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P C Wilkins

Publications and source records attributed to P C Wilkins.

13 recordsLinked to original sources
PubMed1998

The hydroxylase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath) exists in several forms as shown by electrospray-ionisation mass spectrometry.

The hydroxylase of the soluble methane monooxygenase from the bacterium Methylococcus capsulatus (Bath) has been investigated by means of electrospray-ionisation mass spectrometry (ESI-MS) and liquid chromatography ESI-MS (LC/ESI-MS). The hydroxylase is a non-heme diiron protein consisting of three pairs of non-identical subunits (alpha approximately 60 kDa, beta approximately 45 kDa and gamma approximately 20 kDa). Liquid chromatographic separation of the hydroxylase subunits was required before MS analysis in order to detect the alpha-subunit. The masses measured for the three subunits were found to disagree with those calculated from their gene sequences. Experiments involving the use of CNBr and trypsin cleavage followed by LC/ESI-MS and MS/MS analyses permitted the location and correction of errors in the sequences deduced from the use of cDNA. The ESI-MS results also showed that the alpha-subunit of the hydroxylase exists in multiple forms which result from cleavage of the protein. This observation explains a number of enigmatic features of the protein previously reported in the literature and illustrates the pivotal role of ESI-MS in complementing data obtained from molecular biology for the characterisation of the primary sequence of proteins.

Cyanogen Bromide↗
PubMed1996

Direct electrochemistry of the hydroxylase of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

The redox properties of the hydroxylase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath) have been thoroughly investigated. Previous studies used redox indicator titrations and spectroscopic methods for the determination of the concentrations of reduced species. Herein we report, for the first time, direct electrochemistry (i.e. without the use of mediators) of the diiron centers of the hydroxylase from M. capsulatus (Bath) at a modified gold electrode giving rise to two waves at 4(+/- 10) mV and -386(+/- 14) mV versus saturated calomel electrode (SCE). In addition, the effects of proteins B and B' on the redox reactions were determined. The redox potentials of the complex with protein B are -25(+/- 14) mV and -433(+/- 8) mV versus SCE whereas protein B' had no effect though it did alter the effect of protein B on the redox potentials.

Electrochemistry↗
PubMed1994

Further evidence for multiple pathways in soluble methane-monooxygenase-catalysed oxidations from the measurement of deuterium kinetic isotope effects.

The data from the deuterium isotope experiments in this study show that the primary kinetic isotope effect for methane oxidation catalysed by soluble methane monooxygenase from Methylococcus capsulatus (Bath) is very small, < 2. In contrast, the primary kinetic isotope effect for -CH3 group oxidation in toluene is large, > 7. A mechanistic pathway in which a substrate radical is formed from hydrogen atom abstraction by a ferryl species is believed to operate for CH4, the toluene -CH3 group and similar alkanes. Direct oxygen atom addition, rather than H atom abstraction, is indicated for aromatic ring oxidations in benzene and toluene and for styrene oxide formation from styrene. Thus, more than one mechanistic pathway appears to operate in soluble methane-monooxygenase-catalysed reactions and, in some cases, the pathway chosen may be dictated by the substrate. In the soluble methane-monooxygenase-catalysed oxidation of toluene the rates of: (a) substrate dissociation from the enzyme-substrate complex, (b) product formation and (c) product release (benzyl alcohol and p-cresol) from the enzyme-product complex are comparable in magnitude. Therefore all three of these steps are partially rate-determining in the soluble methane monooxygenase catalytic cycle for toluene oxidation.

Benzene↗
PubMed1993

Abduction of iron(III) from the soluble methane monooxygenase hydroxylase and reconstitution of the binuclear site with iron and manganese.

The apo-form of the soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath) was prepared via chelation of iron(III) with 3,4-dihydroxybenzaldehyde. The apohydroxylase was reconstituted by the anaerobic addition of Fe(II) followed by air oxidation. The enzyme thus prepared regained 85-90% of its original catalytic activity. The incorporation of two manganese(II) ions/mol of apohydroxylase was monitored by EPR spectroscopy. The Mn(II) ions occupy the native diiron active site and remain in the +2 oxidation state. The EPR data suggest strong coupling between the two Mn(II) ions and retention of the mu-hydroxo (alkoxo) bridge. The results of this study indicate that the M. capsulatus (Bath) hydroxylase contains a single diiron site.

Apoenzymes↗
PubMed1993

Resistance of human squamous carcinoma cells to transforming growth factor beta 1 is a recessive trait.

Because most human squamous carcinoma cell lines of the aerodigestive and genital tracts are refractory to the antiproliferative action of transforming growth factor beta 1 (TGF beta 1) in vitro, we have begun to identify the causes for resistance of squamous carcinoma cell lines to TGF beta 1 by using somatic cell genetics. Two stable hybrid cell lines (FaDu-HKc.1 and FaDu-HKc.2) were obtained by fusing a TGF beta 1-resistant human squamous carcinoma cell line, FaDu-HygR, with a human papilloma virus 16-immortalized, TGF beta 1-sensitive, human foreskin keratinocyte cell line, HKc-neoR. Whereas TGF beta 1 did not inhibit DNA synthesis in parental FaDu-HygR cells, it reduced DNA synthetic activity of HKc-neoR, FaDu-HKc.1, and FaDu-HKc.2 cells by 75-85% (IC50, 2-5 pM). Although squamous carcinoma cells express lower than normal levels of TGF beta 1 type II receptors on their cell surface, TGF beta 1 type II receptor mRNA was detected in all four cell lines. Recessive genes involved in TGF beta 1 signaling may be localized to the distal portion of chromosome 18q, as this was the sole chromosomal region of homozygous deletion in parental FaDu-HygR cells. Furthermore, our previous observation that mutant p53 decreases sensitivity of keratinocytes to TGF beta 1 was supported by the finding that the level of the mutant p53 protein expressed by the hybrid cell lines was greatly reduced. In summary, TGF beta 1 resistance of FaDu cells appears to be recessive and is presumably due to the loss of one or more post-receptor elements of the signaling pathway.

Base Sequence↗
PubMed1993

Activation of the hydroxylase of sMMO from Methylococcus capsulatus (Bath) by hydrogen peroxide.

Hydrogen peroxide can activate the non-heme binuclear iron-containing hydroxylase of soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) in the catalysis of oxidation of methane and other sMMO substrates. The reductase, protein B, O2 and NADH are normally required for catalytic activity, but can be replaced by H2O2 serving as the source of both oxygen and electrons for the reaction. Similar results have been observed in a different strain of MMO from Methylosinus trichosporium OB3b (Andersson, K.K., Froland, W.A., Lee, S.-K. and Lipscomb, J.D. (1991) New J. Chem. 15, 410-415). The Km,app for H2O2 was found to be 66 mM. Labelled oxygen experiments show that the oxygen atom in the product in the peroxide driven system is derived from H2O2 and not O2. Using C2-C5 alkanes and 2-butene as substrates it was shown that the product distribution differed in the complete sMMO and H2O2-driven systems, indicating that more than one pathway is available to the enzyme. Protein B, which is required for catalytic activity in the complete system, was found to be an inhibitor of the hydroxylase/H2O2 system. It was also observed that protein B not only affected the activity, but also the selectivity of carbon hydroxylation with 2-methylbutane as substrate.

Dose-Response Relationship, Drug↗
PubMed1992

Biological methane activation involves the intermediacy of carbon-centered radicals.

The spin-trapping technique has demonstrated that carbon-centered radicals are produced during soluble-methane-monooxygenase catalysis of the hydroxylation of several different types of substrate. The resulting spin-adducts were identified from the hyperfine splitting constants in their EPR spectra. Isotopic labelling showed unequivocally that the trapped radicals were derived from substrate. The carbon-centered substrate radicals are believed to result from hydrogen-atom abstraction by a ferryl species in a cytochrome-P-450-like mechanism. No hydroxy radical nor an oxygen-based radical of any kind was detected in any of the spin-trapping experiments.

Carbon↗
PubMed1991

Metastatic properties of the human prostatic cell line, PPC-1, in athymic nude mice.

Limitation in the number of human prostatic cell lines has created a gap in knowledge regarding the in-vivo progression of this common cancer. The recently isolated primary prostatic carcinoma cell line, PPC-1, has been shown to be tumorigenic in athymic nude mice. These cells are now shown to form metastases to secondary sites in 10 of 12 animals in this initial study. Metastases were localized to lung and lymph nodes, and the tumor histology closely resembled that of the undifferentiated, rapidly dividing primary tumors. This is the first report describing the metastatic properties of a primary prostatic cancer cell line. PPC-1 cells are therefore likely to represent a good model system for the study of human prostate cancer progression.

Animals↗
PubMed1989

Kinetics and thermodynamics of the reaction of deoxyhemerythrin with nitric oxide.

Deoxyhemerythrin reacts with NO to form a 1:1 adduct shown spectrophotometrically. The kinetics of the formation have been studied directly by stopped-flow measurements at four different temperatures (0.0-23.6 degrees C). The kinetics of the dissociation have been studied, also by stopped-flow techniques, at five different temperatures (4.0-35.1 degrees C) using three different scavengers [Fe(II)(edta)2-, O2 and sperm whale deoxymyoglobin], which gave similar values. For the formation kf = (4.2 +/- 0.2) x 10(6) M-1 s-1, delta Hf not equal = 44.3 +/- 2.3 kJ mol-1, delta Sf not equal to = 30 +/- 8 J mol-1 K-1 and for the dissociation kd = 0.84 +/- 0.02 s-1, delta Hd not equal to 95.6 +/- 2.1 kJ mol-1 delta Sd not equal to = 74 +/- 7 J mol-1 K-1 (25 degrees C, I = 0.2 M and pH 7-8.1). From the kinetic data the thermodynamic data for the formation of HrNO were calculated: Kf = (5.0 +/- 0.3) x 10(6) M-1, delta H = -51.3 +/- 3.1 kJ mol-1 and delta S = -44 +/- 11 J mol-1 K-1 (25 degrees C). The kinetic data suggest that NO occupies the same iron(II) site in deoxyhemerythrin as oxygen does. The equilibrium constant for the formation of Fe(II)(edta)(NO)2- has been redetermined: K1 = (1.45 +/- 0.07) x 10(7) M-1, delta H = -77.5 +/- 1.5 kJ and mol-1 and delta S = -123.5 J mol-1 K-1 (25 degrees C).

Biotransformation↗
PubMed1987

Acid-assisted anion interaction with deoxyhemerythrin.

The interaction of N3-, CNO- and F- with deoxyhemerythrin was monitored from pH 6 to 9 and 3 degrees C to 25 degrees C at an ionic strength 0.5 M (Na2SO4) by using competition with O2. A proton is involved in forming the adduct and is not lost from the protein even at pH values as high as 9. Thermodynamic and kinetic parameters for the interaction of each anion are obtained. It is considered that the proton is introduced as the acid HX and the corresponding parameters for the reaction of HX with deoxyhemerythrin are calculated. These are compared with those for the O2 reaction with deoxyhemerythrin. The adducts are remarkably inert and have dissociation half-lives ranging from 7 s (N3-) to 70 s (CNO- and F-) at 25 degrees C. This stability is ascribed to protonation of a bridged hydroxy group or even bridge-breakage.

Acids↗