interactions of lysozyme in solution

The effect of two physiological cosolutes (urea and trimethylamine-N-oxide) and of KCl on the intermolecular interactions in concentrated lysozyme solutions were studied by synchrotron radiation small angle x-ray scattering. The evolution of the structure factors as a function of cosolute and/or salt concentration was modeled using pair potentials following an approach recently described in the literature. It was found that the structure factors for salt and/or cosolute concentration series at a fixed protein concentration can best be described using a variable depth attractive potential and a constant effective charge rather than a constant attractive potential and a variable effective charge as done in previous work.     

肠道菌群及其代谢产物氧化三甲胺——冠心病治疗的新靶点

冠心病 (Coronary artery disease,CAD) 是全球发病率和死亡率最高的一种心血管疾病,冠心病和肠道菌群失调密切相关,肠道菌群可能是未来冠心病的重要诊断标志物,改善肠道菌群微环境有望成为治疗冠心病的新途径。作为肠道菌群参与合成的活性代谢产物,氧化三甲胺 (Trimethylamine-N-oxide,TMAO) 水平的升高与心血管疾病患病风险、全因死亡率的增加有关;基础研究表明TMAO可能具有促动脉粥样硬化特性;这些研究提示TMAO可作为预防和治疗冠心病的潜在靶点。文中分析了当前调控肠道菌群及其代谢产物TMAO治疗冠心病的临床及基础性研究,以期为冠心病的治疗提供帮助。     

Partial purification and characterization of an anion-activated ATPase from radish microsomes

Previous investigation showed two distinct ATP-dependent proton-transporting systems in microsomal vesicle from radish seedlings, one inhibited by vanadate and one inhibited by NO-3. On the bases of the effects of these inhibitors we could discriminate two distinct ATPase activities in the same material. The NO-3 sensitive activity was separated from the vanadate-sensitive activity and partially purified by a single-step chromatographic method, which lead to approx 35-fold purification from the microsomes and to a specific activity of 2.3 mumol Pi X min-1 X mg protein-1, at 30 degrees C. The partially purified activity was specific for ATP, some activity being observed toward GTP, and even less toward CTP, UTP and ITP. No significant Pi hydrolysis was found with ADP, AMP, p-nitrophenylphosphate and glucose 6-phosphate. ADP but not AMP was inhibiting in the presence of ATP. The activity was dependent on divalent cations in the order of preference: Mg2+ greater than Mn2+ greater than Co2+ greater than Ca2+ greater than Zn2+. The activity was unaffected by monovalent cations, strongly activated by Cl-, inhibited by 90% by 50 mM NO-3, virtually unaffected by oligomycin and NaN3. At least 90% of the activity was abolished in the presence of each: 10 microM N,N'-dicyclohexylcarbodiimide, 10 microM erythrosin B, 10 mu mersalyl, 100 microM trimethyltin, 100 microM diethylstilbestrol, 100 microM N-ethylmaleimide. No inhibition has been found in the presence of Ca2+, at a concentration blocking the vanadate-sensitive activity. Nigericin, gramicidin and carbonylcyanide p-trifluoromethoxyphenylhydrazone stimulated the activity of this preparation after it was incubated in the presence of sonicated phospholipids, suggesting the capacity of the ATPase to function as a H+-transporting system. All characteristics mentioned were closely similar to those described in the vacuolar ATPases.     

Partial purification and characterization of phospholipid N-methyltransferases from murine thymocyte microsomes

Significant amounts of phospholipid N-methyltransferase activity in murine thymocytes were found to be distributed on the plasma membrane. The enzyme activity had an optimum pH of 9. The presence of divalent cations, Mg2+ (10 mM) or Ca2+ (1 mM), and EGTA separately in the assay had only a small effect on the enzyme activity. However, addition of both 10 mM Mg2+ and 1 mM Ca2+ increased the enzyme activity. The presence of two enzymes for each conversion of phosphatidylethanolamine (PE) to phosphatidylmonomethylethanolamine (PME) and PME to phosphatidylcholine (PC) was suggested by the result of the determination of the incorporated radioactivity into PME, phosphatidyldimethylethanolamine (PDE) and PC; the apparent Km values for S-adenosyl-L-methionine were 20 and 400-500 microM for the conversion of PE to PME and for the conversion of PME to PC they were 5 microM and 40 microM. S-Adenosyl-L-homocysteine (AdoHcy), a known inhibitor of enzymatic methylation, competitively inhibited [14C]methyl incorporation into total lipid. The apparent Ki value for AdoHcy was 44.7 microM. Two phospholipid N-methyltransferases were partially purified by extraction with sodium deoxycholate, gel filtration on Sephadex G-75, and affinity column chromatography on AdoHcy-Sepharose. One enzyme, mainly catalyzing the formation of PME, was purified approximately 1548-fold and the other catalyzing the formation of PDE and PC, was purified approximately 629- to 703-fold. However, the former still contained a little activity for PDE and PC formation and the latter contained a little activity for PME formation. In these partially purified phospholipid N-methyltransferase preparations, little contaminating protein O-carboxylmethyltransferase activity was observed; however, significant PC-phospholipase A2 activity was detected. This result may suggest that phospholipid N-methyltransferases associate with phospholipase A2 in the thymocyte plasma membrane.     

Partial purification and characterization of lathosterol 5-desaturase from rat liver microsomes

The terminal oxidase of the NADH-dependent lathosterol 5-desaturation system was solubilized from rat liver microsomes with 2% Triton X-100, and partially purified approximately 18-fold with 19% yield after DEAE-cellulose and 6-aminohexyl-Sepharose column chromatography. The final enzyme preparation was free from other electron transfer components and phospholipids in microsomes, and the desaturation reaction was reconstituted with the following components: NADH, molecular oxygen, phospholipids and three proteins, i.e., NADH-cytochrome b5 reductase, cytochrome b5 and the terminal oxidase. Omission of one of these components led to an almost complete loss of the desaturase activity. Under the reconstitution conditions, the desaturase activity was significantly inhibited by potassium cyanide but was not affected by -SH reagents such as N-ethylmaleimide and dithiothreitol.     

Partial purification and characterization of 7 alpha-hydroxysteroid dehydrogenase from rat liver microsomes

An NADPH-dependent 7 alpha-hydroxysteroid dehydrogenase acting on 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid was partially purified 160-fold with a yield of 13% from rat liver microsomes using DEAE-cellulose, hydroxyapatite and Affi-Gel Blue column chromatography. The specific activity of the purified enzyme was 91.3 nmol chenodeoxycholic acid formed/min per mg of protein. The reaction was reversible, and the optimum pH of the enzyme for the oxidation was about 8.5, whereas that for the reduction was about 5.0 A molecular weight of the enzyme was estimated to be about 130,000 by Superose 6TM gel filtration chromatography. The apparent Km value for 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid was 35.7 microM and that for NADPH was 90.9 microM. The preferred substrate for the enzyme was 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid rather than 3 alpha,12 alpha-dihydroxy-7-keto-5 beta-cholanoic acid, a 7-keto-bile acid analogue. The enzyme also preferred the unconjugated form to the conjugated forms. The enzyme activity was inhibited by p-chloromercuribenzoate; however, the inhibition was prevented by addition of reduced form of glutathione to the reaction mixture, indicating that the enzyme requires a sulfhydryl group for activity.     

Extraction of 11 beta-hydroxysteroid dehydrogenase from rat liver microsomes by detergents

In these studies our goal was to solubilize the microsomal enzyme, 11 beta-hydroxysteroid dehydrogenase (11-HSD) as the first step in its purification. Enzyme was extracted from rat liver microsomes with representative detergents (Zwittergents, Tritons, modified sterols). Oxidation-reduction (O-R) ratios of extracts varied with detergent used and ranged from 0.18 (CHAPS) to 3.8 (Zwittergent 3-14) relative to a ratio of 1.7 in intact microsomes. All detergents solubilized 11-HSD using lack of sedimentation during high speed centrifugation as criterion. With Triton DF-18 and Triton X-100, optimum extraction of 11-HSD occurred in the detergent-protein ratio range of 0.1 to 0.2 O-R ratios decreased with increased Triton X-100, but were constant as Triton DF-18 was varied. The pH optimum of enzyme extraction was 9 at a detergent-protein ratio of 0.05 and 7.5-8.0 at a ratio of 0.2. Sodium chloride increased enzyme extraction by detergents; in the absence of detergent, salt extracted protein, but not enzyme. In aqueous solution at 0 degrees C or -15 degrees C, microsomal 11-oxidation activity rose within 24 h, then decreased; reductase activity consistently decreased. Oxidation and reduction activities were inversely related in the microsomal bound enzyme. No relationship between these activities appeared in detergent-solubilized enzymes. Possible mechanisms to account for the unexpected behavior of this enzyme are discussed.     

Solubilization,purification and reconstitution of Ca-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: Preservation of native structure and function of the enzyme by DHPC

The properties of Ca²⁺-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C₁₂E₈) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca²⁺-ATPase with a greater specific activity than solubilization with C₁₂E₈ or Triton X-100. DHPC was determined to be superior to C₁₂E₈; while that the C₁₂E₈ was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca²⁺-ATPase retained the E1Ca−E1*Ca conformational transition as that observed for native microsomes; whereas the C₁₂E₈ and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca²⁺ transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C₁₂E₈ and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca²⁺-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C₁₂E₈ and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca²⁺ uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca²⁺-ATPase retained more organized and native secondary conformation compared to C₁₂E₈ and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C₁₂E₈ and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca²⁺-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C₁₂E₈ and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein–lipid interactions in the function of the membrane-bound enzyme.     

Partial purification of locust flight muscle lipoprotein lipase (LpL): apparent differences from mammalian LpL

1. An attempt was made to purify lipoprotein lipase (LpL) from the flight muscle of the migratory locust based on affinity for heparin, which is known to avidly bind mammalian LpL. 2. However, locust LpL appeared to completely lack this property, which indicates that the suggested membrane-binding of locust LpL is very different from that of mammalian LpL: a heparin-like glycosaminoglycan is not involved. 3. Since locust LpL lacks heparin affinity, other purification methods were assayed. Solubilization of locust LpL was obtained by the detergent Tween 20. 4. Though both anion and cation exchange chromatography resulted in the complete loss of enzyme activity, partial purification of locust LpL was achieved by gel filtration chromatography.     

Partial purification of pisatin demethylase,a cytochrome P-450 from the pathogenic fungus Nectria haematococca

The plant pathogen Nectria haematococca can demethylate pisatin, a phytoalexin from pea. Demethylation is apparently necessary for virulence on pea and is catalyzed by a microsomal cytochrome P-450 monooxygenase system. The cytochrome P-450 and NADPH-cytochrome P-450 reductase of this system were solubilized with sodium cholate and partially purified by chromatography on blue A-agarose and -aminohexyl-agarose. The reductase was further purified by chromatography on 2,5-ADP-agarose to a specific activity of about 16 moles cytochrome c reduced per min per mg protein. Upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the reductase fraction contained one major band of molecular weight 84,000. The partially purified cytochrome P-450 fraction contained a number of minor bands and three major bands of molecular weights 52,000, 56,000 and 58,000. This fraction lost all demethylase activity during concentration after -aminohexyl-agarose chromatography, so it could not be purified further. The purified reductase could reconstitute demethylase activity of cytochrome P-450 fractions and appeared to be rate-limiting for demethylase activity in microsomal extracts.     

Solubilization, purification and reconstitution of Ca(2+)-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: preservation of native structure and function of the enzyme by DHPC

The properties of Ca(2+)-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C(12)E(8)) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca(2+)-ATPase with a greater specific activity than solubilization with C(12)E(8) or Triton X-100. DHPC was determined to be superior to C(12)E(8); while that the C(12)E(8) was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca(2+)-ATPase retained the E1Ca-E1*Ca conformational transition as that observed for native microsomes; whereas the C(12)E(8) and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca(2+) transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C(12)E(8) and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca(2+)-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C(12)E(8) and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca(2+) uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca(2+)-ATPase retained more organized and native secondary conformation compared to C(12)E(8) and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C(12)E(8) and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca(2+)-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C(12)E(8) and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein-lipid interactions in the function of the membrane-bound enzyme.     

Quantification of the influence of trimethylamine-N-oxide (TMAO) on the heat resistance of Escherichia coli K12 at lethal temperatures

Aim: To quantify the influence of trimethylamine‐N‐oxide (TMAO) on the heat resistance of Escherichia coli K12 MG1655 cells at static temperatures. Methods and Results: Stationary‐phase E. coli cells were inactivated at 52, 54 and 58°C. The heat resistance is described as reduction in the inactivation rate, k_max, and/or an increase in the time for one decimal reduction, D, and/or an increase in the time for the fourth decimal reduction, t_4D. Conclusions: Resistance of E. coli changed – increased – at all temperatures under study. Generally, the addition of TMAO to the growth medium protected E. coli cells, leading to an increase in their heat resistance, i.e. reduced k_max and increased D and t_4D values are obtained. Significance and Impact of the Study: Additional knowledge on the reaction of E. coli to heat in the presence of the organic osmolyte TMAO at lethal temperatures is provided. This work contributes to an improved understanding of the level of the resistance of bacteria to heat in the presence of osmolytes.