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)

A character of rat liver mitochondria degradation after the heat treatment of animals is studied. It is found that mitochondria under the effect of elevated temperature do not considerably change their functional characteristics and thus they are capable to provide the normal rate of ATP synthesis, the rate of succinate oxidation being slightly increased. At the same time the heating caused the degradation of mitochondria which results in the decrease of their thermostability, in the increased susceptibility to lytic effect of trypsin and phospholipase D, and in the activation of succinate dehydrogenase and cytochrome c oxidase. The mitochondria degradation is due to the formation of "latent impairments" in the structure of mitochondria.
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PMID:[Some characteristics of mitochondrial multienzyme systems from rat liver mitochondria after heating of rats]. 20 Feb 83

Applying the technique of 'tip-dip' to mitochondria, we have shown the existence in this organelle of a cationic channel of large conductance, which is blocked by a 13-residue peptide possessing the sequence of the N-terminal extremity of the cytochrome c oxidase subunit IV precursor. To study the submitochondrial localization of the channel, the effect of trypsin on isolated channels and on entire mitochondria were compared. One side of isolated channels is sensitive to trypsin, which eliminates the voltage dependence. Channels isolated from trypsinized mitochondria were devoid of voltage dependence and were blocked by the peptide. This suggests a localization of the channel on the outer membrane. Consistent with this hypothesis, the channel was observed with the highest frequency in outer membrane fractions purified by different procedures, either from bovine adrenal cortex or from rat liver mitochondria. Such a localization is also consistent with digitonin solubilization experiments. The channel was solubilized before the inner membrane marker, cytochrome c oxidase. The orientation of the channel was inferred from its trypsin sensitivity and its potential dependence: a transmembrane potential (inside negative) will close the channel.
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PMID:A peptide-sensitive channel of large conductance is localized on mitochondrial outer membrane. 170 87

The arrangement of three subunits of beef heart cytochrome c oxidase, subunits Va, VIa, and VIII, has been explored by chemical labeling and protease digestion studies. Subunit Va is an extrinsic protein located on the C side of the mitochondrial inner membrane. This subunit was found to label with N-(4-azido-2-nitrophenyl)-2-aminoethane[35S]sulfonate and sodium methyl 4-[3H]formylphenyl phosphate in reconstituted vesicles in which 90% of cytochrome c oxidase complexes were oriented with the C domain outermost. Subunit VIa was cleaved by trypsin both in these reconstituted vesicles and in submitochondrial particles, indicating a transmembrane orientation. The epitope for a monoclonal antibody (mAb) to subunit VIa was lost or destroyed when cleavage occurred in reconstituted vesicles. This epitope was localized to the C-terminal part of the subunit by antibody binding to a fusion protein consisting of glutathione S-transferase (G-ST) and the C-terminal amino acids 55-85 of subunit VIa. No antibody binding was obtained with a fusion protein containing G-ST and the N-terminal amino acids 1-55. The mAb reaction orients subunit VIa with its C-terminus in the C domain. Subunit VIII was cleaved by trypsin in submitochondrial particles but not in reconstituted vesicles. N-Terminal sequencing of the subunit VIII cleavage product from submitochondrial particles gave the same sequence as the untreated subunit, i.e., ITA, indicating that it is the C-terminus which is cleaved from the M side. Subunits Va and VIII each contain N-terminal extensions or leader sequences in the precursor polypeptides; subunit VIa is made without an N-terminal extension.
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PMID:Topology of subunits of the mammalian cytochrome c oxidase: relationship to the assembly of the enzyme complex. 170 66

Transverse-plane topography of mitochondrial outer-membrane long-chain acyl-CoA synthetase was investigated using proteases as probes for exposure of crucial domains, i.e. domains containing the active site or otherwise required for enzymatic activity. Incubation of intact mitochondria with the nonspecific proteases proteinase K and subtilisin resulted in a time-dependent loss of 90% or more of the long-chain acyl-CoA synthetase activity compared to control incubations. The integrity of the outer membrane before and during this treatment was shown by cytochrome c oxidase latency as well as the stability of adenylate kinase activity in the presence of protease. After a 15-min incubation in these conditions, site-specific proteases such as trypsin and chymotrypsin had only a limited inhibitory effect (29 and 58% loss of activity, respectively); however, treatment of hypotonically disrupted mitochondria with these proteases resulted in increased (71 and 77%, respectively) loss of activity. Exposure of trypsin-sensitive crucial domains on the inner surface of the membrane was directly demonstrated by incubation of trypsin-loaded outer-membrane vesicles. Together, these results suggest that mitochondrial long-chain acyl-CoA synthetase is a transmembrane enzyme, possessing crucial domains on both sides of the outer membrane. However, the cytosolic exposure of the enzyme does not appear to be affected by a change in the medium ionic strength as seen previously for other outer-membrane enzymes. In an experiment investigating the topography of the active site of the enzyme, an immobilized substrate analog, desulfo-CoA-agarose, was preincubated with intact mitochondria. This resulted in up to a 42% loss of the activity of long-chain acyl-CoA synthetase, consistent with a cytosolic exposure for at least the CoA-binding domain of the active site.
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PMID:Transverse-plane topography of long-chain acyl-CoA synthetase in the mitochondrial outer membrane. 218 22

A voltage-dependent cationic channel of large conductance is observed in phospholipid bilayers formed at the tip of microelectrodes from proteoliposomes derived from mitochondrial membranes. This channel was blocked by a 13-residue peptide with the sequence of the amino terminal extremity of the nuclear-coded subunit IV of cytochrome c oxidase. The blockade was reversible, voltage- and dose-dependent. The peptide did not affect the activity of a Torpedo chloride channel observed under the same conditions. From experiments with phospholipid monolayers, it is unlikely that the peptide inserts into bilayers under the experimental conditions used. The blockade was observed from both sides of the membrane, being characterized by more frequent transitions to the lower conductance states, and a maximum effect was observed around 0 mV. Channels, the gating mechanism of which had been eliminated by exposure to trypsin, were also blocked by the peptide. For trypsinized channels, the duration of the closure decreased and the blockade saturated at potentials below -30 mV. These observations are consistent with a translocation of the peptide through the channel. Dynorphin B, which has the same length and charge as the peptide, had some blocking activity. Introduction of negative charges in the peptide by succinylation suppressed the activity.
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PMID:Blockade of a mitochondrial cationic channel by an addressing peptide: an electrophysiological study. 248 95

Role of supernumerary subunits of bovine heart cytochrome c oxidase has been investigated by examining the influence on the enzymatic activity of their removal by chromatographic procedures or controlled digestion by trypsin. Is has been shown that partial proteolytic cleavage of subunit IV results in depression of respiratory activity and of redox-linked proton translocation. Selective removal by gel-filtration of subunit Vlb has no significant influence on the redox and protonmotive activity of the oxidase.
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PMID:Role of supernumerary subunits in mitochondrial cytochrome c oxidase. 255 39

The role of zinc in beef heart cytochrome c oxidase has been studied by using x-ray absorption spectroscopy, zinc depletion and secondary structure predictions of subunits of beef heart cytochrome c oxidase. The stoichiometry of zinc in cytochrome oxidase has been determined in 35 different preparations and found to be one-half of copper (Cu:Zu = 2:1). Zinc is tightly bound to this enzyme and cannot be removed by dialysis against EDTA. However, zinc could be partially (up to 50%) depleted by treating the enzyme with either dipicolinic acid or by trypsin digestion. This partial depletion of zinc does not change the O2 uptake rate. X-ray absorption spectroscopy shows that the atom is in a distorted tetrahedral environment with mostly sulfur ligands. Since subunit VIa removed by the digestion removes about one-half the zinc, a possible binding site involves the two S sites present in that subunit with an appropriate folding in a structural role.
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PMID:On the environment of zinc in beef heart cytochrome c oxidase: an x-ray absorption study. 284 29

Vesicles reconstituted with bovine heart cytochrome c oxidase and dioleoylphosphatidylcholine can be resolved into two populations by column chromatography in DEAE-Sephacryl (Madden, T.D. and Cullis, P.R. (1984) J. Biol. Chem. 259, 7655-7658). These two fractions (I and II) were treated with two proteases. These are trypsin, which has been found to cleave subunit IV in the M domain of the cytochrome c oxidase molecule, and chymotrypsin, which has been found to cleave subunit III in the C domain. These studies show that fraction I vesicles contain cytochrome c oxidase orientation with the M domain outside, i.e., in the same topology as in submitochondrial particles, while fraction II vesicles contain enzyme molecules with their C domain outside, and thus in the same orientation as in mitochondria.
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PMID:Orientation of cytochrome c oxidase molecules in the two populations of reconstituted vesicles resolved by column chromatography on DEAE-Sephacryl. 298 11

Bovine heart mitochondrial cytochrome c oxidase has been treated with trypsin in order to investigate the role of components a, b, and c (nomenclature of Capaldi) in cytochrome c binding, electron transfer, and proton-pumping activities. Cytochrome c oxidase was dispersed in nondenaturing detergent solution (B. Ludwig, N. W. Downer, and R. A. Capaldi (1979) Biochemistry 18, 1401) and treated with trypsin. This treatment inhibited electron transfer activity by 9% when compared to a similarly treated control in a polarographic assay (493 s-1) and had no large effect on the high affinity (Km = 6.1 X 10(-8) M) or low affinity (Km = 2.2 X 10(-6) M) sites of cytochrome c interaction with cytochrome c oxidase. Direct thermodynamic binding experiments with cytochrome c showed that neither the high affinity (1.04 +/- 0.06 mol cytochrome c/mol cytochrome c oxidase) nor the high-plus-low affinity (2.21 +/- 0.15 mol cytochrome c/mol cytochrome c oxidase) binding sites of cytochrome c on the enzyme were perturbed by the trypsin treatment. Control and trypsin-treated enzyme incorporated into phospholipid vesicles (prepared by the cholate dialysis method) exhibited respiratory control ratios of 6.5 +/- 0.7 and 6.3 +/- 0.6, respectively. The vectorial proton translocation activity in the phospholipid vesicles was unaffected by trypsin treatment with proton translocated to electron transferred ratios being equivalent to the control. NaDodSO4-PAGE showed that components a, b, and c were completely removed by the trypsin treatment. [14C]Iodoacetamide labeling experiments showed that the content of component c in the enzyme was depleted by 85% and that greater than 50% of component a was cleaved upon the trypsin treatment. These results suggest that components a, b, and c are not required for maximum electron transfer and proton translocation activities in the isolated enzyme.
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PMID:Characterization of electron transfer and proton translocation activities in trypsin-treated bovine heart mitochondrial cytochrome c oxidase. 300 79

Isolated beef heart cytochrome c oxidase was reconstituted in liposomes by the cholate dialysis method with 85% of the binding site for cytochrome c oriented to the outside. Trypsin cleaved specifically subunit VIa and half of subunit IV from the reconstituted enzyme. The kinetic properties of the reconstituted enzyme were changed by trypsin treatment if measured by the spectrophotometric assay but not by the polarographic assay. It is concluded that subunit VIa and/or subunit IV participate in the electron transport activity of cytochrome c oxidase.
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PMID:Effect of trypsin on the kinetic properties of reconstituted beef heart cytochrome c oxidase. 300 49


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