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

Microdomains of high cytosolic free Ca(2+) concentration in the proximity of mitochondria might have an important role in the stimulation of steroidogenesis in bovine adrenal glomerulosa cells. In the present study we have investigated local changes of free Ca(2+) concentration near the outer mitochondrial membrane ([Ca(2+)](om)) under stimulation with angiotensin II (Ang II) and K(+). Glomerulosa cells in primary culture were transfected with a recombinant cDNA encoding the N-terminal region of the human translocase protein 20 of the outer mitochondrial membrane, in frame with the Ca(2+)-sensitive photoprotein aequorin. This chimaeric aequorin (TomAeq) was associated with mitochondria-enriched subcellular fractions of transfected COS-7 cells and was susceptible to proteinase K, showing that it was targeted to the outer mitochondrial membrane, facing the cytosolic space. In bovine adrenal glomerulosa cells transfected with TomAeq cDNA, Ang II induced a transient [Ca(2+)](om) peak reaching 1.42+/-0.28 microM, which decreased immediately to the basal resting value. The peak response to Ang II was strikingly lower than the peak response of mitochondrial free Ca(2+) concentration, which increased to 5.4+/-1.2 microM. The smaller response of [Ca(2+)](om) to Ang II compared with the elevated matrix response did not result from buffering effects of the organelle, from altered mechanisms of intramitochondrial Ca(2+) transport or from differences in the affinity of the chimaeric aequorins for Ca(2+). This approach has allowed us to follow perimitochondrial Ca(2+) homeostasis in bovine glomerulosa cells under stimulation with Ca(2+)-mobilizing agonists and to reveal a strong gradient of Ca(2+) concentration between the mitochondrial matrix and the immediate environment of the organelle.
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PMID:Measurement of perimitochondrial Ca2+ concentration in bovine adrenal glomerulosa cells with aequorin targeted to the outer mitochondrial membrane. 1041 40

Despite growing evidence for a mitochondrial localization of nitric oxide (NO) synthase and a broadening spectrum of NO actions on mitochondrial respiration and apoptosis, the basis for interaction between the enzyme and the organelle remain obscure. Here we investigated mitochondrial localization of endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells and human embryonic kidney cells transfected or infected with eNOS expression vectors. Copurification of eNOS with mitochondria was observed in both human umbilical vein endothelial cells and eNOS-expressing human embryonic kidney cells. Immunodetectable eNOS was cleaved from mitochondria by proteinase K treatment, suggesting eNOS association with the outer mitochondrial membrane. Localization of eNOS to a proteinase K-cleavable site on the cytoplasmic face of the outer membrane was confirmed by immunogold labeling of non-permeabilized mitochondria. Markers for mitochondrial subfractions ruled out the possibility of eNOS association with an intramitochondrial site or inverted mitochondrial particles. Denaturation of eNOS did not attenuate association with mitochondria. Mutant eNOS lacking a pentabasic amino acid sequence within the autoinhibitory domain (residues 628-632 of the bovine eNOS) showed dramatically reduced binding to the mitochondrial but not to the plasma membrane, which was associated with increased oxygen consumption. Collectively, these findings argue in favor of eNOS localization to the outer mitochondrial membrane in endothelial cells and identify elements of a novel anchoring mechanism.
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PMID:Docking of endothelial nitric oxide synthase (eNOS) to the mitochondrial outer membrane: a pentabasic amino acid sequence in the autoinhibitory domain of eNOS targets a proteinase K-cleavable peptide on the cytoplasmic face of mitochondria. 1476 67

This study examines the role of c-jun N-terminal kinase (JNK) in mitochondrial signaling and bioenergetics in primary cortical neurons and isolated rat brain mitochondria. Exposure of neurons to either anisomycin (an activator of JNK/p38 mitogen-activated protein kinases) or H2O2 resulted in activation (phosphorylation) of JNK (mostly p46(JNK1)) and its translocation to mitochondria. Experiments with mitochondria isolated from either rat brain or primary cortical neurons and incubated with proteinase K revealed that phosphorylated JNK was associated with the outer mitochondrial membrane; this association resulted in the phosphorylation of the E(1alpha) subunit of pyruvate dehydrogenase, a key enzyme that catalyzes the oxidative decarboxylation of pyruvate and that links two major metabolic pathways: glycolysis and the tricarboxylic acid cycle. JNK-mediated phosphorylation of pyruvate dehydrogenase was not observed in experiments carried out with mitoplasts, thus suggesting the requirement of intact, functional mitochondria for this effect. JNK-mediated phosphorylation of pyruvate dehydrogenase was associated with a decline in its activity and, consequently, a shift to anaerobic pyruvate metabolism: the latter was confirmed by increased accumulation of lactic acid and decreased overall energy production (ATP levels). Pyruvate dehydrogenase appears to be a specific phosphorylation target for JNK, for other kinases, such as protein kinase A and protein kinase C did not elicit pyruvate dehydrogenase phosphorylation and did not decrease the activity of the complex. These results suggest that JNK mediates a signaling pathway that regulates metabolic functions in mitochondria as part of a network that coordinates cytosolic and mitochondrial processes relevant for cell function.
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PMID:c-Jun N-terminal kinase regulates mitochondrial bioenergetics by modulating pyruvate dehydrogenase activity in primary cortical neurons. 1794 12

Recently, we have reported the presence of calpain-calpastatin system in mitochondria of bovine pulmonary smooth muscle [P. Kar, T. Chakraborti, S. Roy, R. Choudhury, S. Chakraborti, Arch. Biochem. Biophys. 466 (2007) 290-299]. Herein, we report its localization in the mitochondria. Immunoblot, immunoelectron microscopy and casein zymographic studies suggest that mu-calpain and calpastatin are present in the inner mitochondrial membrane; but not in the outer mitochondrial membrane or in the inter membrane space or in the matrix of the mitochondria. Co-immunoprecipitation studies suggest that mu-calpain-calpastatin is associated in the inner mitochondrial membrane. Additionally, the proteinase K and sodium carbonate treatments of the mitoplasts revealed that mu-calpain is integrally and calpastatin is peripherally embedded to the outer surface of inner mitochondrial membrane. These studies indicate that an association between mu-calpain and calpastatin occurs in the inner membrane towards the inter membrane space of the mitochondria, which provides better insight about the protease regulation towards initiation of apoptotic processes mediated by mitochondria.
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PMID:Submitochondrial localization of associated mu-calpain and calpastatin. 1808 16

Although PTEN has been widely described as a nuclear and cytosolic protein, in the last 2 years, alternative organelles, such as the endoplasmic reticulum (ER), pure mitochondria, and mitochondria-associated membranes (MAMs), have been recognized as pivotal targets of PTEN activity.Here, we describe different methods that have been used to highlight PTEN subcellular localization.First, a protocol to extract nuclear and cytosolic fractions has been described to assess the "canonical" PTEN localization. Moreover, we describe a protocol for mitochondria isolation with proteinase K (PK) to further discriminate whether PTEN associates with the outer mitochondrial membrane (OMM) or resides within the mitochondria. Finally, we focus our attention on a subcellular fractionation protocol of cells that permits the isolation of MAMs containing unique regions of ER membranes attached to the outer mitochondrial membrane (OMM) and mitochondria without contamination from other organelles. In addition to biochemical fractionations, immunostaining can be used to determine the subcellular localization of proteins; thus, a detailed protocol to obtain good immunofluorescence (IF) is described. The employment of these methodological approaches could facilitate the identification of different PTEN localizations in several physiopathological contexts.
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PMID:Methods to Study PTEN in Mitochondria and Endoplasmic Reticulum. 2703 78