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J T McFarland

Publications and source records attributed to J T McFarland.

At least 19 recordsLinked to original sources
PubMed2001

Efficacy of digital rectal examination after radiotherapy for prostate cancer.

PURPOSE: Digital rectal examination is widely performed for following patients with localized prostate cancer after definitive therapy. This examination has marginal efficacy for detecting initial prostate cancer and postoperative recurrence. To determine the efficacy of digital rectal examination in terms of new information provided after radiotherapy we analyzed the results of digital rectal examination in the followup of patients with prostate cancer after radiotherapy. MATERIALS AND METHODS: We performed a nonrandomized study in 235 consecutive patients with prostate cancer followed at a large tertiary care military hospital between January 1, 1995 and December 31, 1999. All patients had been treated with prostate radiotherapy and had no evidence of metastatic disease at the first visit within that interval. Digital rectal examination was done at followup and the main outcome measure was new information provided by that examination. RESULTS: A total of 1,544 digital rectal examinations were performed in 1,627 visits. New information was provided by digital rectal examination in only 30% of 286 abnormal examinations, of which more than three-quarters were related to bleeding and would otherwise have been noted on routine examination by the primary care provider. All 8 persistent recurrent prostate nodules were noted in the context of increasing prostate specific antigen. CONCLUSIONS: Routine digital rectal examination in patients with prostate cancer after radiotherapy may be omitted from followup protocols.

Combined Modality Therapy↗
PubMed1989

Spectral observation of an acyl-enzyme intermediate of lipoprotein lipase.

There have been several studies indicating that hydrolysis reactions of fatty acid esters catalyzed by lipases proceed through an acyl-enzyme intermediate typical of serine proteases. In particular, one careful kinetic study with the physiologically important enzyme lipoprotein lipase (LPL) is consistent with rate-limiting deacylation of such an intermediate. To observe the spectrum of acyl-enzyme and study the mechanism of LPL-catalyzed hydrolysis of substrate, we have used a variety of furylacryloyl substrates including 1,2-dipalmitoyl-3-[(beta-2-furylacryloyl)triacyl]glyceride (DPFATG) to study the intermediates formed during the hydrolysis reaction catalyzed by the enzyme. After isolation and characterization of the molecular weight of adipose LPL, we determined its extinction coefficient at 280 nm to quantitate the formation of any acyl-enzyme intermediate formed during substrate hydrolysis. We observed an intermediate at low pH during the enzyme-catalyzed hydrolysis of (furylacryloyl)imidazole. This intermediate builds early in the reaction when a substantial amount of substrate has hydrolyzed but no product, furylacrylate, has been formed. The acyl-enzyme has a lambda max = 305 nm and a molar extinction coefficient of 22,600 M-1 cm-1; these parameters are similar to those for furylacryloyl esters including the serine ester. These data provide the first spectral evidence for a serine acyl-enzyme in lipase-catalyzed reactions. The LPL hydrolysis reaction is base catalyzed, exhibiting two pKa values; the more acidic of these is 6.5, consistent with base catalysis by histidine. The biphasic rates for substrate disappearance or product appearance and the absence of leaving group effect indicate that deacylation of intermediate is rate limiting.

Acylation↗
PubMed1987

Electrostatic effect upon association of reduced nicotinamide adenine dinucleotide and equine liver alcohol dehydrogenase.

The rate of association of equine liver alcohol dehydrogenase and its coenzymes exhibits a large pH dependence with slower rates at basic pH and an observed kinetic pKa value of approximately 9-9.5. This pH dependence has been explained by invoking local active site electrostatic effects which result in repulsion of the negatively charged coenzyme and the ionized hydroxyl anion form of the zinc-bound water molecule. We have examined a simpler hypothesis, namely, that the pH dependence results from the electrostatic interaction of the coenzyme and the enzyme which changes from an attractive interaction of the negatively charged coenzyme and the positively charged enzyme to a repulsive interaction between the two negatively charged species at the isoelectric point for the enzyme (pH 8.7). We have tested this proposal by examining the ionic strength dependence of the association rate constant at various pH values. These data have been interpreted by using the Wherland-Gray equation, which we have shown can be applied to the kinetics of enzyme-coenzyme association. Our results indicate that the shielding of the buffer electrolyte changes from a negative to a positive value as the charge on the protein changes at the isoelectric point. This result is exactly that which is predicted for electrostatic effects that depend on the charge of the protein molecule and is not consistent with predictions based upon the local active site effects. At low ionic strength values of 10 mM or less, approximately 75% of the observed pH dependence results from the enzyme electrostatic effects; the remaining pH dependence may result from active site effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗
PubMed1986

Energetics of beta-oxidation. Reduction potentials of general fatty acyl-CoA dehydrogenase, electron transfer flavoprotein, and fatty acyl-CoA substrates.

We have determined reduction potentials for porcine mitochondrial general fatty acyl-CoA dehydrogenase (GAD) and electron transfer flavoprotein (ETF) using an anaerobic spectroelectrochemical titration method. Computer simulation techniques were used to analyze the absorbance data. Nernst plots of the simulated data gave E'0, 7.1, quinone/semiquinone = -0.014 V and E'0, 7.1, semiquinone/hydroquinone = -0.036 V for ETF and E'0, 7.1, quinone/semiquinone = -0.155 V and E'0, 7.1, semiquinone/hydroquinone = -0.122 V for GAD. Using these techniques we have also determined a conditional reduction potential of -0.156 V for the chromophore producing fatty acyl-CoA substrate beta-2-furylpropionyl-CoA. From this value and our previous determination of the equilibrium constant for the transhydrogenation reaction between beta-2-furylpropionyl-CoA and the oxidized substrate crotonyl-CoA (Keq = 10.4), we have determined a reduction potential of -0.126 V for the butyryl-CoA/crotonyl-CoA couple. In light of the structural similarity between butyryl-CoA and octanoyl-CoA, the optimal substrate for GAD, the reduction potential for octanoyl-CoA should be similar to that for butyryl-CoA; i.e. fatty acyl-CoA substrates and GAD are essentially isopotential. The ability of octanoyl-CoA to reduce GAD quantitatively (Keq = 9.0) poses a dilemma in light of the nearly equal reduction potentials. We postulate that the stable charge-transfer complex formed between enzyme and optimal product is significantly lower in energy than enzyme and product and thus is responsible for pulling the reaction toward completion.

Acyl Coenzyme A↗
PubMed1986

The EH2 reduced intermediate of glutathione reductase contains oxidised flavin-while EH4 does not.

Glutathione reductase is a flavoprotein whose x-ray structure has been established. Functional data and the x-ray structure are consistent with a mechanism of reaction in which NADPH reacts with the enzyme to produce a two electron, EH2, and four electron, EH4, intermediate. The former is competent for the transfer of electrons to the substrate glutathione. Several structures are possible for the two NADPH intermediates; in order to aid in the determination of the structure of these intermediates, we have determined their resonance Raman spectra at two excitation frequencies. These studies establish that the EH2 intermediate is an oxidized flavin species while the EH4 species is not. Furthermore, the most likely structure for EH2 involves a charge transfer donation of electrons from the anion of cys-63 to the N5 position of flavin.

Flavins↗
PubMed1985

Structure-function correlation of fatty acyl-CoA dehydrogenase and fatty acyl-CoA oxidase.

We have employed a new pseudosubstrate, beta-(2-furyl)propionyl coenzyme A (FPCoA), to study the functional properties of two enzymes, fatty acyl-CoA dehydrogenase from porcine liver and fatty acyl-CoA oxidase from Candida tropicalis, involved in the oxidation of fatty acids. Previous studies from our laboratory have shown that the dehydrogenase exhibits oxidase activity at the rate of dissociation of the product charge-transfer complex. This raises the question of the difference in functionality between these two flavoproteins. To investigate these differences, we have compared the pH dependence of product formation, the isotope effects using tetradeuterio-FPCoA, and the spectral properties and chemical reactivity of the product charge-transfer complexes formed with the two enzymes. The pH dependencies of the reaction of FPCoA with electron-transfer flavoprotein (ETF) for the dehydrogenase and of the reaction of FPCoA with O2 for the oxidase are quite similar. Both reactions proceed more rapidly at basic pH values while substrate binds more tightly at acidic pH values. These data for both enzymes are consistent with a mechanism in which enzyme is involved in protonation of the carbonyl group of substrate followed by base-catalyzed removal of the C-2 proton from substrate. The C-2 anion of substrate may then serve as the active species in reduction of enzyme-bound flavin. The deuterium isotope effects for both enzyme systems are primary across the entire pH range, assuring that the chemically important step of substrate oxidation is rate limiting in these steady-state kinetic experiments. The two enzymes differ in the chemical reactivity of their product charge-transfer complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl-CoA Dehydrogenase↗
PubMed1983

Resonance Raman studies of ETE dehydrogenase (an iron sulfur flavoprotein).

ETF Dehydrogenase is an iron sulfur flavoprotein responsible for the transfer of electrons between electron transfer flavoprotein (ETF) and CoQ of the electron transport chain. We have determined the resonance Raman spectrum of this enzyme observing in the process at least seven of thirteen flavin bands in the 1100cm-1-1600 cm-1 region of the Raman spectrum. The positions of three of these bands, II, IX, and X (see Figure I and Table I for band numbering system) in ETF dehydrogenase is very similar to their positions in aqueous solution of flavins in which water is hydrogen bonded to N-1, N-5, C=0(2), C=0(4), and N-H(3) of flavin. Conversely the positions of the flavin Raman bands are considerably shifted from those of flavin in nonhydrogen bonding solvent. The positions of bands II, IX, and X are nearly identical to those in the flavoprotein glutathione reductase; x-ray structural investigations on this enzyme indicate that there is extensive hydrogen bonding between FAD and protein in this molecule. A previous study in our laboratory has demonstrated that metal complexation at N-5 and C=0(4) with either Ru or Ag produces large shifts in the positions of Raman bands II, VI, IX, and X. None of these shifts are observed in ETF dehydrogenase indicating that there is no direct inner sphere coordination of Fe to flavin. In addition to the Raman bands of flavin observed in our spectrum, we also observe one band that is in the Fe-S stretching region observed for a variety of Fe-S proteins. This band is located at 331 cm-1. The frequency of the band corresponds to the 335 cm-1 band associated with the strongest Fe-S stretching mode in the 4Fe-4S protein ferrodoxin from C. pasterianum. The observed frequency is quite different from that of the 3Fe-3S proteins such as ferrodoxin(II) from D. gigas. Finally, ETF dehydrogenase shows no loss of activity or visual evidence of photodegradation in the laser beam as most other FeS proteins do.

Animals↗
PubMed1983

Kinetic methods for the study of the enzyme systems of beta-oxidation.

Kinetic methods for studying the reactions of the "general" fatty acyl CoA dehydrogenase under three sets of substrate and enzyme concentration conditions have been developed. The reaction of butyryl-CoA and electron transfer flavoprotein (ETF) can be studied either under steady-state conditions with enzyme at catalytic concentration or under single-turnover conditions with enzyme in excess. Under the latter conditions, acyl-CoA dehydrogenase acts both as a catalyst and an ultimate electron-transfer acceptor. The reductive half-reaction of butyryl-CoA and enzyme can also be studied in a separate kinetic experiment. Comparison of the pH dependences of the rate constants and isotope effects of the steady-state reaction of butyryl-CoA and ETF with the same parameters for the reductive half-reaction is consistent with a mechanism involving transfer of electrons from butyryl-CoA to ETF within a ternary complex. An alternative mechanism in which the reductive half-reaction takes place prior to the binding and reaction of ETF seems unlikely because the pH 8.5 isotope effect on the reductive half-reaction is much larger than that on the complete reaction in spite of the fact that the rates of the reactions are comparable. The pH dependence of the Km for substrate and KI for inhibitor is consistent with a mechanism for transfer of electrons within the ternary complex which involves protonation of the C = O group of substrates. The protonation labilizes the C-2 proton and base catalysis of the removal of the C-2 proton results in the production of the active enzyme-substrate species, namely the C-2 anion of substrate.

Acyl-CoA Dehydrogenase↗
PubMed1981

Mechanistic studies on fatty acyl-CoA dehydrogenase.

It has previously been shown that the "partial" reaction between fatty acyl-CoA dehydrogenase and acyl-CoA substrate is pH-dependent (larger rate constants at basic pH) and shows a biphasic rate profile indicative of formation of an initial charge transfer complex between the C-2 anion of substrate and enzyme. The present investigation indicates that the complete reaction between acyl-CoA and electron transfer flavoprotein shows a pH profile dependent upon ionization of a single basic group with pKa = 7.7. these facts are consistent with electron transfer which occurs through an obligatory charge transfer complex between the C-2 anion of substrate and oxidized FAD at the enzyme active site. The anion of acetoacetyl-CoA forms a charge transfer complex with enzyme which serves as a model for the putative catalytically active complex mentioned above. Resonance Raman investigation of this acetoacetyl-CoA-enzyme complex indicates that the 1586 cm-1 band is coupled strongly to the charge transfer electronic transition. Since this vibrational band is associated with vC=N at N-5, C-4a of the flavin ring, we suggest that electron transfer takes place at this site.

Acyl Coenzyme A↗
PubMed1980

Metal-flavin complexation. A resonance Raman investigation.

Several groups have recently shown that high quality resonance Raman spectra can be obtained for flavin species in spite of their intense fluorescence. We are interested in obtaining the resonance Raman spectra of flavins in various chemical environments in order to determine whether the spectra are useful in probing the chemical interaction between flavins and protein in flavoenzymes. We have obtained the resonance Raman spectrum of a nonfluorescent Ag+ complex of FMN. Several large changes occur in the FMN resonance Raman spectrum upon Ag+ complexation; among these are changes in the 1580 cm-1 region of the FMN spectrum (assigned to nu C=N at N-5 and C-4a), the 1410 cm-1 region and the 1260 cm-1 region (associated with a vibration having some delta N-N-H character at N-3). Similar changes are observed in the same region of a Ru2+-FMN complex. Since these spectral changes occur in two metal flavin complexes with very different electronic spectra, they would seem to be due to vibrational changes induced by metal complexation at N-5 and the oxygen at C-4 of flavin rather than the details of the vibronic interactions which give rise to the resonance enhancement of the spectrum. A structure for the Ag+-FMN complex is suggested. This study has potential physiological significance, because it illustrates the possible role of resonance Raman spectroscopy as a tool for the determination of direct flavin metal interaction in dilute aqueous solution of metalloflavoproteins.

Chemical Phenomena↗
PubMed1980

The deuterium isotope effect upon the reaction of fatty acyl-CoA dehydrogenase and butyryl-CoA.

Reduction of the oxidized FAD at the active site of porcine liver fatty acyl-CoA dehydrogenase by butyryl-CoA results in bleaching of 30 to 60% of the 450 nm absorbance of flavin and in the production of a new absorbance band at 565 nm. The wavelength of the maximum absorbance of this new band (lambda max) is dependent on the chemical nature of the substrate, e.g. this band occurs at 645 nm when beta-2-furylpropionyl-CoA (a pseudosubstrate) reacts with enzyme. Since lambda max for this band is substrate-dependent, the band is most likely the result of charge transfer complex formation between oxidized fatty acyl-CoA, and the reduced flavin of the enzyme. The rate profile for the reaction of butyryl-CoA and enzyme is biphasic at 450 nm but consists of a single exponential process at 565 nm. The rate constant for reaction at 565 nm is approximately 12 s-1 ((butyryl-CoA) = 2.5 x 10(-5) M, pH 8.6), and the 450 nm rate profile can be fit to a rate equation for two sequential reactions of rate constant 12 s-1 and 3.4 s-1, the amount of flavin reduction in each kinetic step being approximately 50%. The deuterium isotope effect measured on each step of the biphasic time course of the 450 nm reaction is very large, in the range kH/kD = 30 to 50. The rate profile at 565 nm for perdeuterobutyryl-CoA is markedly different than that for the protiobutyryl-CoA in that it is biphasic. It appears that two rate processes have been separated by virtue of different isotope effects; the first process shows kH/kD = 2 while the second shows kH/kD = 50. The data are interpreted in terms of a mechanism involving an obligatory charge transfer complex.

Acyl Coenzyme A↗