PCR-based detection of genetically modified soybean and maize in raw and highly processed foodstuffs.

The PCR method has proved to be an invaluable tool for the specific and sensitive detection of genetically modified material (e.g., Roundup Ready Soybean and Bt-176 "Maximizer" Maize) in foodstuffs. The first step in the procedure, namely the purification of nucleic acids from the sample, is often the deciding factor in the production of meaningful results. In this study, we present two procedures that enable an efficient isolation of trace amounts of genetic material from both raw and highly processed foodstuffs. We show that for optimal, PCR-ready DNA purification from highly processed foodstuffs and PCR inhibitor-rich substances--such as cocoa-containing products--adapted protocols for the QIAGEN QIAamp DNA Stool Mini Kit can be utilized. For complete DNA isolation from raw foodstuffs, a protocol using the DNeasy Plant Mini Kit is presented.

Purification of a two-subunit cytochrome-b-containing heterodisulfide reductase from methanol-grown Methanosarcina barkeri.

Heterodisulfide reductase catalyzes the terminal step in the energy-conserving electron-transport chain in methanogenic Archaea. The heterodisulfide reductase activity of the membrane fraction of methanol-grown Methanosarcina barkeri was solubilized by Chaps. Chromatography on Q-Sepharose and Superdex-200 yielded a high-molecular-mass fraction (> 700 kDa) which was dissociated by dodecyl beta-D-maltoside. After chromatography on Q-Sepharose, an active heterodisulfide reductase preparation was obtained which was composed of only two different subunits of apparent molecular masses 46 kDa and 23 kDa. For each 69 kDa, the enzyme contained 0.6 mol cytochrome b, 0.2 mol FAD, 20 mol non-heme iron and 20 mol acid-labile sulfur. The 23-kDa subunit possessed heme-derived peroxidase activity, showing that this polypeptide is the cytochrome b. The purified enzyme contained the cytochrome b in the reduced form. Upon addition of the heterodisulfide of coenzyme M and N-7-mercaptoheptanoylthreonine phosphate the cytochrome was instantaneously oxidized, indicating that the cytochrome b served as electron donor for heterodisulfide reduction.

H2: heterodisulfide oxidoreductase complex from Methanobacterium thermoautotrophicum. Composition and properties.

The reduction of the heterodisulfide (CoM-S-S-HTP) of coenzyme M (H-S-CoM) and N-7-mercaptoheptanoylthreonine phosphate (H-S-HTP) with H2 is an energy-conserving step in most methanogenic Archaea. In this study, we show that in Methanobacterium thermoautotrophicum (strain Marburg) this reaction is catalyzed by a stable H2-heterodisulfide oxidoreductase complex of F420-non-reducing hydrogenase and heterodisulfide reductase. This complex, which was loosely associated with the cytoplasmic membrane, was purified 17-fold with 80% yield to apparent homogeneity. The purified complex was composed of six different subunits of apparent molecular masses 80, 51, 41, 36, 21 and 17 kDa, and 1 mol complex, with apparent molecular mass 250 kDa, contained approximately 0.6 mol nickel, 0.9 mol FAD, 26 mol non-heme iron and 22 mol acid-labile sulfur. In 25 mM Chaps, the complex partially dissociated into two subcomplexes. The first subcomplex was was composed of the 51-, 41- and 17-kDa subunits; 1 mol trimer contained 0.7 mol nickel, 10 mol non-heme iron and 9 mol acid-labile sulfur and exhibited F420-non-reducing hydrogenase activity. The other subcomplex was composed of the 80-, 36- and 21-kDa subunits; 1 mol trimer contained 0.8 mol FAD, 22 mol non-heme iron and 15 mol acid-labile sulfur and exhibited heterodi-sulfide-reductase activity. The stimulatory effects of potassium phosphate, a membrane component, uracil derivatives and coenzyme F430 on the H2:heterodisulfide-oxidoreductase activity of the purified complex are described.

Si-face stereospecificity at C5 of coenzyme F420 for F420-dependent N5,N10-methylenetetrahydromethanopterin dehydrogenase, F420-dependent N5,N10-methylenetetrahydromethanopterin reductase and F420H2:dimethylnaphthoquinone oxidoreductase.

Coenzyme F420-dependent enzymes catalyze the reversible reduction of F420 by stereospecific hydride transfer to C5 of 5-deazaflavin. Two F420-dependent enzymes have been investigated with respect to the stereochemistry of hydride transfer, the F420-dependent NADP reductase and the F420-reducing hydrogenase. Both enzymes were found to be Si-face specific. In this study we report that three additional F420-dependent enzymes are also Si-face specific: N5,N10-methylenetetrahydromethanopterin dehydrogenase, N5,N10-methylenetetrahydromethanopterin reductase and coenzyme F420H2: dimethylnaphthoquinone oxidoreductase (F420H2 dehydrogenase). Thus, all five characterized F420-dependent enzymes are Si-face specific, which is noteworthy since coenzyme F420 is functionally similar to pyridine nucleotides and both Si-face specific and Re-face specific pyridine-nucleotide-dependent enzymes exist.

Purification of a cytochrome b containing H2:heterodisulfide oxidoreductase complex from membranes of Methanosarcina barkeri.

The reduction of CoM-S-S-HTP, the heterodisulfide of coenzyme M (H-S-CoM) and N-7-mercaptoheptanoylthreonine phosphate (H-S-HTP), with H2 is an energy-conserving step in methanogenic archaea. We report here that in Methanosarcina barkeri this reaction is catalyzed by a membrane-bound multienzyme complex, designated H2:heterodisulfide oxidoreductase complex, which was purified to apparent homogeneity. The preparation was found to be composed of nine polypeptides of apparent molecular masses 46 kDa, 39 kDa, 28 kDa, 25 kDa, 23 kDa, 21 kDa, 20 kDa, 16 kDa, and 15 kDa and to contain 3.2 nmol cytochrome b, 70 to 80 nmol non-heme iron and acid-labile sulfur, 5 nmol Ni, and 0.6 nmol FAD per mg protein. The 23 kDa polypeptide possessed heme-derived peroxidase activity indicating that this polypeptide is the cytochrome b. The purified H2:heterodisulfide oxidoreductase complex catalyzed the reduction of CoM-S-S-HTP with H2 at a specific activity of 6 U/mg protein (1 U = 1 mumol.min-1), the reduction of benzylviologen with H2 at a specific activity of 66 U/mg protein and the reduction of CoM-S-S-HTP benzylviologen with H2 at a specific activity of 66 U/mg protein and the reduction of CoM-S-S-HTP HTP with reduced benzylviologen at a specific activity of 24 U/mg protein. The complex did not mediate the reduction of coenzyme F420 with H2 nor the oxidation of reduced coenzyme F420 with CoM-S-S-HTP. The reduced cytochrome b in the enzyme complex could be oxidized by CoM-S-S-HTP and re-reduced by H2. The specific rates of cytochrome oxidation and reduction were too high to be resolved under our experimental conditions. The findings suggest that the H2:heterodisulfide oxidoreductase complex is composed of a F420-non-reducing hydrogenase, a cytochrome b and heterodisulfide reductase and that cytochrome b is a redox carrier in the electron transport chain involved in CoM-S-S-HTP reduction with H2.