Intracellular singlet oxygen generation by phagocytosing neutrophils in response to particles coated with a chemical trap.

To determine if singlet oxygen (O2(1 delta g)) is produced by neutrophils (PMNs) during the process of phagocytosis, glass beads were coated with a specific chemical trap for O2(1 delta g), 9,10-diphenylanthracene (DPA). Singlet oxygen, but not other reactive oxygen species, reacts rapidly with DPA at a rate of kr = 1.3 x 10(6) M-1 s-1 to form a stable product, DPA-endoperoxide (Corey, E. J., and Taylor, W. C. (1964) J. Am. Chem. Soc. 86, 3881-3882; Wasserman, H. H., Scheffer, J. R., and Cooper, J. L. (1972) J. Am. Chem. Soc. 94, 4991-4996; Turro, N. J., Chow, M.-F., and Rigaudy, J. (1981) J. Am. Chem. Soc. 103, 7218-7224). The production of DPA-endoperoxide was determined by ultraviolet spectroscopy as a decrease in DPA absorbance at 355 nm. The absorbance of DPA was normalized to the absorbance of perylene, which was included in the coating on the beads as a nonreactive, internal standard. In the present study, DPA- and perylene-coated beads were initially allowed to adhere to fibronectin-coated coverslips. PMNs were then added to the bead-coated coverslips and allowed to adhere and phagocytose the beads for 1 h at 37 degrees C. In some experiments, 4B-phorbol-12-myristate-13-acetate (PMA) (1 ng/2.5 x 10(7) cells/ml), a known activator of the PMN NADPH-oxidase, was added as a co-stimulant. The amount of O2(1 delta g) produced by phagocytically stimulated PMNs was calculated to be 11.3 +/- 4.9 nmol of O2(1 delta g)/1.25 x 10(6) cells. Low dose PMA co-stimulation increased the production of O2(1 delta g) to 14.1 +/- 4.1 nmol/1.25 x 10(6) cells. Averaged together these amounts represent approximately 19 +/- 5.0% of the total oxygen consumed by PMNs in response to DPA- and perylene-coated beads. The specificity of the DPA reaction with O2(1 delta g) was confirmed by warming to 120 degrees C, which releases O2(1 delta g) from the DPA-endoperoxide, regenerating the parent DPA compound (Wasserman et al., 1972; Turro et al., 1981) and the absorbance at 355 nm. In addition, beta-carotene, an avid quencher of O2(1 delta g), was included in the coating of some bead preparations; assays in which these beads were used showed no change in the absorbance at 355 nm. Singlet oxygen production by myeloperoxidase was also measured using the coated bead assay and the results suggest that this is a major pathway by which singlet oxygen is generated in phagocytically stimulated PMNs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Perturbation of sarcoplasmic reticulum calcium release and phenol red absorbance transients by large concentrations of fura-2 injected into frog skeletal muscle fibers

Intact single twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with two indicator dyes: phenol red, to monitor a previously described signal (denoted delta pHapp; Hollingworth and Baylor. 1990. J. Gen. Physiol. 96:473-491) possibly reflective of a myoplasmic pH change following action potential stimulation; and fura-2, to monitor the associated change in the myoplasmic free calcium concentration (delta[Ca2+]). Additionally, it was expected that large myoplasmic concentrations of fura-2 (0.5-1.5 mM) might alter delta pHapp, since it was previously found (Baylor and Hollingworth. 1988. J. Physiol. 403:151-192) that the Ca2(+)-buffering effects of large fura-2 concentrations: (a) increase the estimated total concentration of Ca2+ (denoted by delta[CaT]) released from the sarcoplasmic reticulum (SR), but (b) reduce and abbreviate delta[Ca2+]. The experiments show that delta pHapp was increased at the larger fura-2 concentrations; moreover, the increase in delta pHapp was approximately in proportion to the increase in delta[CaT]. At all fura-2 concentrations, the time course of delta pHapp, through time to peak, was closely similar to, although probably slightly slower than, that of delta[CaT]. These properties of delta pHapp are consistent with an hypothesis proposed by Meissner and Young (1980. J. Biol. Chem. 255:6814-6819) and Somlyo et al. (1981. J. Cell Biol. 90:577-594) that a proton flux from the myoplasm into the SR supplies a portion of the electrical charge balance required as Ca2+ is released from the SR into the myoplasm. A comparison of the amplitude of delta pHapp with that of delta[CaT] indicates that, in response to a single action potential, 10-15% of the charge balance required for Ca2+ release may be carried by protons.     

A high performance micro-dual-wavelength-spectrophotometer (MDWS)

The dual wavelength spectrophotometer (DWS) has proven to be the most sensitive device to monitor minute optical absorbance changes, which are inaccessible to conventional single or double beam spectrophotometers. The typical set ups, e.g. extensively used for Ca2+ or phytochrome measurements, are huge, expensive and cumbrous. Therefore, a novel high performance micro-dual-wavelength spectrophotometer (MDWS) was developed. It is miniaturized and no moving parts such as vibrational mirrors or rotating filter wheels involved. Its specifications are superior compared to the conventional set up being capable of detecting minute optical changes (reflection, absorbance, transmittance) at particular wavelengths.     

Cardioselective sulfonylthiourea HMR 1098 blocks mitochondrial uncoupling induced by a KATP channel opener,P-1075, in beating rat hearts

We investigated effects of blockade of cardiac ATP-sensitive potassium channels (KATP) with a novel cardioselective sulfonylthiourea, HMR 1098, on metabolic uncoupling caused by a potent KATP opener, P-1075, in Langendorff-perfused rat hearts. We used (1) 87Rb-NMR to detect activation-deactivation of sarcolemmal KATP, (2) 31P-NMR to monitor high-energy phosphates, (3) oxygen uptake measurements to monitor cellular respiration, and (4) myocardial optical absorbance measurements at 603 nm to follow changes in cytochrome c oxidase redox state. Activation of sarcolemmal KATP by P-1075 (5 microM) and a mitochondrial uncoupler 2,4-dinitrophenol (DNP) (50 microM) stimulated Rb+ efflux from the hearts by 130% and 60%, respectively. HMR 1098 (5 and 30 microM) blocked activation of sarcolemmal KATP in situ. HMR 1098 also prevented cardiac arrest and mitochondrial uncoupling induced by P-1075, such as (a) depletion of phosphocreatine and ATP by 40%, (b) two-fold decrease in venous oxygen, and (c) reduction of cytochrome c oxidase (demonstrated by an increase in 603 nm optical absorbance). The metabolic effects of P-1075 can be readily explained by activation of putative mitochondrial KATP. We concluded that blockade of mitochondrial uncoupling by HMR 1098 included an inhibiting effect of HMR 1098 on sarcolemmal and mitochondrial KATP in beating rat hearts.     

Characterization of the cycle of iron-mediated electron transfer from Adriamycin to molecular oxygen

The anticancer drug adriamycin binds iron and these complexes cycle to reduce molecular oxygen (Zweier, J. L. (1984) J. Biol. Chem. 259, 6056-6058). Optical absorption, EPR, and M?ssbauer spectroscopic data are correlated with polarographic O2 consumption and chemical Fe2+ extraction measurements in order to characterize each step in this cycle. Fe3+ binds to adriamycin at physiologic pH forming a complex with an optical absorbance maximum at 600 nm. EPR signals at g = 4.2 and g = 2.01, and a doublet M?ssbauer spectrum with isomer shift delta = 0.57 mm/s and quadrupole splitting delta EQ = 0.74 mm/s are observed indicating that the Fe3+ bound to adriamycin is high spin S = 5/2. Under anaerobic conditions the absorbance maximum at 600 nm decreases with an exponential decay constant = 0.77 h-1, and the EPR and M?ssbauer spectra of Fe3+-adriamycin similarly decrease as the Fe3+ is reduced to EPR silent Fe2+. The Fe2+-adriamycin complex which is formed exhibits a M?ssbauer spectrum with delta = 1.18 mm/s and delta EQ = 1.82 mm/s indicative of high spin Fe2+. As the EPR spectra of Fe3+-adriamycin decrease on reduction of the Fe3+ to Fe2+ a signal of the oxidized adriamycin free radical appears at g = 2.004 with line width of 8 G. On exposure to O2 the absorption maximum at 600 nm, the Fe3+ EPR, and the Fe3+ M?ssbauer spectra all return. Polarographic measurements demonstrate that O2 is consumed and that H2O2 is formed. Addition of high affinity Fe2+ chelators block O2 consumption indicating that Fe2+ formation is essential for O2 reduction. This cycle of iron-mediated O2 reduction can explain the formation of the reactive reduced oxygen and adriamycin radicals which are thought to mediate the biological activity of adriamycin.     

Changes in phenol red absorbance in response to electrical stimulation of frog skeletal muscle fibers

Intact single twitch fibers from frog muscle were stretched to long sarcomere length, micro-injected with the pH indicator dye phenol red, and activated by action potential stimulation. Indicator-related absorbance changes (denoted by delta A0 and delta A90) were measured with 0 degree and 90 degrees polarized light (oriented, respectively, parallel and perpendicular to the fiber axis). Two components of delta A were detected that had generally similar time courses. The "isotropic" component, calculated as the weighted average (delta A0 + 2 delta A90)/3, had the wavelength dependence expected for a change in myoplasmic pH. If calibrated in pH units, this signal's peak amplitude, which occurred 15-20 ms after stimulation, corresponded to a myoplasmic alkalization of average value 0.0025 +/- 0.0002 (+/- SEM; n = 9). The time course of this change, as judged from a comparison with that of the fibers' intrinsic birefringence signal, was delayed slightly with respect to that of the myoplasmic free [Ca2+] transient. On average, the times to half-peak and peak of the phenol red isotropic signal lagged those of the birefringence signal by 2.4 +/- 0.2 ms (+/- SEM; n = 8) and 8.4 +/- 0.5 ms (+/- SEM; n = 4), respectively. The other component of the phenol red signal was "dichroic," i.e., detected as a difference (delta A0-delta A90 greater than 0) between the two polarized absorbance changes. The wavelength dependence of this signal was similar to that of the phenol red resting dichroic signal (Baylor and Hollingworth. 1990. J. Gen. Physiol. 96:449-471). Because of the presence of the active dichroic signal, and because approximately 80% of the phenol red molecules appear to be bound in the resting state to either soluble or structural sites, the possibility exists that myoplasmic events other than a change in pH underlie the phenol red isotropic signal.     

The ratio of the two light reactions and their coupling in chloroplasts.

The kinetics of the absorbance changes of chlorophyll alphaI (P-700) and plastoquinone induced by xenon flashes of saturating intensity were studied in spinach chloroplasts. 1. The total amount of chlorophyll alphaI is compared with that amount being reduced via the rate-limiting step between the light reactions. This is based on the amplitudes of the absorbance changes of chlorophyll alphaI after chemical reduction and after a group of flashes following far-red preillumination. It is concluded that only 75% of chlorophyll alphaI is coupled to chlorophyll alphaII via linear electron transport and that the remaining 25% is functionally isolated. 2. A ratio of 0.85 for coupled chlorophyll alphaI to chlorophyll alphaII is estimated from the time course of the absorbance changes of plastoquinone and chlorophyll alphaI in two independent ways. 3. The oxygen yield per flash is used to calculate the difference extinction coefficient of chlorophyll alphaI at the maximum of the red absorbance band in spinach chloroplasts: delta xi703 = (6.7 +/- 0.7)-10(4) M-1-cm-1. The assumption of a quantitative electron transfer from water via plastoquinone to coupled chlorophyll alphaI is supported by the same extinction coefficient reported by Hiyama and Ke for Photosystem I particles. The location and function of the different chlorophylls alphaI is discussed in detail.     

Indications to an NADPH oxidase as a possible pO2 sensor in the rat carotid body

The rat carotid body superfused with low pO2 exhibited an optical absorbance spectrum which resembles the reduced spectrum of the NADPH oxidase in neutrophils. Diphenylene iodonium (DPI) as a specific inhibitor of the oxidase attenuated the reduced absorbance spectrum in the carotid body. Also absorbance bleaching by low doses of cyanide (50 and 100 microM) was inhibited by DPI, whereas higher doses of cyanide (300 microM) caused an absorbance spectrum typical for reduced cytochromes. It is concluded that an NADPH oxidase acts as a pO2 sensor in the carotid body with low affinity for oxygen and high affinity for cyanide.     

Quantitative analysis of hemoglobin oxygenation state of rat brain in situ by near-infrared spectrophotometry

The light in the near-infrared region (700-900 nm) was illuminated on the rat head, and absorption spectra were measured with the transmitted light under various conditions. The absorbance changes less than 780 nm were attributable to hemoglobin in the brain tissue, whereas those greater than 780 nm were associated with both hemoglobin and cytochrome oxidase. The changes of oxy- and total (oxy- plus deoxy-) hemoglobin content in the rat head could be monitored quantitatively by expressions of delta A700--1.20 delta A730 and delta A700--1.52 delta A730, respectively. The oxyhemoglobin content in the tissue was decreased as the O2 tension in inspired gas was lowered. At 10% O2 approximately 50% of hemoglobin was deoxygenated. The total hemoglobin content was increased under anoxic conditions. Inhalation of 5% CO2 and intravenous injection of a Ca2+ blocker nicardipine increased the O2 saturation of hemoglobin in the brain. These conclusions were confirmed by measuring the difference absorption spectra in the near-infrared region.     

Broadband Optical Mammography: Chromophore Concentration and Hemoglobin Saturation Contrast in Breast Cancer

This study reports the optical characterization and quantitative oximetry of human breast cancer using spectrally-resolved images collected with a broadband, continuous-wave optical mammography instrument. On twenty-six cancer patients, we collected two-dimensional optical mammograms and created maps of the concentrations of hemoglobin, water, and lipids, as well as the oxygen saturation of hemoglobin. For each cancerous breast, we analyzed the difference between the tumor region (as identified by x-ray and optical mammography) and the remainder of breast tissue. With respect to the surrounding tissue, we found that cancer regions have significantly higher concentrations of total hemoglobin (+2.4±0.4 μM) and water (+7±1% v/v), and significantly lower lipid concentration (8±2% v/v) and oxygen saturation of hemoglobin (5±1%). We also found a significant correlation between the tumor optical contrast and the grade of breast cancer as quantified by the Nottingham histologic score; this demonstrates how optical signatures may be representative of metabolic and morphological features, as well as the aggressive potential of the tumor.     

Rapid spectrophotometric determination of oxygen consumption using hemoglobin, in vitro: light scatter correction and expanded dynamic range

The method of using absorbance in conjunction with hemoglobin (Hb) to monitor rapid changes in oxygen consumption in vitro was improved by using a non-linear calibration technique and multiwavelength spectroscopy. The O(2) dependence of Hb absorbance was effectively linearized using the current technique (R(2) = 0.990+/-0.002, n = 3), and extended the dynamic range of [O(2)] determinations by 1.6-fold over previous approaches. The association/dissociation rates of O(2) and Hb were evaluated using the current approach and were not significant on the 100-ms time domain. A method was also developed for compensating for large amplitude light scattering changes in turbid media using multiwavelength analysis. Both the nonlinear calibration curve and light scattering corrections were validated in isolated porcine heart mitochondrial preparations.     

Kinetic studies of Rhus vernicifera laccase. Evidence for multi-electron transfer and an oxygen intermediate in the reoxidation reaction.

1. The reoxidation of reduced Rhus vernicifera laccase (monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) by molecular oxygen has been studied by optical absorption and EPR methods. 2. The reoxidation by oxygen of the type 1 Cu+ and the two-electron acceptor is characterized by a second-order rate constant of about 5-10(6) M-1-s-1. 3. The appearance of an optical intermediate (with an absorbance maximum around 360 nm) parallels the reoxidation of type 1 Cu+ and the two-electron acceptor. It disappears in a first-order reaction with a half-time of 20 s. A similar intermediate is formed during normal turnover. 4. The type 2 Cu+ appears to be reoxidized in an intramolecular reaction with a half-time of about 20 s, suggesting a correlation between the reoxidation of this site and the disappearance of the optical intermediate. 5. The results suggest that three electrons are rapidly transferred to oxygen leading to the formation of an enzyme-bound oxygen intermediate.     

Spectral evidence for sub-picosecond iron displacement after ligand detachment from hemoproteins by femtosecond light pulses.

We have measured spectral and kinetic differences in protoheme, sperm whale or horse heart myoglobin and human hemoglobin following photodissociation induced by optical pulses of 80 fs duration. Full ligation was performed with oxygen or carbon monoxide. Femtosecond kinetics and transient difference spectra revealed the appearance of a deoxy species with tau approximately equal to 250-300 fs. The transient deoxy species in myoglobin and hemoglobin evidenced a 3-4 nm red shift of their delta A spectra compared with the equilibrium delta A spectrum. This shift was not observed after photodissociation of the carbon monoxide liganded protoheme. We proposed that the 250 fs time constant corresponding to the appearance of the deoxy-like species is related to the displacement of the ferrous iron out of the heme plane. Consequently, the small red shift of the delta A spectra observed in photodissociated hemoproteins may be tentatively attributed to changes in the vibrational modes of either the proximal histidine-Fe2+ bond and/or of the N4 porph-Fe-N epsilon His (F8) bent.     

Purification and partial characterization of the b-type cytochrome from human polymorphonuclear leukocytes

Polymorphonuclear leukocytes contain an oxidase system that can be activated to produce superoxide radicals and hydrogen peroxide. A nonmitochondrial b cytochrome, functioning in the generation of these oxygen species, has been purified to apparent homogeneity from human polymorphonuclear phagocytes. After solubilization of the cytochrome with Triton X-100, the cell extract was subsequently chromatographed on Blue Sepharose and Sephacryl S-300. The final preparation was maximally purified 170-fold with a specific content of 5.33 +/- 2.03 nmol mg-1 of protein (mean +/- S.D.; n = 7) and a yield of 21 +/- 13% (n = 5). The apparent molecular mass of the nondenatured cytochrome was estimated by gel filtration to be 235 kDa. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single polypeptide was found with a molecular mass of 127 kDa. From the pyridine hemochrome spectrum 1 protoheme IX/polypeptide was calculated. The light absorbance bands of the dithionite-reduced cytochrome were found to be at 558.5 (alpha), 529 (beta), and 426 nm (Soret), and that of the oxidized cytochrome at 413.5 nm. The difference absorbance coefficients are delta epsilon (426.5 - 440 nm) = 160.6 +/- 11 mM-1 cm-1 and delta epsilon (558.5 - 542 nm) = 29.3 +/- 2 mM-1 cm-1 (mean +/- S.D.; n = 5). Carbon monoxide binds to the cytochrome in a time-dependent fashion (maximum binding after 50-60 min). The midpoint potential of the solubilized nonpurified cytochrome is identical to the cytochrome in situ (Em7.0 = -218 +/- 7 mV (mean +/- S.D.; n = 5)). However, purified cytochrome b shows a significantly decreased midpoint potential, estimated at -407 +/- 18 mV (n = 4). The protein does not contain noncovalently bound FAD or FMN, and no spectral evidence was obtained for the presence of covalently bound flavin. Preliminary amino acid analysis of the cytochrome shows a high content of hydrophilic residues.     

Chlorination of pyridinium compounds. Possible role of hypochlorite, N-chloramines, and chlorine in the oxidation of pyridinoline cross-links of articular cartilage collagen type II during acute inflammation

Reactive oxygen species produced by activated neutrophils and monocytes are thought to be involved in mediating the loss of collagen and other matrix proteins at sites of inflammation. To evaluate their potential to oxidize the pyridinoline (Pyd) cross-links found in collagen types I and II, we reacted hydrogen peroxide (H(2)O(2)), hypochlorous acid/hypochlorite (HOCl/OCl(-)), and singlet oxygen (O(2)((1)delta g)) with the Pyd substitutes, pyridoxamine dihydrochloride and vitamin B(6), which share the same chemical structure and spectral properties of Pyd cross-links. Neither H(2)O(2) (125-500 microm) nor O(2)((1)delta g) (10-25 microm) significantly changed the spectral properties of pyridoxamine or vitamin B(6). Reaction of HOCl/OCl(-) (12.5-50 microm) with pyridoxamine at pH 7.2 resulted in a concentration-dependent appearance of two new absorbance peaks and a decrease in fluorescence at 400 nm (excitation 325 nm). The new absorbance peaks correlated with the formation of an N-chloramine and the product of its subsequent reaction with pyridoxamine. In contrast, the extent to which HOCl reacted with vitamin B(6), which lacks a primary amine group, was variable at this pH. At lysosomal pH 5.5, Cl(2)/HOCl/OCl(-) reacted with both pyridoxamine and vitamin B(6). Four of the chlorinated products of this reaction were identified by gas chromatography-mass spectrometry and included 3-chloropyridinium, an aldehyde, and several chlorinated products with disrupted rings. To evaluate the effects of Cl(2)/HOCl/OCl(-) on Pyd cross-links in collagen, we exposed bone collagen type I and articular cartilage type II to HOCl. Treatment of either collagen type with HOCl at pH 5. 0 or 7.2 resulted in the oxidation of amine groups and, for collagen type II, the specific decrease in Pyd cross-link fluorescence, suggesting that during inflammation both oxidations may be used by neutrophils and monocytes to promote the loss of matrix integrity.     

The fulvic acid fraction as it changes in the mature phase of vegetable oil-mill sludge and domestic waste composting

Sludge resulting from the treatment of effluent from a vegetable oil mill, was composted mixed with domestic waste in a pile for five months. Different proportions of sludge and dry waste were mixed: M1 (1v/2v) and M2 (1v/1v). Monitoring different physical-chemical parameters showed the effect of the substrate on the microbiological activity and on the formation of fulvic acids, affecting the maturity of the final compost. Elemental analysis revealed that the fulvic acids of mixes M1 and M2 presented very low concentrations of carbon, hydrogen, and oxygen and a high level of nitrogen. The FTIR spectroscopy results showed a decrease during composting of the intensity of absorbance of the easily assimilable compounds that are predominant in the initial mixtures i.e. the carbohydrates (1170-1080 cm(-1)) in M1 and long aliphatic chains (2920 cm(-1)) in M2. For mix M1 there was enrichment in compounds bearing oxygen-containing moieties. In M2 it was the nitrogen-containing compounds (in the form of stable amides) which predominated at the end of composting. The first component of PCA analysis, PC1, accounted for 83% of the difference between two distinct groups of parameters governing degradation and restructuration of the fulvic acids during composting. PC2 (17%) explained the variance due to the level of free or less polycondensed compounds in the two mixtures. Oxidised polyphenolic and polysaccharide structures were the least free, or most polycondensed, in the fulvic structures of M1. In M2 fulvic acids however, it was the polyphenols and peptide structures that were involved in the bonding, most likely of the polyphenol-peptide type.     

The hydrophobic amino-terminal sequence of bovine 17 alpha-hydroxylase is required for the expression of a functional hemoprotein in COS 1 cells

The endoplasmic reticulum is a major site of localization for eukaryotic cytochrome P-450 mixed-function oxidase complexes. Previous studies have shown that the microsomal forms of P-450 insert into the membrane via their hydrophobic amino terminus through the signal recognition particle-dependent pathway. We have examined the insertion of bovine 17 alpha-hydroxylase (P45017 alpha) into the endoplasmic reticulum of COS 1 cells to evaluate the functional role of its hydrophobic amino-terminal sequence and membrane insertion. An NH2-terminal truncated protein, P450 delta 2-17, which lacked amino acids 2-17 was expressed in COS 1 cells, subcellular fractions were isolated, and P450 delta 2-17 was localized by immunoblot analysis. Compared to the full-length P45017 alpha, the NH2-terminal truncation resulted in a 2.5-fold decrease in P45017 alpha protein recovered with the microsomal fraction, 50% of which was an integral membrane protein as defined by resistance to Na2CO3 extraction. Despite correct membrane localization, P450 delta 2-17 was not a functional enzyme in COS 1 cells. A CO difference spectrum of microsomes containing P450 delta 2-17 did not give a typical 450 nm absorbance. We conclude that the hydrophobic amino terminus is required for the expression of a functionally competent P45017 alpha in COS 1 cells and suggest that the insertion of the amino terminus into the membrane is necessary for the folding of this protein into its correct structural form.     

Site of inhibition of rat liver microsomal fatty acid chain elongation system by dec-2-ynoyl coenzyme A. Possible mechanism of inhibition

The present study examines the effect of the acetylenic thioester dec-2-ynoyl-CoA (delta 2 10 identical to 1-CoA) on the microsomal fatty acid chain elongation pathway in rat liver. When the individual reactions of the elongation system were measured in the presence of delta 2 10 identical to 1-CoA, the trans-2-enoyl-CoA reductase activity was markedly inhibited (Ki = 2.5 microM), whereas the activities of the condensing enzyme, the beta-ketoacyl-CoA reductase, and the beta-hydroxyacyl-CoA dehydrase were not affected. The absence of inhibition of total microsomal fatty acid elongation was attributed to the significant accumulation of the intermediates, beta-hydroxyacyl-CoA and trans-2-enoyl-CoA, without formation of the saturated elongated product, indicating that the trans-2-enoyl-CoA reductase-catalyzed reaction was the only site affected by the inhibitor. The nature of the inhibition was noncompetitive. In contrast to the delta 2 10 identical to 1-CoA, delta 3 10 identical to 1-CoA did not inhibit trans-2-enoyl-CoA reductase activity, suggesting that the mode of inhibition was not via formation of the 2,3-allene derivative. Based on the observation (a) that p-chloromercuribenzoate markedly inhibits reductase activity, (b) that dithiothreitol protects the enzyme against inactivation by delta 2 10 identical to 1-CoA, (c) of the spectral manifestation of the interaction between thiol reagents and delta 2 10 identical to 1-CoA depicting an absorbance peak similar to that of the beta-ketoacyl thioester-Mg2+ enolate complex, (d) of a similar absorbance spectrum formed by the interaction between delta 2 10 identical to 1-CoA and liver microsomes, and (e) of the absence of formation of a similar spectrum by delta 3 10 identical to 1-CoA, trans-2-10:1-CoA, or delta 2 10 identical to 1 free acid with liver microsomes, we propose that delta 2 10 identical to 1-CoA inactivates trans-2-enoyl-CoA reductase by covalently binding to a critical sulfhydryl group at or in close proximity to the active site of the enzyme.     

The effect of hormones on proton compartmentation in hepatocytes

Liver mitochondria isolated from rats treated acutely with glucagon exhibit higher respiration-dependent H+ ion gradients across the mitochondrial inner membrane than mitochondria from control rats. It has been suggested that similar increases in mitochondrial delta pH in situ could stimulate gluconeogenesis, chiefly because the transport of pyruvate into mitochondria would increase in response to the increase in mitochondrial matrix pH. In order to determine whether the increased delta pH observed in vitro in isolated mitochondria also occurs in situ, the effect of glucagon on the pH in the cytosol and mitochondria matrix spaces of isolated hepatocytes was determined. For qualitative results, the spectral responses of intracellularly trapped 6-carboxyfluorescein was used to monitor cytosol pH, while fluorescein-loaded hepatocytes were used to monitor the mitochondrial pH. Hepatocytes were incubated with the diacetate ester derivatives of these dyes. The esters are permeable to the cell membranes, but are rapidly hydrolyzed in the cells. The free unesterified dyes are relatively impermeable to the cell membranes. After being trapped in the cell, 6-carboxyfluorescein remains localized in the cell cytosol, whereas fluorescein is taken up by the mitochondria as a function of the mitochondrial delta pH. In order to quantitate the actual pH in these compartments, the spectral responses (490-465 nm) of 6-carboxyfluorescein-loaded hepatocytes were used to determine the cytosolic pH. Calibration of these responses was obtained within the cell by determination of the dye's differential absorption coefficient (epsilon 490-465 nm) in various high K+ buffers after equilibration of the internal and external pH with valinomycin and the uncoupler 1799. All absorbance values were corrected for dye leakage. Equal hematocrits of unloaded cells were used to correct for absorbance contributions from cellular constituents. The mitochondrial pH was determined by a combination of the indicator dye and [14C]5,5'-demethyloxazolidine-2, 4-dione (DMO) distribution ratio methods. The weak acid DMO freely distributes across the plasma membrane and mitochondrial membrane in whole cells according to the pH gradient across each membrane. Knowledge of the cytoplasmic pH from the 6-carboxyfluorescein data allows the expected distribution of DMO across the plasma membrane to be calculated. The excess accumulation of DMO in intact hepatocytes over that predicted from the plasma membrane pH gradient alone was then used to calculate the pH gradient across the mitochondrial inner membrane. The effects of valinomycin, uncouplers, and hormones on the pH in cytosolic and mitochondrial compartm     

The interaction of nitric oxide with ascorbate oxidase.

1. The reaction of nitric oxide with oxidized and reduced ascorbate oxidase (L-ascorbate: oxygen oxidoreductase, EC 1.10.3.3) has been investigated by optical absorption measurements and electron paramagnetic resonance, and the results are compared with those of ceruloplasmin. 2. Upon anaerobic incubation of oxidized ascorbate oxidase with nitric oxide a decrease of the absorbance at 610 nm is found, which is due to an electron transfer from nitric oxide to Type-1 copper. 3. In the presence of nitric oxide the EPR absorbance of ascorbate oxidase decreases and shows predominatly a signal with characteristics of Type-2 copper (g parallel = 2.248; A parallel = 188 G), whereas the type-1 copper signal has vanished. 4. Comparison of the intensities of the EPR signals before and after NO-treatment points to the presence of one Type-2 and three Type-1 copper atoms per molecule of ascorbate oxidase. 5. It is shown that the changes in the optical and the EPR spectrum of ascorbate oxidase induced by nitric oxide are reversible. No difference in enzymic activity is found between the native enzyme and the NO-treated enzyme after removal of nitric oxide.