Kinetic model-based factor analysis of dynamic sequences for 82-rubidium cardiac positron emission tomography.

PURPOSE: Factor analysis has been pursued as a means to decompose dynamic cardiac PET images into different tissue types based on their unique temporal signatures to improve quantification of physiological function. In this work, the authors present a novel kinetic model-based (MB) method that includes physiological models of factor relationships within the decomposition process. The physiological accuracy of MB decomposed (82)Rb cardiac PET images is evaluated using simulated and experimental data. Precision of myocardial blood flow (MBF) measurement is also evaluated. METHODS: A gamma-variate model was used to describe the transport of (82)Rb in arterial blood from the right to left ventricle, and a one-compartment model to describe the exchange between blood and myocardium. Simulations of canine and rat heart imaging were performed to evaluate parameter estimation errors. Arterial blood sampling in rats and (11)CO blood pool imaging in dogs were used to evaluate factor and structure accuracy. Variable infusion duration studies in canine were used to evaluate MB structure and global MBF reproducibility. All results were compared to a previously published minimal structure overlap (MSO) method. RESULTS: Canine heart simulations demonstrated that MB has lower root-mean-square error (RMSE) than MSO for both factor (0.2% vs 0.5%, p < 0.001 MB vs MSO, respectively) and structure (3.0% vs 4.7%, p < 0.001) estimations, as with rat heart simulations (factors: 0.2% vs 0.9%, p < 0.001 and structures: 3.0% vs 6.7%, p < 0.001). MB blood factors compared to arterial blood samples in rats had lower RMSE than MSO (1.6% vs 2.2%, p =0.025). There was no difference in the RMSE of blood structures compared to a (11)CO blood pool image in dogs (8.5% vs 8.8%, p =0.23). Myocardial structures were more reproducible with MB than with MSO (RMSE=3.9% vs 6.2%, p < 0.001), as were blood structures (RMSE=4.9% vs 5.6%, p =0.006). Finally, MBF values tended to be more reproducible with MB compared to MSO (CV= 10% vs 18%, p =0.16). The execution time of MB was, on average, 2.4 times shorter than MSO (p < 0.001) due to fewer free parameters. CONCLUSIONS: Kinetic model-based factor analysis can be used to provide physiologically accurate decomposition of (82)Rb dynamic PET images, and may improve the precision of MBF quantification.

Purification and characterization of an acid phosphatase from the commercial mushroom Agaricus bisporus.

Acid phosphatase [AP; EC 3.1.3.2], a key enzyme involved in the synthesis of mannitol in Agaricus bisporus, was purified to homogeneity and characterized. The native enzyme appeared to be a high molecular weight type glycoprotein. It has a molecular weight of 145 kDa and consists of four identical 39-kDa subunits. The isoelectric point of the enzyme was found at 4.7. Maximum activity occurred at 65 degrees C. The optimum pH range was between 3.5 and 5.5, with maximum activity at pH 4.75. The enzyme was unaffected by EDTA, and inhibited by tartrate and inorganic phosphate. The enzyme exhibits a Km for p-nitrophenylphosphate and fructose-6-phosphate of 370 microM and 3.1 mM, respectively. A broad substrate specificity was observed with significant activities for fructose-6-phosphate, glucose-6-phosphate, mannitol-1-phosphate, AMP and beta-glycerol phosphate. Only phosphomonoesters were dephosphorylated. Antibodies raised against the purified enzyme could precipitate AP activity from a cell-free extract in an anticatalytic immunoprecipitation test.

Purification and characterization of dimethylamine:5-hydroxybenzimidazolyl-cobamide methyltransferase from Methanosarcina barkeri Fusaro.

Dimethylamine:5-hydroxybenzimidazolylcobamide methyltransferase (DMA-MT) was purified from cells of Methanosarcina barkeri Fusaro grown on trimethylamine. In the presence of methylcobalamine:coenzyme M methyltransferase isoenzyme II [MT2(II)] the enzyme quite specifically catalyzed the stoichiometric conversion of dimethylamine (apparent Km = 0.45 mM) and 2-mercaptoethane-sulfonate (coenzyme M) to monomethylamine and methyl-coenzyme M. Monomethylamine was a competitive inhibitor of the reaction (Ki = 4.5 mM). The apparent molecular mass of DMA-MT was 100 kDa and the enzyme was found to be a dimer, composed of identical 50-kDa subunits. A corrinoid content of 0.9 +/- 0.1 mol B12/mol holoenzyme was calculated from HPLC analysis. The as-isolated methyltransferase was inactive, but it could be reductively reactivated. Activation required the presence of methyltransferase-activating protein, ATP and dimethylamine. Incubation with these compounds resulted in the methylation of the corrinoid prosthetic group.

Activation and reaction kinetics of the dimethylamine/coenzyme M methyltransfer in Methanosarcina barkeri strain Fusaro.

Dimethylamine/5-hydroxybenzimidazolylcobamide methyltransferase (DMA-MT) from Methanosarcina barkeri Fusaro catalyzes (Vmax = 4700 nmol x min(-1) x mg(-1) protein; k(cat) = 7.8 s(-1)) the transfer of a methyl group from dimethylamine (apparent Km = 0.45 mM) to its corrinoid prosthetic group to yield monomethylamine (MMA) and the methylated enzyme. The product, MMA, is a competitive inhibitor of the reaction (apparent Ki = 5.5 mM). The methyl group bound to the corrinoid prosthetic group of DMA-MT is subsequently transferred to coenzyme M in a reaction mediated by methylcobalamin/coenzyme M methyltransferase isoenzyme II [MT2(II)], which binds with high affinity to DMA-MT (apparent Km = 0.22 microM). As isolated, DMA-MT is inactive, but it can enzymically be reactivated by methyltransferase activating protein (MAP), ATP, and hydrogenase. Apart from the established role in corrinoid activation, ATP was found to act as a powerful allosteric effector on the methyltransferase reaction. The results of kinetic studies, supported by the resolution of as-yet partially purified auxiliary protein fractions, demonstrate that DMA-MT, MT2(II), MAP, and hydrogenase are the only enzymic components involved in the dimethylamine/coenzyme M methyltransfer in M. barkeri Fusaro.

Involvement of methyltransferase-activating protein and methyltransferase 2 isoenzyme II in methylamine:coenzyme M methyltransferase reactions in Methanosarcina barkeri Fusaro.

The enzyme systems involved in the methyl group transfer from methanol and from tri- and dimethylamine to 2-mercaptoethanesulfonic acid (coenzyme M) were resolved from cell extracts of Methanosarcina barkeri Fusaro grown on methanol and trimethylamine, respectively. Resolution was accomplished by ammonium sulfate fractionation, anion-exchange chromatography, and fast protein liquid chromatography. The methyl group transfer reactions from tri- and dimethylamine, as well as the monomethylamine:coenzyme M methyltransferase reaction, were strictly dependent on catalytic amounts of ATP and on a protein present in the 65% ammonium sulfate supernatant. The latter could be replaced by methyltransferase-activating protein isolated from methanol-grown cells of the organism. In addition, the tri- and dimethylamine:coenzyme M methyltransferase reactions required the presence of a methylcobalamin:coenzyme M methyltransferase (MT2), which is different from the analogous enzyme from methanol-grown M. barkeri. In this work, it is shown that the various methylamine:coenzyme M methyltransfer steps proceed in a fashion which is mechanistically similar to the methanol:coenzyme M methyl transfer, yet with the participation of specific corrinoid enzymes and a specific MT2 isoenzyme.

Purification and properties of an enzyme involved in the ATP-dependent activation of the methanol:2-mercaptoethanesulfonic acid methyltransferase reaction in Methanosarcina barkeri.

In Methanosarcina barkeri the transfer of the methyl group from methanol to 2-mercaptoethanesulfonic acid is catalyzed by the concerted action of two methyltransferases. The first one is the corrinoid-containing methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT1), which binds the methyl group of methanol to its corrinoid prosthetic group. MT1 is only catalytically active when the cobalt atom of the corrinoid is present in the highly reduced Co(I) state. In the course of its purification and even during catalysis, MT1 becomes oxidatively inactivated. The enzyme, however, may be reductively reactivated by a suitable reducing system (hydrogen and hydrogenase), ATP, and an enzyme called methyltransferase activation protein (MAP). In order to elucidate its role in the reactivation process, MAP was purified to apparent homogeneity. The protein had an Mr = 60,000. Preincubation of the enzymic components involved with 8-azido-ATP or with ATP demonstrated MAP to be the primary site of action of ATP. In agreement herewith, the protein was autophosphorylated by [gamma-32P]ATP in a 1:1 stoichiometry. Phosphorylated MAP substituted for ATP in the activation of MT1, and the addition of increasing amounts of MAP phosphate resulted in a corresponding increase of active MT1. However, in the presence of limiting amounts of MAP, maximal activation of MT1 could be achieved during a lag phase provided ATP was present, indicating that MAP acts as a catalyst. This paper is the first to report on the presence, isolation, and function of a phosphorylated protein in a methanogenic archaeon.