Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.23.5 (cathepsin D)
4,130 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Vascular smooth muscle (VSM) cells from hypertensive and normotensive rat aortae and caudal arteries were isolated by enzymatic techniques, homogenized, and fractionated by differential pelleting. By these techniques, only mitochondria could be enriched more than fivefold in any one fraction. The other organelles were distributed heterogeneously in almost all fractions. Hypertensive smooth muscle enzyme distribution patterns were different from the normotensive, suggesting that changes in sedimentation characteristics had occurred. Activity of the enzyme 5'-nucleotidase increased in whole tissue homogenates and in the 'microsomal' fraction of aortic and caudal artery of hypertensive VSM. The lysosomal protease, cathepsin D, of hypertensive animals decreased in activity for both vascular smooth muscles while N-acetyl-beta-glucosaminidase and pNPPase (acid phosphatase) increased. The possibility of a functional deficiency in protein degradation causing lysosomal overloading is discussed.
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PMID:Alterations in lysosomes, catalase-containing organelles, mitochondria and plasma membrane fragments from hypertensive rat aorta and caudal artery. 21 41

Isoelectric focusing was used to investigate the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase, beta-galactosidase and beta-N-acetylhexosaminidase in the following, previously characterized subcellular fractions from rat kidney: a special rough microsomal fraction, enriched up to 9-fold over the homogenate in acid hydrolases; a smooth microsomal fraction; a Golgi membrane fraction enriched about 2.5-fold in acid hydrolases and 10- to 20-fold in several glycosyl transferases; and a lysosomal fraction enriched up to 25-fold in acid hydrolases. The electro-focusing behavior of the hydrolases in these fractions was markedly sensitive to the autolytic changes that occur under acidic conditions, even at 4 degrees C. Autolysis was minimized by extracting fractions in an alkaline medium (0.2% Triton X-100, 0.1 M sodium glycinate buffer, pH 10, 0.1 % p-nitrophenyloxamic acid) and adding p-nitrophenyloxamic acid (0.1 %), AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND cathepsin D, to the pH gradient. The enzymes in the lysosomal fraction displayed a characteristic bimodal or trimodal distribution. Arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in an acidic form with an isoelectric point of 4.4, and a basic form with an isoelectric point of 6.2, 6.7 and 8.0, respectively. Acid phosphatase and beta-galactosidase occurred in an acidic, intermediate and basic form with isoelectric points of about 4. 1, 5.6 and 7.4, respectively. In the special rough microsomal fraction these enzymes were mostly in a basic form with isoelectric points between 7.5 and 9; these were 1-2 units higher than the corresponding basic forms in the lysosomal fraction. Treatment of extracts of the rough microsomal fraction with bacterial neuraminidase raised the isoelectric points of all five hydrolases by 1-2.5 units, indicating the presence of some N-acetylneuraminic acid residues in these basic glycoenzymes. The hydrolases in the Golgi fraction were largely in an acidic form with isoelectric points similar to or lower than those of the corresponding acidic components in the lysosomal fraction. The hydrolases in the smooth microsomal fraction showed isoelectric-focusing patterns intermediate between those in the rough microsomal and the Golgi fractions. These findings support the following scheme for the synthesis, transport and packaging of the lysosomal enzymes. Each hydrolase is synthesized in a restricted portion of the r
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PMID:Changes in electronegativity of lysosomal hydrolases during intracellular transport. An isoelectric-focusing study in subcellular fractions of rat kidney. 23 56

Phenobarbital was given to male rats as a single injection and as repetitive injections for 7 days. The effects of treatment on the lysosomal hydrolases acid phosphatase, cathepsin D, and aryl sulfatase were analyzed at different intervals ranging from 1 to 15 days after seven injections, and from 1 to 48 h after a single injection. In both cases, microsomal protein and NADPH-cytochrome c reductase were measured to ensure proper induction. After a single injection, a slight decrease in hydrolytic activities was observed. Repetitive administration of phenobarbital gave rise to a marked decrease of lysosomal enzyme activities 1 day after cessation of treatment. This decrease was followed by a continuous increase in activity up to day 3 and 4. One or 2 weeks after treatment, enzyme activities declined to control values. The increase in activity of lysosomal hydrolytic enzymes was correlated with the onset of induced autophagy of endoplasmic reticulum membranes described as occurring in liver upon cessation of phenobarbital exposure. It is concluded that phenobarbital treatment per se decreases lysosomal enzyme activities, whereas the induced autophagy following cessation of exposure is associated with enhanced levels of lysosomal hydrolases in rat liver.
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PMID:Induction of liver lysosomal enzymes during the autophagic phase following phenobarbital treatment of rat. 40 31

A study was done to determine whether the Ca2+-activated muscle protease (CAF) that removes Z disks from myofibrils in the presence of Ca2+ is located in a sedimentable subcellular organelle. Porcine skeletal muscle cells were diced finely with a scalpel and were suspended in 0.25 M sucrose, 4 mM EDTA with a VIRTIS homogenizer. Filtration of the suspended muscle through four layers of cheesecloth removed most of the myofibrils and stromal protein. Nuclear (1,000 gavg for 15 min), mitochondrial-microsomal (50,000 gavg for 60 min), and supernatant fractions were assayed for succinic dehydrogenase, acid ribonuclease, cathepsin D, and CAF activities. Approximately 96% of total succinic dehydrogenase activity, 81% of cathepsin D activity, and 45% of acid ribonuclease activity, but only 14% of total CAF activity, were found in the nuclear and mitochondrial-microsomal fractions. Cathepsin D activity in the nuclear and mitochondrial-microsomal fractions was decreased if assays were done without prior treatment to rupture membranous structures; hence, our cell rupture and homogenization procedures preserved some intact lysosomal organelles. The results indicate that the small amount of CAF activity in the nuclear and mitochondrial-microsomal fractions was due to contamination by supernate and that CAF is not located in a membrane-bounded subcellular particle. Because CAF is active at the intracellular pH and temperature of living skeletal muscle cells and is in direct contact with the cytoplasm of muscle cells, its activity must be regulated by intracellular cellular Ca2+ concentration to prevent continuous and indiscriminate degradation of myofibrils.
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PMID:A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Subcellular localization of the protease in porcine skeletal muscle. 94 76

The biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase was studied using an in vitro cell-free translation system, pulse-chase experiments with primary cultured rat hepatocytes and subcellular fractionation techniques of rat liver after pulse-labeling with [35S]methionine in vivo. The single polypeptide of 45 kDa translated in the cell-free system from membrane-bound polysomal RNAs was converted to the 64 kDa form when the translation was carried out in the presence of microsomal vesicles. Pulse-chase experiments using cultured rat hepatocytes showed that acid phosphatase is initially synthesized as an endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form of 64 kDa, and processed via an Endo H-sensitive intermediate form of 62 kDa to an Endo H-resistant form with a 67 kDa mass. Phase separation with Triton X-114 showed that both the 64 and 67 kDa forms have hydrophobic properties. Treatment of the cells with chloroquine or tunicamycin, drugs which enhance the secretion of lysosomal hydrolases, had no effect on the normal transport of acid phosphatase to lysosomes. Acid phosphatase did not contain the phosphorylated high mannose type of oligosaccharide chains observed in cathepsin D. Subcellular fractionation experiments in conjunction with pulse-labeling in vivo showed that the acid phosphatase of the 67 kDa form was present in the Golgi heavy fraction (GF3) and the Golgi light fraction (GF1+2) enriched in cis and trans Golgi elements, respectively, at 30 min after the administration of [35S]methionine. Simultaneously, this polypeptide was also found in the lysosomal membrane fraction, thereby indicating that acid phosphatase is delivered to lysosomes in a membrane-bound form, immediately after reaching the trans-Golgi region.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase in rat hepatocytes. 169 35

Our recent studies have shown that cathepsin L is first synthesized as an enzymatically inactive proform in endoplasmic reticulum and is successively converted into an active form during intracellular transport and we postulated that aspartic proteinases would be responsible for the intracellular propeptide-processing step of procathepsin L accompanied by the activation of enzyme (Y. Nishimura, T. Kawabata, and K. Kato (1988) Arch. Biochem. Biophys. 261, 64-71). To better understand this proposed mechanism, we investigated the effect of pepstatin, a potent inhibitor of aspartic proteinases, on the intracellular processing kinetics of cathepsin L analyzed by pulse-chase experiments in vivo with [35S]methionine in the primary cultures of rat hepatocytes. In the pepstatin-treated cells, the proteolytic conversion of cellular procathepsin L of 39 kDa to the mature enzyme was significantly inhibited and considerable amounts of proenzyme were found in the cell after 5-h chase periods. Further, the subcellular fractionation experiments demonstrated that the intracellular processing of procathepsin L in the high density lysosomal fraction was significantly inhibited and that considerable amounts of the procathepsin L form were still observed in the light density microsomal fraction after 2 h of chase. These results suggest that pepstatin treatment caused a significant inhibitory effect on the intracellular processing and also on the intracellular movement of procathepsin L from the endoplasmic reticulum to the lysosomes. These findings provide the first evidence showing that aspartic proteinase may play an important role in the intracellular proteolytic processing and activation of lysosomal cathepsin L in vivo. Therefore, we suggest that cathepsin D, a major lysosomal aspartic proteinase, is more likely to be involved in this proposed model in the lysosomes.
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PMID:Evidence that aspartic proteinase is involved in the proteolytic processing event of procathepsin L in lysosomes. 265 11

We have detected, solubilized, and purified to near-homogeneity a membrane-bound acid protease from rabbit reticulocytes. Chemical, physical, immunological, and catalytic characterization demonstrate that the enzyme is cathepsin D. With cytochrome b5 as substrate, the enzyme shows a surprisingly high pH optimum and is stimulated by ATP and DPG. Possible roles for the protease include protein processing of microsomal enzymes, degradation of subcellular organelles, and destruction of excess hemoglobin chains. The possible role of cathepsin D in protein processing of microsomal enzymes will be best assessed by the molecular biological approaches described in the following two presentations.
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PMID:Cathepsin D in erythroid cells. 269 41

Cathepsin D was assessed in C6 glioma cells grown in medium with an intermediate- or low-percent composition of serum. The amount, form, and subcellular location of cathepsin D differed after treatment with cyanate or monensin in cells grown in a low-serum, growth-factor-supplemented medium. Immunoblotting showed that cathepsin D in the lysosomal fraction of the C6 cell line had a molecular weight (Mr) of 42 kD, whereas that in the microsomal fraction had Mr's of 42, 47, and 78 kD. After treatment for 1 to 16 hr with 4 mmol/L cyanate and subcellular fractionation, the molecular weight of lysosomal cathepsin D was the same in treated and untreated cells, but more enzyme was found in lysosomes of treated cells at 8 and 16 hr. In the microsomal fraction, the amounts of both the 42 and 47 kD forms were increased after 1 to 16 hr of treatment. When exposed to 20 mmol/L cyanate, C6 cells remained viable, but compared with untreated cells, they showed 25% less lysosomal cathepsin D, with increased amounts found in the microsomal fraction. The 78 kD protein detected by immunoblotting was present in both the lysosomal and microsomal fractions but was predominant in the latter. The apparent molecular weight of this protein was the same after cyanate but differed with monensin, where Mr's of 39, 42, and 73 kD were found. Monensin-treated cells had less lysosomal cathepsin D and relatively more microsomal enzyme. The differing molecular weights of cathepsin D from cyanate- and monensin-treated cells suggest that their inhibitions occur at different processing loci in distal elements of the Golgi stacks. The differences in the pI of cathepsin D and the number of its forms from cyanate- and monensin-treated cells are also consistent with interference in the late stages of glycoprotein maturation. In this paper we show that the amount, molecular form, and consequent intracellular location of cathepsin D in cells of the C6 line can be affected by agents that selectively disrupt stages in Golgi-related protein modification and transport.
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PMID:Alterations of the posttranslational processing of a lysosomal enzyme in C6 glioma cells. 304 14

Procathepsin H in kidney and liver microsomal lumen was identified to have a molecular mass of 41 kDa by immunoblot analysis. The proenzyme was then concentrated by applying the microsomal contents to a concanavalin A-Sepharose column. When the concanavalin A-adsorbed fraction was incubated at pH 4.0 at 20 degrees C, the activity measured with synthetic substrate increased 3.5 times over that of the control after 24 h incubation. Immunoblot analysis showed that acidic treatment caused the disappearance of procathepsin H. Thus the proenzyme might be processed to the mature enzyme under acidic conditions. The marked increase of enzymatic activity and the conversion of proenzyme were completely blocked with pepstatin which is a potent inhibitor of aspartic proteases. These results suggested that a protease for processing procathepsin H might be cathepsin D, a major lysosomal aspartic protease. Therefore, procathepsin H seems to be synthesized first in the enzymatically inactive form in endoplasmic reticulum and successively converted into the active form in lysosomes during biosynthesis.
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PMID:Identification of latent procathepsin H in microsomal lumen: characterization of proteolytic processing and enzyme activation. 327 36

The regulation of the lysosomal pathway by burn injury was investigated using a local burn model in which one hindlimb of a rat is scaled at 85 degrees C for 3.6 s. By two days postinjury the rate of net protein breakdown in the incubated soleus muscle from the burned leg is doubled. The activity of cathepsin D increases 40% to 50% and that of cathepsins B and L increase 80% to 100%. The activity of a lysosomal nonprotease activity, N-acetylglucosaminidase, is not significantly increased. The latency of lysosomal enzymes (an estimate of cellular autophagy) did not change at any time postburn. Inhibitors of autophagy (ie, leucine and 3-methyladenine) inhibited net protein breakdown and increased latency to a similar extent in muscles from control and burned legs. Thus, there is no evidence that a change in cellular autophagy is responsible for the increased proteolysis seen in intact muscle. However, burn-induced changes in alternative routes of protein sequestration cannot be excluded. Burn did not increase either receptor-mediated or fluid phase endocytosis by incubated soleus muscle. Burn injury also did not reduce the inhibitory action of cytoplasmic inhibitors of cathepsins B and L in skeletal muscle. However, burn injury markedly stimulated the synthesis of glycoproteins in the microsomal fraction without affecting overall protein synthesis. This increase in synthesis preceded the rise in lysosomal protease activity. These results support the possibility that induction of lysosomal protease synthesis may underlie burn-induced increases in muscle proteolysis.
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PMID:Regulation of lysosomal proteolysis in burn injury. 329 35


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