المؤلفون: Trond Olav Berg, Per Ottar Seglen, Per E. Stromhaug, Trond Berg

المصدر: European Journal of Biochemistry. 221:595-602

مصطلحات موضوعية: Male, Leupeptins, Centrifugation, Vacuole, Cell Fractionation, Vinblastine, Biochemistry, Cathepsin C, chemistry.chemical_compound, Methionine, Phagosomes, Lysosome, Autophagy, Centrifugation, Density Gradient, medicine, Animals, Rats, Wistar, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases, Phagosome, L-Lactate Dehydrogenase, biology, Leupeptin, Acid phosphatase, Dipeptides, Rats, Cytosol, medicine.anatomical_structure, Liver, chemistry, biology.protein, Cell fractionation, Lysosomes

الوصف: In density-gradient analyses of autophagic vacuoles from isolated rat hepatocytes, autophagosomes could be recognized by the presence of an autophagically sequestered cytosolic enzyme, lactate dehydrogenase (LDH). Lysosomes were identified by marker enzymes such as acid phosphatase, or by degradation products from 125I-tyramine-cellobiose-asialoorosomucoid (125I-TC-AOM) loaded into the lysosomes by an intravenous injection in vivo 18 h prior to cell isolation. Autophagosomes and lysosomes showed similar, largely overlapping, density distributions both in hypertonic sucrose gradients and in isotonic Nycodenz gradients. As a step towards the purification of autophagosomes, we investigated the possibility of using lysosomal enzyme substrates to achieve selective destruction of lysosomes by swelling. Hepatocytes were first incubated for 2 h at 37 degrees C with vinblastine (50 microM) to obtain an accumulation of autophagosomes (to 3-5-times above the control level). The cells were then electrodisrupted and the disruptates incubated with a variety of substrates for lysosomal enzymes. Among these, glycyl-phenylalanine-2-naphthylamide (GPN), a cathepsin-C substrate, and methionine-O-methylester (MetOMe), an esterase substrate, turned out to induce extensive rupture of lysosomes, as measured by a strongly reduced sedimentability of acid phosphatase and a nearly complete loss of 125I-TC-AOM sedimentability in substrate-treated preparations from control or vinblastine-treated cells. The lysosomes of cells treated with leupeptin or asparagine were largely resistant to the action of GPN, probably as a result of interference with cathepsin-C activity or lysosomal function in general. Autophagosomes were partially destroyed by MetOMe, as indicated by a reduction in sedimentable LDH, but GPN had no effect on either autophagosomes or mitochondria. The ability of GPN to selectively destroy lysosomes without affecting the autophagosomes of vinblastine-treated cells should make GPN treatment a useful aid in the purification of rat liver autophagosomes.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::2fc1e421dad7ff5a6e07b706e6cff75bTest
https://doi.org/10.1111/j.1432-1033.1994.tb18771.xTest

المؤلفون: Per Ottar Seglen, H. Hoyvik, P. B. Gordon

المصدر: Experimental cell research. 166(1)

مصطلحات موضوعية: Male, Sucrose, Density gradient, Lactose, Vacuole, Biology, Vinblastine, chemistry.chemical_compound, Phagocytosis, Lysosome, Phagosomes, Organelle, medicine, Autophagy, Animals, Phagosome, Adenine, Hydrolysis, Chloroquine, Rats, Inbred Strains, Cell Biology, Rats, Cytosol, medicine.anatomical_structure, Biochemistry, chemistry, Liver, Lysosomes

الوصف: [14C]Lactose, introduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, is sequestered autophagically in the same way as the established sequestration probe, [14C]sucrose. However, unlike the inert sucrose molecule, lactose is rapidly hydrolysed in the lysosomes, and can therefore be used to probe the last step of the autophagic pathway (i.e. fusion with the lysosome). During autophagy lactose is present only at a low, steady-state level in pre-lysosomal vacuoles (probably autophagosomes), serving as a useful marker for these organelles. If autophagosome-lysosome fusion is blocked with vinblastine (Kovacs et al., Exp cell res 137 (1982) 191), [14C]lactose will accumulate continuously as a function of the sequestration rate, and reach a high level in the pre-lysosomal vacuoles. Density gradient analysis, using chloroquine (CLQ) to alter lysosomal density, suggests that these organelles have a broad density distribution (1.08-1.13 g/ml), thus differing significantly from the distribution of lysosomes.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::c46f164170981e9a73cc29b88622bbe1Test
https://pubmed.ncbi.nlm.nih.gov/3743649Test