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: UMLS:C0272170 (SDS)
50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The codon of the catalytic serine in the active site of the vacuolar serine proteinase yscB (PrB) was changed to alanine, yielding the mutant gene prb1-Ala519. Following replacement of the wild-type PRB1 allele with prb1-Ala519, only a 73-kDa molecule was detected by immunoprecipitation with PrB-specific antiserum. The size of the mutant molecule corresponds to the unprocessed cytoplasmic precursor (pre-super-pro-PrB), as detected in sec61 mutants, when translocation into the endoplasmic reticulum is blocked. However, the mutant molecule is completely translocated into the secretory pathway, as indicated by protection from proteinase K digestion in spheroplast lysates in the absence of detergent. When N-glycosylation was inhibited in prb1-Ala519 mutant cells by tunicamycin, a smaller molecule of about 71 kDa appeared consistent with single N-glycosylation and signal-sequence cleavage of the translocated mutant PrB molecule in the endoplasmic reticulum. Thus, the active-site mutation prevents the wild-type processing of the N-glycosylated 73-kDa precursor of PrB to the 41.5 kDa pro-PrB in the endoplasmic reticulum. In order to characterize the processing of wild-type super-pro-PrB in more detail, we generated antibodies against the non-enzymatic superpeptide domain of the 73-kDa precursor expressed in Escherichia coli. We find that, in addition to pro-PrB, a distinct protein (superpeptide) with a mobility of about 41 kDa in SDS/PAGE is generated in the endoplasmic reticulum. Pulse-chase experiments indicate rapid degradation of the 41-kDa superpeptide in wild-type cells. Correspondingly, the superpeptide was virtually undetectable by immunoblotting wild-type cell extracts. In contrast, no degradation of radioactively labeled 41-kDa superpeptide was observed within 60 min in mutant strains deficient in the vacuolar proteinase yscA (PrA), in which maturation of vacuolar pro-PrB to active PrB is blocked. Accordingly, superpeptide antigenic material was readily detected by immunoblotting cell extracts and enriched in vacuolar preparations of PrA deficient mutant cells. These results indicate that the superpeptide and pro-PrB travel to the vacuole, where the superpeptide is rapidly degraded upon pro-PrB activation to PrB. Using purified vacuoles, rapid degradation of the superpeptide was reconstituted in vitro by addition of either mature PrA or mature PrB. However, the PrA-triggered in vitro degradation of the superpeptide required PrB activity, as this process was inhibited in the presence of the PrB inhibitor chymostatin.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Biogenesis of the yeast vacuole (lysosome). Mutation in the active site of the vacuolar serine proteinase yscB abolishes proteolytic maturation of its 73-kDa precursor to the 41.5-kDa pro-enzyme and a newly detected 41-kDa peptide. 173 47

Brefeldin A (BFA) inhibited in a dose-dependent manner secretion of the cell-surface enzyme acid phosphatase (APase) into the periplasm of Candida albicans and caused intracellular accumulation of enzyme protein. Cells grown in the presence of BFA became more dense, implying that cell-surface growth was also blocked by BFA treatment. The APase that was accumulated intracellularly migrated faster on SDS-PAGE, suggesting less N-linked glycosylation compared with the mature, periplasmic APase produced in the absence of BFA. Pulse-chase experiments and gel-filtration of oligosaccharides released by Endo H treatment suggested that the core-glycosylated precursor form of APase accumulated in the presence of BFA. These results strongly suggested that endoplasmic reticulum (ER)-to-Golgi transport in C. albicans was inhibited by BFA. Aberrant membrane structures were observed in BFA-treated cells. Within 1 h of BFA removal these structures were replaced with rough ER membranes, suggesting that the accumulated membranes were derived from the ER.
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PMID:Brefeldin A blocks an early stage of protein transport in Candida albicans. 191 3

Processing of the measles virus haemagglutinin (H) protein was analysed by the pulse-chase method, immunoprecipitation with an anti-H monoclonal antibody and SDS-polyacrylamide gel electrophoresis, combined with the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or monensin (inhibitors of intracellular processing of secretory proteins) to cultures and digestion of the protein with endoglycosidase H or neuraminidase. The apparent Mr of the H protein was increased from 74K to 78K during the chase period. Addition of either CCCP or monensin to the chase medium inhibited the appearance of the 78K H protein, but not the immunoreactivity of the H protein or dimer formation, suggesting that these two events occur in the rough endoplasmic reticulum. The 74K H protein processed in the presence of CCCP was fully sensitive to endoglycosidase H digestion, whereas the 74K H protein processed in the presence of monensin was partially resistant to endoglycosidase H. In experiments using 3H-labelled sugars, [3H]galactose was incorporated into the 74K H protein in the presence of monensin. Neuraminidase treatment increased the electrophoretic mobility of the 78K H protein to 74K. Only the 78K H protein was detected on the surface of untreated cells, and it was resistant to endoglycosidase H digestion. These data suggest that after galactose addition sialic acid is added to the H protein in the trans-Golgi complex and then the mature 78K H protein is transported to the cell surface.
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PMID:Glycosylation of measles virus haemagglutinin protein in infected cells. 194 Aug 65

Transport of the lysosomal enzyme cathepsin C was studied in Morris hepatoma 7777 cells. Subcellular fractions obtained after isopyenic centrifugation in sucrose gradients of labelled cell homogenates were sequentially extracted by hypo-osmotic shock, Na2CO3 and Triton X-100. Polypeptides related to cathepsin C were immunoprecipitated and analysed by SDS/PAGE and fluorography. At early times after synthesis and for up to 60 min, precursor polypeptides of cathepsin C are distributed in endoplasmic reticulum and Golgi fractions, in membrane-associated form, as Triton X-100 is necessary for their extraction. At 2 h and later after synthesis, intermediate and mature forms of the enzyme can be totally extracted by hypo-osmotic shock from gradient fractions corresponding to the lysosomes of Morris hepatoma 7777 cells.
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PMID:Transient membrane association of the precursors of cathepsin C during their transfer into lysosomes. 203 58

Kopsinine is an indole alkaloid. Oral administration of kopsinine 200 mg/kg once daily for 3 d significantly increased liver microsomal protein, cytochrome P-450, cytochrome b5, NADPH-cytochrome C reductase, aminopyrine demethylase and benzo(a)pyrine hydroxylase activities in mice. kopsinine only induced cytochrome P-450 in rough endoplasmic reticulum of liver. SDS-polyamine gel electrophoresis analysis showed that the protein bands of microsomes from kopsinine treated mice were similar to that induced by phenobarbital in mice. Metyrapon, a specific inhibitor of cytochrome P-450, partially antagonized aminopyrine demethylase activity of microsomes from mice treated with kopsinine. The results suggest that kopsinine yields a pentobarbital-like induction on liver mixed function oxidase in mice. In addition, kopsinine was found to shorten the barbital-induced sleeping time in mice.
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PMID:[Induction by kopsinine of hepatic mixed-function oxidase in mice]. 208 3

Exposure to a sublethal concentration of methyl parathion (MEP) reduced the activity of cytoplasmic malate dehydrogenase, mitochondrial malate dehydrogenase, and lactate dehydrogenase by 30 to 49% in the liver and the skeletal muscle of the freshwater catfish. Clarias batrachus, after 7 days. The activities then began to recover and reached the control levels on the 28th day of MEP exposure. A complete recovery occurred on the 7th day when MEP was withdrawn from the medium after an exposure for 1 week. The withdrawal-dependent recovery in the activities was inhibited partially or completely by actinomycin D and cycloheximide, suggesting de novo synthesis of the enzymes during the recovery period. A conjoint treatment of MEP and triiodothyronine (T3) restored the activities to control levels, indicating T3 protection against the pesticide toxicity. SDS-PAGE of the cytoplasmic fraction of the liver showed some noticeable changes in the protein pattern after an exposure to MEP. Ultrastructural studies on MEP-treated liver cells showed disappearance of the glycogen granules and appearance of numerous smooth endoplasmic reticulum, lysosomal dense bodies, and swollen mitochondria. These changes in the liver are an indication of hepatic toxicity leading toward necrosis.
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PMID:Enzymatic and ultrastructural studies in a freshwater catfish: impact of methyl parathion. 209 89

The low density lipoprotein receptor-related protein (LRP) is a cell surface glycoprotein that binds and transports plasma lipoproteins enriched in apolipoprotein E. It is synthesized in the endoplasmic reticulum as a transmembrane glycosylated precursor that migrates with an apparent molecular mass of about 600 kd on SDS-polyacrylamide gels. After it reaches the Golgi complex, the protein is cleaved to generate two subunits with apparent molecular masses of approximately 515 and 85 kd respectively. The larger NH2-terminal alpha-subunit lacks a membrane-spanning region. It remains attached to the membrane through noncovalent association with the smaller COOH-terminal beta-subunit. Proteolysis occurs at the sequence RHRR, which resembles the sequence RKRR at the proteolytic site in the receptors for insulin and insulin-like growth factor-1 (IGF-1), the only other cell surface receptors known to undergo proteolytic processing. Proteolysis of LRP occurs coincident with the conversion of the N-linked carbohydrates to the mature endoglycosidase H-resistant, neuraminidase-sensitive form. Proteolysis is prevented by brefeldin A, which blocks transport to the Golgi complex. These data raise the possibility that LRP and the receptors for insulin and IGF-1 are processed by a specific endoprotease that recognizes protein with extended basic sequences and resides in the trans-Golgi complex or in post-Golgi vesicles of the constitutive secretory pathway.
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PMID:Proteolytic processing of the 600 kd low density lipoprotein receptor-related protein (LRP) occurs in a trans-Golgi compartment. 211 85

The plasma membrane fraction of chicken osteoclasts was purified utilizing 20% continuous Percoll gradients. Biochemical marker enzyme analysis (ouabain-sensitive Na+,K(+)-ATPase and 5'-nucleotidase) indicated that plasma membrane enrichment was 11.87-fold and 7.25-fold, respectively, and contamination with mitochondria, endoplasmic reticulum, and lysosomes was low as determined by succinic dehydrogenase, NADH dehydrogenase, and N-acetylglucosaminidase activities, respectively. SDS latency of Na+,K(+)-ATPase and 5'-nucleotidase activities of the isolated plasma membranes revealed that 43-50% of vesicles were sealed, with 10-16% in the inside-out orientation, depending on the membrane fraction used. Electron microscopy confirmed the vesicular nature of the plasma membrane fraction. The plasma membrane Ca2(+)-ATPase had a high-affinity (KCa = 0.22 microM; Vmax = 0.16 mumol/mg per min) and a low-affinity (KCa = 148 microM; Vmax = 0.37 mumol/mg per min) component. Calmodulin (0.12 microM) had no effect on Ca2(+)-ATPase activity. However, trifluoperazine (0.1 mM), a calmodulin antagonist, strongly inhibited especially the high-affinity component of the enzyme. Vanadate and lanthanum also caused inhibition. In the presence of CDTA, a potent Ca2+ and Mg2+ chelating agent, high-affinity Ca2(+)-ATPase activity was abolished, indicating that trace Mg2+ was essential for activity. The Ca2(+)-ATPase substrate curve using ATP showed a high-affinity (Km = 12.3 microM; Vmax = 0.022 mumol/mg per min) and a low-affinity (Km = 43.8 microM; Vmax = 0.278 mumol/mg per min) component. These results demonstrate that osteoclasts have a plasma membrane Ca2(+)-ATPase with characteristics similar to the enzyme responsible for active calcium extrusion in other cells.
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PMID:Characterization of a Ca2(+)-ATPase in osteoclast plasma membrane. 214 47

Rat hepatic lipase is a glycoprotein bearing two N-linked oligosaccharide chains. The importance of glycosylation in the secretion of hepatic lipase was studied using freshly isolated rat hepatocytes. Various inhibitors of oligosaccharide synthesis and processing were used at concentrations that selectively interfere with protein glycosylation. Secretion of hepatic lipase activity was abolished by tunicamycin, castanospermine, and N-methyldeoxynojirimycin. No evidence was found by ELISA or Western blotting for secretion of inactive protein. Inhibition of secretion became apparent after a 30-min lag, corresponding to the time of intracellular transport of pre-existing protein. Simultaneously, intracellular hepatic lipase activity ws depleted. Secretion of hepatic lipase protein and activity was not affected by deoxymannojirimycin and swainsonine. Upon SDS-polyacrylamide gel electrophoresis, hepatic lipase secretion by deoxymannojirimycin- or swainsonine-treated cells showed an apparent Mr of 53 kDa and 55 kDa, respectively, which was distinct from hepatic lipase secreted by untreated cells (Mr = 58 kDa). We conclude that glycosylation and subsequent oligosaccharide processing play a permissive role in the secretion of hepatic lipase. As secretion is prevented by the glucosidase inhibitors castanospermine and N-methyldeoxynojirimycin, but not by inhibitors of subsequent oligosaccharide trimming, the removal of glucose residues from the high-mannose oligosaccharide intermediate in the rough endoplasmic reticulum appears the determining step.
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PMID:Secretion of rat hepatic lipase is blocked by inhibition of oligosaccharide processing at the stage of glucosidase I. 215 Apr 12

We have identified collagen-binding proteins in detergent extracts of metabolically labelled bovine aortic endothelial cells (BAEC) by collagen type IV-Sepharose affinity chromatography. The major collagen type IV-binding protein identified by SDS/PAGE had a molecular mass of 48 kDa, which we term the 'collagen-binding 48 kDa protein' (CB48). The pI of CB48 was 8.0-8.3 in a two-dimensional gel system, running non-equilibrium pH gel electrophoresis in the first dimension and SDS/PAGE in the second dimension. Under these conditions CB48 separated into two major (a and b) and one minor isoform (c); a was the most basic of the three isoforms. Two-dimensional chymotryptic peptide maps derived from each individual isoform were virtually identical. The charge differences between the isoforms were due in part to differential H3(32)PO4 incorporation by the protein. CB48 bound to intact collagen type IV and the collagenous region of collagen type IV, but not to the globular NC1 domain. Cell-surface labelling and indirect immunofluorescence experiments localized the bulk of CB48 intracellularly in the endoplasmic reticulum Golgi region, with a minor population of molecules on the cell surface. A specific rabbit polyclonal anti-CB48 serum did not inhibit the attachment or spreading of BAEC to collagen type IV in an 'in vitro' adhesion assay, suggesting that the cell-surface population of CB48 is not involved in BAEC adhesion. We conclude that CB48 is a collagen-binding phosphoprotein that interacts with the collagenous domain of collagen type IV and may be involved in intracellular transport of collagen molecules.
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PMID:A 48 kDa collagen-binding phosphoprotein isolated from bovine aortic endothelial cells interacts with the collagenous domain, but not the globular domain, of collagen type IV. 215 86


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