Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The complete amino acid sequences of subunits VII and VIIa from yeast cytochrome c oxidase are reported. Subunits VII and VIIa are 57 residues (Mr = 6603) and 54 residues (Mr = 6303) in length, respectively. Both polypeptides are amphiphilic, have an internal hydrophobic section and hydrophilic NH2 and COOH termini, and terminate at their COOH termini with a basic amino acid. This structural motif is similar to that possessed by subunit VIII of yeast cytochrome c oxidase. All three polypeptides have hydrophobic sections which are long enough to span the inner membrane; all three polypeptides lack methionine at their NH2 termini; and all three polypeptides have COOH termini which could result from proteolysis by a protease with trypsin or cathepsin B-like activity. These observations raise the interesting possibility that subunits VII, VIIa, and VIII are transmembranous polypeptides which are processed at both their NH2 and COOH termini during their biogenesis.
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PMID:The nuclear-coded subunits of yeast cytochrome c oxidase. The amino acid sequences of subunits VII and VIIa, structural similarities between the three smallest polypeptides of the holoenzyme, and implications for biogenesis. 301 77

Micromolar concentrations of GDP or GTP stimulate protein synthesis by isolated yeast mitochondria 3- to 10-fold even if alpha-ketoglutarate and an ATP-regenerating system are present. No stimulation is observed with GMP, UTP, CTP, TTP, and the nonhydrolyzable GTP analogues guanyl(beta, gamma-methylene) diphosphate and guanyl imidodiphosphate. This stimulatory effect of exogenously added guanyl nucleotides may answer the long standing question why protein synthesis by isolated mitochondria is so slow. It can also explain previous reports by two other laboratories that a high speed supernatant from yeast cells stimulates protein synthesis by isolated mitochondria. The supernatant contains nondialyzable GMP which is converted to GDP under the conditions used for assaying mitochondrial protein synthesis. The stimulatory effect of high speed supernatants is abolished by 5'-nucleotidase (which degrades GMP) or by trypsin (which destroys supernatant protein(s) necessary for converting GMP to GDP). No evidence was obtained that the stimulatory effect of high speed supernatants was caused by precursors to cytoplasmically made cytochrome c oxidase subunits.
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PMID:Stimulation of in vitro mitochondrial protein synthesis by yeast cytoplasmic extracts is caused by guanyl nucleotides. 624 10

We have shown that aq. 100% (w/v) chloral hydrate (2,2,2-trichloroethane-1,1-diol) dissociates bovine heart cytochrome c oxidase. We have developed new procedures of polyacrylamide-gel electrophoresis in the presence of chloral hydrate that permit variation in the pH of the separation, and, by using these procedures, we have observed 15 components in preparations of the enzyme. This number contrasts with the eight bands that were seen on electrophoresis in the presence of SDS (sodium dodecyl sulphate) and urea. We have isolated material from these eight bands and have characterized each by electrophoresis in the presence of chloral hydrate. Twelve of the fifteen components that were seen by electrophoresis in chloral hydrate were identified as constituents of the eight bands seen by electrophoresis in the presence of SDS and urea. Two-dimensional electrophoretic separations confirmed these identifications ans showed that the other three components which were resolved as discrete bands by electrophoresis in the presence of chloral hydrate appeared to be diffusely present in the electrophoretic separations performed in the presence of SDS and urea, which suggested anomalous behaviour in that detergent. Trypsin treatment of cytochrome c oxidase caused total loss, as observed by electrophoretic separations in the presence of chloral hydrate, of a number of components. The trypsin-sensitive components included all of those that behaved anomalously in the presence of SDS and urea. Chloral hydrate is a potent non-ionic dissociating agent for cytochrome c oxidase and its use in polyacrylamide-gel electrophoresis, with variation in the pH of the gel, permits charge-dependent separations that should have general application in the analysis of membrane proteins.
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PMID:Additional components of bovine heart cytochrome c oxidase demonstrated by high-resolution polyacrylamide-gel electrophoresis in the presence of chloral hydrate. 627 32

A method for preparing two-dimensional crystals from highly purified beef heart cytochrome c oxidase is described. This involves mixing the enzyme with phosphatidylcholine and then extracting excess lipid with deoxycholate. The reconstituted crystals show PI symmetry. Alternating rows of monomers are more closely packed (along the b axis) than in the previously described P121 crystals. However, the monomer structure in projection is the same in the two crystal forms. The P121 crystal form has been reacted with trypsin. This treatment did not alter the crystals but removed most of the impurities present in these cytochrome c oxidase preparation of low purity.
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PMID:Preparation of two-dimensional arrays from purified beef heart cytochrome c oxidase. 628 3

The arrangement of subunit IV in beef heart cytochrome c oxidase has been explored by chemical labeling and protease digestion studies. This subunit has been purified from four samples of cytochrome c oxidase that had been reacted with N-(4-azido-2-nitrophenyl)-2-aminoethyl[35S]-sulfonate (NAP-taurine), diazobenzene[35S]sulfonate, 1-myristoyl-2-[12-[(4-azido-2-nitrophenyl)amino]lauroyl]-sn-glycero-3- [14C]phosphocholine (I), and 1-palmitoyl-2-(2-azido-4-nitrobenzoyl)-sn-glycero-3-[3H]phosphocholine (II), respectively. The labeled polypeptide was then fragmented by cyanogen bromide, at arginyl side chains with trypsin (after maleylation), and the distribution of the labeling within the sequence was analyzed. The N-terminal part of subunit IV (residues 1-71) was shown to be heavily labeled by water-soluble, lipid-insoluble reagents but not by the phospholipid derivatives. These latter reagents labeled only in the region of residues 62-122, containing the long hydrophobic and putative membrane-spanning stretch. Trypsin cleavage of native cytochrome c oxidase complex at pH 8.2 was shown to clip the first seven amino acids from subunit IV. This cleavage was found to occur in submitochondrial particles but not in mitochondria or mitoplasts. These results are interpreted to show that subunit IV is oriented with its N terminus on the matrix side of the mitochondrial inner membrane and spans the membrane with the extended sequence of hydrophobic lipid residues 79-98 buried in the bilayer.
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PMID:Arrangement of subunit IV in beef heart cytochrome c oxidase probed by chemical labeling and protease digestion experiments. 631 39

Tubulin purified from rat brain was labeled by conjugation with N-succinimidyl 3-(4-hydroxy[5-125I]iodophenyl)propionate. Mitochondrial fraction prepared by centrifugation on sucrose density gradient was enriched about 4-fold in cytochrome c oxidase as compared to total liver homogenate. Contamination by plasma membranes was estimated to be about 5%. Radioiodinated pure tubulin bound to purified rat liver mitochondria; binding was time- and temperature-dependent: maximum binding was obtained after 45 min of incubation at 37 degrees C. Under conditions of binding, mitochondria retained their normal characteristics for phosphate accumulation. That binding actually occurs on mitochondria was demonstrated by the co-sedimentation of the tubulin binding and cytochrome c oxidase activities on sucrose gradient. Radioiodinated tubulin binding to mitochondria was specific and saturable. Saturation of binding was obtained using tubulin concentration ranging from 0.02 to 200 micrograms/ml. Hill plot and double reciprocal plot of binding data yielded values of 6 X 10(-8) M for an apparent KD and a maximal binding capacity of 1.4 nmol of tubulin/mg of mitochondrial protein. The Hill coefficient was 0.98 indicating that radioiodinated tubulin bound to a single class of noninteracting sites. The interaction between tubulin and mitochondria was reversible. Dissociation of the complex was obtained by dilution and by lowering the temperature. The dissociation of tubulin-mitochondria complexes was insensitive to ionic strength (0.1 to M NaCl). Mild treatment of mitochondria by trypsin (5 min at 37 degrees C) decreased of tubulin binding, suggesting that protein component(s) of membranes are involved in the interaction of tubulin with mitochondria.
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PMID:Interaction of tubulin with rat liver mitochondria. 708 18

Activity of NADH-, succinate- and cytochrome c oxidase systems of respiratory chain from rat liver mitochondrial membranes was studied in animals with various type of liver impairment under conditions of chronic allergic ulcerous colitis. Activity of the polyenzymatic systems did not exhibit marked differences as compared with the controls in fatty and chronic dystrophy but the activity was considerably increased if chronic dystrophy progressed and hepatitis developed. In chronic allergic ulcerous colitis resistance of the polyenzymatic oxidase systems to heat treatment was decreased in liver mitochondrial membranes. Under conditions of the pathology proteins and phospholipids from mitochondrial membranes were especially sensitive to the effect of trypsin and phospholipase; the rate of reactions correlated well with the severity of the liver impairment. Development of latent impairments in liver mitochondrial membranes was shown to depend on the severity of the pathological process.
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PMID:[Activity and stability of liver mitochondrial membrane polyenzyme systems in chronic allergic ulcerative colitis]. 731 85

The role of supernumerary subunits of bovine heart cytochrome c oxidase has been investigated by examining the effect on the enzymatic activity of limited proteolysis by chymotrypsin, thermolysin, and trypsin. All three proteases, when added to the soluble oxidase, digested subunits III, VIa, and VIb and caused inhibition of electron flow in the oxidase. In addition, trypsin and thermolysin also digested subunit IV. Trypsin cleaved off an N-terminal segment of seven residues; thermolysin cleaved only the first four residues at the N-terminus of subunit IV. Digestion of the soluble oxidase by trypsin but not by thermolysin caused decoupling of redox-linked proton pumping in the oxidase. It is concluded that the sequence V5-V6-K7 of the hydrophilic N-terminus of subunit IV, which protrudes out of the matrix side of the mitochondrial membrane, mediates the access of protons into the transmembrane proton translocating pathway in the oxidase.
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PMID:Role of nuclear-encoded subunits of mitochondrial cytochrome c oxidase in proton pumping revealed by limited enzymatic proteolysis. 791 75

Bovine heart cytochrome c oxidase is a multisubunit enzyme whose oligomeric state is dependent on its detergent or phospholipid environment. We have utilized the cleavable, heterobifunctional cross-linking reagent N-succinimidyl 3-[(4-azidophenyl)dithio]propionate (SADP) to detect cytochrome c oxidase dimers. Monomeric or dimeric enzyme dispersed in Triton X-100 (as assessed by sedimentation velocity measurements) was reacted with SADP. A unique intersubunit cross-link having an apparent molecular mass of 136 kDa was identified in the dimeric enzyme; this product was insensitive to limited proteolysis by trypsin and contained a cross-link between two adjacent monomers. Two-dimensional NaDodSO4-PAGE (the second dimension containing beta-mercaptoethanol to cleave the cross-linking reagent) indicated that subunit I was the major component of the dimer-specific cross-link. The dimer-specific cross-link created by SADP was observed in phospholipid vesicles [cardiolipin/phosphatidylcholine (1:20, w/w)] containing dimeric (2 microM heme aa3) enzyme; a low yield of dimer-specific cross-link was observed in liposomes containing 6 microM (heme aa3) monomeric enzyme. The 136-kDa cross-link was not observed in liposomes containing 2 microM (heme aa3) monomeric enzyme. These results indicate that subunit I from each monomer may provide one site of interaction between monomers in the dimeric form of the enzyme and that cytochrome c oxidase monomers may reassociate to form dimeric complexes in phospholipid vesicles.
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PMID:Detection of bovine heart mitochondrial cytochrome c oxidase dimers in Triton X-100 and phospholipid vesicles by chemical cross-linking. 824 Nov 83

Horse cytochrome c was reacted with the spin label (succinimidyl-2,2, 5,5-tetra-methyl-3-pyrroline-1-oxyl-carboxylate) using optimized conditions and the reaction products were separated by a combination of cation-exchange chromatography and HPLC. The purified cytochrome c derivatives were digested with TPCK treated trypsin and the resulting peptides were separated by reverse phase HPLC. The modified Lys residues were subsequently characterized by Edman degradation and mass spectrometry. These analyses showed that five distinct cytochrome c derivatives had been produced which were modified at the specific Lys residues including Lys8, Lys25, Lys72, Lys86 or Lys87, respectively. The electron paramagnetic resonance (EPR) spectra for each cytochrome c derivative revealed that for the spin label attached to Lys8 and Lys87 only one component contributes to the spectrum whereas for Lys25, Lys72 and Lys86 the spectrum consists of two components. The highest mobility with the rotational correlation time, tauB, of 0.38 ns was observed for Lys87. The longest tauB of 1.84 ns was obtained for Lys72. An attempt to correlate the spin label mobility with the local protein structure is presented. These mono derivatized cytochrome c molecules provide a unique tool for EPR studying the interaction between cytochrome c and the lipid bilayer, as well as cytochrome c oxidase and reductase.
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PMID:Preparation and electron paramagnetic resonance characterization of spin labeled monoderivatives of horse cytochrome c. 967 42


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