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:3.2.1.31 (beta-glucuronidase)
7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Biosynthesis of myeloperoxidase (MPO), a myeloid lysosomal hemoprotein critical for the optimal oxygen-dependent microbicidal activity of human neutrophils, is incompletely understood. The primary translation product undergoes cotranslational N-linked glycosylation with subsequent insertion of the Fe-containing prosthetic group into the peptide backbone, thereby converting the enzymatically inactive, heme-free apoproMPO into the peroxidatively active precursor, proMPO. Eventually, proMPO undergoes proteolytic processing into native, lysosomal MPO, with subunits of 59 and 13.5 Kd. We studied three unanswered questions regarding MPO biosynthesis: (1) At what point during MPO biosynthesis is the heme moiety inserted into the apoenzyme? (2) What consequences does heme-insertion have on subsequent processing events? (3) What role does the mannose-6-phosphate receptor (M6PR) system play in the delivery of MPO to the lysosome? Disruption of Golgi by brefeldin A (BFA) produced two major changes in MPO biosynthesis: (1) processing of the 89-Kd precursor to mature MPO was blocked and (2) constitutive secretion of the MPO precursor was inhibited. Inhibition of heme synthesis with succinyl acetone (SA) reduced peroxidase activity and profoundly blocked processing of proMPO to mature MPO. This inhibition of processing was not a generalized effect on all lysosomal enzymes, because the maturation of a non-heme-containing lysosomal enzyme, beta-glucuronidase, was not altered. Electron microscopy showed that, although the normal peroxidase staining of endoplasmic reticulum was absent in SA-treated cells, there were MPO-related peptides in the ER. The role of the M6PR system was assessed by immunoprecipitating fractions obtained from M6PR affinity column chromatography. The 89-Kd proMPO failed to adhere to the M6PR affinity column, whereas the 59-Kd heavy subunit of mature MPO was specifically eluted from the column. We interpret these data to indicate that: (1) processing of proMPO to mature MPO occurs in a post-ER compartment that is itself BFA-sensitive or is distal to a BFA-sensitive compartment and (2) heme insertion into apoproMPO precedes and may be a prerequisite for proteolytic processing to enzymatically active mature MPO. Our analysis of the M6PR system in MPO biosynthesis led to the unanticipated finding that there were phosphomannosyl residues on mature MPO, but none on proMPO. We suggest that the bulk of proMPO at any time is not phosphorylated, but, when generated, the phosphorylated proMPO is quickly processed to the phosphorylated 59-Kd subunit of mature MPO. Thus, if the M6PR is important in the intracellular transport of MPO, it is the phosphorylated mature MPO that is directed to the lysosomal compartment by this system.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Roles of heme insertion and the mannose-6-phosphate receptor in processing of the human myeloid lysosomal enzyme, myeloperoxidase. 133 78

Gastric mucosal PG E2 receptors are the common antisecretory working point of all prostanoid types and may also be involved in "protective" effects. We investigated the subcellular localization of these receptors, as measured by displaceable 3H-PG E2 binding, and identified different organelles by monitoring the activities of specific marker enzymes. Porcine mucosal homogenates were subdivided by differential centrifugation into fractions P1 (1000 x g), P2 (20,000 x g), P3 (300,000 x g) and the supernatant S1. P3 was further fractionated over a series of sucrose step gradients. Mitochondria and lysosomes were enriched in P2 (maximum specific activities of cytochrome-c-oxidase of beta-glucosidase, beta-glucuronidase, beta-galactosidase, respectively). Plasma membranes (alkaline phosphatase, gamma-glutamyl-transpeptidase, 5-nucleotidase), tubulovesicles (H+/K(+)-ATPase) and rough endoplasmic reticulum (NADPH-cytochrome-c-reductase) were mainly found in P3, which also contained the majority of 3H-PG E2 binding sites. In contrast, prostanoid binding was barely detectable in S1. Density fractionation of P3 revealed that 3H-PG E2 binding sites shared a similar sedimentation profile with plasma membranes and tubulovesicular markers. No or negative correlation was found with lysosomes, rough endoplasmic reticulum and mitochondria. We conclude that mucosal PG E2 receptors are predominantly located at the cell surface. This supports the view that prostanoids inhibit gastric secretion through membrane receptors, but gives no clue for intracellular "protective" working points.
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PMID:Subcellular localization of prostaglandin E2 receptors in the gastric mucosa. 134 83

Thionins cause the irreversible inactivation of beta-glucuronidase (GUS) in vitro in a dose- and time-dependent manner. The enzyme is also sensitive to externally added thionins when expressed in the cytoplasmic compartment of tobacco protoplasts transformed with the Gus gene under the 35S promoter of the cauliflower mosaic virus. In protoplasts transformed with the Gus gene fused to a signal peptide, where GUS is translocated into the lumen of the endoplasmic reticulum, the activity is significantly increased both by externally-added and by transiently-expressed thionin, suggesting that it interferes with GUS secretion.
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PMID:Effects of thionins on beta-glucuronidase in vitro and in plant protoplasts. 153 4

A hybrid protein system was used for the study of protein transport in plant cells. A nucleotide sequence (vic) encoding a putative signal peptide of 15 amino acid residues, derived from the published aa sequence of one Pisum vicilin, was synthesized and fused in frame to the gus gene encoding a bacterial cytosolic beta-glucuronidase (GUS). When the hybrid vic::gus gene was expressed in tobacco cells using the cauliflower mosaic virus 35S promoter, the hybrid GUS protein was targeted to, and glycosylated inside the rough endoplasmic reticulum. Glycosylation could be blocked with the antibiotic tunicamycin. The study of transient expression in protoplasts showed that extracellular secretion efficiency was low, which may be due to the nature of the GUS protein.
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PMID:Use of the signal peptide of Pisum vicilin to translocate beta-glucuronidase in Nicotiana tabacum. 155 71

The lysosomal compartment has been examined in activated T-lymphocytes by immunogold electron microscopy and subcellular fractionation. Immunoprecipitation and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of radiolabelled extracts of the T-cells showed that they contained three antigens which are fundamental to normal lysosomal function: a representative lysosomal enzyme beta-glucuronidase, a lysosomal associated membrane protein (LAMP-1), and the cation-independent mannose 6-phosphate lysosomal enzyme targeting receptor (MPR). Immunogold labelling showed that beta-glucuronidase was present in the rough endoplasmic reticulum, the Golgi complex and Golgi-associated vesicles. The enzyme was also found to accumulate in distinct, non-Golgi organelles in which LAMP-1 was co-localized, probably lysosomes. LAMP-1 was also found in tubular elements of the Golgi and in a complex of vesicles clustered near the nucleus where MPR was also present at high density. Fractionation of homogenates from lymphocytes on Percoll gradients revealed that beta-glucuronidase was distributed throughout the low density region containing rough endoplasmic reticulum, Golgi and plasma membrane components, and the high density region which contained only lysosomal activity. Multiple immunogold electron microscopy of the latter fraction showed the presence of homogenous vesicles which had large amounts of beta-glucuronidase within the lumen, LAMP-1 at the periphery and no MPR. These vesicles were probably mature lysosomes, arising from pre-lysosomal organelles enriched for LAMP-1 and MPR.
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PMID:Localization of lysosomal antigens in activated T-lymphocytes. 174 96

Egasyn (esterase-22), a member of the nonspecific carboxylesterase multigene family (E.C. 3.1.1.1), is the endoplasmic reticulum (ER)-targeting protein of beta-glucuronidase. We utilized the polymerase chain reaction (PCR) in the eventual isolation of murine egasyn cDNAs. PCR primers were based upon: (1) partial amino acid sequences derived from egasyn peptides and (2) a conserved active site region shared by carboxylesterases. The amino acid sequence deduced from the PCR product matched that obtained from egasyn protein. This product was utilized as a probe to screen a cDNA library. Two cDNAs whose composite sequence encoded an open reading frame of 562 amino acids were isolated. A message size of 1700-2000 bp was revealed by RNA blot hybridization analysis. S1 nuclease protection analyses detected mRNA in liver, kidney, lung, and submandibular gland, but not in spleen, brain, and testes. Genetic mapping confirmed the location of an egasyn cDNA fragment in cluster 1 of the esterase region on chromosome 8. Transfection of COS cells with the 2022-bp cDNA resulted in the expression of esterase activity, which comigrated on native gels with liver esterase-22. The features of the deduced amino acid sequence of the egasyn cDNA are compared with previously characterized carboxylesterases and with other lumenal ER proteins.
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PMID:Characterization and functional expression of a cDNA encoding egasyn (esterase-22): the endoplasmic reticulum-targeting protein of beta-glucuronidase. 178 3

To study protein secretion in plant cells, we established and evaluated a model system based on transient synthesis of heterologous proteins in tobacco protoplasts. We show that the nonsecretory enzymes phosphinothricin acetyl transferase, neomycin phosphotransferase II, and beta-glucuronidase are secreted when targeted to the lumen of the endoplasmic reticulum by signal peptide-mediated translocation. These data are consistent with the view that secretion can occur independent of active sorting mechanisms by nonspecific migration through the exocytic pathway. However, the rate of secretion differs significantly among these enzymes. Furthermore, the presence of signal sequences was found to be correlated with a reduction of the levels of the encoded gene products. This is the result of post-transcriptional events that limit either synthesis or stability of the proteins in vivo.
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PMID:Protein secretion in plant cells can occur via a default pathway. 196 50

Mouse egasyn cDNA was inserted into expression vector pCDpoly and transfected into mammalian cell lines. Transfected human HepG2 cells, monkey COS-1 cells, and mouse L cells expressed egasyn-esterase catalytic activity. Within COS-1 cells, egasyn was localized to the endoplasmic reticulum. Although individual cells produced large amounts of egasyn, no secretion was observed. No beta-glucuronidase-egasyn complexes were formed in transfected HepG2 or COS-1 cells. However, these complexes were readily detected in transfected L cells. Although the signal for retention of egasyn in the endoplasmic reticulum appears to be species independent, the signal for association with beta-glucuronidase is species restricted.
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PMID:Expression of egasyn-esterase in mammalian cells. Sequestration in the endoplasmic reticulum and complexation with beta-glucuronidase. 200 90

It has been documented that when furnished with an endomembrane signal sequence for the endoplasmic reticulum, beta-glucuronidase (GUS) is N-glycosylated, resulting in the nearly complete loss of enzymatic activity. To enable use of beta-glucuronidase as a reporter protein in secretory and vacuolar targeting studies, one of the two putative N-linked glycosylation sites within the GUS gene was altered by site-directed mutagenesis. The second N-linked glycosylation site was not altered because sequence analysis of nucleotide sequences around the second putative glycosylation site revealed that the published sequence was incorrect, and that no such site existed.
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PMID:Manipulation of beta-glucuronidase for use as a reporter in vacuolar targeting studies. 210 75

The degradative activity of lymphocytes plays by important role in a number of essential immune functions. In the present study we have examined how the activation of resting lymphocytes, by the mitogen concanavalin A (Con A), affects three major components of the lysosomal compartment: the lysosomal enzyme beta-glucuronidase (Gus); an integral lysosomal membrane protein (LAMP-1); and the mannose 6-phosphate receptor (MPR) which directs lymphocyte enzyme transport. Resting T cells were found to contain only very low levels of these proteins, but they were actively synthesized by, and far more abundant in, stimulated lymphoblasts. Although the lysosomal antigens did not have a distinct cytoplasmic localization in the resting lymphocytes, in the activated T lymphoblasts they were present in several highly developed intracellular structures, including the rough endoplasmic reticulum and the Golgi complex. Furthermore, in these latter cells Gus was also found to be accumulated within the lumen of large vesicles which we characterized as lysosomes by the presence of LAMP-1 at the periphery and by the absence of MPR. Subcellular fractionation confirmed that these organelles were present in the activated lymphocytes only, and not in the resting T cells. Our results demonstrate that lymphocyte activation is accompanied by the synthesis of the enzymic and structural components of the lysosomal compartment which are sorted and assembled into distinct organelles in the activated cell.
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PMID:The activation of resting lymphocytes is accompanied by the biogenesis of lysosomal organelles. 217 61


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