acid-phosphatase and methylamine

We investigated the effect of recombinant human tumor necrosis factor (TNF) on the lysosomal enzyme activity of various established cell lines in vitro. Incubation of 1 x 10(6) TNF-sensitive mouse tumorigenic fibroblasts (L-M cells) in the presence of TNF (100 U/ml) for 48 h increased the total (the sum of the enzyme activities in the lysosomes and the cytoplasm) acid phosphatase and beta-glucuronidase activities by 3.7- and 4.2-fold, respectively. The same increase was observed even when 1 U/ml of TNF was added to some cultures and no further augmentation occurred at 10 or 100 U/ml. Measurement of total and free enzyme activities showed that TNF stimulation not only enhanced the total intracellular enzyme activity but also accelerated the conversion into free (cytoplasmic) enzyme activity. Addition of a lysosomotropic agent (methylamine) suppressed both the enhancement of lysosomal enzyme activity and the cytotoxicity of TNF. A similar enhancement of lysosomal enzyme activities was also detected in various TNF-sensitive tumor cell lines, and a strong correlation (acid phosphatase: r = 0.836, beta-glucuronidase: r = 0.910) was observed between the enhancement of enzyme activity and sensitivity to TNF. No such increase was detected in TNF-resistant human diploid cells. These results show that TNF induces the activation and release of lysosomal enzymes in TNF-sensitive cells, and suggest that such events may play an important role in TNF-mediated cytotoxicity.

Topics: Acid Phosphatase; Animals; Cell Compartmentation; Cell Line; Dose-Response Relationship, Drug; Enzyme Activation; Glucuronidase; Humans; In Vitro Techniques; Lysosomes; Methylamines; Mice; Recombinant Proteins; Tumor Necrosis Factor-alpha

Previous studies have shown that up to a half of infused triacylglycerol does not exit the intestine via lymphatics. This suggests the presence of a mucosal lipase which could provide fatty acids for potential transport via the portal vein. The present study describes an acid-active lipase in rat intestinal mucosa. Acid lipase was assayed using a glyceryl tri[14C]oleate emulsion (pH 5.8). Mucosal homogenates were differentially centrifuged to yield cellular organelles and cytosol. Cells were sequentially released from villi using citrate and EDTA. The enzyme was found to be most active in the proximal quarter intestine and in the upper third of villi. Its greatest activity was in the lysosomal fraction. Esophageal diversion demonstrated that lingual lipase was not the precursor of the mucosal acid lipase. Bile salts stimulated activity 3- to 5-fold, but other neutral or anionic detergents were inhibitory. Of the detergents tested, taurocholate at super critical micellar concentrations could restore activity only with SDS. Sepharose 6B chromatography suggested that the enzyme partitioned into an SDS and taurocholate mixed micelle. We conclude that mucosal acid lipase is a distinct, intrinsic enzyme of the intestinal mucosa. It is predominantly lysosomal in origin. The location of its greatest activity in the villus tips of the proximal intestine suggests that it is potentially involved in mucosal triacylglycerol disposal.

Topics: Acid Phosphatase; Animals; Bile Acids and Salts; Chloroquine; Intestinal Mucosa; Lipase; Lipid Metabolism; Male; Methylamines; Rats; Rats, Inbred Strains

Sub-microgram quantities of bacterial lipopolysaccharide (LPS) have been found to substantially reduce the intracellular catalytic activities of three representative lysosomal enzymes (namely, acid phosphatase, hexosaminidase, and beta-glucuronidase) in human monocyte-derived macrophages. This response was not associated with a concurrent increase in enzyme catalytic activity in the culture supernatant, and hence, could not be explained by mobilization of preformed material. By conducting experiments in the presence and absence of indomethacin, a cyclooxygenase inhibitor, the reduction in lysosomal enzyme catalytic activities was shown not to be dependent on the ability of LPS to induce prostaglandin E2 production. The response was not found to be the result of a more generalized LPS-dependent reduction in the ability of the cells to synthesize protein, since the presence of LPS in macrophage cultures did not appreciably affect the amount of [35S]methionine incorporated into total cellular proteins. A kinetic analysis of the effect of LPS on the down-regulation of enzyme catalytic activities indicated that this was an early response of the cells to LPS exposure. An investigation of the effects of blockade of enzyme catabolism (using the lysosomotropic weak-base, methylamine) indicated that the reduction of catalytic enzyme activities in response to LPS was probably due to a decreased rate of production of active product, rather than an enhanced rate of enzyme catabolism. This suggestion was confirmed by experiments in which the synthesis of pro-hexosaminidase (measured by biosynthetic labeling with [35S]methionine and specific immunoprecipitation of labeled pro-hexosaminidase) was found to be reduced by 42% after a 24-h exposure to LPS (although the synthesis of complement component C3 was stimulated by a factor of 4.5). It is suggested that the ability of LPS to regulate the functional expression of protein products contributes to changes in the overall functional status of these cells in response to this bacterial product.

Topics: Acid Phosphatase; Complement C3; Dose-Response Relationship, Drug; Glucuronidase; Hexosaminidases; Humans; In Vitro Techniques; Indomethacin; Lipopolysaccharides; Lysosomes; Macrophages; Methylamines; Protein Biosynthesis; Time Factors

Incubation of isolated rat hepatocytes with 10 mM methylamine resulted in an inhibition of endogenous protein degradation and a microscopically visible enlargement of the lysosomes. Lysosomes from methylamine-treated cells exhibited increased buoyancy in metrizamide gradients and increased fragility as measured by the release of acid phosphatase activity in vitro, despite the fact that no methylamine remained in the gradient-isolated organelles. When methylamine was extracted from intact cells, the inhibition of protein degradation was immediately relieved, whereas the lysosomal enlargement (and to a certain extent also the increased fragility) persisted for some time. The methylamine-induced osmotic swelling of the lysosomes would thus seem to involve not merely a passive stretching of the lysosomal membrane, but rather some structural change (e.g., an increased amount of membrane material) which is relatively slowly reversible, but without effect on lysosomal function.

Topics: Acid Phosphatase; Animals; Centrifugation, Density Gradient; In Vitro Techniques; Kinetics; Liver; Lysosomes; Male; Methylamines; Proteins; Rats; Rats, Inbred Strains; Vacuoles