Tissue and subcellular distribution of enzymes inactivating leupeptin

1983 ◽  
Vol 3 (2) ◽  
pp. 179-184 ◽  
Author(s):  
Catherine P. Brown ◽  
Robert J. Beynon
Keyword(s):  
Subcellular Distribution ◽  
Proteinase Inhibitor ◽  
Soluble Fraction ◽  
Streptomyces Sp ◽  
Culture Filtrates ◽  
Mammalian Tissues

Leupeptin, a proteinase inhibitor obtained from culture filtrates of Streptomyces sp., has been proposed as a valuable agent for the restriction of proteolysis in vivo. We have previously shown that mammalian tissues possess an enzymic system that is capable of inactivating leupeptin (Beynon R39 Brown CP & Butler PEr 1981, Acta. Biol. Med. Germ. 40, 1539–1546). This paper demonstrates the ubiquitous distribution of the inactivating system throughout mammalian tissues and provides evidence for the location of the enzyme(s) in the soluble fraction of the cell.

scholarly journals Subcellular distribution of glycosylphosphatidylinositol-specific phospholipase D in rat liver

1996 ◽  
Vol 320 (1) ◽  
pp. 315-319 ◽  
Author(s):  
Thomas HARI ◽  
Hans KUNZE ◽  
Ernst BOHN ◽  
Urs BRODBECK ◽  
Peter BÜTIKOFER
Keyword(s):  
Rat Liver ◽  
Phospholipase D ◽  
Subcellular Distribution ◽  
Lysosomal Enzymes ◽  
Body Fluids ◽  
Intracellular Distribution ◽  
Lysosomal Fraction ◽  
Mammalian Tissues

Glycosylphosphatidylinositol (GPI)-hydrolysing enzymes have been described in many mammalian tissues and body fluids; however, their site(s) of action and in vivo functions have remained unclear. In order to identify a possible intracellular site of GPI hydrolysis, we studied the subcellular distribution of GPI-hydrolysing activity in rat liver. We found that purified fractions from rat liver hydrolysed the GPI moieties of two GPI-anchored proteins with the specificity of a phospholipase D. This GPI-specific phospholipase D (GPI-PLD) activity was found to be highly enriched in a lysosomal fraction and showed a similar intracellular distribution to that of typical lysosomal enzymes. Our results indicate that lysosomes may represent a possible intracellular site of GPI-PLD action.

Cigarette smoking, emphysema, and damage to alpha 1-proteinase inhibitor

1994 ◽  
Vol 266 (6) ◽  
pp. L593-L611 ◽  
Author(s):  
M. D. Evans ◽  
W. A. Pryor
Keyword(s):  
Cigarette Smoke ◽  
Proteinase Inhibitor ◽  
Inflammatory Cells ◽  
Lavage Fluid ◽  
Lung Lavage ◽  
Tissue Destruction ◽  
Phagocytic Cells ◽  
Lung Fluid

The proteinase-antiproteinase theory for the pathogenesis of emphysema proposes that the connective tissue destruction associated with emphysema arises from excessive proteinase activity in the lower respiratory tract. For this reason, the relative activities of neutrophil elastase and alpha 1-proteinase inhibitor (alpha 1-PI) are considered important. Most emphysema is observed in smokers; therefore, alpha 1-PI has been studied as a target for smoke-induced damage. Damage to alpha 1-PI in lung fluid could occur by several mechanisms involving species delivered to the lung by cigarette smoke and/or stimulated inflammatory cells. Oxidative damage to alpha 1-PI has received particular attention, since both cigarette smoke and inflammatory cells are rich sources of oxidants. In this article we review almost two decades of research on mechanistic studies of damage to alpha 1-PI by cigarette smoke and phagocytic cells in vitro, studies emphasizing the importance of elastinolytic activity in the pathogenesis of emphysema in vivo and studies of human lung lavage fluid to detect defects in alpha 1-PI at the molecular and functional levels.

scholarly journals MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

2018 ◽  
Vol 116 (1) ◽  
pp. 303-312 ◽  
Author(s):  
Erol C. Bayraktar ◽  
Lou Baudrier ◽  
Ceren Özerdem ◽  
Caroline A. Lewis ◽  
Sze Ham Chan ◽  
...  
Keyword(s):  
Metabolite Profiling ◽  
Cultured Cells ◽  
Transgenic Animals ◽  
Cell Types ◽  
Tissue Level ◽  
Specific Cell ◽  
Mammalian Tissues ◽  
Cell Type Specific ◽  
Untargeted Metabolite Profiling

Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially localized 3XHA epitope tag (MITO-Tag) for the fast isolation of mitochondria from cultured cells to generate MITO-Tag Mice. Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology, and our strategy should be generally applicable for studying other mammalian organelles in specific cell types in vivo.

scholarly journals MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

2018 ◽  
Author(s):  
Erol Can Bayraktar ◽  
Lou Baudrier ◽  
Ceren Özerdem ◽  
Caroline A. Lewis ◽  
Sze Ham Chan ◽  
...  
Keyword(s):  
Metabolite Profiling ◽  
Cultured Cells ◽  
Transgenic Animals ◽  
Cell Types ◽  
Tissue Level ◽  
Specific Cell ◽  
Mammalian Tissues ◽  
Cell Type Specific ◽  
Untargeted Metabolite Profiling

ABSTRACTMitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell-types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially-localized 3XHA epitope-tag (“MITO-Tag”) for the fast isolation of mitochondria from cultured cells to now generate “MITO-Tag Mice.” Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology and our strategy should be generally applicable for studying other mammalian organelles in specific cell-types in vivo.

scholarly journals An atlas of protein-protein interactions across mammalian tissues

2018 ◽  
Author(s):  
Michael A. Skinnider ◽  
Nichollas E. Scott ◽  
Anna Prudova ◽  
Nikolay Stoynov ◽  
R. Greg Stacey ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Cellular Mechanisms ◽  
Stable Isotope Labelling ◽  
Proteomic Approach ◽  
Mouse Tissues ◽  
Mammalian Tissues ◽  
Genetically Manipulated

SummaryCellular processes arise from the dynamic organization of proteins in networks of physical interactions. Mapping the complete network of biologically relevant protein-protein interactions, the interactome, has therefore been a central objective of high-throughput biology. Yet, because widely used methods for high-throughput interaction discovery rely on heterologous expression or genetically manipulated cell lines, the dynamics of protein interactions across physiological contexts are poorly understood. Here, we use a quantitative proteomic approach combining protein correlation profiling with stable isotope labelling of mammals (PCP SILAM) to map the interactomes of seven mouse tissues. The resulting maps provide the first proteome-scale survey of interactome dynamics across mammalian tissues, revealing over 27,000 unique interactions with an accuracy comparable to the highest-quality human screens. We identify systematic suppression of cross-talk between the evolutionarily ancient housekeeping interactome and younger, tissue-specific modules. Rewiring of protein interactions across tissues is widespread, and is poorly predicted by gene expression or coexpression. Rewired proteins are tightly regulated by multiple cellular mechanisms and implicated in disease. Our study opens up new avenues to uncover regulatory mechanisms that shape in vivo interactome responses to physiological and pathophysiological stimuli in mammalian systems.

scholarly journals MicroRNA-130a Represses Transcriptional Activity of Aquaporin 4 M1 Promoter

2012 ◽  
Vol 287 (15) ◽  
pp. 12006-12015 ◽  
Author(s):  
Sugunavathi Sepramaniam ◽  
Lim Kai Ying ◽  
Arunmozhiarasi Armugam ◽  
E. M. Wintour ◽  
Kandiah Jeyaseelan
Keyword(s):  
Translational Regulation ◽  
Infarct Volume ◽  
Luciferase Reporter ◽  
Alternative Promoters ◽  
Stem Loop ◽  
Mammalian Tissues ◽  
Fluid Regulation ◽  
Reporter Assays ◽  
The Central Nervous System

Aquaporins (AQPs) are transmembrane water channels ubiquitously expressed in mammalian tissues. They play prominent roles in maintaining cellular fluid balance. Although expression of AQP1, -3, -4, -5, -8, -9, and -11 has been reported in the central nervous system, it is AQP4 that is predominately expressed. Its importance in fluid regulation in cerebral edema conditions has been highlighted in several studies, and we have also shown that translational regulation of AQP4 by miR-320a could prove to be useful in infarct volume reduction in middle cerebral artery occluded rat brain. There is evidence for the existence of two AQP4 transcripts (M1 and M23) in the brain arising from two alternative promoters. Because the AQP4 M1 isoform exhibits greater water permeability, in this study, we explored the possibility of microRNA-based transcriptional regulation of the AQP4 M1 promoter. Using RegRNA software, we identified 34 microRNAs predicted to target the AQP4 M1 promoter region. MicroRNA profiling, quantitative stem-loop PCR, and luciferase reporter assays revealed that miR-130a, -152, -668, -939, and -1280, which were highly expressed in astrocytes, could regulate the promoter activity. Of these, miR-130a was identified as a strong transcriptional repressor of the AQP4 M1 isoform. In vivo studies revealed that LNATM anti-miR-130a could up-regulate the AQP4 M1 transcript and its protein to bring about a reduction in cerebral infarct and promote recovery.

scholarly journals THE MECHANISM OF ACTION OF COLCHICINE

1967 ◽  
Vol 34 (2) ◽  
pp. 525-533 ◽  
Author(s):  
G. G. Borisy ◽  
E. W. Taylor
Keyword(s):  
Soluble Fraction ◽  
Noncovalent Complex ◽  
Binding Activity ◽  
Chromatographic Behavior ◽  
Mitotic Apparatus ◽  
Vitro Assay ◽  
Culture Cells ◽  
Antimitotic Activity

The majority of the colchicine-3H bound by tissue culture cells (KB or Hela) was found to be present as a noncovalent complex with a macromolecule which appears in the soluble fraction after homogenization. Similar binding was demonstrated in vitro and was confined to a component of the soluble fraction. The binding-equilibrium constant and the kinetic constants were essentially the same in vivo and in vitro. Bound radioactivity was reisolated and shown to be present in a molecule with the same chromatographic behavior and specific antimitotic activity as colchicine. In vitro assay of binding activity of a variety of cells and tissues showed a correlation with the presence of microtubules. High binding activity was given by dividing cells, mitotic apparatus, cilia, sperm tails, and brain tissue. Binding to extracts of slime mold or to purified muscle proteins was very low or undetectable. The binding site had a sedimentation constant of 6S and it is suggested that the protein is a subunit of microtubules.

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