Mimicking respiratory phosphorylation using purified enzymes

The enzymes of oxidative phosphorylation are a striking example of the functional association of multiple enzyme complexes, working together to form ATP from cellular reducing equivalents. These complexes, such as cytochrome c oxidase or the ATP synthase, are typically investigated individually and therefore, their functional interplay is not well understood. Here, we present methodology that allows the co-reconstitution of purified terminal oxidases and ATP synthases in synthetic liposomes. The enzymes are functionally coupled via proton translocation where upon addition of reducing equivalents the oxidase creates and maintains a transmembrane electrochemical proton gradient that energizes the synthesis of ATP by the F1F0 ATP synthase. The method has been tested with the ATP synthases from Escherichia coli and spinach chloroplasts, and with the quinol and cytochrome c oxidases from E. coli and Rhodobacter sphaeroides, respectively. Unlike in experiments with the ATP synthase reconstituted alone, the setup allows in vitro ATP synthesis under steady state conditions, with rates up to 90 ATP×s(-1)×enzyme(-1). We have also used the novel system to study the phenomenon of "mild uncoupling" as observed in mitochondria upon addition of low concentrations of ionophores (e.g. FCCP, SF6847) and the recoupling effect of 6-ketocholestanol. While we could reproduce the described effects, our data with the in vitro system does not support the idea of a direct interaction between a mitochondrial protein and the uncoupling agents as proposed earlier.

Adenosine 5'-Phosphosulfate Sulfotransferase and Adenosine 5'-Phosphosulfate Reductase Are Identical Enzymes

Adenosine 5′-phosphosulfate (APS) sulfotransferase and APS reductase have been described as key enzymes of assimilatory sulfate reduction of plants catalyzing the reduction of APS to bound and free sulfite, respectively. APS sulfotransferase was purified to homogeneity from Lemna minor and compared with APS reductase previously obtained by functional complementation of a mutant strain of Escherichia coli with an Arabidopsis thaliana cDNA library. APS sulfotransferase was a homodimer with a monomer M r of 43,000. Its amino acid sequence was 73% identical with APS reductase. APS sulfotransferase purified from Lemna as well as the recombinant enzyme were yellow proteins, indicating the presence of a cofactor. Like recombinant APS reductase, recombinant APS sulfotransferase used APS (K m = 6.5 μM) and not adenosine 3′-phosphate 5′-phosphosulfate as sulfonyl donor. TheV max of recombinant Lemna APS sulfotransferase (40 μmol min−1 mg protein−1) was about 10 times higher than the previously published V max of APS reductase. The product of APS sulfotransferase from APS and GSH was almost exclusively SO3 2−. Bound sulfite in the form ofS-sulfoglutathione was only appreciably formed when oxidized glutathione was added to the incubation mixture. Because SO3 2− was the first reaction product of APS sulfotransferase, this enzyme should be renamed APS reductase.

Optimized on-line enantioselective capillary electrophoretic method for kinetic and inhibition studies of drug metabolism mediated by cytochrome P450 enzymes.

Pharmacokinetic and pharmacodynamic properties of a chiral drug can significantly differ between application of the racemate and single enantiomers. During drug development, the characteristics of candidate compounds have to be assessed prior to clinical testing. Since biotransformation significantly influences drug actions in an organism, metabolism studies represent a crucial part of such tests. Hence, an optimized and economical capillary electrophoretic method for on-line studies of the enantioselective drug metabolism mediated by cytochrome P450 enzymes was developed. It comprises a diffusion-based procedure, which enables mixing of the enzyme with virtually any compound inside the nanoliter-scale capillary reactor and without the need of additional optimization of mixing conditions. For CYP3A4, ketamine as probe substrate and highly sulfated γ-cyclodextrin as chiral selector, improved separation conditions for ketamine and norketamine enantiomers compared to a previously published electrophoretically mediated microanalysis method were elucidated. The new approach was thoroughly validated for the CYP3A4-mediated N-demethylation pathway of ketamine and applied to the determination of its kinetic parameters and the inhibition characteristics in presence of ketoconazole and dexmedetomidine. The determined parameters were found to be comparable to literature data obtained with different techniques. The presented method constitutes a miniaturized and cost-effective tool, which should be suitable for the assessment of the stereoselective aspects of kinetic and inhibition studies of cytochrome P450-mediated metabolic steps within early stages of the development of a new drug.

Novel mechanisms for PKC family enzymes to regulate PI3K signaling pathway

Phosphatidylinositol 3-kinase (PI3K) generates membrane phospholipids that serve as second messengers to recruit signaling proteins to plasma membrane consequently regulating cell growth and survival. PI3K is a heterodimer consisting of a catalytic p110 subunit and a regulatory p85 subunit. Association of the p85 with other signal proteins is critical for induced PI3K activation. Activated PI3K, in turn, leads to signal flows through a variety of PI3K effectors including PDK1, AKT, GSK3, BAD, p70 S6K and NFκB. The PI3K pathway is under regulation by multiple signal proteins representing cross-talk between different signaling cascades. In this study, we have evaluated the role of protein kinase C family kinases on signaling through PI3K at multiple levels. Firstly, we observed that the action of PKC specific inhibitors like Ro-31-8220 and GF109203X was associated with an increased AKT phosphorylation and activity, suggesting that PKC kinases might play a negative role in the regulation of PI3K pathway. Then, we demonstrated the stimulation of AKT by PKC inhibition was dependent on functional PI3K enzyme and able to be transmitted to the AKT effector p70 S6K. Furthermore, we showed an inducible physical association between the PKCζ isotype and AKT, which was accompanied by an attenuated AKT activity. However, a kinase-dead form of PKC failed to affect AKT. In the second part of our research we revealed the ability of a different PKC family member, PKCδ to bind to the p85 subunit of PI3K in response to oxidative stress, a process requiring the activity of src tyrosine kinases. The interaction was demonstrated to be a direct and specific contact between the carboxyl terminal SH2 domain of p85 and tyrosine phosphorylated PKCδ. Several different types of agonists were capable to induce this association including tyrosine kinases and phorbol esters with PKCδ tyrosine phosphorylation being integral components. Finally, the PKCδ-PI3K complex was related to a reduction in the AKT phosphorylation induced by src. A kinase-deficient mutant of PKCδ was equally able to inhibit AKT signal as the wild type, indicative of a process independent of PKCδ catalytic activity. Altogether, our data illustrate different PKC isoforms regulating PI3K pathway at multiple levels, suggesting a mechanism to control signal flows through PI3K for normal cell activities. Although further investigation is required for full understanding of the regulatory mechanism, we propose that complex formation of signal proteins in PI3K pathway and specific PKC isoforms plays important role in their functional linkage. ^

The evidence behind the level of a random urine protein:creatinine in the diagnosis of preeclampsia: A systematic review

Objective. To determine the accuracy of the urine protein:creatinine ratio (pr:cr) in predicting 300 mg of protein in 24-hour urine collection in pregnant patients with suspected preeclampsia. ^ Methods. A systematic review was performed. Articles were identified through electronic databases and the relevant citations were hand searching of textbooks and review articles. Included studies evaluated patients for suspected preeclampsia with a 24-hour urine sample and a pr:cr. Only English language articles were included. The studies that had patients with chronic illness such as chronic hypertension, diabetes mellitus or renal impairment were excluded from the review. Two researchers extracted accuracy data for pr:cr relative to a gold standard of 300 mg of protein in 24-hour sample as well as population and study characteristics. The data was analyzed and summarized in tabular and graphical form. ^ Results. Sixteen studies were identified and only three studies met our inclusion criteria with 510 total patients. The studies evaluated different cut-points for positivity of pr:cr from 130 mg/g to 700 mg/g. Sensitivities and specificities for pr:cr of 130mg/g -150 mg/g were 90-93% and 33-65%, respectively; for a pr:cr of 300 mg/g were 81-95% and 52-80%, respectively; for a pr:cr of 600-700mg/g were 85-87% and 96-97%, respectively. ^ Conclusion. The value of a random pr:cr to exclude pre-eclampsia is limited because even low levels of pr:cr (130-150 mg/g) may miss up to 10% of patients with significant proteinuria. A pr:cr of more than 600 mg/g may obviate a 24-hour collection.^

INACTIVATION OF MUTAGENIC DRUGS AND THEIR METABOLITES IN THE URINE OF PATIENTS ADMINISTERED ANTINEOPLASTIC THERAPY

Urines from patients administered mutagenic antineoplastic drugs were significantly mutagenic in the Ames assay, and hence may pose a genotoxic hazard to hospital personnel or family members caring for the patient. The urines were tested for mutagenicity in several different strains of Salmonella typhimurium that were uvr positive or negative (TA98, TA100, TA102, UTH8413, UTH8414). The urines were fractionated by high pressure liquid chromatography (HPLC) and the fractions assayed for mutagenicity in the strains in which the whole urine was mutagenic. Only fractions of urines containing the parent compound (cisplatin, doxorubicin, or mitomycin) were mutagenic; no other fraction showed significant mutagenicity. However, urine containing cyclophosphamide had two fractions that were mutagenic. One fraction, the fraction containing cyclophosphamide, required metabolic activation for mutagenicity. The other fraction did not require activation for mutagenicity.^ The chemical and mutagenic stability of these urines at room temperature was assayed over a 14 day period. The parent compound degraded within the first seven days, but the urines remained mutagenic. Cis-platinum was chemically stable in the urine; however, the urine decreased in mutagenicity. The decrease was probably the result of stable ligands binding to the platinum.^ Inactivation methods were developed to reduce the genotoxic hazard. Urine containing cisplatin was inactivated by complexing the cisplatin with diethyldithiocarbamate (DDTC). Oxidation with NaOCl of urines containing mitomycin and doxorubicin (sodium thiosulfate must be added to the doxorubicin urine) results in mutagenic inactivation. Inactivation of urine containing cyclophosphamide requires oxidation with alkaline potassium permaganate and trapping of active degradation products with sodium thiosulfate. Urines containing these drugs can be inactivated, but not always by the same method that inactivates the drug alone in solution. Therefore, in the future development of inactivation methods, both chemical and mutagenic assays are necessary to determine effectiveness. Methods of inactivation of mutagenic excreta developed in this study are both effective and practical. ^

Activity of ETS enzymes and phosphatase in the Sea of Okhotsk and Kuril region in summer 1992 and 1993

An estimate of rate of transformation of organic matter and regeneration of nutrients (in particular phosphorus) was calculated for different regions of the Sea of Okhotsk. The rate was estimated by means of rate of complete oxidation of organic matter to CO2 and H2O catalyzed by enzymes of the electron transport system (ETS) and rate of hydrolytic splitting of phosphate from organic phosphorus compounds catalyzed by alkaline phosphatase. Organic matter destruction rate was at its maximum on the shelf of Kamchatka and Sakhalin, as well as in the layer of maximum oxygen gradients in deep waters. It was found that zones of intensive primary production were characterized by high rates of phosphorus regeneration, which provided for 80% of primary production when concentration of mineral phosphorus was low.

Enzyme activities of digestive and metabolic enzymes of Calanus finmarchicus from Maria S. Merian cruise MSM26 in spring 2013

The present study aimed to contribute to the knowledge on the intraspecific variations of enzyme activities in populations of Calanus finmarchicus from different longitudes across the North Atlantic Ocean and their relation to changing environmental conditions. C. finmarchicus was sampled across the North Atlantic in basins with decreasing temperature regimes from east to west (Iceland Basin, Irminger Basin and Labrador Basin) in late March/early April 2013. Potential maximum enzyme activities of digestive (proteinases and lipases/esterases) and metabolic (citrate synthase) enzymes of copepods from all sampling stations were analysed and thermal profiles (5-50°C) of enzyme activities were determined. In order to investigate its acclimation potential, C. finmarchicus were acclimated to 4°C and 15°C for two weeks and thermal profiles of enzyme activities were compared afterwards.