Lysosomal alpha-D-mannosidase of rat liver. Purification and comparison with the golgi and cytosolic alpha-D-mannosidases

Rat liver contains alpha-D-mannosidases in lysosomes, Golgi membranes, and cytosol. The lysosomal enzyme has now been purified approximately 30,000-fold over the crude extract and is free of at least 13 other lysosomal hydrolases. The enzyme has an apparent molecular weight of 335,000 by molecular sieve chromatography and 200,000 by sucrose density centrifugation. It is a glycoprotein, as evidenced by its binding to a concanavalin A affinity column and by a positive periodic acid-Schiff stain. The enzyme has a pH optimum near 4.6. Although it is generally insensitive to a large variety of inorganic salts, chelating agents, and sulfhydryl reagents, prolonged exposure to ethylenediaminetetraacetic acid caused loss of activity, which could be restored by the addition of ZnSO4. Substrate specificity studies were performed on the purified lysosomal alpha-D-mannosidase, as well as on the purified Golgi and cytosolic alpha-D-mannosidases. The three enzymes exhibited only very limited activity on native glycoproteins, but were found to be active on glycopeptides and oligosaccharides, hydrolyzing 1 yields 2 and 1 yields 3 linkages, except that the Golgi enzyme had negligible activity towards the latter linkage. Immunological comparisons by antibody precipitation tests and double-diffusion plates indicated that the three enzymes are not immunologically related. The alpha-D-mannosidase isolated from rat epididymis was found to be immunologically very similar, if not identical, to the lysosomal enzyme isolated from rat liver.     

Aflatoxin inhibition of rat liver mitochondria

When aflatoxin B₁ (AFB₁) is added to an actively respiring rat liver mitochondrial preparation, 25–44% inhibition of electron transport is produced with concentrations ranging from 2.5–4.8 middot; 10^?4M, respectively. The degree of inhibition levels off at 4.8 middot; 10^?4M, which was shown to be in agreement with the critical micelle concentration. Submitochondrial or Gregg particles exhibit a maximum of 63% inhibition. Weanling rats maintained on a 5% casein semipurified diet for 15 days showed an approximate 30–50% reduction in the degree of aflatoxin inhibition for both mitochondria and Gregg particles compared to control animals fed a 20% casein diet ad libitum. The mitochondria of the protein-deprived animals had similar respiratory control ratios to normal animals. Dietary protein deficiency appears to exert its effect primarily at the site of action of aflatoxin rather than to alterations in membrane transport. The major site of inhibition of electron transport appeared to be between cytochromes b and c (c₁) as indicated by comparison of systems employing various substrates which donate their electrons to various portions of the electron transport system. At concentrations just below critical micelle formation, AFB₁ also reduced the ADP:O ratio, which was partially relieved by protein deficiency. The relevance of these findings to liver cell necrosis promoted by aflatoxin is discussed.     

Forms and intracellular distribution of alpha-D-mannosidases in murine liver and spleen

1. The intracellular distribution of alpha-D-mannosidase in homogenates of murine liver and spleen was investigated by differential and gradient density centrifugation. 2. In both tissues an enzyme with a neutral pH optimum was found in the cytosol together with an alpha-D-mannosidase with optimal activity between pH 5.5 and 6.0 which was also partially membrane-bound. 3. In liver the acidic alpha-D-mannosidase was obtained almost entirely in a particulate form distributed equally between a heterogeneous low density region and heavy density lysosomes. 4. The lysosomal form of the liver enzyme was purified to electrophoretic homogeneity and shown to be a glycoprotein composed of four identical subunits of molecular weight 65 kDa. 5. Antibody raised against the purified liver alpha-D-mannosidase immunoprecipitated a polypeptide from spleen which had the same molecular size. This acidic enzyme was the predominant type of alpha-D-mannosidase in spleen, but in contrast to liver, it was obtained mainly in a cytosoluble form, the remaining activity being present in the heterogeneous light density compartment. 6. Although both tissues contain the same molecular form of the acidic alpha-D-mannosidase, in murine spleen this enzyme does not appear to be associated with stable heavy density lysosomes.     

Calcium inhibition of rat liver microsomal calcium-dependent ATPase

Measurement of the inward rate of Ca2+ transport by rat liver microsomes under conditions of varying free intravesicular Ca2+ (1 microM to 5 mM) revealed that inward transport rate is maximum at low intravesicular Ca2+, and that transport rate decreases with an apparent inhibition constant of about 250-350 microM as intravesicular Ca2+ accumulates. This relationship is confirmed by measurement of Ca2+-dependent ATPase activity; activity is greatest when intravesicular Ca2+ is 1 microM, is lower when intravesicular Ca2+ is 60 microM, and is minimum when intravesicular Ca2+ is 5 mM. Unexpectedly, the ratio of Ca2+ transport rate to Ca2+-dependent ATP hydrolysis rate appears to be significantly greater than 2:1.     

Phosphoinositide inhibition of acetyl-CoA carboxylase from rat liver

When purified acetyl-CoA carboxylase was incubated with various phospholipids, the effects on carboxylase activity were quite diverse. Phosphatidic acid, phosphatidylcholine, and phosphatidylinositol were slightly stimulatory, whereas carboxylase was inhibited by polyphosphoinositides in a time- and concentration-dependent manner. Phosphatidylinositol 4,5-bisphosphate (TPI) was the most effective inhibitor; carboxylase activity was inhibited 50% after incubation with 1.5 μm TPI for 30 min. Incubation of carboxylase with citrate reduced the susceptibility to inhibition by TPI. The inhibition was reversed by removal of TPI from the inhibited enzyme. Incubation of TPI with divalent metal cations removed its ability to inhibit carboxylase. Sedimentation studies showed that TPI treatment shifts carboxylase to a less-polymerized form. The K_m for ATP, 24 μm, was not affected by the inhibitor. However, the apparent K_m for acetyl-CoA was decreased from 44 to 11 μm following incubation with TPI. The possibility that polyphosphoinositides may play a role in acetyl-CoA carboxylase regulation is discussed.     

Aflatoxin inhibition of rat liver mitochondrial cytochrome oxidase activity

Aflatoxins B1, B2, G1, G2, and M1 have been evaluated for activity toward cytochrome oxidase in isolated rat liver mitochondria employing ferrocytochrome c and p-phenylene diamine as reductants. The aflatoxins inhibited the cytochrome oxidase activity to a greater extent when monitored by O2 uptake measurements than by substrate oxidation. AFG2 and AFM1 were the most potent (50-70%). Using oligomycin and 2,4-DNP as respiratory inhibitor and uncoupler, respectively, the aflatoxins appear to inhibit e- rather than energy transfer reactions. These toxins did not uncouple cytochrome oxidase activity.     

Disulfiram inhibition of aldehyde reductase from the rat liver

Disulphiram (tetraethylthiuram disulphide teturam, antabus), the known antialcoholic preparation, is studied for its effect on the aldehyde reductase activity (EC 1.1.1.1) in the rats' liver. Apparent Km and V are calculated for acetylaldehyde and NADH as well as Ki of disulphiram relative to the substrate and cofactor of the enzyme. The obtained data permit considering disulphiram a high-specific inhibitor of aldehyde reductase in rats' liver.     

Effects of mitogenic stimulation on lymphocyte alpha-D-mannosidases

Three types of alpha-D-mannosidase are present in human and murine lymphocytes. Their levels increased substantially when the cells were activated by T-cell mitogens, concanavalin A (Con A) and phytohaemagglutinin (PHA), and in the murine cells also by lipopolysaccharide (LPS), a B-cell mitogen. The intracellular localization of the alpha-D-mannosidases in the non-stimulated and activated murine cells was investigated by fractionation of lymphocyte lysates on colloidal silica (Percoll) and discontinuous sucrose gradients. In both types of cell, an enzyme having optimal activity at neutral pH was obtained in the cytosolic fraction and another alpha-D-mannosidase most active at an intermediate pH was obtained partly in membrane-bound form. In contrast, an acidic alpha-D-mannosidase, which was particularly elevated in the activated murine spleen cells, had a distribution in these lymphoblasts which was markedly different from that in non-stimulated lymphocytes. In the latter, the major proportion of the activity was obtained in a cytosolic fraction and the remainder in a particulate fraction of light density, whereas the enzyme in activated lymphocytes was distributed between vesicles of light and heavy density comparable with lysosomal organelles. Moreover, the acidic alpha-D-mannosidase still remained membrane bound even when cell lysates were prepared under hypotonic conditions which disrupt lysosome integrity. These results suggest that lymphocyte activation involves either stabilization of fragile lysosomes present in resting cells or de novo synthesis of lysosome-like structures. The acidic alpha-D-mannosidase present within isolated, intact lysosomes was found to be in a form, A, whereas a different form, B, was most prominent in whole-cell extracts of both types of lymphocyte.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and comparison of the structures of human liver acidic alpha-D-mannosidases A and B.

Human liver alpha-D-mannosidases A and B were purified 11 500-fold and 2000-fold respectively. Both showed microheterogeneity when analysed by isoelectric focusing. Alpha-D-Mannosidases A and B are immunologically identical but differ in their range of pI values, molecular masses, uptake into fibroblasts and subunit compositions. Alpha-D-Mannosidase A consists of equimolar proportions of subunits of molecular masses 62 kDa and 26 kDa, which are linked by disulphide bridges in the intact enzyme. Alpha-D-Mannosidase B also contains a small subunit, of molecular mass 26 kDa, and a variable mixture of larger subunits, of molecular masses 58 kDa and 62 kDa. The 62 kDa and 58 kDa subunits, but not the 26 kDa one, contain concanavalin A-recognizing glycans. The 58 kDa subunit has a lower pI, contains less high-mannose glycans but probably contains more mannose 6-phosphate than the 62 kDa subunit. It is postulated that the differences in structure and properties of alpha-D-mannosidases A and B are due to differences in the state of processing of the large subunit. This suggestion is consistent with a single locus on chromosome 19 for lysosomal alpha-D-mannosidase.     

Aflatoxin inhibition of glucocorticoid binding capacity of rat liver nuclei

The effect of aflatoxin B₁ on the binding capacity of rat liver cytoplasmic glucocorticoid receptors and the nuclear binding of the activated receptor complex was investigated. No alterations in the kinetics of [³H]dexamethasonccytosol receptor complex formation were noted 2 h after treatment with 1 mg/kg aflatoxin B₁. However, a 33% decrease in the concentration of nuclear acceptor sites and a 24% decrease in the glucocorticoid receptor-nuclear binding equilibrium constant of dissociation was observed. This response was near maximal at 2 h and persisted for at least 36 h. Inhibition of nuclear binding capacity was directly related to aflatoxin B₁ dose, with a correlation coefficient of 0.99. Actinomycin D treatment (0.1 mg/kg) resulted in a slight reduction (16%) in the concentration of nuclear acceptor sites but had no effect on the nuclear binding dissociation constant.Administration of [³H]dexamethasone to aflatoxin B₁-treated rats produced a similar pattern of glucocortocoid binding distribution in vivo to that observed in vitro. No differences in [³H]dexamethasone-cytoplasmic receptor binding between control and aflatoxin B₁-treated rats were found, whereas nuclear [³H]dexamethasone binding was reduced 34% by aflatoxin B₁ treatment.     

NADPH-dependent inhibition of lipid peroxidation in rat liver microsomes.

Microsomal NADPH-driven electron transport is known to initiate lipid peroxidation by activating oxygen in the presence of iron. This pro-oxidant effect can mask an antioxidant function of NADPH-driven electron transport in microsomes via vitamin E recycling from its phenoxyl radicals formed in the course of peroxidation. To test this hypothesis we studied the effects of NADPH on the endogenous vitamin E content and lipid peroxidation induced in liver microsomes by an oxidation system independent of iron: an azo-initiator of peroxyl radicals, 2,2'-azobis (2,4-dimethylvaleronitrile), (AMVN), in the presence of an iron chelator deferoxamine. We found that under conditions NADPH: (i) inhibited lipid peroxidation; (ii) this inhibitory effect was less pronounced in microsomes from vitamin E-deficient rats than in microsomes from normal rats; (iii) protected vitamin E from oxidative destruction; (iv) reduced chromanoxyl radicals of vitamin E homologue with a 6-carbon side-chain, chromanol-alpha-C-6. Thus NADPH-driven electron transport may function both to initiate and/or inhibit lipid peroxidation in microsomes depending on the availability of transition metal catalysts.     

Substrate inhibition of rat liver and kidney arginase with fluoride

Fluoride is an uncompetitive inhibitor of rat liver arginase. This study has shown that fluoride caused substrate inhibition of rat liver arginase at substrate concentrations above 4 mM. Rat kidney arginase was more sensitive to inhibition by fluoride than liver arginase. For both liver and kidney arginase preincubation with fluoride had no effect on the inhibition. When assayed with various concentrations of L-arginine, rat kidney arginase did not have Michaelis-Menten kinetics. Lineweaver-Burk and Eadie-Hofstee plots were nonlinear. Kidney arginase showed strong substrate activation at concentrations of L-arginine above 4 mM. Within narrow concentrations of L-arginine, the inhibition of kidney arginase by fluoride was uncompetitive. Fluoride caused substrate inhibition of kidney arginase at L-arginine concentrations above 1 mM. The presence of fluoride prevented the substrate activation of rat kidney arginase.     

Thyroid hormone inhibition of phosphatidylinositol-specific phospholipase C in rat liver

We investigated the effect of thyroid hormone on phosphatidylinositol-specific phospholipase C activity in rat liver. Thyroidectomy increased the activity of the enzyme. Thyroid hormone (T4, 40 micrograms) administration to thyroidectomized-rats decreased phospholipase C activity. The inhibition induced by thyroid hormone was of a non-competitive type. The higher concentration of Ca2+ strongly inhibited the activity of the enzyme obtained from thyroidectomized-rats' liver in vitro. The diminished activity of the enzyme obtained from thyroxine-treated-thyroidectomized-rats was recovered by pretreatment of the enzyme with EGTA. The activity of the enzyme derived from thyroidectomized-rats was not affected by EGTA treatment. These results suggest that thyroid hormone decreases the activity of phosphatidylinositol-specific phospholipase C activity through the mobilization of Ca2+ in the intracellular space.     

Mechanism of inhibition of rat liver bilirubin UDP-glucuronosyltransferase by triphenylalkyl derivatives

A series of potent and competitive inhibitors of UDP-glucuronosyltransferase derived from 7,7,7-triphenylheptanoic acid has been synthesized in order to probe the active site of the isozyme involved in the glucuronidation of the endogenous toxic compound, bilirubin IXα. Like triphenylalkylcarboxylic acids, triphenyl alcohols were found to be very effective competitive inhibitors of the reaction (K_i 12 to 180 μM). Superimposition of the best inhibitors with bilirubin by computer modeling showed a marked spatial similarity, which accounts for the observed competitive-type inhibition. The bulky triphenylmethyl moiety of the inhibitor superimposed well on the part of the bilirubin molecule containing three of the four pyrrole rings. In agreement, substitution of the triphenylmethyl moiety by planar structures such as fluorenyl or indenyl rings completely suppressed the inhibition. In addition, the weak inhibition exerted by the shortest carboxylic acids could be related to the higher acidity of these molecules. The inhibition potency depended on the acidity of the molecules; the more acidic, the less inhibitory, suggesting that the presence of a negative charge on the inhibitor molecule prevents bilirubin glucuronidation. Based on these results, a reaction mechanism for bilirubin glucuronidation is postulated. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 19–27, 1998