[Studies on metabolites and the structural modification of beta-caryophyllene].
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Animals↗
Biomedical subjects
L Yu
Publications and source records attributed to L Yu.
Characterization of purified cytochrome c1 from Rhodobacter sphaeroides R-26.
Cytochrome c1 from a photosynthetic bacterium Rhodobacter sphaeroides R-26 has been purified to homogeneity. The purified protein contains 30 nmol heme per mg protein, has an isoelectric point of 5.7, and is soluble in aqueous solution in the absence of detergents. The apparent molecular weight of this protein is about 150,000, determined by Bio Gel A-0.5 m column chromatography; a minimum molecular weight of 30,000 is obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The absorption spectrum of this cytochrome is similar to that of mammalian cytochrome c1, but the amino acid composition and circular dichroism spectral characteristics are different. The heme moiety of cytochrome c1 is more exposed than is that of mammalian cytochrome c1, but less exposed than that of cytochrome c2. Ferricytochrome c1 undergoes photoreduction upon illumination with light under anaerobic conditions. Such photoreduction is completely abolished when p-chloromercuriphenylsulfonate is added to ferricytochrome c1, suggesting that the sulfhydryl groups of cytochrome c1 are the electron donors for photoreduction. Purified cytochrome c1 contains 3 +/- 0.1 mol of the p-chloromercuriphenylsulfonate titratable sulfhydryl groups per mol of protein. In contrast to mammalian cytochrome c1, the bacterial protein does not form a stable complex with cytochrome c2 or with mammalian cytochrome c at low ionic strength. Electron transfer between bacterial ferrocytochrome c1 and bacterial ferricytochrome c2, and between bacterial ferrocytochrome c1 and mammalian ferricytochrome c proceeds rapidly with equilibrium constants of 49 and 3.5, respectively. The midpoint potential of purified cytochrome c1 is calculated to be 228 mV, which is identical to that of mammalian cytochrome c1.
Cytochrome c Group↗
Influence of smoking fewer cigarettes on exposure to tar, nicotine, and carbon monoxide.
In the hope of reducing the adverse health consequences of smoking, physicians frequently advise their patients who cannot quit to smoke fewer cigarettes. Habitual smokers may compensate for the reduced number of cigarettes, however, by taking in more smoke per cigarette. We measured the intake of tar (estimated as mutagenic activity of the urine), nicotine, and carbon monoxide during short-term cigarette restriction. With a reduction from an average of 37 cigarettes to an average of 5 cigarettes per day, the intake of tobacco toxins per cigarette increased roughly threefold and daily exposure to tar and carbon monoxide declined only 50 percent. We conclude that smoking fewer cigarettes may reduce exposure to toxins and related adverse health consequences. However, consistent with a tendency to maintain intake of nicotine, the magnitude of the benefit is much less than expected. Whether "oversmoking" persists during long-term restriction of cigarettes requires further investigation.
Adult↗
Spin-label electron paramagnetic resonance and differential scanning calorimetry studies of the interaction between mitochondrial succinate-ubiquinone and ubiquinol-cytochrome c reductases.
The interaction between succinate-ubiquinone and ubiquinol-cytochrome c reductases in the purified, dispersed state and in embedded phospholipid vesicles was studied by differential scanning calorimetry and by electron paramagnetic resonance (EPR). When the purified, detergent-dispersed succinate-ubiquinone reductase, ubiquinol-cytochrome c reductase, and cytochrome c oxidase undergo thermodenaturation, they show an endothermic transition. However, when these isolated electron-transfer complexes are embedded in phospholipid vesicles, they undergo exothermodenaturation. The energy released could result from the collapse of the strained interaction between unsaturated fatty acyl groups of phospholipids and an exposed area of the complex formed by removal of interacting proteins. The exothermic enthalpy change of thermodenaturation of a protein-phospholipid vesicle containing both succinate-ubiquinone and ubiquinol-cytochrome c reductases was smaller than that of a mixture of protein-phospholipid vesicles formed from the individual electron-transfer complexes. This suggests specific interaction between succinate-ubiquinone reductase and ubiquinol-cytochrome c reductase in the membrane. This idea is supported by saturation transfer EPR studies showing that the rotational correlation time of spin-labeled ubiquinol-cytochrome c reductase is increased when mixed with succinate-ubiquinone reductase prior to embedding in phospholipid vesicles. These results indicate that succinate-ubiquinone reductase and ubiquinol-cytochrome c reductase are indeed present in the membrane as a supermacromolecular complex. No such supermacromolecular complex is detected between NADH-ubiquinone and ubiquinol-cytochrome c reductases or between succinate-ubiquinone and NADH-uniquinone reductases.
Animals↗
Use of an azido-ubiquinone derivative to identify subunit I as the ubiquinol binding site of the cytochrome d terminal oxidase complex of Escherichia coli.
The radiolabeled, photoreactive azido-ubiquinone derivative (azido-Q), 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl-[3H]octyl)- 1,4-benzoquinone, was used to investigate the active site of ubiquinol oxidase activity of the cytochrome d complex, a two-subunit terminal oxidase of Escherichia coli. The azido-Q, when reduced by dithioerythritol, was shown to support enzymatic oxygen consumption by the cytochrome d complex that was 8% of the rate observed with ubiquinol-1. This observation provided the rationale behind further studies of the possible photoinactivation and labeling of the active site by this azido-Q. Ten min of photolysis of the purified cytochrome d complex in the presence of the azido-Q resulted in a 60% loss of the ubiquinol-1 oxidase activity. Uptake of the radiolabeled azido-Q by the cytochrome d complex was correlated to the photoinactivation of the ubiquinol-1 oxidase activity. Both increased linearly during the first 4 min of photolysis and reached 90% of the maximum within 10 min. Photolysis times longer than 10 min resulted in no increase in the maximum of 2 mol of azido-Q incorporated per mol of enzyme. The rate of azido-Q uptake by subunit I, but not subunit II, correlated well with the rate of loss of ubiquinol oxidase activity. Use of ubiquinol-0, which is not oxidized by the enzyme, to competitively inhibit radiolabeling of nonspecific binding sites, resulted in a significant decrease (42%) of azido-Q labeling of subunit II while it did not affect the labeling of subunit I. After photolysis for 4 min, the ratio of radiolabeled azido-Q in subunits I to II of the complex was 4.3 to 1.0. These observations support the conclusion that the ubiquinol substrate binding site is located on subunit I of the cytochrome d complex.
Azides↗
Immunological comparison of the b and c1 cytochromes from bovine heart mitochondria and the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26.
Antibodies against cytochromes b and c1 of bovine heart mitochondria and the photosynthetic bacterium, Rhodopseudomonas sphaeroides R-26, were raised in rabbits. The purified antibodies showed high titers against their respective antigens in enzyme-linked immunosorbent assays. Less than 15% cross-reactivity between the mitochondrial and bacterial cytochromes was detected. Although antibodies against mitochondrial cytochrome b did not inhibit the mitochondrial cytochrome b-c1 complex, a 70% inhibition was obtained when these antibodies were incubated with delipidated mitochondrial cytochrome b-c1 complex prior to reconstitution with phospholipids indicating that the catalytic site(s) of mitochondrial cytochrome b are masked by phospholipids. On the other hand, antibodies against bacterial cytochrome b showed significant inhibition of the intact bacterial cytochrome b-c1 complex, indicating that some of the catalytic site epitopes of bacterial cytochrome b are exposed to the hydrophilic environment. Similar to antibodies against mitochondrial cytochrome b, antibodies against bacterial cytochrome b inhibited 50% activity of the mitochondrial cytochrome b-c1 complex only when they were incubated with the delipidated mitochondrial cytochrome b-c1 complex prior to reconstitution with phospholipids, indicating that the common epitopes between the cytochromes b are masked by phospholipids. Antibodies against mitochondrial and bacterial cytochromes c1 completely inhibited their respective cytochrome b-c1 complexes but no cross-immunoinhibition was observed. However, when antibodies against bacterial cytochrome c1 were incubated with the delipidated mitochondrial cytochrome b-c1 complex before reconstitution with phospholipids, a 65% inhibition was observed, indicating that the common epitopes between the cytochromes c1 were also somewhat masked by phospholipids. Antibodies against mitochondrial cytochrome c1 inhibited 70% of the succinate oxidase activity in the intact mitochondria preparation, but no inhibition was observed in submitochondrial particles, indicating that some mitochondrial cytochrome c1 epitopes are exposed to the cytoplasmic side.
Animals↗
Reduced tar, nicotine, and carbon monoxide exposure while smoking ultralow- but not low-yield cigarettes.
An unresolved public health issue is whether some modern cigarettes are less hazardous than others and whether patients who cannot stop smoking should be advised to switch to lower-yield cigarettes. We studied "tar" (estimated by urine mutagenicity), nicotine, and carbon monoxide exposure in habitual smokers switched from their usual brand to high- (15 mg of tar), low- (5 mg of tar), or ultralow-yield (1 mg of tar) cigarettes. There were no differences in exposure comparing high- or low-yield cigarettes, but tar and nicotine exposures were reduced by 49% and 56%, respectively, and carbon monoxide exposure by 36% while smoking ultralow-yield cigarettes. Similarly, in 248 subjects smoking their self-selected brand, nicotine intake, estimated by blood concentrations of its metabolite cotinine, was 40% lower in those who smoked ultralow but no different in those smoking higher yields of cigarettes. Our data indicate that ultralow-yield cigarettes do deliver substantial doses of tar, nicotine, and carbon monoxide, but that exposures are considerably less than for other cigarettes.
Adult↗
The EPR spectra of the cytochrome b-c1 complex of Rhodopseudomonas sphaeroides.
The purified cytochrome b-c1 complex of Rhodopseudomonas sphaeroides has two b cytochromes distinguishable by optical, thermodynamic and electron paramagnetic resonance criteria (gz values are approximately equal to 3.75 and approximately equal to 3.4). EPR features typical of a Rieske iron sulfur cluster (g values of 2.03 1.90 and 1.81) and a c1 type cytochrome (g approximately equal to 3.4) were also observed. The b and c1 cytochromes were individually purified from the complex. The cytochrome c1 retained its native EPR spectrum. The b cytochrome lost over 90% of the intensity from the 'b566 type' heme site (g approximately equal to 3.75), while the 'b561 type' heme site (g approximately equal to 3.4) retained its native EPR spectrum.
Ascorbic Acid↗
Mouse muscle nicotinic acetylcholine receptor gamma subunit: cDNA sequence and gene expression.
Clones coding for the mouse nicotinic acetylcholine receptor (AChR) gamma subunit precursor have been selected from a cDNA library derived from a mouse myogenic cell line and sequenced. The deduced protein sequence consists of a signal peptide of 22 amino acid residues and a mature gamma subunit of 497 amino acid residues. There is a high degree of sequence conservation between this mouse sequence and published human and calf AChR gamma subunits and, after allowing for functional amino acid substitutions, also to the more distantly related chicken and Torpedo AChR gamma subunits. The degree of sequence conservation is especially high in the four putative hydrophobic membrane spanning regions, supporting the assignment of these domains. RNA blot hybridization showed that the mRNA level of the gamma subunit increases by 30 fold or more upon differentiation of the two mouse myogenic cell lines, BC3H-1 and C2C12, suggesting that the primary controls for changes in gene expression during differentiation are at the level of transcription. One cDNA clone was found to correspond to a partially processed nuclear transcript containing two as yet unspliced intervening sequences.
Amino Acid Sequence↗
Identification of ubiquinone-binding proteins in yeast mitochondrial ubiquinol-cytochrome c reductase using an azido-ubiquinone derivative.
An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone, was used to study the ubiquinone-protein interaction and to identify the ubiquinone-binding proteins in yeast mitochondrial ubiquinone-cytochrome c reductase. The phospholipids and Q6 in purified reductase were removed by repeated ammonium sulfate precipitation in the presence of 0.5% sodium cholate. The resulting phospholipid- and ubiquinone-depleted reductase shows no enzymatic activity; activity can be completely restored by the addition of phospholipids and Q6 or Q2. The ubiquinone- and phospholipid-replenished ubiquinonol-cytochrome c reductase is also fully active upon reconstituting with bovine succinate-ubiquinone reductase to form succinate-cytochrome c reductase. When an azido-ubiquinone derivative was added to the ubiquinone and phospholipid-depleted reductase in the dark, followed by the addition of phospholipids, partial reconstitutive activity was restored, while full ubiquinol-cytochrome c reductase activity was observed when Q2H2 was used as substrate in the assay mixture. Apparently, the large amount of Q2H2 present in the assay mixture displaces the azido-ubiquinone in the system. Photolysis of the azido-Q-treated reductase with long-wavelength ultraviolet light abolishes about 70% of both the restored reconstitutive activity and Q2H2-cytochrome c reductase activity. The activity loss is directly proportional to the covalent binding of [3H]azido-ubiquinone to the reductase protein. When the photolyzed, [3H]azido-ubiquinone-treated sample was subjected to SDS-polyacrylamide gel electrophoresis followed by analysis of the distribution of radioactivity among the subunits, the cytochrome b protein and a protein with an apparent molecular weight of 14 000 were heavily labeled. The amount of radioactive labeling in both these proteins was affected by the presence of phospholipids.
Affinity Labels↗
Determination of nicotine N-oxide by gas chromatography following thermal conversion to 2-methyl-6-(3-pyridyl)tetrahydro-1,2-oxazine.
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Chemical Phenomena↗
Psychosocial factors in urinary incontinence.
This article reviews psychosocial factors involved in the development and maintenance of urinary incontinence. Four aspects of this relationship are examined: the perception of and response to urinary incontinence; the development and/or exacerbation of incontinence symptoms; the psychosocial consequences for individuals, families, and caregivers; and the role of psychosocial factors in treatment. Areas for future research are identified and clinical implications of existing findings are presented.
Aged↗
Studies of protein-phospholipid interaction in isolated mitochondrial ubiquinone-cytochrome c reductase.
The interaction between phospholipids, ubiquinone and highly purified ubiquinol-cytochrome c reductase was studied using differential scanning calorimetry. The enzyme complex and its delipidated forms undergo thermodenaturation at 337.3 and 322.7 K, respectively. The reduced reductase is more stable toward thermodenaturation than is the oxidized enzyme. While phospholipids restored enzymatic activity to the delipidated enzyme complex and stabilized the enzyme toward thermodenaturation, ubiquinone showed little effect on the thermostability of ubiquinol-cytochrome c reductase. The effect of phospholipids on the thermotropic properties of ubiquinol-cytochrome c reductase is dependent upon the molecular properties of the phospholipid. When ubiquinol-cytochrome c reductase was embedded in closed asolectin vesicles, an exothermic transition peak was observed upon thermodenaturation. When the asolectin concentration in the reconstituted preparation was less than 0.3 mg/mg protein, an amorphous structure was observed in the electron micrograph and the preparation showed an endothermic transition upon thermodenaturation. The thermotropic properties of the enzyme-phospholipid vesicles were affected by the phospholipid head groups as well as the fatty-acyl chains, with those phospholipids having the most highly unsaturated fatty-acyl chains having the greatest effect. The energy for the exothermic transition may be derived from the collapse, upon thermodenaturation, of a strained interaction between the unsaturated fatty-acyl groups of phospholipids and protein molecules resulting from vesicle formation. The exothermic transition of the enzyme-phospholipid vesicle was abolished when cholesterol was included in the vesicles and when reductase was treated with a proteolytic enzyme prior to incorporation into the phospholipid vesicles.
Animals↗
Effect of alkyl side chain variation on the electron-transfer activity of ubiquinone derivatives.
The effect of the alkyl side chain of the ubiquinone molecule on the electron-transfer activity of ubiquinone in mitochondrial succinate-cytochrome c reductase is studied by using synthetic ubiquinone derivatives that possess the basic ubiquinone structure of 2,3-dimethoxy-5-methyl-1,4-benzoquinone with different alkyl side chains at the 6-position. The alkyl side chains vary in chain length, degree of saturation, and location of double bonds. When a ubiquinone derivative is used as an electron acceptor for succinate-ubiquinone reductase, an alkyl side chain of six carbons is needed to obtain the maximum activity. However, when it serves as an electron donor for ubiquinol-cytochrome c reductase or as a mediator in succinate-cytochrome c reductase, an alkyl side chain of 10 carbons gives maximal efficiency. Introduction of one or two isolated double bonds into the alkyl side chain of the ubiquinone molecule has little effect on electron-transfer activity. However, a conjugated double bond system in the alkyl side chain drastically reduces electron-transfer efficiency. The effect of the conjugated double bond system on the electron-transferring efficiency of ubiquinone depends on its location in the alkyl side chain. When location is far from the benzoquinone ring, the effect is minimal. These observations together with the results obtained from photoaffinity-labeling studies lead us to conclude that flexibility in the portion of the alkyl side chain immediately adjacent to the benzoquinone ring is required for the electron-transfer activity of ubiquinone.
Alkylation↗
Identification of the binding site on cytochrome c1 for cytochrome c.
The reagent 1-ethyl-3-(3-[14C]trimethylaminopropyl)carbodiimide (ETC) was used to identify specific carboxyl groups on the cytochrome bc1 complex (ubiquinol-cytochrome c reductase, EC 1.10.2.2) involved in binding cytochrome c. Treatment of the cytochrome bc1 complex with 2 mM ETC led to inhibition of the electron transfer activity with cytochrome c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both the cytochrome c1 heme peptide and the Mr = 9175 "hinge" peptide were radiolabeled by ETC. In addition, a new band appeared at a position consistent with a 1:1 cross-linked cytochrome c1-hinge peptide species. Treatment of a 1:1 cytochrome bc1-cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bc1, but to decreased radiolabeling of the cytochrome c1 heme peptide. Two new cross-linked species corresponding to cytochrome c-hinge peptide and cytochrome c-cytochrome c1 were formed in place of the cytochrome c1-hinge peptide species. In order to identify the specific carboxyl groups labeled by ETC, a purified cytochrome c1 preparation containing both the heme peptide and the hinge peptide was dimethylated at all the lysines to prevent internal cross-linking. The methylated cytochrome c1 preparation was treated with ETC and digested with trypsin and chymotrypsin, and the resulting peptides were separated by high pressure liquid chromatography. ETC was found to label the cytochrome c1 peptides 63-81, 121-128, and 153-179 and the hinge peptides 1-17 and 48-65. All of these peptides are highly acidic and contain one or more regions of adjacent carboxyl groups. The only peptide consistently protected from labeling by cytochrome c binding was 63-81, demonstrating that the carboxyl groups at residues 66, 67, 76, and 77 are involved in binding cytochrome c. These residues are relatively close to the heme-binding cysteine residues 37 and 40 and indicate a possible site for electron transfer from cytochrome c1 to cytochrome c.
Amino Acid Sequence↗
Studies on protein-lipid interactions in cytochrome c oxidase by differential scanning calorimetry.
The interaction between cytochrome c oxidase and phospholipids was studied by differential scanning calorimetry. The active, lipid-sufficient cytochrome c oxidase undergoes thermodenaturation at 336 K with a relatively broad and concentration dependent endothermic transition. The delipidated enzyme shows an endothermic denaturation temperature at 331.3 K. When the delipidated cytochrome c oxidase was treated with chymotrypsin, a lowered thermodenaturation temperature was observed. When the delipidated cytochrome c oxidase was reconstituted with asolectin to form a functionally active enzyme complex, the thermodenaturation shifted to a higher temperature, with a sharper transition thermogram. The increase in thermotransition temperature and enthalpy change of thermodenaturation of the asolectin-reconstituted enzyme is directly proportionate to the amount of asolectin used, up to 0.5 mg asolectin per mg protein. The thermotransition temperature and enthalpy changes of thermodenaturation for the phospholipid-reconstituted cytochrome c oxidase are affected by the phospholipid headgroup and the fatty acyl groups. Among phospholipids with the same acyl moiety but different head groups, phosphatidylethanolamine was found to be more effective than phosphatidylcholine in protecting cytochrome c oxidase from thermodenaturation. An exothermic transition thermogram was observed for delipidated cytochrome c oxidase embedded in phospholipid vesicles formed with phospholipids containing unsaturated fatty acyl groups. The increase in exothermic transition temperature and exothermic enthalpy change of thermodenaturation of the oxidase-cytochrome c-cytochrome c oxidase complex destabilized cytochrome c but not cytochrome c oxidase toward thermodenaturation.
Animals↗
Interaction and identification of ubiquinone-binding proteins in ubiquinol-cytochrome c reductase by azido-ubiquinone derivatives.
Various azido-ubiquinone derivatives were synthesized and characterized. 3-Azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone was found to be suitable for the study of specific interaction between ubiquinone (Q) and protein. It was synthesized with high specific radioactivity and used to identify the Q-binding proteins in purified ubiquinol-cytochrome c reductase. This azido-Q derivative showed partial efficiency in restoring activity to the Q- and phospholipids-depleted ubiquinol-cytochrome c reductase in the absence of light. Azido-Q derivative treated samples, however, became completely inactivated upon photolysis, and the inactivation was not reversed by addition of Q derivatives. The redox state of the azido-Q derivative has little effect on the Q-binding affinity. Two protein subunits with Mr = 37,000 and 17,000 were found to be heavily labeled when depleted ubiquinol-cytochrome c reductase was treated with [3H] azido-Q derivative followed by photolysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amount of radioactive labeling of the Mr = 17,000 protein was proportional to the degree of inactivation and affected by the presence of phospholipids. The radioactive labeling of the Mr = 37,000 protein subunit, however, showed no correlation with degree of inactivation and was not affected by phospholipids. Since the radiolabeling at the Mr = 17,000 protein subunit was affected by phospholipids and correlated with the enzymatic activity, this subunit is probably the Q-binding protein in this enzyme complex (QPc). The inhibition of enzymatic activity by n-heptyl-4-hydroxyquinoline-N-oxide was easily reversed by addition of the azido-Q derivative. The distribution of radioactivity among the subunits of ubiquinol-cytochrome c reductase was not affected by the presence of antimycin A, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole or n-heptyl-4-hydroxyquinoline-N-oxide, suggesting that the binding site(s) of these inhibitors are not the Q-binding site.
Affinity Labels↗
The clinical value of free phenytoin levels.
The relationship between total and free phenytoin levels and drug toxicity was studied in 80 patients. Twenty-four were taking phenytoin alone. Drug toxicity was assessed by a "blind" rater using an eight-point standardized scoring system. The mean free phenytoin fraction was 0.076 in patients taking phenytoin alone or phenytoin and carbamazepine and 0.11 in patients taking valproic acid (p less than 0.001). The free fraction did not change with the total level over the range tested (6.7 to 39.9 micrograms/ml total phenytoin). There was a strong correlation between free and total levels (r = 0.84). Both free (r = 0.59) and total (r = 0.49) phenytoin levels were positively correlated with the toxicity score. Only total phenytoin levels showed a weak positive correlation with decreasing seizure frequency. Our results suggest that routine free phenytoin level monitoring is not necessary in most clinical situations.
Adolescent↗