Localization Of Lysosomal Enzymes Research Articles

Streptozotocin-induced diabetes mellitus affects lysosomal enzymes in rat liver.

It has been previously shown that dextran sulfate administered to diabetic rats accumulates in the liver and kidney, and this could be due to a malfunction of the lysosomal digestive pathway. The aim of the present study was to evaluate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in the livers of diabetic rats. Diabetes mellitus was induced by streptozotocin in 26 male Wistar rats (12 weeks old), while 26 age-matched controls received only vehicle. The livers were removed on either the 10th or the 30th day of the disease, weighed, and used to evaluate the activity, expression, and localization of lysosomal enzymes. A 50-60% decrease in the specific activities of cysteine proteases, especially cathepsin B, was observed in streptozotocin-induced diabetes mellitus. Expression (mRNA) of cathepsins B and L was also decreased on the 10th, but not on the 30th day. Sulfatase decreased 30% on the 30th day, while glycosidases did not vary (or presented a transitory and slight decrease). There were no apparent changes in liver morphology, and immunohistochemistry revealed the presence of cathepsin B in hepatocyte granules. The decrease in sulfatase could be responsible for the dextran sulfate build-up in the diabetic liver, since the action of sulfatase precedes glycosidases in the digestive pathway of sulfated polysaccharides. Our findings suggest that the decreased activities of cathepsins resulted from decreased expression of their genes, and not from general lysosomal failure, because the levels of glycosidases were normal in the diabetic liver.

Changes in Localization of Lysosomal Enzymes in Muscle Induced by Conditioning or High Pressure Treatment

Changes in Localization of Lysosomal Enzymes in Muscle Induced by Conditioning or High Pressure Treatment

Localization of lysosomal enzymes in the retina and retinal pigment epithelium of RCS rats

Using ultrahistochemical and immunohistochemical techniques, localization of acid phosphatase and cathepsin D was demonstrated in the retina and pigment epithelium of 1 to 42 day old RCS rats and its nonaffected congenic rat strain. Both enzymes are present in the pigment epithelium of the normal and dystrophic rat eye. As early as the age of 1 week, it was found that the lysosomes in the dystrophic rat retina are less stable in releasing acid phosphatase than those of control animals. Infiltration of cathepsin D into the subretinal space could first be detected with certainty in 2-week-old animals. The fragility of the lysosomal membrane and, therefore, the release of both enzymes became more pronounced as the animals aged. The findings of this study indicate that the instability of the lysosomal membrane in the RCS rat pigment epithelium may initiate degeneration of photoreceptors and pigment epithelium. The demonstration of cathepsin D activity has proved very helpful in revealing the physiological or pathophysiological condition of retinal pigment epithelium.

Induction of avascular yolk sac due to reduction of basic fibroblast growth factor by retinoic acid in mice

Vasculogenesis depends on autocrine secretion of basic fibroblast growth factor (bFGF) from capillary endothelial cells. Retinoic acid (RA) induced avascular yolk sac (AVY) of mouse embryos of dams given 60 mg/kg of RA orally on Day 8 of gestation and sacrificed 3 days later. We studied the localization and transcriptional expression of bFGF and FGF-receptor (flg), heparin-binding growth factor (HBGF) activity, localization of lysosomal enzymes and α 1-antitrypsin (AAT), and electron microscopy of the normal mouse visceral yolk sac (VYS) and AVY. bFGF, which is normally present in the endoderm of the VYS of 8-day-old embryos and in all components of the VYS by Day 11 of gestation, was reduced in the AVY. However, in the presence of bFGF in vitro capillary nets were restored in the AVY. The mRNA for bFGF was not detectable in either VYS or AVY, while flg mRNA was detected equally in both organs in Northern blotting. The characteristic distribution pattern of lysosomal enzymes, acid phosphatase, lysozyme, and cathepsin D, and AAT was altered in the AVY. The level of acid phosphatase and AAT was reduced to 10% in the AVY. Electron microscopy revealed a partial or total loss of lysosomal membranes where the contents of lysosomes fused with adjacent lysosomes and the external organelles. These results suggest that vitelline blood vessels are not developed by endogenous autocrine bFGF but by exogenous transcellular bFGF from absorptive endodermal cells. Retinoic acid does not affect the angiogenic capacity of the VYS mesenchyme but destroys lysosomes, which release hydrolytic enzymes, leading to degradation of AAT in the endodermal cells and then digestion of endocytosed bFGF.

Chapter 7 Defining the Intracellular Localization Pathways followed by Lysosomal Enzymes in Dictyostelium discoideum

This chapter describes the biochemical and genetic methods used to define both the intracellular pathways followed by lysosomal enzymes and the molecular nature of the “sorting” signals found on the proteins. Using biochemical methods, the lysosomal enzymes α-man and β-glu in Dictyostelium are synthesized as precursor polypeptides that are cotranslationally translocated into the lumen of the endoplasmic reticulum (ER) and N-glycosylated. The precursors move at different rates from the RER to the Golgi complex, where they are sulfated and sorted into two classes. One class, containing the majority of the precursor polypeptides, is directed to lysosomes where they are rapidly cleaved to mature forms of the enzymes. The other class of precursors is rapidly secreted from cells. The different intracellular transport rates for the α-man and β-glu precursors support the existence of a transport-mediating receptor. The genetic approach involved the isolation and characterization of mutants altered in the secretion, modification, processing, and/or localization of lysosomal enzymes. Through biochemical analysis of these mutants, one can identify and characterize the molecular components involved in the processing and transport of this group of enzymes.

Lysosomal enzymes in Dictyostelium discoideum are transported to lysosomes at distinctly different rates.

We are investigating the molecular mechanisms involved in the localization of lysosomal enzymes in Dictyostelium discoideum, an organism that lacks any detectable mannose-6-phosphate receptors. The lysosomal enzymes alpha-mannosidase and beta-glucosidase are both initially synthesized as precursor polypeptides that are proteolytically processed to mature forms and deposited in lysosomes. Time course experiments revealed that 20 min into the chase period, the pulse-labeled alpha-mannosidase precursor (140 kD) begins to be processed, and 35 min into the chase 50% of the polypeptides are cleaved to mature 60 and 58-kD forms. In contrast, the pulse-labeled beta-glucosidase precursor (105 kD) begins to be processed 10 min into the chase period, and by 30 min of the chase all of the precursor has been converted into mature 100-kD subunits. Between 5 and 10% of both precursors escape processing and are rapidly secreted from cells. Endoglycosidase H treatment of immunopurified radioactively labeled alpha-mannosidase and beta-glucosidase precursor polypeptides demonstrated that the beta-glucosidase precursor becomes resistant to enzyme digestion 10 min sooner than the alpha-mannosidase precursor. Moreover, subcellular fractionation studies have revealed that 70-75% of the pulse-labeled beta-glucosidase molecules move from the rough endoplasmic reticulum (RER) to the Golgi complex less than 10 min into the chase. In contrast, 20 min of chase are required before 50% of the pulse-labeled alpha-mannosidase precursor exits the RER. The beta-glucosidase and alpha-mannosidase precursor polypeptides are both membrane associated along the entire transport pathway. After proteolytic cleavage, the mature forms of both enzymes are released into the lumen of lysosomes. These results suggest that beta-glucosidase is transported from the RER to the Golgi complex and ultimately lysosomes at a distinctly faster rate than the alpha-mannosidase precursor. Thus, our results are consistent with the presence of a receptor that recognizes the beta-glucosidase precursor more readily than the alpha-mannosidase precursor and therefore more quickly directs these polypeptides to the Golgi complex.

Tissue content and localization of lysosomal enzymes in cholestatic rats.

Levels of the lysosomal enzymes beta-glucuronidase and beta-hexosaminidase were determined in tissues from rats at 1-7 weeks after permanent bile duct occlusion (28 animals) and sham operation (16 animals). With increasing duration of bile duct occlusion, increasing levels were found in the liver and spleen, whereas levels were unaffected in the pancreas, kidneys, brain, muscle, and skin. Histochemical investigation showed that the lysosomal enzymes accumulated in the hepatocytes and in the increasing numbers of reticuloendothelial cells of the liver and spleen during cholestasis. No significant accumulation of lysosomal enzymes was seen in the pancreas. In preparations of isolated hepatocytes and non-parenchymal cells increased specific activity of lysosomal enzymes in cholestasis appeared in both cellular fractions. It is assumed that the serum increase of lysosomal enzymes in late cholestasis is partly due to a release from activated macrophages in the liver and spleen.

Radiolabelled colloid uptake distribution and pulmonary contents and localization of lysosomal enzymes in cholestatic rats.

Studies of the activity uptake of radiolabelled 99mTc2S7 colloid in different tissues showed a moderately increased uptake in the lung 1 week and a greatly increased uptake 3 weeks after permanent bile duct occlusion. Three weeks after bile duct occlusion the activity uptake was also slightly increased in the spleen and moderately reduced in the liver. In pulmonary tissue of cholestatic rats, the levels of alkaline and acid phosphatase, beta-glucuronidase, and beta-hexosaminidase were determined after 6 weeks. The pulmonary contents of beta-glucuronidase was increased tenfold, and that of beta-hexosaminidase was increased fourfold in cholestasis. Histochemical investigation showed that the lysosomal enzymes were located in the macrophages, which in cholestasis were abundant in alveolar walls and inside alveoli. Macrophages were also frequently seen to engorge pulmonary veins.

Localization of Lysosomal Enzymes in Herpesvirus-Infected Cells by Electron Microscope Cytochemistry

Evidence of increased lysosomal enzyme content and activity has been reported following infection by a number of herpesviruses, but its significance remains unclear. One hypothesis holds that lysosomal enzymes, as part of host cell defenses. inactivate cytoplasmic viral progeny so that most released particles are noninfectious and infectivity remains highly cell associated. Such a pattern is characteristic of varicellazoster (VZ), and indeed electron microscope cytochemical studies have revealed that cytoplasmic VZ particles are often located within lysosomes (1). It was of interest to determine whether there is a less direct relationship between lysosomes and progeny virus in herpesvirus systems where infectivity is not closely cell-restricted. The present paper describes acid phosphatase and aryl sulfatase localizations in WI-38 cells (diploid human lung fibroblasts) infected with either herpes simplex virus (HSV), strain HF, or human cytomegalovirus, strain AD-169.

Influence of starvation on the activities and localization of cathepsin D and other lysosomal enzymes in hearts of rabbits and mice

An investigation was made in mice and rabbits of the effects of starvation on the activities and cellular localization of lysosomal enzymes in cardiac muscle of the left ventricle. The specific activity of cathepsin D was increased in the atrophying hearts of mice, by 14% after one day and by 26% after 3 days. Simultaneously, the proportion of the enzyme activity that was nonsedimentable during centrifugation at 40 000 g rose significantly, suggesting increased fragility of lysosomes or release of enzyme from lysosomes into the cytosol. In the same hearts protein concentration and the activities of acid phosphatase and beta-acetylglucosaminidase remained unchanged. Changes were similar in rabbit hearts except that specific cathepsin D activity was increased more (46%) and changes in the proportion of the activity that was nonsedimentable were less marked. Immunofluorescent staining for rabbit cathepsin D revealed that this acid proteinase was localized in lysosome-like particles in both interstitial cells and muscle fibres in hearts of normal animals. After 3 days starvation there was no detectable change in interstitial cells, but in the muscle fibres particulate staining was greater and, in addition, intense diffuse cytoplasmic staining was observed. The results indicate that cardiac muscle, like liver but unlike skeletal muscle, develops a significant increase in the activity of the lysosomal protease cathepsin D during starvation. The change occurs specifically in myocytes rather than in interstitial cells. The results suggest a possible role for cathepsin D in contributing to the development of cardiac atrophy and net protein catabolism during prolonged starvation.

The localization of lysosomal enzymes in chromaffin tissue.

1. An homogenate of bovine adrenal medulla contains significant amounts of six acid hydrolases: acid ribonuclease, acid deoxyribonuclease, cathepsin, acid phosphatase, beta-glucuronidase and arylsulphatase. Most of the activity of each enzyme could be sedimented in the large-granule fraction at 242,000 g-min.2. Differential centrifugation indicated the presence of three populations of particles, which sedimented at slightly different rates; these are, in order of decreasing sedimentation rate, mitochondria, particles containing the acid hydrolases, and chromaffin granules.3. The three types of particle could be separated by ultracentrifuging the large-granule fraction in a sucrose density gradient. Most of the activity of each hydrolase was recovered in a layer intermediate between those formed by mitochondria and chromaffin granules.4. The large-granule fraction therefore contains particles which are defined by their enzyme content as lysosomes.5. Highly purified chromaffin granules, containing less than 5% of the activity of each acid hydrolase, were obtained from the gradient.