Acid phosphatase, lysosomal activity

A search for lysosomal hydrolases and related enzymes has been made in haemolysates from human and rabbit red cells.Apart from acid phosphatases, significant activities were found only for a-D-mannosidase, neutral a-D-glucosidase and 3-D-2-acetamido-2-deoxyhexosidase. 

A search for lysosomal hydrolases and related enzymes has been made in haemolysates from human and rabbit red cells. Apart from acid phosphatases, significant activities were found only for a-D-mannosidase, neutral o-D-glucosidase, and -D-2-acetamido-2-deoxyhexosidase. a-D-Mannosidase activity per cell in human red blood cells was 200-times lower than in white cells. The optimal pH was 5.5-6.0. Electrophoresis on cellulose acetate showed three bands. Haemolysates from four patients with mannosidosis were not deficient in a-D-mannosidase. Curves of pH activity and electrophoretic patterns were similar to those of controls. From its biochemical and genetic properties, it is concluded that red cell a-D-mannosidase differs from the lysosomal acid a-D-mannosidase. 

Second, most purple acid phosphatases require activation by reducing agents. Although intracellular metabolites such as NADH and NADPH have redox potentials sufficiently low to reduce the purple phosphatases, it is not yet clear whether they, or other reductants, are present in effective concentration in the lysosome-like organelles to which the enzymes are localized. 

Similarly, Chow et al. observed an increase in the lysozyme activity of a soluble lung fraction and of plasma after continuous exposure of rats to ozone at 0.8 ppm for 8 days. However, no difference in lung or plasma lysozyme activity from control values was present in rats continuously exposed to 0.2 or 0.5 ppm or intermittently exposed (0.2-0.8 ppm, 8 h/day for 7 days). Histochemical evidence of an increase in lung acid phosphatase, a lysosomal enzyme, has also been reported. ... 

The lysosomes are the cell s stomach, serving to break down various cell components. For this purpose, they contain some 40 different types of hydrolases, which are capable of breaking down every type of macromolecule. The marker enzyme of lysosomes is acid phosphatase. The pH optimum of lysosomal enzymes is adjusted to the acid pH value and is also in the range of pH 5. At neutral pH, as in the cytoplasm, lysosomal enzymes only have low levels of activity. This appears to be a mechanism for protecting the cells from digesting themselves in case lysosomal enzymes enter the cytoplasm at any time. In plants and fungi, the cell vacuoles (see p. 43) have the function of lysosomes. 

Lakritz et al. (32) reported that radiation doses of less than 10 kGy (at 0 to 4 C) produced minimal changes in the micro structure of bovine longissimus dorsi muscle. At doses of 30 kGy or higher, myofibril fragmentation and decreased tensile strength were noted. Lakritz and Maerker (33) reported reductions of 8% and 42% in the activities of lysosomal enzymes and acid phosphatase of irradiated (10 kGy) bovine longissimus dorsi muscle tissue. 

The lysosomal acid phosphatase was cytochemically shown to be present in dense bodies of chondrocytes but not in the nearly matrix vesicles461,462). Subsequent studies have confirmed that the amount of acid phosphatase in isolated vesicles is low and also that the activities of -glucuronidase and cathepsin D in the isolated vesicles were negligible463). The evidence indicates that matrix vesicles are not lysosomal. Isolated vesicles contain comparatively little mitochondrial succinic dehydrogenase, suggesting that the matrix vesicles and mitochondria were not identical458). 

The acid phosphatase activity of the ameba, Chaos chaos, is largely confined to particulate bound enzyme which exhibits latency. A noncompetitive heat-stable inhibitor is present in the particulate fraction. The role of this inhibitor in the mechanism of lysosomal activation is not clear (12b, 12c). 

The HPLC method has been used to assay a number of activities usually found associated with lysosomal vesicles. All these assays utilize the fluorometric compound 4-methylumbelliferone (4-MU). When carbohydrates, lipids, phosphates, or sulfates are conjugated with 4-MU, these compounds can be used as substrates for glycosidase, lipases, acid phosphatases, and arylsulfatases. The activity is determined by the release of 4-MU. 

A 336-fold purification by column chromatography was achieved by Brightwell and Tappel (B32) from lysosomes obtained by differential and density sucrose gradients. The lysosomes were frozen and thawed several times, then centrifuged. The resulting soluble acid phosphatase fraction was dialyzed against suitable buffers and then applied to a DEAE-cellulose column or to a CM-cellulose column. Each column was eluted with a linear 0-1 M NaCl solution, the former at pH 7.2 and the latter at pH 5.6. Two peaks of acid phosphatase activity were ob-... 

Using electron microscopy, Novikoff et al. (N6) found that rat liver fractions rich in these lysosomal enzymes, particularly acid phosphatase, showed the presence of mitochondria but had a predominance of single-membrane-limited bodies which were generally electron dense. Fractions with a low acid phosphatase activity rarely showed dense bodies. This observation provided some correlation between the biochemical concept of lysosomes and the existence of a structural unit within the cell (S28). 

The yield of acid phosphatase was 11%, as compared with 30% for aryl sulfatase and 15% for ribonuclease. Obviously, in the attempt to obtain pure preparations of intracellular components, it is inevitable that losses be encountered. Such procedures are therefore not suited for obtaining an estimate of the quantitative distribution of intracellular components. Approximately 5-9% of the lysosomal enzymes—aryl sulfatase, acid phosphatase, and ribonuclease—were present in the free form. The remaining 91-95% of the activities of these enzymes were in the latent form and required alteration of the permeability or disruption of the lysosomal membrane to become active. 

The lysosomal and the soluble fractions of acid phosphatase were compared in several other ways. The values (milf) for the lysosomal fractions on various substrates were -glycerophosphate, 1.6 fructose 1,6-diphosphate, 2.0 p-nitrophenyl phosphate, 1.6 AMP, 0.43 they were not significantly different from those obtained with the soluble fraction. The pH-activity curves with these substrates were similar for the two fractions. Inhibition of phosphatase activity of the lysosomal and soluble fractions occurred at approximately the same concentration of fluoride or n- (+) -tartrate when j8-glycerophosphate, AMP, or fructose 1,6-diphosphate were used as substrates. However, with p-nitrophenyl phosphate... 

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