Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.23.5 (cathepsin D)
 4,130 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Golgi-membrane-bound Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase (CMP-N-acetylneuraminate:beta-galactoside alpha 2-6-sialyltransferase, EC 2.4.99.1) behaves as an acute-phase reactant increasing about 5-fold in serum in rats suffering from inflammation. The mechanism of release from the Golgi membrane is not understood. In the present study it was found that sialyltransferase could be released from the membrane by treatment with ultrasonic vibration (sonication) followed by incubation at reduced pH. Maximum release occurred at pH 5.6, and membranes from inflamed rats released more enzyme than did membranes from controls. Galactosyltransferase (UDP-galactose:N-acetylglucosamine galactosyltransferase; EC 2.4.1.38), another Golgi-located enzyme, which does not behave as an acute-phase reactant, remained bound to the membranes under the same conditions. Release of the alpha 2-6-sialyltransferase from Golgi membranes was substantially inhibited by pepstatin A, a potent inhibitor of cathepsin D-like proteinases. Inhibition of release of the sialyltransferase also occurred after preincubation of sonicated Golgi membranes with antiserum raised against rat liver lysosomal cathepsin D. Addition of bovine spleen cathepsin D to incubation mixtures of sonicated Golgi membranes caused enhanced release of the sialyltransferase. Intact Golgi membranes were incubated at lowered pH in presence of pepstatin A to inhibit any proteinase activity at the cytosolic face; subsequent sonication showed that the sialyltransferase had been released, suggesting that the proteinase was active at the luminal face of the Golgi. Golgi membranes contained a low level of cathepsin D activity (EC 3.4.23.5); the enzyme was mainly membrane-bound, since it could only be released by extraction with Triton X-100 or incubation of sonicated Golgi membranes with 5 mM-mannose 6-phosphate. Immunoblot analysis showed that the transferase released from sonicated Golgi membranes at lowered pH had an apparent Mr of about 42,000 compared with one of about 49,000 for the membrane-bound enzyme. Values of Km for the bound and released enzyme activities were comparable and were similar to values reported previously for liver and serum enzymes. The work suggests that a major portion of sialyltransferase containing the catalytic site is released from a membrane anchor by a cathepsin D-like proteinase located at the luminal face of the Golgi and that this explains the acute-phase behaviour of this enzyme.
 ...
PMID:The role of a cathepsin D-like activity in the release of Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase from rat liver Golgi membranes during the acute-phase response. 314 77

The transport of newly synthesized cathepsin D in fibroblasts at 16-28 degrees C was compared to that at 37 degrees C. At 37 degrees C newly synthesized cathepsin D passes the trans Golgi within 30-60 min, becomes segregated from the secretory route into prelysosomal organelles within 1-2 h and processed to mature forms in dense lysosomes within 1.5-3 h after synthesis. The small fraction of cathepsin D that escapes transport into lysosomes is secreted within less than 2 h. At 16-28 degrees C the transport of cathepsin D to lysosomes is inhibited in a temperature-dependent manner. At 16-28 degrees C cathepsin D precursors are slowly transported to the trans Golgi. The cathepsin D precursors accumulate at a site that is in continuity with the secretory pathway and located within or distal of the trans Golgi and proximal to the site where cathepsin D precursors leave the secretory pathway as complexes with mannose 6-phosphate receptors. The arrest at this site is not complete. The receptor-dependent segregation of the cathepsin D precursors released from the block is impaired at less than or equal to 26 degrees C. The inhibition of segregation results in an increased, albeit retarded secretion of cathepsin D. The fraction of cathepsin D precursors that is segregated from the secretory pathway encounters a further low temperature block in prelysosomal organelles. There cathepsin D precursors are proteolytically processed to an intermediate form, which accumulates transiently.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Low temperature blocks transport and sorting of cathepsin D in fibroblasts. 320 52

Previously we identified an acid protease activity which was located in the endosomes of rabbit alveolar macrophages (Diment, S., and Stahl, P.D. (1985) J. Biol. Chem. 260, 15311-15317). In this study, the endosomal protease is identified as cathepsin D by immunoprecipitation with polyclonal antibodies raised against rabbit cathepsin D and by NH2-terminal sequence. In order to elucidate the mechanism for targeting of cathepsin D to endosomes, we first examined the membrane association of cathepsin D with light (rho = 1.05 g/ml) and heavy density (rho = 1.1 g/ml) vesicles from Percoll density gradients. After sequential washes, 8.4 and 21.9% of cathepsin D activity remained associated with heavy and light density vesicles, respectively. This membrane-associated cathepsin D could not be solubilized in either buffer at pH 5.0 containing mannose 6-phosphate and EDTA or in buffer at pH 10.6. Solubilization required the detergent Triton X-100. To determine whether membrane-associated cathepsin D was found in endosomes, the enzyme was radioiodinated within endosomes and lysosomes with internalized lactoperoxidase. The membrane-associated form was detected in endosomes, but much less in lysosomes. Biosynthetic studies combined with the same extraction procedure revealed that macrophage cathepsin D is first synthesized as an inactive membrane-associated precursor. The precursor is processed to an active, membrane-associated form and then to the active soluble form found in lysosomes. Our studies provide evidence that 1) cathepsin D is in endosomes of macrophages; 2) cathepsin D is transported to endosomes as a membrane-associated form; and 3) the membrane-associated form is a biosynthetic precursor for the soluble form found in endosomes and lysosomes.
 ...
PMID:Cathepsin D is membrane-associated in macrophage endosomes. 336 Aug 12

We have studied the posttranslational modifications of the 52-kD protein, an estrogen-regulated autocrine mitogen secreted by several human breast cancer cells in culture (Westley, B., and H. Rochefort, 1980, Cell, 20:353-362). The secreted 52-kD protein was found to be phosphorylated mostly (94%) on high-mannose N-linked oligosaccharide chains, and mannose-6-phosphate signals were identified. The phosphate signal was totally removed by alkaline phosphatase hydrolysis. The secreted 52-kD protein was partly taken up by MCF7 cells via mannose-6-phosphate receptors and processed into 48- and 34-kD protein moieties as with lysosomal hydrolases. By electron microscopy, immunoperoxidase staining revealed most of the reactive proteins in lysosomes. After complete purification by immunoaffinity chromatography, we identified both the secreted 52-kD protein and its processed cellular forms as aspartic and acidic proteinases specifically inhibited by pepstatin. The 52-kD protease is secreted in breast cancer cells under its inactive proenzyme form, which can be autoactivated at acidic pH with a slight decrease of molecular mass. The enzyme of breast cancer cells, when compared with cathepsin D(s) of normal tissue, was found to be similar in molecular weight, enzymatic activities (inhibitors, substrates, specific activities), and immunoreactivity. However, the 52-kD protein and its cellular processed forms of breast cancer cells were totally sensitive to endo-beta-N-acetylglucosaminidase H (Endo H), whereas several cellular cathepsin D(s) of normal tissue were partially Endo H-resistant. This difference, in addition to others concerning tissue distribution, mitogenic activity and hormonal regulation, strongly suggests that the 52-kD cathepsin D-like enzyme of breast cancer cells is different from previously described cathepsin D(s). The 52-kD estrogen-induced lysosomal proteinase may have important functions in facilitating the mammary cancer cells to proliferate, migrate, and metastasize.
 ...
PMID:Phosphorylation, glycosylation, and proteolytic activity of the 52-kD estrogen-induced protein secreted by MCF7 cells. 354 22

Effect of diltiazem on subcellular distribution of lysosomal enzymes, high-energy phosphate metabolism and mechanical function in the ischemic heart was studied. Ischemia was induced by lowering the afterload pressure of the perfused working rat heart. The activities of cathepsin D, beta,N-acetylglucosaminidase and acid phosphatase were determined in the nonsedimentable and sedimentable fractions after centrifugation of the tissue extract to assess the subcellular distribution of lysosomal enzymes. After ischemia, decreases in the mechanical function and the tissue level of high-energy phosphates were observed. In addition, ischemia caused subcellular redistribution of lysosomal enzymes from the lysosomes to the cytoplasm. Reperfusion of the ischemic heart did not restore the mechanical function and the level of high-energy phosphates completely. Diltiazem (2.21 X 10(-6), 1.11 X 10(-5) and 2.21 X 10(-5) M) was provided for the heart 5 min before the onset of ischemia. Diltiazem preserved high-energy phosphates in the ischemic heart, and inhibited the subcellular redistribution of lysosomal enzymes being caused by ischemia, depending on its concentration. Reperfusion after ischemia with diltiazem recovered the mechanical function that had been decreased by ischemia. These results may indicate that diltiazem can protect the myocardium against ischemic damage.
 ...
PMID:Inhibition of ischemia-induced subcellular redistribution of lysosomal enzymes in the perfused rat heart by the calcium entry blocker, diltiazem. 365 10

We have obtained expression of a cDNA clone for human cathepsin D in Xenopus laevis oocytes. Biosynthetic studies with [35S]methionine labeling demonstrated that most of the cathepsin D remained intracellular and underwent proteolytic cleavage, converting a precursor of Mr 47,000 D to a mature form of Mr 39,000 D with processing intermediates of Mr 43,000-41,000 D. greater than 90% of the cathepsin D synthesized by oocytes bound to a mannose 6-phosphate (Man-6-P) receptor affinity column, indicating the presence of phosphomannosyl residues. An analysis of [2-3H]mannose-labeled oligosaccharides directly demonstrated phosphomannosyl residues on cathepsin D. Sucrose-gradient fractionation, performed to define the membranous compartments that cathepsin D traversed during its biosynthesis, demonstrated that cathepsin D is targeted to a subpopulation of yolk platelets, the oocyte equivalent of a lysosome. Xenopus oocytes were able to endocytose lysosomal enzymes from the medium and this uptake was inhibited by Man-6-P, thus demonstrating the presence of Man-6-P receptors in these cells. Therefore, the entire Man-6-P dependent pathway for targeting of lysosomal enzymes is present in the oocytes. Xenopus oocytes should be a useful system for examining signals responsible for the specific targeting of lysosomal enzymes to lysosomes.
 ...
PMID:Expression of human cathepsin D in Xenopus oocytes: phosphorylation and intracellular targeting. 368 Mar 68

The effects of the phosphate analogues, vanadate and molybdate, on the ATP-activated enzyme, cathepsin D, were investigated. Both were found to inhibit proteolysis but this appeared to be the result of non-specific interactions with the protein substrates which result in precipitation, rather than interactions with the enzyme. Inhibition of proteolysis was induced by the same concentration of inhibitors as that which induced precipitation (measured by turbidity), and was dependent on the concentration of substrate. Precipitation did not occur at neutral pH but was maximal below pH 5. High concentrations of salt (greater than 1M KC1) prevented precipitation of proteins by vanadate and molybdate and under these conditions little inhibition of proteolysis was observed even at high inhibitor concentrations. Nonetheless, ATP was found to activate proteolysis catalyzed directly by lysosomal enzymes at acid pH, while vanadate and molybdate inhibited proteolysis in this system and induced precipitation of substrate. These results indicate that inhibition of proteolysis at acid pH by vanadate (or molybdate) has no relationship to inhibition of proteases and/or ATP dependence of such enzymes. However, direct activation of cathepsin D in lysosomes by ATP remains a viable hypothesis.
 ...
PMID:The effects of vanadate and molybdate on cathepsin D; relationship to ATP activation of lysosomal proteolysis. 385 94

Coated vesicles were isolated from metabolically labeled human fibroblasts with the aid of affinity-purified antibodies against human brain clathrin and Staphylococcus aureus cells. The material adsorbed to the S. aureus cells was enriched in clathrin. When the S. aureus cells bearing the immunoadsorbed material were treated with 0.5% saponin, extracts containing the precursor form of cathepsin D were obtained. The extraction of the precursor was promoted in the presence of mannose 6-phosphate. Material adsorbed to S. aureus cells coated with control immunoglobulins was nearly free of clathrin and contained a small amount of the cathepsin D precursor (less than 20% of that adsorbed with anti-clathrin antibodies). The extraction of this cathepsin D precursor was independent of mannose 6-phosphate and was complete after a brief exposure to saponin. The amount of cathepsin D precursor in coated membranes varied between 0.4 and 2.5% of total precursor. Analysis of pulse chase-labeled fibroblasts revealed that cathepsin D was only transiently associated with coated membranes. The mean residence time of cathepsin D precursor in coated membranes was estimated to be 2 min. These observations support the view that coated membranes participate in the transfer of precursor forms of endogenous lysosomal enzymes to lysosomes.
 ...
PMID:Cathepsin D precursors in clathrin-coated organelles from human fibroblasts. 392 34

Proteoglycans from bovine nasal septa and from the Swarm rat chondrosarcoma were isolated as aggregates (PGC) and as monomers (PGS). Portions of the PGC preparations were degraded with cathepsin D or chondroitinase AC. Chondroitin sulfates were isolated by differential precipitation from alkaline digests of the PGS from bovine nasal septa. The effects of these preparations at concentrations up to 2 mg/ml on the precipitation of tricalcium phosphate in vitro at pH 7.8 in 16 hours at 25 degrees C were ascertained. To this end, the amounts of calcium and phosphate in the precipitates and in the supernates were determined. The PGC preparations were found to be very effective inhibitors; in the presence of 2 mg/ml, precipitate did not form. The PGS preparations were less effective than the PGC preparations; in the presence of 2 mg/ml, about 20% as much calcium phosphate precipitated as in their absence. The chondroitinase AC-degraded preparations at concentrations equivalent to 2 mg/ml of the PGC preparations were approximately as effective as the PGS preparations. On the other hand, the cathepsin D-degraded PGC preparations and the chondroitin sulfate chains were relatively poor inhibitors. Although the viscosity of the solutions may have influenced the rate at which the precipitates settled to the bottom of the tubes, the amounts of the tricalcium phosphate formed were related to the composition and concentration of the proteoglycan preparations.
 ...
PMID:Role of proteoglycans in endochondral ossification: inhibition of calcification. 393 96

The incorporation of [3H]leucine and [32P]phosphate into three lysosomal enzymes, cathepsin D, beta-hexosaminidase and arylsulfatase A by fibroblasts from six patients affected with mucolipidosis III was determined. In the mutant cells the incorporation of 32P in the enzymes was reduced by 70-97% as compared to controls. The residual phosphorylation of lysosomal enzymes is definitely higher than in fibroblasts from patients with mucolipidosis II, where apparently non-phosphorylated enzymes are formed. In mucolipidosis III the major part of the newly formed enzymes accumulated extracellularly and the cellular enzymes were recovered mainly in their processed forms. In mucolipidosis III arylsulfatase A and the processed forms of cathepsin D exhibited a heterogeneity that was not observed in controls. beta-Hexosaminidase and cathepsin D secreted by mucolipidosis III fibroblasts contained only a small amount of phosphorylated oligosaccharides with either one or two phosphate groups per oligosaccharide. As in controls the major fraction of phosphate was present as acid-labile phosphodiester resistant to alkaline phosphatase. The residual phosphorylation of lysosomal enzymes may be related to the partial intracellular retention and processing of these enzymes in fibroblasts from patients with mucolipidosis III.
 ...
PMID:Impaired phosphorylation of lysosomal enzymes in fibroblasts of patients with mucolipidosis III. 612 Aug 34


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>