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L Yu

Publications and source records attributed to L Yu.

At least 775 records · Page 43Linked to original sources
PubMed1985

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↗
PubMed1985

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↗
PubMed1985

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↗
PubMed1985

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↗
PubMed1985

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↗
PubMed1985

Organization and chromosomal specificity of autosomal homologs of human Y chromosome repeated DNA.

The human Y chromosome contains a group of repeated DNA elements, identified as 3.4-kilobase pair (kb) fragments in Hae III digests of male genomic DNA, which contain both Y-specific and non-Y-specific sequences. We have used these 3.4-kb Hae III Y fragments to explore the organizational properties and chromosomal distribution of the autosomal homologs of the non-Y-specific (NYS) 3.4-kb Hae III Y elements. Three distinct organizations, termed domains, have been identified and shown to have major concentrations on separate chromosomes. We have established that domain K is located on chromosome 15 and domain D on chromosome 16 and suggested that domain R is on chromosome 1. Our findings suggest that each domain is composed of a tandemly arrayed cluster of a regularly repeating unit containing two sets of repeated sequences: one that is homologous to the NYS 3.4-kb Hae III Y sequences and one that does not cross-react with the 3.4-kb Hae III Y repeats. Thus, these autosomal repeated DNA domains, like their Y chromosome counterparts, consist of a complex mixture of repeated DNA elements interspersed among each other in ways that lead to defined periodicities. Although each of the three identified autosomal domains cross-reacts with 3.4-kb Hae III Y fragments purified from genomic DNA, the length periodicities and sequence content of the autosomal domains are chromosome specific. The organizational properties and chromosomal distribution of these NYS 3.4-kb Hae III homologs seem inconsistent with stochastic mechanisms of sequence diffusion between chromosomes.

Animals↗
PubMed1984

Purification and properties of cytochrome b from photosynthetic bacterium Rhodopseudomonas sphaeroides R-26.

Cytochrome b of R. sphaeroides R-26 has been purified from the isolated cytochrome b-c1 complex to homogeneity. The purification procedure involves Triton X-100 and urea solubilization, calcium phosphate column chromatography at different pH values, and ammonium sulfate fractionation. The purified protein contains 23 nmol heme per mg protein and has an apparent molecular weight of 43,000, as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis. The spectral characteristics of purified cytochrome b are similar to those of cytochrome b in the active cytochrome b-c1 complex but with a lower absorbance. The amino acid composition has been determined and compared with cytochrome b purified from other sources.

Amino Acids↗
PubMed1984

Characterization of purified cytochrome b-c1 complex from Rhodopseudomonas sphaeroides R-26.

A highly purified cytochrome b-c1 complex from Rhodopseudomonas sphaeroides R-26 was isolated by a procedure involving Triton X-100 solubilization, calcium phosphate column chromatography, and ammonium sulfate fractionation. The purified enzyme complex contains, in nanomoles/mg of protein, cytochrome b, 8.3; cytochrome c1, 8.3; iron-sulfur protein, 15; phospholipids, 182; and ubiquinone, 5. Four major polypeptides with apparent molecular weights of 48,000, 30,000, 24,000, and 12,000 were detected in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mr = 48,000 and 30,000 proteins are cytochromes b and c1, respectively. The enzyme complex catalyzes electron transfer from ubiquinol to cytochrome c with a specific activity of 12.6 mumol of cytochrome c reduced per min/mg of protein at 23 degrees C. This is lower than that of the mitochondrial enzyme, although both systems have similar essential redox components and a similar Km for ubiquinol. The activity is fully sensitive to antimycin A and 5-n-undecyl-6-hydroxy-4, 7-dioxobenzothiazole. The enzyme complex is stable at neutral pH and at lower temperatures, but became less stable when the incubation temperature was raised. At 37 degrees C, the half-life is 15 min. The enzymatic activity was insensitive to treatment with N',N'-dicyclohexylcarbodiimide. No p-chloromercuriphenylsulfonate-alkylable sulfhydryl groups were detected. The major phospholipids associated with the purified enzyme complex are phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol with molar per cent distributions of 25, 21, and 35, respectively. About 60% of the enzymatic activity was abolished upon treatment with phospholipase A2. The phospholipase A2-inactivated activity can be partially restored by the addition of EDTA followed with phospholipids prepared from either the cytochrome b-c1 complex of the same source or a mixture of phosphatidylglycerol and asolectin.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron Transport↗
PubMed1984

Isolation and characterization of cytochrome C1 from photosynthetic bacterium Rhodopseudomonas sphaeroides R-26.

Cytochrome c1 of photosynthetic bacterium R. sphaeroides R-26 has been purified from isolated cytochrome b-c1 complex to a single polypeptide, using a procedure involving Triton X-100 and urea solubilization, calcium phosphate column chromatography and ammonium sulfate fractionation. The purified protein contains 30 nmoles heme per mg protein and has an apparent molecular weight of 30,000, as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis. Bacterial cytochrome c1 is soluble in aqueous solution in the absence of detergent and has spectral characteristics similar to mammalian cytochrome c1. The amino acid compositions of these two proteins, however, are not comparable.

Amino Acids↗
PubMed1983

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids.

Thermotropic properties of purified cytochrome c1 and cytochrome c have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome c1 are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome c is rather insignificant. The formation of a complex between cytochromes c and c1 at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome c upon mixing with cytochrome c1 was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome c was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.

Calorimetry, Differential Scanning↗
PubMed1983

Syntheses and biological activities of azido ubiquinone derivatives.

A general method for the synthesis of azido-ubiquinone derivatives has been developed directly by substituting one hydrogen atom on the benzoquinone ring with an azido group under weakly acidic conditions. The reaction takes several hours and the yield is generally low. The azido-ubiquinone was purified by preparative thin layer chromatography, and identified by NMR, IR and mass spectra. All the synthesized azido-ubiquinone derivatives show partial activity in mediating biological electron transfer in the dark, and show partial or complete inhibition upon photolysis.

Chromatography, Thin Layer↗
PubMed1983

The existence of an antimycin A insensitive ubiquinol-cytochrome c reductase activity in the photosynthetic apparatus.

A nonproteinaceous, antimycin A insensitive ubiquinol-cytochrome c reductase activity is detected in and purified from chromatophores of Rhodopseudomonas sphaeroides, R-26. This activity is about 5 times the antimycin A sensitive reductase activity in chromatophores and the two are not interconvertable. The purification involved chloroform:methanol (2:1), and hexane extractions and florisil column chromatography. The purified preparation contains some bacteriochlorophyll-like pigments and phospholipids, and is stable in organic solvent. It catalyzes the oxidation of ubiquinol by cytochrome c with substrate specificity and pH optimum.

Antimycin A↗