Inhibition of proteolytic degradation of low density lipoprotein in human fibroblasts by chloroquine, concanavalin A, and Triton WR 1339.

1,255

480

~z~I-labeled human epidermal growth factor (hEGF) binds in a specific and saturable manner to human fibroblasts. At 37~ the cell-bound ~25I-hEGF initially may be recovered in a native form by acid extraction; upon subsequent incubation, the cell-bound ~25I-hEGF is degraded very rapidly, with the appearance in the medium of 125I-monoiodotyrosine. At 0~ cell-bound nSI-hEGF is not degraded but slowly dissociates from the cell.The data are consistent with a mechanism in which ~zSI-hEGF initially is bound to the cell surface and subsequently is internalized before degradation. The degradation is blocked by inhibitors of metabolic energy production (azide, cyanide, dinitrophenol), some protease inhibitors (Tos-Lys-

Section: Discussionmentioning

confidence: 99%

125I-labeled human epidermal growth factor. Binding, internalization, and degradation in human fibroblasts.

Carpenter¹,

Cohen²

1976

1,255

480

“…46), suggesting that the lysosome was important in this degradation . Lysosomal degradation of other endocytosed proteins such as low-density lipoprotein and polypeptide hormones is also blocked by ammonium chloride and other lysosomotropic weak bases (50)(51)(52).…”

Section: Inhibition By Lysosomotropicagentsmentioning

confidence: 99%

“…Degradation of Semliki Forest virus was, however, only partially blocked by chloroquine. Lysosomotropic weak bases have been shown to prevent degradation of low-density lipoprotein, insulin, and epidermal growth factor (50,52,66) which enter lysosomes after receptor-mediated endocytosis .…”

Section: Role Of Lysosomes In Infectionmentioning

confidence: 99%

Infectious entry pathway of influenza virus in a canine kidney cell line.

Matlin¹,

Reggio²,

Helenius³

et al. 1981

649

483

The entry of fowl plague virus, an avian influenza A virus, into Madin-Darby canine kidney (MDCK) cells was examined both biochemically and morphologically . At low multiplicity and 0°C, viruses bound to the cell surface but were not internalized . Binding was not greatly dependent on the pH of the medium and reached an equilibrium level in 60-90 min. Over 90% of the bound viruses were removed by neuraminidase but not by proteases. When cells with prebound virus were warmed to 37°C, part of the virus became resistant to removal by neuraminidase, with a half-time of 10-15 min. After a brief lag period, degraded viral material was released into the medium . The neuraminidase-resistant virus was capable of infecting the cells and probably did so by an intracellular route, since ammonium chloride, a lysosomotropic agent, blocked both the infection and the degradation of viral protein.When the entry process was observed by electron microscopy, viruses were seen bound primarily to microvilli on the cell surface at 0°C and, after warming at 37°C, were endocytosed in coated pits, coated vesicles, and large smooth-surfaced vacuoles . Viruses were also present in smooth-surfaced invaginations and small smooth-surfaced vesicles at both temperatures .

“…After its uptake by the cells, the "'I-labeled protein component of the acetyl-LDL was degraded to mono[ 'z''Iliodotyrosine, which was excreted into the culture medium (26) . In the presence of the lysosomal enzyme inhibitor chloroquine (14,22), the uptake of "'I-acetyl-LDL continued but the proteolytic degradation was blocked, suggesting that this degradation was occurring in lysosomes (26) . In contrast to the protein component of acetyl-LDL whose hydrolysis products were released into the culture medium, the cholesterol component of acetyl-LDL accumulated progressively within the macrophages, producing a 38-fold increase in the cellular cholesterol content.…”

Section: Abstract Macrophages -Cholesteryl Esters -Lipoproteins -Lysosomescell Surface Receptorsmentioning

confidence: 99%

“…This lysosomal enzyme hydrolyzes the cholesteryl esters that enter the cell through the receptor-mediated uptake of LDL (11,25). If this lysosomal cholesterol esterase is blocked, as is the case in normal fibroblasts incubated with chloroquine (22) or in mutant fibroblasts from patients with a genetic deficiency of this enzyme (11,25), LDL-derived cholesteryl esters accumulate in lysosomes (11,23) . These cholesteryl ester-filled lysosomes appear in electron micrographs as large MBV (23) .…”

Section: Influence Of Acetyl-ldl On Cholesteryl Ester Removalmentioning

confidence: 99%

Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins.

Brown¹,

Goldstein²,

Krieger³

et al. 1979

493

260

Mouse peritoneal macrophages accumulate large amounts of cholesteryl ester when incubated with human low-density lipoprotein that has been modified by chemical acetylation (acetyl-LDL) . This accumulation is related to a high-affinity cell surface binding site that mediates the uptake of acetyl-LDL by adsorptive endocytosis and its delivery to lysosomes. The current studies demonstrate that the cholesteryl ester accumulation can be considered in terms of a two-compartment model: (a) the incoming cholesteryl esters of acetyl-LDL are hydrolyzed in lysosomes, and (b) the resultant free cholesterol is re-esterified in the cytosol where the newly formed esters are stored as lipid droplets .The following biochemical and morphologic evidence supports the hydrolysisre-esterification mechanism: (a) Incubation of macrophages with acetyl-LDL markedly increased the rate of cholesteryl ester synthesis from ['4C]oleate, and this was accompanied by an increase in the acyl-CoA :cholesteryl acyltransferase activity of cell-free extracts .