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PubMed1999

The modulatory effect of spermine on the glutamate-NMDA receptor is regionally variable in normal human adult cerebral cortex.

The MK-801, glutamate and polyamine binding sites on the N-methyl-D-aspartate class of glutamate receptors labelled with [3H]MK-801 were characterized in four cortical areas (sensorimotor, superior temporal, mid-frontal and occipital) from seven human adult control cases. Age, post-mortem delay, tissue storage time and sex had no significant effects on any of the parameters measured. Dissociation constants (K(D) values) for MK-801 showed similar mean values in the four cortical areas, whereas receptor densities (B(max) values) showed significant differences between sensorimotor or occipital and superior temporal or mid-frontal cortex. There were marked regional differences in the profiles of the spermine- and glutamate-incremented enhancement of specific [3H]MK-801 binding. The EC(50) for the glutamate enhancement was significantly higher in the occipital than in the mid-frontal and sensorimotor cortex, whereas maximal glutamate-enhanced binding values did not differ. The maximal enhancement of [3H]MK-801 binding by spermine and glutamate varied between the cases, ranging from zero to 40.4+/-9.3 fmol x mg protein(-1) for spermine, and from 85+/-5 to 111+/-10 fmol x mg protein(-1) for glutamate. Maximal spermine enhancement of [3H]MK-801 binding was significantly more variable in superior temporal or mid-frontal than in sensorimotor or occipital cortex. The results suggest that N-methyl-D-aspartate receptor sites, especially the polyamine site, are heterogeneous in human cerebral cortex, and show a high degree of regional and individual variability.

Age Factors↗
PubMed2005

The effect of landuse on soil organic carbon chemistry and sorption of pesticides and metabolites.

Earlier studies had shown significant differences in sorption of nine pesticides in soils collected from two landuses (native vegetation and market gardens), which could not be explained on the basis of organic carbon content alone. Consequently it was hypothesised that the differences in sorption behaviour between the two landuses may be due to variation in the chemistry of the organic carbon. In this study the relationship between sorption behaviour of the nine chemicals and soil organic carbon chemistry, as determined by solid-state (13)C NMR spectroscopy, was investigated. No significant differences were found between the two landuses in the distribution of the four main spectral regions of the (13)C NMR spectra of soil OC, except for the carbonyl fraction (165-220ppm), which may reflect the low OC content of the soils from both landuses. For all chemicals, except prometryne, the most significant (P<0.01 or P<0.001) relationship between K(d) values and types of OC was found with the aromatic (110-165ppm) or the alkyl (0-45ppm) fraction. A comparison was made of the variability of K(d) values normalized over OC (i.e. K(oc)), alkyl, aromatic and alkyl+aromatic fractions. Expressing K(d) values for all chemicals, except azinphos methyl, in soils under native vegetation as K(alkyl) or K(aromatic) greatly decreased the variability compared with the K(oc) value. However in the cultivated soils only the sorption coefficients for DEA, DIA and fenamiphos showed a decrease in variability when expressed as K(alkyl) or K(aromatic). This reflected the stronger relationship between sorption coefficients and the alkyl and aromatic fraction of soil OC in soils from native vegetation compared with those determined from the market garden soils. The different relationships between sorption coefficients and types of OC of the two landuses also suggests that the type of aromatic and alkyl carbon under the two landuses is different and NMR characterisation of the OC was not sufficient to distinguish these differences.

Adsorption↗
PubMed1996

Effects of methimazole in the early and established phases of NG-nitro-L-arginine methyl ester hypertension.

In the present study we evaluated the effects of methimazole, an antithyroid drug, on blood pressure and other variables in the early and established phases of hypertension induced by the inhibition of nitric oxide synthesis with the oral administration of NG-nitro-L-arginine methyl ester (L-NAME), 75 mg/100 ml in the drinking water. Moreover, we also evaluated the acute pressor effect of L-NAME on systemic blood pressure in control and rats treated chronically with methimazole, administered via drinking water (30 mg/100 ml). Oral administration of methimazole maintained the blood pressure of L-NAME-treated rats at normal levels 25 days after induction of hypertension. However, after 25 days of methimazole treatment in rats made hypertensive with L-NAME (for 25 days), high blood pressure was similar in methimazole-treated and non-treated L-NAME rats, despite the fact that a hypothyroid state had been achieved in the methimazole-treated rats. Acute intravenous injection of L-NAME caused a similar increase in mean arterial pressure in control and methimazole-treated rats at the lowest dose; however, smaller pressor responses were observed with increasing doses in hypothyroid rats. These results clearly demonstrate that hypothyroidism induced by methimazole prevents, but does not reverse, L-NAME hypertension and reduces the acute pressor responsiveness to L-NAME administration.

Administration, Oral↗
PubMed1990

Transient expression of polysynaptic NMDA receptor-mediated activity during neocortical development.

During a restricted period of early postnatal development, rat neocortical neurons receive a powerful N-methyl-D-aspartate (NMDA) receptor-mediated synaptic input of variable onset latency and duration. These large-amplitude excitatory postsynaptic potentials are especially pronounced in supragranular layers and are generated by activities in polysynaptic circuits. Their occurrence in cortical slices from juvenile (postnatal (P) days 11-20), but not neonatal (P5-10) or adult (greater than or equal to P28) animals, appears to be in part a consequence of the relative immaturity of gamma-aminobutyric acid (GABA)-mediated inhibition, at a time when the requisite functional excitatory circuitry has been established. The transient manifestation of strong NMDA receptor-mediated potentials coincides temporally with a 'developmental window' within which there is enhanced sensitivity for epileptogenesis and for induction of long-term synaptic modifications in rat cortex.

2-Amino-5-phosphonovalerate↗
PubMed2005

The role of nitric oxide in regulation of the cardiovascular system in reptiles.

The roles that nitric oxide (NO) plays in the cardiovascular system of reptiles are reviewed, with particular emphasis on its effects on central vascular blood flows in the systemic and pulmonary circulations. New data is presented that describes the effects on hemodynamic variables in varanid lizards of exogenously administered NO via the nitric oxide donor sodium nitroprusside (SNP) and inhibition of nitric oxide synthase (NOS) by l-nitroarginine methyl ester (l-NAME). Furthermore, preliminary data on the effects of SNP on hemodynamic variables in the tegu lizard are presented. The findings are compared with previously published data from our laboratory on three other species of reptiles: pythons (), rattlesnakes () and turtles (). These five species of reptiles possess different combinations of division of the heart and structural complexity of the lungs. Comparison of their responses to NO donors and NOS inhibitors may reveal whether the potential contribution of NO to vascular tone correlates with pulmonary complexity and/or with blood pressure. All existing studies on reptiles have clearly established a potential role for NO in regulating vascular tone in the systemic circulation and NO may be important for maintaining basal systemic vascular tone in varanid lizards, pythons and turtles, through a continuous release of NO. In contrast, the pulmonary circulation is less responsive to NO donors or NOS inhibitors, and it was only in pythons and varanid lizards that the lungs responded to SNP. Both species have a functionally separated heart, so it is possible that NO may exert a larger role in species with low pulmonary blood pressures, irrespective of lung complexity.

Animals↗
PubMed1996

Distribution of 7-alkyl-2'-deoxyguanosine adduct levels in human lung.

Human lung tissue is frequently studied as a target organ for DNA damage from carcinogen-DNA adducts. In order to assess the distribution of carcinogen-DNA adducts in human lung, we measured 7-methyl-2'-deoxyguanosine-3'-monophosphate (7-methyl-dGp), 7-ethyl-2'-deoxyguanosine-3'-monophosphate (7-ethyl-dGp) and 4-hydroxy-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts in different lobes. The first two result from exposure to N-nitrosamines, including tobacco-specific nitrosamines, and the latter only from tobacco-specific nitrosamines. Using a chemically-specific 32P-postlabeling assay for 7-alkyl-2'-deoxyguanosines, adducts were measured in eight separate lung segments of ten autopsy donors. 7-Methyl-dGp levels were detected in all eighty samples (range from 0.3 to 11.5 adducts/10(7) dGp; mean 2.5 +/- 2.3 adducts/10(7) dGp). 7-Ethyl-dGp were detected in all but five of the samples (range from <0.1 to 7.1 adducts/10(7) dGp; mean 1.6 +/- 1.7 adducts/10(7) dGp). 7-Methyl-dGp levels were approximately 1.5-fold higher than 7-ethyl-dGp levels, and they were positively correlated with each other in most individuals. There was no consistent pattern of adduct distribution in the different lobar segments. Most individuals, especially those with the lowest levels, had similar levels among the lobes, while those with the highest levels had a widely variable pattern ranging as much as ten-fold. Moreover 7-methyl-dGp and 7-ethyl-dGp levels in all people showed a highly significant inter-individual variation (P = 0.0001). The levels of 7-alkyl-2'-deoxyguanosine among individuals could not be explained by differences in tobacco exposure (measured by serum cotinine), onset of death, gender, age, race, blood ethanol, or ventilation and perfusion variability. In an effort to corroborate 7-alkyl-2'-deoxyguanosine adducts variability among lobes or individuals, we sought to determine a correlation with HPB-releasing DNA adducts as an independent marker of tobacco exposure. However, this tobacco- specific carcinogen-DNA adduct could not be detected in four individuals tested (detection limit: 0.3 adducts per 10(7) dGp). Based upon the lack of 7-alkyl-2'-deoxyguanosine discernible adduct patterns, no conclusions could be drawn regarding a potential relationship to lobar cancer incidence. The results indicate that in studies of carcinogen-DNA adducts, such as 7-alkyl-dGp in human lungs, for most individuals a random lung sample would be representative of other parts of the lungs. Some individuals however might be misclassified due to highly variable 7-alkyl-dGp levels.

Adult↗
PubMed1992

Methylations in hemoglobin from monozygotic twins discordant for cigarette smoking: hereditary and tobacco-related factors.

Dose monitoring of exogenous methylators by measurement of N-methylvaline in hemoglobin (Hb) is rendered difficult due to a relatively high, variable background in unexposed persons. A kinetic study indicates intracellular S-adenosylmethionine to be a main source of these background methylations. A comparison of twin pairs indicates that the variation in methylvaline levels is partly hereditary (P less than 0.001). Therefore, in a study of monozygotic twin pairs discordant for tobacco smoking the resolving power of the monitoring was increased and in vivo doses of methylators from the smoke could be more easily monitored through their adducts to Hb. Probably, twin studies offer a useful tool for the identification and quantification of electrophiles of endogenous and exogenous origin.

Adult↗
PubMed1986

Inheritance of DNA methylation in Coprinus cinereus.

We examined the inheritance of 5-methylcytosine residues at a centromere-linked locus in the basidiomycete Coprinus cinereus. Although methylated and unmethylated tracts were inherited both mitotically and meiotically the lengths of these tracts were variable. This variation was not confined to any one phase of the life cycle of the organism, and it usually involved the simultaneous de novo methylation of at least four HpaII-MspI sites. We also found that the higher levels of methylation at this locus were transmitted through meiosis, regardless of the level of methylation of the homologous chromosome.

5-Methylcytosine↗
PubMed1995

Differential effects of L-NAME on blood pressure and heart rate responses to acetylcholine and bradykinin in cynomolgus primates.

NG-nitro-L-arginine methyl ester (L-NAME) has been reported to have variable effects on the vasodilator response to acetylcholine (ACh) and bradykinin (BK) in vivo. Whether administration of L-NAME affects mean arterial pressure (MAP) or heart rate (HR) responses to ACh or BK was examined in conscious cynomolgus primates. ACh (0.1-10 micrograms/kg i.v.) lowered MAP by 6% to 37%, responses which were inhibited (25-62%) in the presence of L-NAME (1-100 mg/kg i.v.). Although L-NAME increased MAP similarly at doses of 10 and 100 mg/kg, only the 100-mg/kg dose inhibited the hypotensive responses induced by the higher doses of ACh. By comparison, nitroprusside (5 micrograms/kg i.v.)-induced hypotensive responses were not inhibited by L-NAME. Phenylephrine (20 micrograms kg-1 min-1 i.v.) increased MAP and lowered HR to levels statistically similar to that of L-NAME but did not alter ACh-induced hypotensive responses. ACh dose-dependently decreased HR, both in the absence and presence of L-NAME or phenylephrine. In pentobarbital-anesthetized monkeys, ACh-induced hypotensive responses were inhibited by 75% to 94% in the presence of L-NAME; BK (0.3-1 microgram/kg i.v.) responses were only modestly affected (< or = 50%). Therefore, in conscious primates, L-NAME affects the basal release of nitric oxide (NO) at lower doses than those required to inhibit its release stimulated by ACh. Also, L-NAME does not appear to act as a cholinergic antagonist or affect the functional mechanisms that control baroreflex responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed2004

A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines.

BACKGROUND: Tumor cell lines are commonly used as experimental tools in cancer research, but their relevance for the in vivo situation is debated. In a series of 11 microsatellite stable (MSS) and 9 microsatellite unstable (MSI) colon cancer cell lines and primary colon carcinomas (25 MSS and 28 MSI) with known ploidy stem line and APC, KRAS, and TP53 mutation status, we analyzed the promoter methylation of the following genes: hMLH1, MGMT, p16INK4a (CDKN2A alpha-transcript), p14ARF (CDKN2A beta-transcript), APC, and E-cadherin (CDH1). We compared the DNA methylation profiles of the cell lines with those of the primary tumors. Finally, we examined if the epigenetic changes were associated with known genetic markers and/or clinicopathological variables. RESULTS: The cell lines and primary tumors generally showed similar overall distribution and frequencies of gene methylation. Among the cell lines, 15%, 50%, 75%, 65%, 20% and 15% showed promoter methylation for hMLH1, MGMT, p16INK4a, p14ARF, APC, and E-cadherin, respectively, whereas 21%, 40%, 32%, 38%, 32%, and 40% of the primary tumors were methylated for the same genes. hMLH1 and p14ARF were significantly more often methylated in MSI than in MSS primary tumors, whereas the remaining four genes showed similar methylation frequencies in the two groups. Methylation of p14ARF, which indirectly inactivates TP53, was seen more frequently in tumors with normal TP53 than in mutated samples, but the difference was not statistically significant. Methylation of p14ARF and p16INK4a was often present in the same primary tumors, but association to diploidy, MSI, right-sided location and female gender was only significant for p14ARF. E-cadherin was methylated in 14/34 tumors with altered APC further stimulating WNT signaling. CONCLUSIONS: The present study shows that colon cancer cell lines are in general relevant in vitro models, comparable with the in vivo situation, as the cell lines display many of the same molecular alterations as do the primary carcinomas. The combined pattern of epigenetic and genetic aberrations in the primary carcinomas reveals associations between them as well as to clinicopathological variables, and may aid in the future molecular assisted classification of clinically distinct stages.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Comparison of chemical analysis of residual monomer in a chemical-cured dental acrylic material to an FTIR method.

OBJECTIVE: The purpose of this work was to perform quantitative analysis of residual monomer in chemical-cured acrylic using an infrared spectroscopic method and to compare it to an accepted form of quantitative chemical analysis. Identical samples of acrylic were analyzed and compared using Fourier transform infrared (FTIR) spectroscopy with multiple standard additions vs. "wet" chemical analysis of bromination followed by titration. METHODS: Two 6 g disks from a single mix of cold-cured acrylic (Lang Dental Mfg. Co., Inc.) were prepared and cured in room air for 1 hr using a ratio of 12 g of powder to 8 mL of liquid. One of the cold-cured acrylic disks was dissolved in glacial acetic acid and analyzed according to the "wet" technique described by Smith and Bains (Smith and Bains, 1956). The other disk was dissolved in methyl isobutyrate (MIBT). Five aliquots, zero plus four incremental additions of pure methyl methacrylate (MMA), were prepared from the MIBT solution. Absorption spectra were collected for all five aliquots. The data were plotted with the ratio of the mass of methyl methacrylate added to the mass of aliquot of MIBT solution as the independent variable, and the absorption, corrected for dilution, as the dependent variable. Least squares fit of the data yielded the slope and intercept. The ratio of intercept to slope divided by the weight fraction of the acrylic disk dissolved in MIBT yielded the concentration of methyl methacrylate in the disk. RESULTS: The plot of absorbance as a function of mass ratio of MMA added to MIBT solution was linear over the concentration range covered (r = 0.999). Analysis of the spectroscopic data revealed a concentration of residual C = C double bonds of 0.32 +/- 0.01 mol/kg. "Wet" chemical analysis of the acrylic yielded a concentration of residual C = C double bonds of 0.288 +/- 0.003 mol/kg. There was significant difference (students t-test, 99% confidence level) between the two techniques with respect to the amount of residual monomer calculated. SIGNIFICANCE: The multiple standard additions technique works well for the quantitative analysis of small amounts of residual double bonds in the poly(methyl methacrylate) chemical-cured acrylic polymer system, eliminating the need for an internal standard or external calibration.

Absorption↗
PubMed2004

Quantitative analysis of DNA methylation in chronic lymphocytic leukemia patients.

Changes in the genomic DNA methylation level have been found to be closely associated with tumorigenesis. In order to analyze the relation of aberrant DNA methylation to clinical and biological risk factors, we have determined the cytosine methylation level of 81 patients diagnosed with chronic lymphocytic leukemia (CLL). The analysis was based on DNA hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with BODIPY FL EDA. Derivatives were separated by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. We analyzed potential correlations between DNA methylation levels and numerous patient parameters, including clinical observations and biological data. As a result, we observed a significant correlation with the immunoglobulin variable heavy chain gene (VH) mutation status. This factor has been repeatedly proposed as a reliable prognostic marker for CLL, which suggests that the methylation level might be a valuable factor in determining the prognostic outcome of CLL. We are now in the process of refining our method to broaden its application potential. In this context, we show here that the oxidation of the fluorescence marker in the samples and the evaporation of methanol in the electrolytes can be prevented by a film of paraffin oil. In summary, our results thus establish capillary electrophoresis as a valuable tool for analyzing the DNA methylation status of clinical samples.

Boron Compounds↗
PubMed1976

The effect of magnesium ions on action spectra for reactions mediated by photosystems I and II in spinach chloroplasts.

Action spectra were measured for positive changes in variable fluorescence (emission greater than 665 nm) excited by a beam of 485 nm chopped at 75 HZ. The action of two further beams were compared, one being variable, the other (reference) constant with respect to wavelength and intensity. Comparison was achieved by alternating the reference and the variable wavelength beams at 0.3 HZ and adjusting the intensity of the latter such as to cancel out any 0.3 HZ component in the 75 HZ fluorescence signal. The relative action then was obtained as the reciprocal of the intensity of the variable wavelength beam. Similarly, action spectra were measured for O2 evolution with ferricyanide/p-phenylenediamine as electron acceptor, and for O2 uptake mediated by methyl viologen with ascorbate 3-(p-chlorophenyl)-1,1-dimethylurea as electron donor in the presence of 2,6-dichlorophenolindophenol. Addition of 5 mM MgCl2 increases the relative action around 480 nm for the change in variable fluorescence and p-phenylenediamine-dependent O2 evolution, and decreases it for methyl viologen-mediated O2 uptake with 2,6-dichlorophenolindo-phenol/ascorbate as electron donor in the presence of 3-(p-chlorophenyl)-1,1-dimethylurea. The change in variable fluorescence and O2 evolution are stimulated by MgCl2, whereas O2 uptake is inhibited by it. The results are discussed in terms of a model assuming a tripartite organization of the photosynthetic pigments (Thornber, J. P. and Highkin, H. R. (1974) Eur. J. Biochem. 41, 109-116; Butler, W. L. and Kitajima, M. (1975) Biochim. Biophys. Acta 396, 72-85). MgCl2 is thought to promote energy transfer to Photosystem II from a light-harvesting pigment complex serving both photosystems.

Chloroplasts↗
PubMed2005

Trophic effect of enteral rehabilitative therapy in rat small bowel transplantation.

INTRODUCTION: We aimed to observe the trophic effects on graft structure and recipient metabolism of enteral rehabilitative therapy in rat small bowel transplantation (SBT). METHODS: Forty-eight recipients of rat allogeneic heterotopic SBT (Sprague-Dawley to Wistar rat) were divided into four groups randomly according to the presence of glutamine or growth hormone in the nutritional support regimen. We compared the histologic indices of graft mucosa and metabolic variables, including changes in body weight, nitrogen balance, urinary 3-methyl histidine excretion, and plasma albumin levels during 14 days after transplantation. RESULTS: Glutamine and growth hormone promoted recovery of graft structure and improved recipient protein metabolism, as evidenced by indices of the enteral rehabilitative therapy. CONCLUSIONS: Glutamine and growth hormone potentiated their effects in enteral rehabilitative therapy, which produced potent trophic effects on graft structure and recipient metabolism in rat SBT.

Animals↗
PubMed1990

Fatty acid profiles in the family Leptospiraceae.

Fatty acid profiles of six leptospira strains representative of genera, species, and serogroups within the family Leptospiraceae were determined by gas liquid chromatography (GLC) of fatty acid methyl ester (FAME) derivatives. The influence of methodological and biological variables on FAME profiles of the same strain was tested. FAME profiles were sharply affected by the fatty acid composition of the culture medium but not by the growth phase. Twenty-four FAME peaks were selected on the basis of their presence in repeated gas chromatographic runs of single strains. Inter-strain divergences of FAME profiles were quantified by linear regression analysis (LR). Step-wise divergences in FAME profiles were observed between strains at serogroup, species, and genus levels.

Chromatography, Gas↗
PubMed2002

Infrared and Raman spectra, conformational stability, ab initio calculations of structure, and vibrational assignment of ethynylmethyl cyclobutane.

The infrared spectra (3200-300 cm(-1)) of the gas and solid and the Raman spectra (3200-30 cm(-1) of the liquid with quantitative depolarization values and the solid have been recorded of ethynylmethyl cyclobutane (cyclobutylmethyl acetylene), c-C4H7CH2C[ triple bond]CH. Both the equatorial and the axial conformers have been identified in the fluid phases and both the gauche and trans conformations of the methyl acetylenic group have been identified for each ring conformer. Variable temperature (-105 to -150 degrees C) studies of the infrared spectra of the sample dissolved in liquid krypton have been carried out. From these data the enthalpy differences have been determined to the 112 +/- 11 cm(-1) (1.34 +/- 0.13 kJ mol) between the most stable equatorial-trans (Et) conformer and the equatorial-gauche (Eg) conformer which is the second most stable conformer and 327 +/- 35 cm(-1) (3.91 + 0.42 kJ/mol) with the axial-gauche (Ag) conformer which is the least stable conformer. The enthalpy difference between the axial-trans (At) and the equatorial-gauche(Eg) is 56 +/- 6 cm(-1). At ambient temperature there is approximately 33% of the Et conformer, 38% of the Eg form, 15% of the At and 14% of the Ag conformer. For the polycrystalline solid the Eg conformer is the only form present which probably results form the packing in the crystal. A complete vibrational assignment is proposed for both equatorial conformers and additionally a few of the fundamentals of the At and Ag conformers have been assigned. The vibrational assignments are supported by normal coordinate calculations utilizing the force constants from ab initio MP2/6-31G(d) calculations. Complete equilibrium geometries have been determined for all four possible rotamers by ab initio calculations employing the 6-31G(d) and 6-311 + + G(d,p) basis sets at levels of restricted Hartree-Fock (RHF) and /or Moller-Plesset (MP2) with full electron correlation by the perturbation method to second order. The results are discussed and compared to those obtained for some similar molecules.

Cyclobutanes↗
PubMed1999

Evidence for a proposed intermediate redox state in the CO/CO(2) active site of acetyl-CoA synthase (Carbon monoxide dehydrogenase) from Clostridium thermoaceticum.

When samples of the enzyme in the C(red1) state were reduced with Ti(3+) citrate, the C-cluster stabilized in an EPR-silent state. Subsequent treatment with CO or dithionite yielded C(red2). The EPR-silent state formed within 1 min of adding Ti(3+) citrate, while C(red2) formed after 60 min. Ti(3+) citrate appeared to slow the rate by which C(red2) formed from C(red1) and stabilize the C-cluster in the previously proposed C(int) state. This is the first strong evidence for C(int), and it supports the catalytic mechanism that required its existence. This mechanism is analogous to those used by flavins and hydrogenases to convert between n = 2 and n = 1 processes. Ti(3+) citrate had a different effect on enzyme in a CO(2) atmosphere; it shifted reduction potentials of metal centers (relative to those obtained using CO) and did not stabilize C(int). Different redox behavior was also observed when methyl viologen and benzyl viologen were used as reductants. This variability was exploited to prepare enzyme samples in which EPR from C(red2) was present without interfering signals from B(red). The saturation properties of B(red) depended upon the redox state of the enzyme. Three saturation "modes", called Sat1-Sat3, were observed. Sat1 was characterized by a sharp g = 1.94 resonance and low-intensity g = 2. 04 and 1.90 resonances, and was observed in samples poised at slightly negative potentials. Sat2 was characterized by weak intensity from all three resonances, and was strictly associated with intermediate redox states and the presence of CO(2). Sat3 was characterized by strong broad resonances with normalized intensities essentially unchanged relative to nonsaturating conditions, and was observed at the most negative potentials. Each mode probably reflects different spatial relationships among magnetic components in the enzyme.

Aldehyde Oxidoreductases↗