Clearance of lysosomal hydrolases following intravenous infusion. Kinetic and competition experiments with beta-glucuronidase and N-acetyl-beta-D-glucosaminidase.

Clearance experiments with highly purified lysosomal glycosidases, β-glucuronidase and N-acetyl-β-d-glucosaminidase, following intravenous infusion revealed widely varying clearance profiles which depended on the tissue source of the enzyme. Normal rat serum β-glucuronidase and epididymal N-acetyl-β-d-glucosaminidase were cleared slowly from the circulation when compared with rat preputial gland β-glucuronidase, liver lysosomal β-glucuronidase, and liver lysosomal N-acetyl-β-d-glucosaminidase, respectively, which were cleared rapidly. Experiments comparing the catalytic properties and molecular dimensions of the enzymes revealed no differences between rapid and slow clearance forms. Kinetic analysis using the rapid clearance forms of β-glucuronidase has allowed the resolution of at least two components, rapid and slow. Clearance of the rapid component is saturable and appears to reflect binding or uptake by a limited number of sites. By contrast, the clearance rate of the slow component increased linearly with respect to dose and may be due to nonspecific or low-affinity binding. Competition experiments with β-glucuronidase-free lysosomal extract and highly purified lysosomal enzymes, but not serum glycoproteins or colloidal silver, suggest that one lysosomal enzyme inhibits clearance of others and that a common mechanism may be involved in their binding.     

Elevated serum beta-glucuronidase reflects hepatic lysosomal fragility following toxic liver injury in rats.

The level of serum beta-glucuronidase increases in various pathological conditions, including liver disorders. The aim of this investigation was to study the changes in liver lysosomal membrane stability during experimentally induced hepatic fibrosis that may result in the elevation of serum beta-glucuronidase. Liver injury was induced by intraperitoneal injections of N-nitrosodimethylamine (NDMA) in adult male albino rats over 3 weeks. The progression of fibrosis was evaluated histopathologically as well as by monitoring liver collagen content. Lipid peroxides and beta-glucuronidase levels were measured in the liver homogenate and subcellular fractions on days 0, 7, 14, and 21 after the start of NDMA administration. Serum beta-glucuronidase levels were also determined. A significant increase was observed in beta-glucuronidase levels in the serum, liver homogenate, and subcellular fractions, but not in the nuclear fraction on days 7, 14, and 21 after the start of NDMA administration. Lipid peroxides also increased in the liver homogenate and the lysosomal fraction. The measurement of lysosomal membrane stability revealed a maximum lysosomal fragility on day 21 during NDMA-induced fibrosis. In vitro studies showed that NDMA has no significant effect on liver lysosomal membrane permeability. The results of this investigation demonstrated that lysosomal fragility increases during NDMA-induced hepatic fibrosis, which could be attributed to increased lipid peroxidation of lysosomal membrane. In this study, we also elucidated the mechanism of increased beta-glucuronidase and other lysosomal glycohydrolases in the serum during hepatic fibrosis.     

The egasyn gene affects the processing of oligosaccharides of lysosomal beta-glucuronidase in liver.

The accumulation of the relatively large amounts of beta-glucuronidase in microsomal fractions of normal mice depends on formation of complexes with the protein egasyn. Unexpectedly, it was found that the egasyn gene also affects the processing of beta-glucuronidase, which is segregated to lysosomes. In egasyn-positive mice lysosomal beta-glucuronidase from liver has a mean pI of 5.9 with a minor proportion at pI 5.4, whereas in egasyn-negative mice the proportion of the two lysosomal forms is reversed. Combined experiments measuring susceptibility to neuraminidase and to endoglycosidase H and specific binding to Ricinus communis lectin-agarose columns showed that the alterations in isoelectric point were associated with a decrease in complex oligosaccharides of lysosomal beta-glucuronidase in egasyn-positive mice. Since this alteration occurs not only in a congenic strain carrying the Eg0 gene but also in several other inbred strains that are homozygous for this gene, it is considered to be a genuine effect of the Eg gene rather than other genes that might regulate oligosaccharide processing. Also, the alteration is likely to be a result of direct physical interaction of the egasyn protein and lysosomal beta-glucuronidase, since a second lysosomal enzyme, beta-galactosidase, which does not form complexes with egasyn, is unaffected. The results suggest a model in which egasyn not only causes accumulation of beta-glucuronidase in the microsomal compartment but also acts upon the precursor to lysosomal beta-glucuronidase to alter its interaction with trans-Golgi-apparatus processing enzymes.     

Plasma clearance of glycoproteins with terminal mannose and N-acetylglucosamine by liver non-parenchymal cells. Studies with beta-glucuronidase, N-acetyl-beta-D-glucosaminidase, ribonuclease B and agalacto-orosomucoid.

The mechanism of bile-pigment formation from haem breakdown was studied by using 18O labelling of the molecular oxygen required for macrocyclic ring cleavage. For haem degradation by the spleen microsomal haem oxygenase system, mass spectrometry of the product bilirubin revealed that cleavage occurred by the Two-Molecule Mechanism, i.e. the terminal lactam oxygen atoms in bilirubin were derived from two different oxygen molecules. Similarly, degradation of myoglobin by coupled oxidation with ascorbate and oxygen proceeded via the Two-Molecule Mechanism. Cobalt and manganese complexes of protoporphyrin IX were not degraded by either the haem oxygenase system or the coupled oxidation system. This result suggests that the iron atom possesses unique properties in facilitating porphyrin breakdown.     

Alterations in cardiac lysosomal hydrolases following induction of the calcium paradox

Although perfusion of the heart with calcium-free medium for a brief period followed by reperfusion with calcium-containing medium results in marked structural derangements (calcium paradox), the mechanisms for this cell damage are far from clear. Since activation of lysosomal enzymes has been associated with pathological damage, it was the purpose of this study to examine alterations in the activities of several lysosomal enzymes in rat hearts subjected to calcium paradox. No significant changes in the activities of beta-acetylglucosaminidase, beta-galactosidase, alpha-mannosidase, or acid phosphatase were seen in the homogenates of hearts exposed to the calcium paradox. However, there were dramatic alterations in the lysosomal enzyme activities in the sedimentable and nonsedimentable fractions during calcium paradox. The lysosomal enzyme activities were also detected in the perfusate collected during reperfusion with calcium-containing medium. These changes occurred during the reperfusion period since no alterations were apparent after calcium-free perfusion and were dependent upon the time of reperfusion with medium containing Ca2+ as well as the time of perfusion with Ca2+ -free medium before inducing Ca2+ paradox. These data indicate that alterations in lysosomal enzymes owing to reinstitution of calcium in Ca2+-deprived hearts may occur as a part of cardiac damage and general cellular disintegration.     

Effects of di-isopropyl phosphorofluoridate on rat liver microsomal and lysosomal beta-glucuronidase

N-Bromosuccinimide completely inactivated the cellulase, and titration experiments showed that oxidation of one tryptophan residue per cellulase molecule coincided with 100% inactivation. CM-cellulose protected the enzyme from inactivation by N-bromosuccinimide. The cellulase was inhibited by active benzyl halides, and reaction with 2-hydroxy-5-nitrobenzyl bromide resulted in the incorporation of 2.3 hydroxy-5-nitrobenzyl groups per enzyme molecule; one tryptophan residue was shown to be essential for activity. Diazocarbonyl compounds in the presence of Cu2+ ions inhibited the enzyme. The pH-dependence of inactivation was consistent with the reaction occurring with a protonated carboxyl group. Carbodi-imide inhibited the cellulase, and kinetic analysis indicated that there was an average of 1 mol of carbodi-imide binding to the cellulase during inactivation. Treatment of the cellulase with diethyl pyrocarbonate resulted in the modification of two out of the four histidine residues present in the cellulase. The modified enzyme retained 40% of its original activity. Inhibition of cellulase activity by the metal ions Ag+ and Hg2+ was ascribed to interaction with tryptophan residues, rather than with thiol groups.     

Purification and chemical properities of mouse liver lysosomal (L form) beta-glucuronidase.

The lysosomal form (L form) of beta-glucuronidase was purified 6,500-fold from the liver of C57BL/6J mice with high yield. Purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence or absence of sodium dodetcyl sulfate. The microsomal forms of beta-glucuronidase were spontaneously converted to the L form. The purified L form is a tetramer of molecular weight of 280,000 to 300,000, composedd of four identical subunits of 75,000 molecular weight. The enzyme contains a high content of arginine and glutamic acid and a very low content of sulfur-containing amino acids. Approximately 7% of the enzyme molecule is compose of carbohydrate. Sugars in the L form are glucosamine, mannose, galactose, and glucose. Sialic acid and fucose are absent in the enzyme.     

The activity of kidney lysosomal hydrolases and renin in hypertensive rats.

In renal cortices of hypertensive rats the activity of renin and β-glucuronidase is significantly enhanced, while the activity of acid phosphatase remains pratically unchanged. A significant decrease in protein content accompanies the rise in enzyme activity. The distribution pattern of renin, β-glucuronidase and acid phosphatase after isopycnic centrifugation of cortical homogenates from hypertensive animals is different from that of controls.     

Quantitative distribution of lysosomal hydrolases in the rat nephron.

The activities of N-acetyl-beta, D-glucosaminidase (NAG, EC 3.2.1.30), beta, D-galactosidase (beta-gal, EC 3.2.1.23) and acid phosphatase (ac-Pase, EC 3.1.3.2) were measured in the glomeruli, five segments of the proximal and four segments of the distal tubule of normal male Wistar rats. The activities of NAG and beta-gal are 3- to 5-fold higher in the first part of the proximal tubule than in other segments and very low in glomeruli. We propose that the distribution of these two glycosidases reflects the contribution of the different tubular segments to the reabsorption of glycoproteins. The maximal activity of ac-Pase was found in the straight part of the proximal tubule. It was only 1.5-fold higher than in the distal tubule. Moreover, the activity in glomeruli is rather high. We conclude that ac-Pase is not primarily involved in the handling of reabsorbed molecules.     

Purification and characterization of microsomal and lysosomal beta-glucuronidase from rat liver by use of immunoaffinity chromatography.

Rat liver beta-glucuronidase (EC 3.2.1.31), both from microsomal and lysosomal fractions, were purified about 9500-fold over the homogenate with high yield using affinity chromatography prepared by coupling purified specific immunoglobulin G against rat preputial gland beta-glucuronidase to Sepharose 2B and isoelectric focusing. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of approximately 310000. In dodecylsulfate polyacrylamide gel electrophoresis, the microsomal beta-glucuronidase showed a single band corresponding to a molecular weight of 79000, while the lysosomal beta-glucuronidase had three distinct bands which consisted of one major and two minor bands corresponding to molecular weight of 79000, 74000, and 70000, respectively. A broad pH activity curve with a single optimum at pH 4.4 was observed in both the microsomal and the lysosomal beta-glucuronidases. Immunological gel diffusion technique with rabbit antiserum against rat liver lysosomal beta-glucuronidase revealed that both enzymes had the same or quite similar antigenic determinants.     

Urinary lysosomal hydrolases in mucolipidosis II and mucolipidosis III.

Investigation of the binding characteristics of acid beta-D-galactosidase, N-acetyl-beta-D-glucosaminidase, alpha-D-galactosidase and alpha-L-fucosidase from patients with mucolipidosis II and mucolipidosis III to concanavalin A--Sepharose 4B revealed a 2--10-fold decrease in the proportion of enzyme activities from patients with mucolipidoses II and III that adsorbed on the lectin. Neuraminidase treatment of the unadsorbed enzyme fraction did not significantly increased the proportion of enzyme activities that bound to the concanavalin A--Sepharose 4B. Characterization of acid beta-D-galactosidase from the adsorbed and unadsorbed enzyme fractions of mucolipidosis II and mucolipidosis III patients demonstrated identical apparent Km values of 0.22 mM with respect to 4-methylumbelliferyl beta-D-galactopyranoside, altered pH--activity profiles and heterogeneous isoelectric-focusing patterns. The results of this study support the suggestion of an alteration of a post-translational modification (possibly glycosylation) occurring in mucolipidosis II and mucolipidosis III common to the lysosomal hydrolases that affects the mannoserelated properties of these enzymes.     

Alkali burns of the rabbit cornea. I. A histochemical study of beta-glucuronidase, beta-galactosidase and N-acetyl-beta-D-glucosaminidase.

In alkali burned rabbit corneas activities of beta-glucuronidase, N-acetyl-beta-D-glucosaminidase and acid beta-galactosidase were studied histochemically in various time intervals after the traumatization. The technic with semipermeable membranes was employed. Within four days after the injury enzyme activities in the traumatized area were almost lacking. The corresponding activities in the unaffected part of the cornea were within the norm. On the 7th day enzyme activities were on an increase (but still subnormal) in the traumatized area. This area was surrounded by a zone of keratocytes with high levels of enzyme activities. This was particularly remarkable in keratocytes subjacent to the epithelium. The activation of all enzymes studied was present in the basal layer of the epithelium and in the endothelium as well. On the 14th day enzyme activities in the traumatized area were nearly restored and on the 32nd day they could not be distinguished from the normal cornea. Beta-galactosidase displayed a relatively maximal increase in the activity of all enzymes investigated.     

Quantitative distribution of lysosomal hydrolases in the rat nephron

Summary The activities of N-acetyl-,d-glucosaminidase (NAG, EC 3.2.1.30),,d-galactosidase (-gal, EC 3.2.1.23) and acid phosphatase (ac-Pase, EC 3.1.3.2) were measured in the glomeruli, five segments of the proximal and four segments of the distal tubule of normal male Wistar rats. The activities of NAG and-gal are 3- to 5-fold higher in the first part of the proximal tubule than in other segments and very low in glomeruli. We propose that the distribution of these two glycosidases reflects the contribution of the different tubular segments to the reabsorption of glycoproteins. The maximal activity of ac-Pase was found in the straight part of the proximal tubule. It was only 1.5-fold higher than in the distal tubule. Moreover, the activity in glomeruli is rather high. We conclude that ac-Pase is not primarily involved in the handling of reabsorbed molecules.This research was supported by the Swiss National Science Foundation, grant 3.904-077     

Characterization of lysosomal hydrolases that are elevated in Gaucher's disease.

Although Gaucher's disease occurs in three distinct forms with greatly varying degrees of severity, there is no correlation between the clinical course of the disease and levels of residual glucocerebrosidase, the fundamental enzymatic deficiency. In an effort to study secondary changes which might contribute to the pathology of Gaucher's disease, homogenates of spleen, liver, and brain tissue, as well as serum from patients with Gaucher's disease were analyzed for their content of a number of lysosomal enzymes. Extracts of 8 Gaucher spleens contained 3- to 4-fold increases in acid phosphatase activity as well as 5-to 10-fold increases in galactocerebrosidase⁵ activity. The marked elevation in galactocerebrosidase activity in Gaucher spleen was documented using various [³H]galactose labeled galactocerebrosides as substrates and with [³H]galactose labeled lactocerebroside under the “lactosylceramidase I”⁵ assay conditions established by Suzuki (Tanaka, H., and Suzuki, K., 1975, J. Biol. Chem., 250, 2324–2332) that measure galactocerebrosidase activity specifically in the presence of G_mi-ganglioside β-galactosidase. Acid phosphatase determinations using extracts of liver from a case of infantile, neuropathic Gaucher's disease revealed a 2-fold elevation in this activity, whereas brain acid phosphatase activity in this case was similar to that of control tissue. Separation of hexosaminidase A and B activities on DEAE-Sephadex columns indicated increases in both forms of the enzyme in Gaucher tissue with the major increase occurring in the hexosaminidase B component. Glucuronidase and nonspecific esterase were observed to be elevated approximately 2-fold. However, not all lysosomal enzyme activities were increased. Levels of splenic arylsulfatase A and B, α-arabinosidase, sphingomyelinase, α-mannosidase, and G_mi-ganglioside β-galactosidase activities in Gaucher spleen were unremarkable. G_mi-ganglioside β-galactosidase was measured using 4-methylumbelliferyl-β-d-galactopyranoside and [³H]galactose labeled lactocerebroside under the specific assay conditions described by Suzuki for the determination of “lactosylceramidase II” activity. Although levels of arylsulfatase A and B in Gaucher spleen were similar to those of control tissue, arylsulfatase A activity was markedly reduced (20% of control) in homogenates of brain from the case of infantile (type 2) Gaucher's disease. The metabolic and pathologic consequences of these changes in lysosomal enzymes in Gaucher's disease are discussed.