Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Incubation of NADH-ubiquinone oxidoreductase (Complex I) with chymotrypsin caused loss of rotenone-sensitive ubiquinone-1 reduction and an increase in rotenone-insensitive ubiquinone reduction. 2. Within the same time-course, NADH-K(3)Fe(CN)(6) oxidoreductase activity was unaffected. 3. Mixing of chymotrypsin-treated Complex I with Complex III did not give rise to NADH-cytochrome c oxidoreductase activity. 4. Gel electrophoresis in the presence of sodium dodecyl sulphate revealed selective degradation of several constituent polypeptides by chymotrypsin. 5. With higher chymotrypsin concentrations and longer incubation times, a decrease in NADH-K(3)Fe(CN)(6) oxidoreductase was observed. The kinetics of this decrease correlated with solubilization of the low-molecular-weight type-II NADH dehydrogenase (subunit mol.wts. 53000 and 27000) and with degradation of a polypeptide of mol.wt. 30000. 6. Phospholipid-depleted Complex I was more rapidly degraded by chymotrypsin. Specifically, a subunit of mol.wt. 75000, resistant to chymotrypsin in untreated Complex I, was degraded in phospholipid-depleted Complex I. In addition, the 30000-mol.wt. polypeptide was also more rapidly digested, correlating with an increased rate of transformation to type II NADH dehydrogenase.
...
PMID:Effects of proteolytic digestion by chymotrypsin on the structure and catalytic properties of reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase from bovine heart mitochondria. 41 83

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.
...
PMID:Identification of the binding site on cytochrome c1 for cytochrome c. 298 91

The binding of [14C]dicyclohexylcarbodiimide (DCCD) to soluble complex III from yeast mitochondria was examined under conditions which resulted in the inhibition of proton ejection but had a minimal effect on cytochrome c reductase activity. Incubation of the complex with 50-100 nmol of [14C]DCCD/nmol of cytochrome b at 12 degrees C did not result in any changes in the appearance of the high-molecular-weight subunits (I-V) after sodium dodecyl sulfate-gel electrophoresis, although a slight broadening of the three lowest molecular-weight subunits (VI-VIII) was observed. The [14C]DCCD was bound preferentially to subunit III (cytochrome b) and a wide band with an apparent low-molecular weight ranging from 8000 to 9000 to less than 2000 depending on the gel system used. Extraction of the [14C]DCCD-treated complex III with chloroform:methanol had no effect on subunit III but completely removed the low-molecular-weight radioactive band. Thin-layer chromatography of the chloroform:methanol extract revealed that the radioactive material extracted from the [14C]DCCD-treated complex III migrated with the same apparent RF as either free [14C]DCCD or cardiolipin. Amino acids were not detectable in an acid hydrolysate of the chloroform:methanol extract, suggesting the absence of protein. Digestion of the [14C]DCCD-treated complex III with either chymotrypsin or Staphylococcus aureus V8 protease resulted in the decrease of both staining intensity and labeling in subunit III but had no effect on the radioactivity in the low-molecular-weight material. These results confirm that DCCD binds preferentially to cytochrome b in complex III from yeast mitochondria and suggest that cytochrome b may play an important role in proton translocation at this site of the respiratory chain.
...
PMID:The preferential binding of dicyclohexylcarbodiimide to cytochrome b and phospholipids in soluble complex III from yeast mitochondria. 608 3

Cytochrome c1 is a subunit of ubiquinol--cytochrome c reductase (EC 1.10.2.2). In Neurospora crassa wild type 74A grown in the presence of chloramphenicol, the subunit is inserted only into the bilayer of the mitochondrial inner membranes without associating with other proteins. From these modified membranes a monodisperse (cytochrome c1)-Triton complex was isolated by subjecting the Triton-solubilized membranes to affinity chromatography on immobilized cytochrome c. A water-soluble pentamer of cytochrome c1 was prepared from the (cytochrome c1)-Triton complex by removing the detergent. By limited proteolytic digestion of the cytochrome c1-Triton complex with chymotrypsin, a water-soluble monomeric cytochrome c1 was prepared which has a molecular weight of only 24 000 as compared to 31 000 of the membrane-bound cytochrome c1. The 24 000-Mr cytochrome c1 and the 31 000-Mr cytochrome c1 have same light absorption spectra and cytochrome-c-binding properties. These results are used to propose the following model. Cytochrome c1 consists of a large hydrophilic part and a small hydrophobic part. The hydrophilic part extends from the mitochondrial inner membrane into the intermembrane space. This part carries the heme and interacts with cytochrome c. The hydrophobic part anchors the cytochrome c1 to the bilayer.
...
PMID:Membrane-bound and water-soluble cytochrome c1 from Neurospora mitochondria. 626 10

The orientation of the different subunits of complex III in the yeast inner mitochondrial membrane has been investigated by several different approaches. Immunoinhibition studies of cytochrome c reductase activity in intact mitoplasts and submitochondrial particles using IgG obtained from specific antisera against complex III, the iron-sulfur protein, core protein I, and core protein II suggested a transmembranous orientation of the complex with the antigenic sites of the iron-sulfur protein exposed on the cytoplasmic surface of the membrane. A lack of immunoinhibition was observed with the IgG against either core protein suggesting that these proteins may not be involved in catalysis. Digestion of mitoplasts with chymotrypsin indicated that the protein mass of cytochromes b and c1 protrudes from the cytoplasmic surface of the membrane; however, the hemes of cytochrome b appear to be buried within the membrane while the heme of cytochrome c1 is partially exposed on the chymotrypsin-sensitive portion of the polypeptide. By contrast, the iron-sulfur protein does not protrude from the membrane as it is completely resistant to chymotrypsin digestion. Labeling with the hydrophilic membrane-impermeant probe diazobenzenesulfonate suggests that core protein II is exposed on both sides of the membrane but protrudes into the matrix; while core protein I is within the membrane. Immunoprecipitation studies of sodium dodecyl sulfate and Triton X-100-solubilized mitochondria with subunit-specific antisera suggest that cytochromes b and c1 and core protein I are tightly associated in complex III. By contrast, the iron-sulfur protein and core protein II are loosely associated with the other subunits of the complex such that they are dissociated by low concentrations of detergent.
...
PMID:Topographical orientation of complex III in the yeast mitochondrial membrane. 631 51

Cytochrome b was identified as one of the ubiquinone-binding proteins in bovine heart mitochondrial ubiquinol-cytochrome c reductase by photoaffinity labeling using 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]-octyl)-1,4-benzoquinone ([3H]azido-Q). The [3H]azido-Q-labeled cytochrome b protein was purified to homogeneity from the azido-Q-labeled ubiquinol-cytochrome c reductase by a procedure involving Triton X-100 and urea treatment, calcium phosphate column chromatography, acetone precipitation, decanoyl-N-methylglucamide-cholate extraction, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified cytochrome b protein containing 0.5 mol of azido-Q/mol of protein was subjected to reductive carboxymethylation and succinylation prior to digestion by chymotrypsin. Two azido-Q-linked peptides with retention times of 47.1 and 49.0 min were obtained by high performance liquid chromatographic separation. Partial amino-terminal amino acid sequences of these two peptides were determined to be GATVI- and ALVADL-, indicating that these two chymotryptic peptides are from amino residues 142-155 and 326-336. Monospecific polyclonal antibodies against two synthetic ubiquinone-binding peptides, NH2-G-A-T-V-I-T-N-L-L-S-COOH (P-47) and NH2-W-A-L-V-A-D-L-L-T-L-T-W-I-COOH (P-49), were generated in rabbits and purified. Western blotting and enzyme-linked immunosorbent assays showed that the purified antibodies against P-47 reacted with cytochrome b-containing reductases and purified cytochrome b protein. Antibodies against P-47 inhibited activities of succinate-cytochrome c and ubiquinol-cytochrome c reductases only when they were incubated with phospholipid-depleted reductases prior to the replenishment with phospholipid. No inhibition was observed with incubation with phospholipid-containing reductases, indicating that this peptide involved in ubiquinone binding is buried in a phospholipid environment.
...
PMID:Ubiquinone binding domains in bovine heart mitochondrial cytochrome b. 829 88

---