Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

The topology of several of the cytoplasmically made subunits of beef heart cytochrome c oxidase has been determined by protease digestion of oriented membrane preparations, using subunit-specific antibodies to identify cleavage products. Reconstituted vesicles of cytochrome c oxidase and asolectin were used as a vesicle preparation with the C domain of the enzyme available for protease digestion. Submitochondrial particles were used as vesicles with the M domain outermost. Trypsin and/or proteinase K cleaved polypeptides CIV, ASA, AED, STA, and IHQ. Cleavage of CIV, STA, and IHQ was from the M domains only and involved the removal of a fragment from the N-terminus in each case. Polypeptide AED was cleaved from the C side in the N-terminal part, while ASA was cleaved from both the C and M domains. Polypeptide fragments were electroblotted from polyacrylamide gels onto derivatized glass paper and sites of proteolytic cleavage determined by N-terminal sequence analysis.
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PMID:Orientation of the cytoplasmically made subunits of beef heart cytochrome c oxidase determined by protease digestion and antibody binding experiments. 283 91

Cytochrome c oxidase from rat liver was incubated with various proteinases of different specificities and the enzymic activity was measured after various incubation times. A loss of catalytic activity was found after digestion with proteinase K, aminopeptidase M and a mitochondrial proteinase from rat liver. In each case the decrease in enzymic activity was compared with the changes in intensities of the polypeptide pattern obtained after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The susceptibilities of the subunit polypeptides of the soluble cytochrome c oxidase to proteinases were very different. Whereas subunit I was most susceptible, subunits V--VII were rather resistant to degradation. From the relative inaccessibility of subunits V--VII to proteinases it is likely that these polypeptides are buried in the interior of the enzyme complex.
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PMID:Catalytic activity and arrangement of subunit polypeptides in rat liver cytochrome c oxidase as studied by proteolysis. 624 69

Peptides with sequences based on the leader sequence of yeast cytochrome c oxidase subunit IV (pCOX IV-(1-25)) activate the electrophoretic uptake of K+ and other cations such as tetraethylammonium and lysine by rat liver mitochondria with EC50 = 11-15 microM. Uptake of these cations is dependent on respiration and is prevented by uncoupling agents, and the Vmax for K+ is 1.2-1.5 micromol/min/mg. Albeit more slowly, the non-electrolytes mannitol and sucrose are also transported by this pathway. Treatment of the peptides with proteinase K eliminates the stimulatory effect. Since the stimulated rate is not inhibited by ATP or by cyclosporin, we conclude that this pathway is not related to the mitochondrial KATP channel or the Ca2+-dependent permeability transition pore. Transport is stimulated by pCOX IV-(1-23), pCOX IV-(1-22), and pCOX IV-(1-12)Y, but not by a 13-amino acid peptide representing the nuclear location sequence of the SV40 large T antigen, which is responsible for directing that protein to the nucleus. Spermine, which has four positive charges, also has no stimulatory effect, and an amphiphilic 22-residue peptide derived from antithrombin III with seven net charges is only one-twentieth as effective as pCOX IV-(1-22). Thus, these data indicate that the sequence/structure is important for activation of transport. We also demonstrate that mitochondrial uncoupling, previously reported to be induced by these peptides, actually reflects coupled accumulation of salt. In view of our findings, it is also likely that the lytic effects attributed to these peptides are secondary to swelling and are not due to membrane damage per se. Finally, we show that, in non-ionic media, the peptide is an inhibitor of cytochrome c oxidase.
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PMID:Effect of leader peptides on the permeability of mitochondria. 915 2

The nuclear PET309 gene of Saccharomyces cerevisiae is necessary for expression of the mitochondrial COX1 gene, which encodes subunit I of cytochrome c oxidase. In a pet309 null mutant, there is a defect both in accumulation of COX1 pre-RNA, if it contains introns, and in translation of COX1 RNAs [Manthey, G. M. & McEwen, J. E. (1995) EMBO J. 14, 4031-4043]. To facilitate identification and intracellular localization of the protein Pet309p, that is encoded by the PET309 gene, Pet309p was tagged at the carboxy terminus with an epitope from the human c-myc protein. A monoclonal antibody against the c-myc epitope detected a 98-kDa protein in mitochondria of yeast cells that expressed the PET309-c-myc fusion protein from a high copy number plasmid. This protein was not detectable in cells that did not express the fusion protein, or that expressed it from a single copy centromeric vector. Additional analyses of mitochondrial subfractions demonstrated that the PET309-c-myc fusion protein is localized specifically in the inner mitochondrial membrane. It could not be extracted by alkaline sodium carbonate, yet it was susceptible to proteinase K digestion in mitoplasts (mitochondria with a disrupted outer membrane). These results indicate that Pet309p spans the inner membrane, with domain(s) exposed to the intermembrane space side of the membrane. How Pet309p may function in concert with other gene products necessary for COX1 RNA translation or accumulation, such as Mss51p or Nam1p, respectively, is discussed.
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PMID:The Saccharomyces cerevisiae Pet309 protein is embedded in the mitochondrial inner membrane. 969 14

Leigh syndrome (LS) associated with cytochrome c oxidase (COX) deficiency is an autosomal recessive neurodegenerative disorder caused by mutations in SURF1. Although SURF1 is ubiquitously expressed, its expression is lower in brain than in other highly aerobic tissues. All reported SURF1 mutations are loss of function, predicting a truncated protein (hSurf1) product. Western blot analysis with anti-hSurf1 antibodies demonstrated a specific 30 kDa protein in control fibroblasts, but no protein in LS patient cells. Steady-state levels of both nuclear- and mitochondrial-encoded COX subunits were also markedly reduced in patient cells, consistent with a failure to assemble or maintain a normal amount of the enzyme complex. An epitope (FLAG)-tagged hSurf1 was targeted to mitochondria in COS7 cells and a mitochondrial import assay showed that the hSurf1 precursor protein (35 kDa) was imported and processed to its mature form (30 kDa) in a membrane potential-dependent fashion. The protein was resistant to alkaline carbonate extraction and susceptible to proteinase K digestion in mitoplasts. Mutant proteins in which the N-terminal transmembrane domain or central loop were deleted, or the C-terminal transmembrane domain disrupted, did not accumulate and could not rescue COX activity in patient cells. Co-expression of the N- and C-terminal transmembrane domains as independent entities also failed to rescue the enzyme deficiency. These data demonstrate that hSurf1 is an integral inner membrane protein with an essential role in the assembly or maintenance of the COX complex and that insertion of both transmembrane domains in the intact protein is necessary for function.
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PMID:Expression and functional analysis of SURF1 in Leigh syndrome patients with cytochrome c oxidase deficiency. 1055 3

Alpha-1,4-galacturonosyltransferase (GalAT) is an enzyme required for the biosynthesis of the plant cell wall pectic polysaccharide homogalacturonan (HGA). GalAT activity in homogenates from pea (Pisum sativum L. var. Alaska) stem internodes co-localized in linear and discontinuous sucrose gradients with latent UDPase activity, an enzyme marker specific for Golgi membranes. GalAT activity was separated from antimycin A-insensitive NADH:cytochrome c reductase and cytochrome c oxidase activities, enzyme markers for the endoplasmic reticulum and the mitochondria, respectively. GalAT and latent UDPase activities were separated from the majority (80%) of callose synthase activity, a marker for the plasma membrane, suggesting that little or no GalAT is present in the plasma membrane. GalAT activities in proteinase K-treated and untreated Golgi vesicles were similar, whereas no GalAT activity was detected after treating Golgi vesicles with proteinase K in the presence of Triton X-100. These results demonstrate that the catalytic site of GalAT resides within the lumen of the Golgi. The products generated by Golgi-localized GalAT were converted by endopolygalacturonase treatment to mono- and di-galacturonic acid, thereby showing that GalAT synthesizes 1-->4-linked alpha-D-galacturonan. Our data provide the first enzymatic evidence that a glycosyltransferase involved in HGA synthesis is present in the Golgi apparatus. Together with prior results of in vivo labeling and immunocytochemical studies, these results show that pectin biosynthesis occurs in the Golgi. A model for the biosynthesis of the pectic polysaccharide HGA is proposed.
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PMID:The catalytic site of the pectin biosynthetic enzyme alpha-1,4-galacturonosyltransferase is located in the lumen of the Golgi. 1155 63

Highly purified rat liver mitochondria (RLM) when exposed to tert-butylhydroperoxide undergo matrix swelling, membrane potential collapse, and oxidation of glutathione and pyridine nucleotides, all events attributable to the induction of mitochondrial permeability transition. Instead, RLM, if treated with the same or higher amounts of H2O2 or tyramine, are insensitive or only partially sensitive, respectively, to mitochondrial permeability transition. In addition, the block of respiration by antimycin A added to RLM respiring in state 4 conditions, or the addition of H2O2, results in O2 generation, which is blocked by the catalase inhibitors aminotriazole or KCN. In this regard, H2O2 decomposition yields molecular oxygen in a 2:1 stoichiometry, consistent with a catalytic mechanism with a rate constant of 0.0346 s(-1). The rate of H2O2 consumption is not influenced by respiratory substrates, succinate or glutamate-malate, nor by N-ethylmaleimide, suggesting that cytochrome c oxidase and the glutathione-glutathione peroxidase system are not significantly involved in this process. Instead, H2O2 consumption is considerably inhibited by KCN or aminotriazole, indicating activity by a hemoprotein. All these observations are compatible with the presence of endogenous heme-containing catalase with an activity of 825 +/- 15 units, which contributes to mitochondrial protection against endogenous or exogenous H2O2. Mitochondrial catalase in liver most probably represents regulatory control of bioenergetic metabolism, but it may also be proposed for new therapeutic strategies against liver diseases. The constitutive presence of catalase inside mitochondria is demonstrated by several methodological approaches as follows: biochemical fractionating, proteinase K sensitivity, and immunogold electron microscopy on isolated RLM and whole rat liver tissue.
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PMID:Catalase takes part in rat liver mitochondria oxidative stress defense. 1757 67