Concentrations Of Lysosomal Enzymes Research Articles

Functions of Lysosomes in Mammalian Female Reproductive System

The lysosome is the most acidic membrane-bound intracellular organelle. Lysosomal acidity is primarily maintained by vacuolar H+-ATPase (V-ATPase) and counter ion channels. There are >60 hydrolytic enzymes in the lysosome for its fundamental digestive role. Lysosomes also play important roles in endocytosis, exocytosis, autophagy, and cell death. Studies that have implicated roles of lysosomes in the female reproductive system are reviewed here. In the ovary, lysosomes are implicated in the preparation of free cholesterol for steroidogenesis and degradation of regulators of steroidogenesis, regulation of follicular atresia, follicle rupture during ovulation, luteal cell survival, and luteal regression. In the oviduct, lysosomes are involved in endocytosis of both serum and oviductal luminal components. In the uterus during the menstrual/estrous cycle, lysosomes are associated with endometrial secretion, apoptosis, and menstruation. In the uterus during early pregnancy, lysosomes are involved in the temporal and directional changes of endocytosis, uterine epithelial acidification upon embryo implantation initiation, and embryo-maternal mutual communications via extracellular vesicles. In the placenta, lysosomes are implicated in nutrient transport and placental separation from the uterus for parturition. In the mammary gland, lysosomes are important for mammary gland development and involution. These findings suggest/demonstrate functions of lysosomes in multiple processes of female reproduction, from ovulation to ovarian steroidogenesis for pregnancy maintenance, and from essential in utero nurturing of developing embryos/fetuses via embryo/fetal-maternal communications, to optional postpartum nurturing of newborns via lactation. Future studies using genetically or modified animal models and pharmacological approaches will provide novel insights into the functions and mechanisms of lysosomes in the mammalian female reproductive system.

Urinary lysosomal enzyme excretion in pregnant women with hypertensive disorders

Background: The authors assessed proximal renal tubular dysfunction and/or damage in pregnant women with various types of hypertension by measuring the three urinary lysosomal enzyme levels: N-acetyl-β-d-glucosaminidase (NAG), arylsulfatase A and β-glucuronidase. Methods: The study consisted of 120 pregnant women divided into four groups: 41 women in 20th week of gestation or more, with pregnancy-induced hypertension (PIH group), 28 pregnant women after 20 weeks of pregnancy with pre-eclampsia (PE group), 21 pregnant women with chronic hypertension, identified before 20th week of pregnancy (CH group) and 30 healthy, pregnant women (healthy controls (HC) group). Results: Statistical analysis showed significantly higher levels of all the three of lysosomal enzymes in the urine of patients with PE compared with the healthy pregnant women, pregnant women with PIH and the ones with chronic hypertension. Additionally, significantly higher values of NAG were found in the group of pregnant women with PIH compared with healthy pregnancies. No correlation was found between the concentration of enzymes in urine and values of blood pressure in any of the analyzed groups of pregnant women. Conclusions: The authors conclude that higher values of all the studied enzymes in PE group, in the comparison with the other groups, indicate proximal tubular damage at the cellular level. The lack of correlation between the concentration of lysosomal enzymes and blood pressure suggests that the damage to these parts of kidney is complex. In addition, mechanisms other than hypertension realizing intracellular enzymes may be involved in this process.

The Hermansky-Pudlak syndrome 1 (HPS1) and HPS2 genes independently contribute to the production and function of platelet dense granules, melanosomes, and lysosomes

Hermansky-Pudlak syndrome (HPS) is an inherited hemorrhagic disease affecting the related subcellular organelles platelet dense granules, lysosomes, and melanosomes. The mouse genes for HPS, pale ear and pearl, orthologous to the human HPS1 and HPS2 (ADTB3A) genes, encode a novel protein of unknown function and the beta(3)A subunit of the AP-3 adaptor complex, respectively. To test for in vivo interactions between these genes in the production and function of intracellular organelles, mice doubly homozygous for the 2 mutant genes were produced by appropriate breeding. Cooperation between the 2 genes in melanosome production was evident in increased hypopigmentation of the coat together with dramatic quantitative and qualitative alterations of melanosomes of the retinal pigment epithelium and choroid of double mutant mice. Lysosomal and platelet dense granule abnormalities, including hyposecretion of lysosomal enzymes from kidneys and depression of serotonin concentrations of platelet dense granules were likewise more severe in double than single mutants. Also, lysosomal enzyme concentrations were significantly increased in lungs of double mutant mice. Interaction between the 2 genes was specific in that effects on organelles were confined to melanosomes, lysosomes, and platelet dense granules. Together, the evidence indicates these 2 HPS genes function largely independently at the whole organism level to affect the production and function of all 3 organelles. Further, the increased lysosomal enzyme levels in lung of double mutant mice suggest a cause of a major clinical problem of HPS, lung fibrosis. Finally, doubly mutant HPS mice are a useful laboratory model for analysis of severe HPS phenotypes.

Defects in the cappuccino (cno) gene on mouse chromosome 5 and human 4p cause Hermansky-Pudlak syndrome by an AP-3–independent mechanism

Defects in a triad of organelles (melanosomes, platelet granules, and lysosomes) result in albinism, prolonged bleeding, and lysosome abnormalities in Hermansky-Pudlak syndrome (HPS). Defects in HPS1, a protein of unknown function, and in components of the AP-3 complex cause some, but not all, cases of HPS in humans. There have been 15 inherited models of HPS described in the mouse, underscoring its marked genetic heterogeneity. Here we characterize a new spontaneous mutation in the mouse, cappuccino (cno), that maps to mouse chromosome 5 in a region conserved with human 4p15-p16. Melanosomes ofcno/cno mice are immature and dramatically decreased in number in the eye and skin, resulting in severe oculocutaneous albinism. Platelet dense body contents (adenosine triphosphate, serotonin) are markedly deficient, leading to defective aggregation and prolonged bleeding. Lysosomal enzyme concentrations are significantly elevated in the kidney and liver. Genetic, immunofluorescence microscopy, and lysosomal protein trafficking studies indicate that the AP-3 complex is intact in cno/cno mice. It was concluded that the cappuccino gene encodes a product involved in an AP-3–independent mechanism critical to the biogenesis of lysosome-related organelles.

Defects in the cappuccino (cno) gene on mouse chromosome 5 and human 4p cause Hermansky-Pudlak syndrome by an AP-3–independent mechanism

AbstractDefects in a triad of organelles (melanosomes, platelet granules, and lysosomes) result in albinism, prolonged bleeding, and lysosome abnormalities in Hermansky-Pudlak syndrome (HPS). Defects in HPS1, a protein of unknown function, and in components of the AP-3 complex cause some, but not all, cases of HPS in humans. There have been 15 inherited models of HPS described in the mouse, underscoring its marked genetic heterogeneity. Here we characterize a new spontaneous mutation in the mouse, cappuccino (cno), that maps to mouse chromosome 5 in a region conserved with human 4p15-p16. Melanosomes ofcno/cno mice are immature and dramatically decreased in number in the eye and skin, resulting in severe oculocutaneous albinism. Platelet dense body contents (adenosine triphosphate, serotonin) are markedly deficient, leading to defective aggregation and prolonged bleeding. Lysosomal enzyme concentrations are significantly elevated in the kidney and liver. Genetic, immunofluorescence microscopy, and lysosomal protein trafficking studies indicate that the AP-3 complex is intact in cno/cno mice. It was concluded that the cappuccino gene encodes a product involved in an AP-3–independent mechanism critical to the biogenesis of lysosome-related organelles.

Enzymatic markers of cyclosporine nephrotoxicity in patients after renal transplantation.

Toxicity of cyclosporine (CsA), an immunosuppressive drug widely used in transplantation, to the transplanted kidney creates a serious side effect. Therefore, searching for sensitive indicators of nephrotoxic action is well worth the effort. In this work we describe the results of estimation of urine concentration of lysosomal enzymes widely present in the kidney: N-acetyl-beta-D-glucosaminidase (NAG), its isoenzyme NAG-B and beta-glucuronidase (beta-Gr). The studies were conducted in various periods after transplantation of kidneys, on patients under various treatments and receiving different doses of CsA. The results indicate a substantial dependence of the activity of NAG and NAG-B on CsA doses and the period after transplantation. The enzyme proved to be also a sensitive indicator of graft rejection. No such dependence was observed in the case of beta-Gr.

Lysosomal Enzyme Trafficking between Phagosomes, Endosomes, and Lysosomes in J774 Macrophages: ENRICHMENT OF CATHEPSIN H IN EARLY ENDOSOMES

In this study we take advantage of recently developed methods using J774 macrophages to prepare enriched fractions of early endosomes, late endosomes, dense lysosomes, as well as phagosomes of different ages enclosing 1-micron latex beads to investigate the steady state distribution and trafficking of lysosomal enzyme activity between these organelles. At steady state these cells appear to possess four different cellular structures, in addition to phagolysosomes, where acid hydrolases were concentrated. The first site of hydrolase concentration was the early endosomes, which contained the bulk of the cellular cathepsin H. This enzyme was acquired by phagosomes significantly faster than the other hydrolases tested. The second distinct site of lysosomal enzyme concentration was the late endosomes which contain the bulk of cathepsin S. The third and fourth large pools of hydrolases were found in two functionally distinct types of dense lysosomes, only one of which was found to be secreted in the presence of chloroquine or bafilomycin. Among this secreted pool was soluble furin, generally considered only as a membrane-bound trans-Golgi network resident protein. Thus, the organelles usually referred to as "lysosomes" in fact encompass a growing family of highly dynamic but functionally distinct endocytic organelles.

Severe persistent visual field constriction associated with vigabatrin

Case 1 –A 22 year old woman with longstanding complex partial epilepsy started taking vigabatrin 2000 mg/day in addition to carbamazepine 600 mg/day and sodium valproate 1400 mg/day. Thirty seven...

Human serum amyloid P is a multispecific adhesive protein whose ligands include 6-phosphorylated mannose and the 3-sulphated saccharides galactose, N-acetylgalactosamine and glucuronic acid.

Carbohydrate recognition by amyloid P component from human serum has been investigated by binding experiments using several glycosaminoglycans, polysaccharides and a series of structurally defined neoglycolipids and natural glycolipids. Two novel classes of carbohydrate ligands have been identified. The first is 6-phosphorylated mannose as found on lysosomal hydrolases, and the second is the 3-sulphated saccharides galactose, N-acetyl-galactosamine and glucuronic acid as found on sulphatide and other acidic glycolipids that occur in neural or kidney tissues or on subpopulations of lymphocytes. Binding to mannose-6-phosphate containing molecules and inhibition of binding by free mannose-6-phosphate and fructose-1-phosphate are features shared with mannose-6-phosphate receptors involved in trafficking of lysosomal enzymes. However, only amyloid P binding is inhibited by galactose-6-phosphate, mannose-1-phosphate and glucose-6-phosphate. These findings strengthen the possibility that amyloid P protein has a central role in amyloidogenic processes: first in formation of focal concentrations of lysosomal enzymes including proteases that generate fibril-forming peptides from amyloidogenic proteins, and second in formation of multicomponent complexes that include sulphoglycolipids as well as glycosaminoglycans. The evidence that binding to all of the acidic ligands involves the same polypeptide domain on amyloid P protein, and inhibition data using diffusible, phosphorylated monosaccharides, is potentially important leads to novel drug designs aimed at preventing or even reversing amyloid deposition processes without interference with essential lysosomal trafficking pathways.

Circadian rhythms of biliary protein and lipid excretion in rats

To gain insight into the mechanisms by which hepatocytes release lipids and proteins into bile, we studied extended, steady-state secretion of bile, lipids, and lysosomal and canalicular membrane proteins in freely moving, unanesthetized rats with chronic bile fistulas. We found circadian rhythms of biliary secretion for all measured constituents. In the basal state (nocturnal feeding), two distinct secretory patterns emerged: type 1, characterized by a peak at midnight and a nadir at noon; and type 2, characterized by a peak at 8 A.M. and a nadir at 8 P.M. We observed parallel, type 1 circadian rhythms of excretion for bile, biliary lipids (bile acid, cholesterol, phospholipid), and a canalicular membrane enzyme (alkaline phosphodiesterase I). In contrast, a type 2 circadian rhythm was observed for the outputs of two lysosomal enzymes. Hepatic lysosomal enzyme concentrations and the number of pericanalicular lysosomes decreased (P less than 0.05) by 15 and 35%, respectively, at the nadir of their biliary output relative to the time of their peak outputs. In response to daytime feeding, major shifts in the circadian rhythms of excretion of biliary constituents occurred such that secretion of bile, lipids, and the canalicular membrane protein adopted a type 2-like rhythm, whereas the biliary secretion of the lysosomal proteins exhibited a type 1-like pattern. These results indicate that bile flow and biliary excretion of individual lipids and proteins exhibit distinct circadian rhythms that are altered by feeding. Secretory events at the canaliculus that depend on the transmembrane flux of bile acids, such as water and lipid movement or the solubilization of membrane proteins, display a common rhythm.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and immunological quantification of rat liver lysosomal glycosidases

Although lysosomal enzyme activities are known to vary in response to numerous physiological and pharmacological stimuli, the relationship between lysosomal enzyme activity and enzyme concentration has not been systematically studied. Therefore we developed radioimmunoassays for two lysosomal glycosidases in order to determine lysosomal enzyme concentration. beta-Galactosidase and beta-glucuronidase were purified from rat liver 2780-fold and 1280-fold respectively, by using differential centrifugation, affinity chromatography, ion-exchange chromatography and molecular-sieve chromatography. Polyclonal antibodies to these enzymes were raised in rabbits, and two radioimmunoassays were established. Antibody specificity was shown by: (i) selective immunoprecipitation of enzyme activity; (ii) identical bands of purified enzyme on SDS/polyacrylamide-gel electrophoresis and immunoelectrophoresis; (iii) single immunoreactive peaks in molecular-sieve chromatography experiments. Sensitivities of the assays were such that 15 ng of beta-galactosidase and 45 ng of beta-glucuronidase decreased the ratio of bound to free radiolabel by 50%; minimal detectable amounts of immunoreactive enzymes were 2 ng and 10 ng respectively. The assays were initially used to assess the effects of physiological perturbations (i.e. fasting and age) on enzyme concentrations in rat liver; these experiments showed that changes in enzyme concentrations do not always correlate with changes in enzyme activities. This represents the first report of radioimmunoassays for lysosomal glycosidases. The results suggest that these radioimmunoassays provide useful technology for the study of regulatory control mechanisms of the concentrations of lysosomal glycosidases in mammalian tissues.

Pancreatic duct obstruction in rabbits causes digestive zymogen and lysosomal enzyme colocalization.

The pancreatic duct of anesthetized rabbits was cannulated and, in some animals, flow of pancreatic exocrine secretions was blocked by raising the cannula to a vertical position. Blockage for 3-7 h caused a rapid and significant rise in serum amylase activity and an increase in amylase activity within the pancreas. The concentration of lysosomal enzymes in the pancreas was not altered but they became redistributed among subcellular fractions and, as a result, an increased amount was recovered in the 1,000-g, 15-min pellet, which was enriched in zymogen granules. Immunofluorescence studies indicated that lysosomal enzymes become localized within organelles which, in size and distribution, resemble zymogen granules. They also contain digestive enzyme zymogens. Blockage of pancreatic secretions also caused lysosomal enzyme-containing organelles to become more fragile and subject to in vitro rupture. These changes noted after short-term pancreatic duct obstruction are remarkably similar to those previously noted to occur during the early stages of diet and secretagogue-induced experimental pancreatitis, observations that have suggested that colocalization of digestive enzyme zymogens and lysosomal hydrolases might result in intracellular digestive enzyme activation and be an important early event in the evolution of those forms of experimental acute pancreatitis.

Effects of the Specific Cholecystokinin Antagonist L 364,718 in Experimental Acute Pancreatitis in the Rat

Cholecystokinin (CCK) has been suggested to be involved in the pathogenesis of acute pancreatitis. To test this hypothesis, we administered the highly selective and specific CCK receptor antagonist L 364,718 to rats in which acute experimental pancreatitis had been induced by the use of transduodenal pancreatic duct injection of taurocholate. It was, however, found that despite the use of L 364,718 at a high dose level (1 mg/kg body weight given three times), and also given prior to induction of pancreatitis, the mortality rate, the serum or ascites amylase activity, the pancreatic concentrations of lysosomal enzymes or the morphology of the pancreas were not affected. This suggests that the CCK receptors are not involved in the pathogenesis of acute pancreatitis in this experimental model, and, consequently, that CCK receptor antagonists have no place in the therapy of this condition.

Pigmentation and lysosomal phenotypes in mice doubly homozygous for both light-ear and pale-ear mutant alleles.

We have developed a new strain of mice homozygous for mutant alleles at both the light-ear locus on chromosome 5 and the pale-ear locus on chromosome 19. The pigmentation pattern of the double mutants, designated light-pale, is indistinguishable from the parental type. Elevated concentrations of lysosomal enzymes observed in certain tissues of the light-ear and pale-ear singly homozygous mice also are present in the double mutants, and are quantitatively indistinguishable from either parent. Although both mutations have pleiotropic effects on organelles in several tissues, neither locus influences the secretion of pancreatic zymogen granules. The close similarity in phenotypes of light ear, pale ear, and light-pale mice suggest that the le and ep loci encode different subunits of a multimeric protein, and that mutations affecting either subunit result in comparable losses of function.

Carcinoembryogenic antigen and lysosomal enzymes in gastric juice as an aid in the diagnosis of gastric cancer.

Malignant transformation in cells is accompanied by enzymatic changes that may be useful markers of malignancy. Studies of oncofetal antigens in gastric juice show an increased concentration in patients with gastric carcinoma. The object of the present study was to test if the concentration of lysosomal enzymes and carcinoembryonic antigen (CEA) were altered in patients with gastric carcinoma compared to patients earlier operated with gastric resection--a supposedly premalignant condition. The results show that no differences could be found in CEA and beta-hexosaminidase levels between the patients with gastric carcinoma and those with gastric resection. However, lysozyme content was significantly higher in gastric carcinoma and might prove useful for screening of patients after gastric resection. Both CEA, beta-hexosaminidase, and lysozyme levels were significantly higher in patients with gastric carcinoma than in a control population, and can therefore have a role in screening of asymptomatic patients.

The Effect of 13-cis Retinoic Acid on Epidermal Lysosomal Hydrolase Activity in Darier's Disease and Pityriasis Rubra Pilaris

Five patients with Darier's disease and 6 patients with pityriasis rubra pilaris were treated with 13-cis-retinoic acid. Extracts of separated epidermis were assayed for extractable protein, lactic dehydrogenase, Cathepsin D, beta glucuronidase and neutral proteinase before beginning therapy and 2, 4 and 8 weeks after therapy had begun. The epidermal extracts from patients with pityriasis rubra pilaris before beginning therapy were similar to extracts from normal control patients. During the course of therapy with 13-cis-retinoic acid, protein extractability, lactic dehydrogenase and neutral proteinase did not change; there was a highly significant decrease in the specific activity of the lysosomal hydrolases Cathepsin D and beta glucuronidase. A similar but less dramatic fall was noted in the Darier's patients taking 13-cis-retinoic acid. Darier's patients also had a decrease in neutral proteinase activity before beginning therapy; the specific activity of this enzyme increased during the course of therapy. 13-cis-retinoic acid does not induce clinical remission by increasing the intracellular concentration of lysosomal enzymes in epidermis in vivo.

Pigmentation and lysosome function in mice homozygous for both pale ear and beige-J pigment genes.

SUMMARYWe have examined mice doubly homozygous for both pale ear (ep/ep) and beige (bgJ/bgJ) mutations in order to detect genetic interactions between these 2 loci affecting pigmentation and lysosome physiology. The doubly homozygous mouse has a new pigmentation phenotype consistent with independent effects ofepandbg. The beige (Brandt, Elliott & Swank, 1975) and pale ear (Novak & Swank, 1979)genes have abnormal kidney lysosomal enzyme accumulation caused by defective secretion into urine. No cumulative effect on these functions was observed in the new double mutant phenotype. The new phenotype has giant lysosomes typical of the beige mutation. Unexpectedly, the beige gene corrects the effect of the pale ear on serum lysosomal enzyme concentration. There is also a gene dosage effect of the beige gene on this serum lysosomal enzyme phenotype. The results suggest that the beige and pale ear genes affect the same pathway(s) of lysosome biosynthesis and/or processing. The action of the beige gene may precede that of the pale ear gene in lysosome physiology.

Lysosomal enzyme activities in pulmonary macrophages from rabbits breathing iron oxide.

The effects of an in vivo exposure to a nontoxic particle, iron oxide, on lysosomal hydrolases in pulmonary macrophages were examined. Rabbits breathed a submicron-sized aerosol of iron oxide for 3 h (mass concentration, 186 to 222 mg/m3). Macrophages were recovered by lung lavage 0, 12, 18, and 24 h later. The number of cells lavaged after iron oxide was significantly greater than the control number, whereas the amount of protein recovered per 10(8) cells decreased, suggesting the influx of smaller cells. Macrophages from animals exposed to iron oxide reacted histochemically for acid phosphatase showed increased stain intensity and redistribution of enzyme within the cytoplasm. Considerable cell-to-cell variability in enzyme activity was evident in these macrophages. Homogenates of cells exposed to iron oxide assayed for 6 lysosomal acid hydrolases showed little change in specific activity or in total enzyme per 10(8) cells when compared to homogenates of cells from control animals. Sucrose density gradient centrifugation demonstrated that exposure to iron oxide caused an increase in lysosome heterogeneity over that seen in control preparations and the appearance of a population of lysosomes of increased density. An aerosol of iron oxide did not greatly alter the average concentrations of lysosomal enzymes in pulmonary macrophages, but did stimulate recruitment of new cells and thus increases the total amount of some enzymes in the lung.

Effect of macrophage activation by immunoadjuvants on serum levels of lysosomal hydrolases in mice.

The effect of pyran copolymer, injected into mice bearing the M109 Madison lung carcinoma, on serum concentrations of lysozyme, beta-glucuronidase, and N-acetyl-beta, D-glucosaminidase was studied and compared with that of other immunoadjuvants. Increases in lysozyme levels ranging from 50 to 100% were observed after injection of pyran, BCG and Bru-Pel; increases in the levels of the other enzymes were less consistent. Other immunoadjuvants were less effective in raising serum concentrations of lysosomal enzymes. The findings were correlated with the results of previous studies on macrophage activation and antineoplastic action produced by these immunoadjuvants and suggest that serum levels of lysozyme can serve as indices of these effects.

The effect of anti-inflammatory compounds on the biochemical changes in the Arthus reaction.

The effect of anti-inflammatory drugs on the biochemical changes in the Arthus reaction have been studied and correlated to changes in the pathology of the reaction. In the Arthus reaction all the non-steroidal anti-inflammatory drugs inhibited the migration of cells into the lesion and reduced the lysosomal enzyme concentration at the Arthus site. The steroids did not inhibit the cellular inflitration or the total lysosomal enzyme concentration in the skin but did reduce the concentration of cathepsin D in the oedema fluid. In addition, prednisolone and, to a lesser extent, hydrocortisone reduced the degree of oedema formation. The results suggest that non-steroidal anti-inflammatory drugs inhibit the Arthus reaction by reducing the cellular infiltration whereas anti-inflammatory steroids act by preventing these cells secreting their lysosomal enzymes and thus causing tissue damage.