The membrane-bound (particulate) form of methane monooxygenase from Methylococcus capsulatus (Bath) has been solubilised using the non-ionic detergent dodecyl-P-D-maltoside. A wide variety of detergents were tested and found to solubilise membrane proteins but did not yield methane monooxygenase in a form that could be subsequently activated. After solubilisation with dodecyl-P-D-maltoside, enzyme activity was recovered using either egg or soya-bean lipids. Attempts to further purify the solubilized methane monooxygenaser protein into its component polypeptides were unsuccessful and resulted in complete loss of enzyme activity. The major polypeptides present in the solubilised enzyme had molecular masses of 49 kDa, 23 kDa and 22 kDa which were similar to those seen in crude extracts [Prior, S. D. & Dalton H. (1985) J. Cen. Microbiol. 131, 155-1631. Studies on substrate and inhibitor specificities indicated that the membrane-associated and solubilised forms of methane rnonooxygenase were quite similar to each other but differed substantially from the well-characterised soluble methane monooxygenase found in cells grown in a low copper regime and synthesised independently of the particulate methane monooxygenase.The enzyme methane monooxygenase (MMO) catalyses the NADHz and 02-dependent oxidation of methane to methanol :Two distinct forms of the enzyme have been observed which differ in cellular location, a soluble methane monooxygenase (sMMO) expressed during growth in conditions of copper limitation and a particulate (membrane-bound) methane monooxygenase (pMMO) expressed during growth in the presence of excess copper [l]. The sMMO from Methylococcus cupsulutus (Bath) has been well characterised and is comprised of three components; protein A, a non-haem-iron-containing protein of molecular mass 210 kDa which contains the site of methane oxidation; protein B (15.7 kDa) which regulates the flow of electrons through the complex and protein C, an FAD/FezS2-containing protein (42 kDa) which interacts with NADH2 and passes electrons to protein A [2 -41. In contrast to the detailed studies which have been performed on sMMO much less information is available on pMMO.Tonge et al. [5] reported that pMMO from Methylosinus trichosporium OB3B could be released from the membrane using a variety of techniques (detergent treatment, sonication or phospholipase treatment) and that this enzyme could utilise ascorbate as well as NADH as the electron donor for the MMO reaction. This solubilised enzyme was later purified [6] and resolved into three components, a copper-containing protein (47 kDa), a protein of molecular mass 9400 kDa and a cytochrome c, , (13 kDa); after purification this enzyme system utilised ascorbate but not NADH as the electron donorCorrespondence to H. Dalton Since the purification of pMMO requires the prior solubilisation of the enzyme, and we were unable to remove pMMO of M. capsulatus (Bath) from the membrane by the method of Tonge et al.[6], we investigated this problem further and describe here methods f...

Mitochondrial glutamine synthetase (EC 6.3.1.2) is the primary ammonia-detoxifying enzyme in avian liver and is therefore analogous in function to carbamoylphosphate synthetase I (ammonia) (EC 6.3.4.16) in mammalian liver. In mammalian liver, glutamine synthetase is cytosolic and its distribution is restricted to a few hepatocytes around the terminal venules. These cells do not express carbamoylphosphate synthetase I. Using immunocytochemistry, we show here that there is little or no zonation of glutamine synthetase in avian liver. Rather, it is broadly distributed to most hepatocytes, much like carbamoyl-phosphate synthetase I in mammalian liver. In situ hybridization with a cloned glutamine synthetase cDNA probe showed the distribution of glutamine synthetase mRNA in both mammalian and avian liver to correspond to the distribution of immunoreactive protein.Neither glutamine synthetase nor carbamoyl-phosphate synthetase I and ornithine transcarbamoylase (EC 2.1.3.3) are strictly zoned in liver of the Texas tortoise or of an Argentine tree frog, both ofwhich possess a complete urea cycle but which may also rely on glutamine synthetase for ammonia detoxication. These latter results suggest that the mutually exclusive expression of either carbamoyl-phosphate synthetase I or glutamine synthetase may be unique to mammalian liver.

Determination of the avidity of immunoglobulin G (IgG) directed against a specific marker has become an established diagnostic tool for identifying or excluding acute infections with pathogens. A novel assay format termed AVIcomp (avidity competition based on mass action) circumventing the conventional chaotropic format has been developed for determination of the avidity of marker-specific IgG in patient specimens. Its applications for cytomegalovirus (CMV) and Toxoplasma gondii are presented. Specific high-avidity IgG from the patient specimen is selectively blocked using a soluble antigen in a sample pretreatment reagent, and the amount of remaining specific low-avidity IgG is determined relative to that in an untreated control. The comparison of the conventional chaotropic format, represented by the Radim CMV IgG Avidity assay, and the newly developed AVIcomp method, as exemplified by the Architect CMV IgG Avidity assay, on blood drawn within 4 months after seroconversion revealed a sensitivity of 100% (97.3% by an alternative calculation) for the AVIcomp format versus 87.5% (75.7% by an alternative calculation) for the chaotropic avidity assay. The specificity on 312 CMV IgG reactive and CMV IgM nonreactive specimens from pregnant women was 100% for the AVIcomp assay and 99.7% for the conventional avidity assay. The Architect Toxo IgG Avidity assay showed an agreement of 97.2% with the bioMérieux Vidas Toxo IgG Avidity Assay employing chaotropic reagents. These performance data suggest that the AVIcomp format shows superior sensitivity and equivalent specificity for the determination of IgG avidity to assays based on the chaotropic method and that the AVIcomp format may also be applicable to other disease states.Over recent years, numerous publications have shown that the avidity of marker-specific immunoglobulin G (IgG) is a suitable tool for distinguishing between acute and recurrent or past infection with a pathogen (7). Avidity tests have been developed for rubella virus (17), Toxoplasma gondii (5,8,18), cytomegalovirus (CMV) (1), varicella-zoster virus (11), human immunodeficiency virus (25), hepatitis viruses (22,26,27,29), Epstein-Barr virus (28), and others. For immunocompetent, untreated individuals, the presence of low-avidity IgG directed against pathogens may indicate a recent infection, whereas the presence of high-avidity IgG excludes a primary infection (13,14). During the early immune response, IgG antibodies are targeting a multiplicity of different epitopes of the pathogen with relatively low avidity. Clonal selection finally results in high-avidity antibodies directed mainly against a limited number of immunodominant epitopes (5).For T. gondii infections, high-avidity IgG serves to rule out a recent infection as well; however, low-avidity results are not indicative of a recent or past infection (16). This is due to the fact that the antibody avidity maturation kinetics for T. gondii

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