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)

A full-length cDNA encoding a calreticulin-like protein was isolated by immune-screening a germinating castor bean endosperm cDNA library with antisera raised to the total lumenal fraction of purified plant endoplasmic reticulum. The calcium-binding properties of the recombinant protein were characterized and shown to be essentially identical to those reported for the mammalian calreticulin. Calcium overlays and immune blot analysis confirmed the endoplasmic lumenal identity of this reticuloplasmin. Probing protein blots of endoplasmic reticulum subfractions with radio-iodinated calreticulin showed specific associations with various polypeptides including one identified as the abundant reticuloplasmin protein disulfide isomerase. Characterization of the corresponding genomic clones revealed that calreticulin is encoded by a single gene of 3 kb in castor. The full genomic sequence reveals the presence of 12 introns, 12 translated exons, and one exon containing the last three amino acids of the translated sequence and the 3'-untranslated region of the gene. Northern blot analysis of RNA isolated from various organ tissues showed a basal constitutive level of expression throughout the plant, but more abundant mRNA being detected in tissues active in secretion. This was confirmed by analysis of transgenic tobacco plants containing 1.8 kb of 5'-untranslated genomic sequence fused to the beta-glucuronidase reporter gene (GUS) showed a more localized pattern of expression. Activity being localized to the vasculature (phloem, root hairs and root tip) in vegetative tissue, and being strongly expressed in the floral organs including the developing and germinating seed.
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PMID:Cloning and characterization of the calreticulin gene from Ricinus communis L. 929 Jun 42

Activation of the cellular immune system and subsequent lysis of vector-transduced cells by adenovirus- or transgene-specific cytotoxic T lymphocytes have been shown to limit transgene expression in animal models. The adenovirus gp19K gene product associates with major histocompatibility complex class I proteins and prevents their maturation by sequestering them in the endoplasmic reticulum. gp19K has been shown to block the ability of adenovirus-specific cytotoxic T lymphocytes to recognize virus-infected cells in vitro. To determine if gp19K expression in an adenovirus vector would increase transgene persistence, a vector that replaces the E1 region of adenovirus with an expression cassette encoding both gp19K and beta-glucuronidase was constructed. This vector produced high levels of functional gp19K in infected cells. RNase protection analysis revealed efficient expression of the gp19K gene in the mouse lung. Enhanced persistence and increased beta-glucuronidase activity were observed in the lung and liver following delivery of the gp19K-expressing adenovirus vector in B10.HTG mice but not in BALB/c mice. Since gp19K binds to both class I alleles on B10.HTG mice but only one allele on BALB/c mice, these results suggest that the major histocompatibility complex class I haplotype of mice is important in determining the effectiveness of gp19K in vivo. Since gp19K has previously been shown to interact with every human major histocompatibility complex class I allele tested, the inclusion of gp19K in gene therapy vectors may increase vector persistence in human gene therapy trials.
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PMID:Expression of gp19K increases the persistence of transgene expression from an adenovirus vector in the mouse lung and liver. 931 44

Signal sequences and endoplasmic reticulum (ER) retention signals are known to play central roles in targeting and translocation in the secretory pathway, but molecular aspects about their involvement are poorly understood. We tested the effectiveness of deduced signal sequences from various genes (hydroxyproline-rich glycoprotein [HRGP] from Phaseolus vulgaris; Serpin from Manduca sexta) to direct a modified beta-glucuronidase (GUS) protein into the secretory pathway in transgenic tobacco (Nicotiana tabacum L.). The reporter protein was not secreted to the cell wall/extracellular space as monitored using extracellular fluid analysis (low- or high-ionic-strength conditions) but occurred in membranes with a density of 1.16 to 1.20 g/mL. Membrane-bound GUS equilibrated with the plasma membrane (PM) and the ER on linear sucrose gradients with or without ethylenediaminetetraacetic acid, suggesting that GUS associates with the ER and the PM. Confocal microscopy of fixed cultured cells prepared from GUS control and HRGP signal peptide (SP)-GUS-expressing plants suggested only cytosolic localization in GUS-expressing plants but substantial peripheral localization in HRGP SP-GUS plants, which is consistent with GUS being associated with the PM. Aqueous two-phase partitioning of microsomal membranes from HRGP SP-GUS and Serpin SP-GUS transgenic leaves also indicated that GUS activity was enriched in the ER and the PM. These observations, together with hydrophobic moment plot analysis, suggest that properties of the SP-GUS protein result in its retention in the secretory pathway and PM.
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PMID:Gene fusions of signal sequences with a modified beta-glucuronidase gene results in retention of the beta-glucuronidase protein in the secretory pathway/plasma membrane. 939 Apr 28

The foregut, stomach, caecum, midgut, and rectum of the digestive tract of Nautilus pompilius L.were investigated with ultrastructural and enzyme-cytological methods. Three different cell types were identified within the lamina epithelialis mucosae: main cells, goblet cells, and cells with secretory granules. The main cell type is the epithelial cell with microvilli, a basal nucleus surrounded by dictyosomes, rough endoplasmic reticulum, mitochondria, and electron-dense granules identified as lysosomes in the apical part of the cell. In the caecum this cell type contains endosymbiotic bacteria. The presence of endocytotic vesicles and the storage of lipids in the caecum indicate that this organ is involved in the process of absorption. In the caecum and the longitudinal groove of the rectum the main cells are, in addition, ciliated, facilitating the transport of food particles and faeces. Two types of goblet cells are found in all organs except in the stomach, forming a gliding path for food particles and protecting the epithelium. In the foregut and rectum, cells with electron-dense granules were recognized as the third type. The conspicuous secretory cells of the rectum represent a delimited rectal gland; its possible biological function is discussed. The tunica muscularis in all organs of the digestive tract consists of obliquely striated muscle cells innervated by axons containing transparent, osmiophilic and dense-cored vesicles. Positive reactions for acid and alkaline phosphatase, monoamine oxidase, beta-glucuronidase, and trypsin- and chymotrypsin-like enzymes are localized in the lamina epithelialis mucosae.
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PMID:Cytological and enzyme-histochemical investigations on the digestive organs of Nautilus pompilius (Cephalopoda, Tetrabranchiata). 966 55

Cinnamyl alcohol dehydrogenase 2 (CAD 2) localization and the cell-specific activity of the eucalyptus CAD 2 promoter were investigated by CAD 2 immunogold localization and promoter beta-glucuronidase (GUS) histochemistry in apical and mature parts of stable transformed poplar (Populus tremula x P. alba) stems. Both CAD 2 protein and GUS activity were found to be confined in the same types of cells in the shoot apices, particularly in the determined meristematic cells in leaf axils and shell zones, procambium and developing tracheids. Within mature stems, CAD 2 and GUS were also identified in cambium and in fully or partially lignified cells derived from it (young xylem, developing phloem fibres, chambered parenchyma cells around phloem). Additionally, GUS activity was found in the scale leaves of apical shoot buds and in the roots (namely in the procambium, cambium, phellogen, young xylem, pericycle) of transformed plants. By employing immunogold cytochemistry, CAD 2 was shown to be localized in the cytoplasm within cambial, ray and young xylem cells in stems, the gold particles being randomly attached to endoplasmic reticulum and Golgi-derived vesicles. These results support a crucial role for CAD 2 in lignification and indicate a new role for this enzyme in branching events within the shoot apex and during lateral root formation.
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PMID:Immunolocalization of cinnamyl alcohol dehydrogenase 2 (CAD 2) indicates a good correlation with cell-specific activity of CAD 2 promoter in transgenic poplar shoots. 968 67

Spermatozoa released from the seminiferous tubules are terminally differentiated cells with no known synthetic activity. Their components are synthesized in the spermatogenic cells during spermatogenesis. In this study, we report the characterization and immunolocalization of beta-glucuronidase in mouse testicular germ cells and spermatozoa. The enzyme is an exoglycohydrolase with dual localization, being present in lysosomes and endoplasmic reticulum of several mouse and rat tissues. The purified germ cell preparations (spermatocytes, round spermatids, and condensed/elongated spermatids) when assayed for beta-glucuronidase activity showed that the spermatocytes contained five times more enzyme activity per cell than the spermatids. Polyacrylamide gel electrophoresis, carried out under native and denaturing conditions, demonstrated that the germ cells express only the lysosomal form of the enzyme (pI 5.5-6.0) with a subunit molecular mass of 74 kDa. Immunocytochemical studies revealed a positive reaction in the Golgi membranes, Golgi-associated vesicles, and lysosomes of late spermatocytes (pachytene spermatocytes) and a stage-specific localization during spermiogenesis. The forming or formed acrosome of the elongated spermatids (stages 9-16) and epididymal spermatozoa was highly immunopositive. Comparison of immunoprecipitation curves and kinetic properties of the enzyme present in spermatocytes and spermatozoa revealed no major differences. Taken together, our results demonstrate that beta-glucuronidase activities present in the lysosomes of spermatocytes and the sperm acrosome are kinetically and immunologically similar.
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PMID:Characterization and immunolocalization of beta-D-glucuronidase in mouse testicular germ cells and spermatozoa. 1004 47

Accumulated evidence links an important signal involved in glucose-stimulated insulin release to the activation of the islet lysosomal glycogenolytic enzyme acid glucan-1,4-alpha-glucosidase. We have analyzed the function of the lysosomal system/lysosomal enzyme activities in pancreatic islets of young (6-8 weeks), spontaneously diabetic, GK (Goto-Kakizaki) rats and Wistar control rats in relation to glucose-induced insulin release. The insulin secretory response to glucose was markedly impaired in the GK rat, but was restored by the adenylate cyclase activator forskolin. Islet activities of classical lysosomal enzymes, e.g.. acid phosphatase, N-acetyl-beta-D-glucosaminidase, beta-glucuronidase, and cathepsin D, were reduced by 20-35% in the GK rat compared with those in Wistar controls. In contrast, the activities of the lysosomal alpha-glucosidehydrolases, i.e.. acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase, were increased by 40-50%. Neutral alpha-glucosidase (endoplasmic reticulum) was unaffected. Comparative analysis of liver tissue showed that lysosomal enzyme activities were of the same magnitude in GK and Wistar rats. Notably, in Wistar rats, the activities of acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase were approximately 15-fold higher in islets than in liver. Other lysosomal enzymes did not display such a difference. Normalization of glycemia in GK rats by phlorizin administered for 9 days did not influence either the lysosomal alpha-glucosidehydrolase activities or other lysosomal enzyme activities in GK islets. Finally, the pseudotetrasaccharide acarbose, which accumulates in the lysosomal system, inhibited acid glucan-1,4-alpha-glucosidase activity in parallel with its inhibitory action on glucose-induced insulin release in intact Wistar islets, whereas no effect was recorded for either parameter in intact GK islets. In contrast, acarbose inhibited the enzyme activity equally in islet homogenates from both GK and Wistar rats, showing that the catalytic activity of the enzyme itself in disrupted cells was unaffected. We propose that dysfunction of the islet lysosomal/vacuolar system is an important defect impairing the transduction mechanisms for glucose-induced insulin release in the GK rat.
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PMID:Dysfunction of the islet lysosomal system conveys impairment of glucose-induced insulin release in the diabetic GK rat. 1038 96

Lysosomal beta-glucuronidase shows a dual localization in mouse liver, where a significant fraction is retained in the endoplasmic reticulum (ER) by interaction with an ER-resident carboxyl esterase called egasyn. This interaction of mouse egasyn (mEg) with murine beta-glucuronidase (mGUSB) involves binding of the C-terminal 8 residues of the mGUSB to the carboxylesterase active site of the mEg. We isolated the recombinant human homologue of the mouse egasyn cDNA and found that it too binds human beta-glucuronidase (hGUSB). However, the binding appears not to involve the active site of the human egasyn (hEg) and does not involve the C-terminal 18 amino acids of hGUSB. The full-length cDNA encoding hEg was isolated from a human liver cDNA library using full-length mEg cDNA as a probe. The 1941-bp cDNA differs by only a few bases from two previously reported cDNAs for human liver carboxylesterase, allowing the anti-human carboxylesterase antiserum to be used for immunoprecipitation of human egasyn. The cDNA expressed bis-p-nitrophenyl phosphate (BPNP)-inhibitable esterase activity in COS cells. When expressed in COS cells, it is localized to the ER. The intracellular hEg coimmunoprecipitated with full-length hGUSB and with a truncated hGUSB missing the C-terminal 18-amino-acid residue when extracts of COS cells expressing both proteins were treated with anti-hGUSB antibody. It did not coimmunoprecipitate with mGUSB from extracts of coexpressing COS cells. Unlike mEg, hEg was not released from the hEg-GUSB complex with BPNP. Thus, hEg resembles mEg in that it binds hGUSB. However, it differs from mEg in that (i) it does not appear to use the esterase active site for binding since treatment with BPNP did not release hEg from hGUSB and (ii) it does not use the C terminus of GUSB for binding, since a C-terminal truncated hGUSB (the C-terminal 18 amino acids are removed) bound as well as nontruncated hGUSB. Evidence is presented that an internal segment of 51 amino acids between 228 and 279 residues contributes to binding of hGUSB by hEg.
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PMID:Human egasyn binds beta-glucuronidase but neither the esterase active site of egasyn nor the C terminus of beta-glucuronidase is involved in their interaction. 1056 16

The physiological function of microsomal beta-glucuronidase is unclear. Substrates may be either glucuronides produced in the lumen of endoplasmic reticulum (ER) or those taken up by hepatocytes. In the latter case, efficient inward transport of glucuronides at the plasma membrane and the ER membrane would be required. Therefore, the potential role of beta-glucuronidase in ER was investigated. Isolated mouse hepatocytes and mouse and rat liver microsomal vesicles were used in the experiments. Selective permeabilization of the plasma membrane of isolated hepatocytes with saponin or digitonin resulted in an almost 4-fold elevation in the rate of beta-nitrophenol glucuronide hydrolysis, while the permeabilization of plasma membrane plus ER membrane by Triton X-100 caused a further 2-fold elevation. In microsomal vesicles, the p-nitrophenol glucuronide or phenolphthalein glucuronide beta-glucuronidase activity showed about 50% latency as revealed by alamethicin or Triton X-100 treatment. A light-scattering study indicated that the microsomes are relatively impermeable to both glucuronides and to glucuronate. On the basis of our results, the role of liver microsomal beta-glucuronidase in the deconjugation of glucuronides taken up by the liver seems unlikely. Hydrolysis of the glucuronides produced in the ER lumen may play a role in substrate supply for ascorbate synthesis or in "proofreading" of glucuronidation.
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PMID:Beta-glucuronidase latency in isolated murine hepatocytes. 1071 38

Geranylgeranyl diphosphate (GGPP) is the precursor for the biosynthesis of gibberellins, carotenoids, chlorophylls, isoprenoid quinones, and geranylgeranylated proteins in plants. There is a small gene family for GGPP synthases encoding five isozymes and one related protein in Arabidopsis, and all homologs have a putative localization signal to translocate into specific subcellular compartments. Using a synthetic green fluorescent protein (sGFP), we studied the subcellular localization of these GGPP synthases. When these fusion proteins were expressed by the cauliflower mosaic virus 35S promoter in Arabidopsis, GGPS1-sGFP and GGPS3-sGFP proteins were translocated into the chloroplast, GGPS2-sGFP and GGPS4-sGFP proteins were localized in the endoplasmic reticulum, and the GGPS6-sGFP protein was localized in the mitochondria. Both GGPS1 and GGPS3 proteins synthesized in vitro were taken up into isolated intact pea chloroplasts and processed to the mature form. RNA-blot and promoter-beta-glucuronidase (GUS) analysis showed that these GGPP synthases genes are organ-specifically expressed in Arabidopsis. GGR and GGPS1 were ubiquitously expressed, while GGPS2, GGPS3, and GGPS4 were expressed specifically in the flower, root, and flower, respectively. These results suggest that each GGPP synthase gene is expressed in different tissues during plant development and GGPP is synthesized by the organelles themselves rather than being transported into the organelles. Therefore, we predict there will be specific pathways of GGPP production in each organelle.
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PMID:Five geranylgeranyl diphosphate synthases expressed in different organs are localized into three subcellular compartments in Arabidopsis. 1075


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