ascorbic-acid and leupeptin

Nitric oxide (NO) releasing drugs (e.g., glyceryl trinitrate) were successfully used in the treatment of cutaneous leishmaniasis in man. In the present study, the effect of NO donors on the catalytic activity of the cysteine proteinase from promastigotes of Leishmania infantum, an agent of Old World visceral and cutaneous leishmaniases, is reported. In particular, one equivalent of NO, released by the NO donors S-nitrosoglutathione, glyceryl trinitrate, (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide, 3-morpholinosydnonimine, S-nitrosoacetylpenicillamine and sodium nitroprusside, inhibited one equivalent of the parasite cysteine proteinase. As expected, NO-deprived compounds did not affect the catalytic activity of the parasite cysteine proteinase. Furthermore, the absorption spectrum of the (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide-treated inactive L. infantum enzyme displayed a maximum in the 330-350 nm wavelength range. The reducing agents dithiothreitol and L-ascorbic acid completely prevented parasite cysteine proteinase inhibition by NO, fully restored the catalytic activity, and reversed the NO-induced absorption spectrum of the inactive enzyme. Moreover, S-nitrosoacetylpenicillamine displayed a leishmanicidal effect, inhibiting the cysteine proteinase activity in vivo. As expected, the NO-deprived compound N-acetylpenicillamine did not affect significantly the parasite viability and the enzyme activity in vivo. These data suggest that the L. infantum cysteine proteinase undergoes NO-mediated S-nitrosylation, thereby representing a possible mechanism of antiparasitic host defence.

Topics: Animals; Ascorbic Acid; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dithiothreitol; Glutathione; Kinetics; Leishmania infantum; Leupeptins; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitro Compounds; Nitroglycerin; Nitroprusside; Nitroso Compounds; Penicillamine; Protozoan Proteins; S-Nitrosoglutathione

Ascorbic acid retards ferritin degradation in K562 erythroleukemia cells leading to an increase in the availability of cellular iron (Bridges, K. R., and Hoffman, K. E. (1986) J. Biol. Chem. 261, 14273-14277). To explore the mechanism of this effect, the influence of ascorbate on subcellular ferritin distribution was examined. Cellular ferritin was pulse-labeled with 59Fe for 2 h, after which the cells were hypotonically lysed and fractionated on an 8% Percoll density gradient. Immediately after the labeling, all of the ferritin was in the cytoplasmic fractions at the top of the gradient. When the labeling was followed by a 24-h period of growth, a portion of the ferritin shifted to the lysosome-associated fractions at the bottom of the gradient, consistent with lysosomal autophagy of cytoplasmic ferritin. When ascorbate was added to the culture medium during the 24-h incubation, the magnitude of the shift was reduced. This process was also examined by size-fractionation of the contents of labeled cells using a Sepharose CL-6B column. Immediately after labeling, ferritin emerged from the column in two peaks, indicating the existence of both ferritin monomer and aggregates within the cytoplasm. After a 24-h period of growth, the monomer peak disappeared, while a new ferritin peak coincident with lysosomes emerged again, indicative of lysosomal autophagy of ferritin. In cells cultured with ascorbate for 24-h, there was a marked attenuation of the shift of ferritin to the lysosomal fractions. The monomer peak disappeared, as in the controls, but there was instead, an accumulation of ferritin as cytoplasmic aggregates. The total ferritin content of the ascorbate-treated cells was increased by 4-fold over that of the control. These experiments indicate that ascorbate blocks the degradation of cytoplasmic ferritin by reducing lysosomal autophagy of the protein. The access to the cell of the potentially toxic iron stored within the ferritin molecule is thereby increased.

Topics: Ascorbic Acid; Cell Line; Cytoplasm; Ferritins; Glucuronidase; Iron; Leupeptins; Lysosomes; Proteins

An important property of ascorbic acid is its ability to increase the availability of storage iron to chelators. To examine the mechanism of this effect, K562 cells were incubated with ascorbate, attaining an intracellular level of 1 nmol/10(7) cells. In contrast to the reductive mobilization of iron seen with isolated ferritin, ascorbate stabilized iron preincorporated into cellular ferritin. Biosynthetic labeling with [35S]methionine demonstrated that ascorbate also retarded the degradation of the ferritin protein shell. Ferritin is normally degraded in lysosomes. The lysosomal protease inhibitors leupeptin and chloroquine produced a qualitatively similar stabilization of ferritin. Ascorbate did not act as a general inhibitor of proteolysis, however, since it did not effect hemoglobin degradation in these cells. The stabilization of cellular ferritin by ascorbate was accompanied by an expansion of the pool of chelatable iron.

Topics: Animals; Ascorbic Acid; Cell Line; Deferoxamine; Dose-Response Relationship, Drug; Ferritins; Iron; Leukemia, Erythroblastic, Acute; Leupeptins; Methionine

Using morphometric methods the effects of the thiol-proteinase inhibitors leupeptin and E-64 on the digestion of intracytoplasmic collagen fibrils were studied in cultured mouse bone explants. Both drugs caused a dose-dependent increase of lysosomal structures containing cross-banded collagen fibrils (CCV) in periosteal fibroblasts. After an incubation period of 48 hours, leupeptin (in a concentration of 65 microM) caused a thirty-fold increase in the volume fraction of CCV. This effect proved to be reversible following upon the withdrawal of the drug. Since the leupeptin-related accumulation of intracellular collagen fibrils was not significantly inhibited by alpha, alpha dipyridyl (a drug that interferes with collagen fibril formation), it is thought unlikely that the fibrils represented newly synthesized collagen. This view is further substantiated by data obtained from explants incubated in the presence of the phagocytosis-inhibiting agent cytochalasin B. This compound completely inhibited the leupeptin-related accumulation of CCV. The data strongly suggest that collagen fibrils found in cytoplasmic vacuoles of periosteal fibroblasts represent collagen taken up by phagocytosis, the integrity of cytoplasmic actin filament systems is a prerequisite for phagocytosis of collagen to occur, and thiol-proteinases, such as cathepsin B, L, and/or N, play an essential role in the digestion of internalized collagen.

Topics: 2,2'-Dipyridyl; Animals; Ascorbic Acid; Bone and Bones; Collagen; Culture Techniques; Cycloheximide; Cytochalasin B; Fibroblasts; Leucine; Leupeptins; Mice; Microscopy, Electron; Oligopeptides; Phagocytosis; Vacuoles