Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.52 (PNGase F)
 1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

CHIP28 occurs naturally in glycosylated and nonglycosylated forms. The purpose of this study was to determine the role of glycosylation in CHIP28 structure and function. A new purification procedure based on phenylboronic acid-agarose (PBA) affinity chromatography was developed to isolate CHIP28. In purified native CHIP28 from erythrocytes, approximately 50% of CHIP28 molecules were glycosylated; each mole of glycosylated CHIP28 contained 5.4 kDa of monosaccharides consisting of 2 mol of Fuc, 8 mol of Gal, 1 mol of GalN, 13 mol of GlcN, 3 mol of Man, and 1 mol of Neu5Ac. The proportions of each monosaccharide and the sensitivity to endo-beta-galactosidase indicated that CHIP28 contained polylactosaminyl oligosaccharides. Glycosylated and nonglycosylated CHIP28 remained tightly associated when solubilized in octyl beta-D-glucoside (OG) and could not be separated by conventional chromatographic procedures. To remove the sugar moiety, CHIP28 was enzymatically deglycosylated by PNGase F and purified by Q-Sepharose anion-exchange and Erythrina cristagalli lectin chromatography. High-performance size-exclusion chromatography revealed that native CHIP28 eluted as an apparent dimer, whereas deglycosylated CHIP28 eluted as an apparent monomer. In reconstituted proteoliposomes, deglycosylated CHIP28 had a single channel water permeability (pf) of 3.1 x 10(-14) cm3/s (10 degrees C), not different from that of 3.2 x 10(-14) cm3/s for native CHIP28. Circular dichroism of native and deglycosylated CHIP28 in OG revealed 45% and 48% alpha-helix, respectively; intrinsic tryptophan fluorescence showed no effects of glycosylation on tryptophan environment. Freeze-fracture electron microscopy with rotary shadowing indicated that native and deglycosylated CHIP28 assembled as tetramers in reconstituted proteoliposomes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and structure-function analysis of native, PNGase F-treated, and endo-beta-galactosidase-treated CHIP28 water channels. 753 4

Acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) is the lysosomal enzyme catalyzing the hydrolysis of ceramide to sphingosine and free fatty acid. Its inherited deficiency causes ceramide accumulation in Farber's disease. The enzyme was purified to apparent homogeneity from human urine by sequential chromatography on octyl-Sepharose, concanavalin A-Sepharose, blue-Sepharose, and DEAE-cellulose. The final preparation, which was enriched approximately 4450-fold over the starting material, resulted in a polypeptide of approximately 50 kDa and could be reduced into two subunits of approximately 13 (alpha) and approximately 40 (beta) kDa. Treatment of the purified enzyme with endoglycosidase H or peptido-N-glycanase F reduced the molecular mass of the beta subunit to approximately 30-35 and approximately 27 kDa, respectively. In contrast, the molecular mass of the alpha subunit was unchanged. The purified enzyme had an apparent Km of 149 microM and a Vmax of 136 nmol/mg/h using N-lauroylsphingosine as substrate. Polyclonal antibodies were raised against the purified urinary enzyme and used to investigate the biosynthesis of acid ceramidase. Immunoprecipitation studies on metabolically labeled skin fibroblasts indicated that both subunits arose from a single precursor of approximately 55 kDa. A minor portion of newly synthesized acid ceramidase was secreted into the medium as a monomeric 47-kDa protein, indicating that generation of the mature heterodimeric enzyme occurred in endosomal and/or lysosomal compartments.
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PMID:Purification, characterization, and biosynthesis of human acid ceramidase. 774 40

Mouse teratocarcinoma F9 cells were induced to primitive endoderm differentiation with retinoic acid, and poly-N-acetyllactosamine-containing surface glycoproteins were identified by radiolabelling endo-beta-galactosidase-cleavable glycans with galactosyltransferase and radiolabelled UDP-galactose. One major radiolabelled band with an apparent size of 250-500 kDa was identified which differed from the known poly-N-acetyllactosamine-containing glycoproteins laminin, fibronectin, lysosome-associated membrane protein (LAMP)-1 and LAMP-2. This acidic glycoprotein, resistant to glycosaminoglycan-degrading enzymes and proteases, was purified by extraction and phase partition with Triton X-114, octyl Sepharose and Helix pomatia lectin chromatography. The purified glycoprotein could be digested by endo-beta-galactosidase and glycopeptide N-glycosidase F to an apparent size of 160-240 kDa. During retinoic-acid-induced differentiation into primitive endoderm cells, the glycoprotein showed a several-fold increase and a broadening to an apparent size of 200- > 700 kDa. The glycoprotein was no longer detected in retinoic-acid and dibutyryl-cAMP-treated cells which had undergone further differentiation to parietal endoderm cells, nor in the permanently differentiated parietal endoderm line F9-AC. The results suggest that the glycoprotein is a major carrier of poly-N-acetyllactosamine chains on differentiating teratocarcinoma F9 cells, and that its expression as revealed by the poly-N-acetyllactosamine labelling method is regulated by the stage of cellular differentiation.
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PMID:Identification of a major poly-N-acetyllactosamine-containing cell-surface glycoprotein of mouse teratocarcinoma cells. Appearance on cells induced to primitive endoderm but not parietal endoderm differentiation. 812 95

The MUC1 glycoprotein, epitectin, a component of the human bladder epithelium, was purified from human urine. Sedimentation equilibrium analysis and gel filtration using polysaccharide or protein standards revealed a polydisperse preparation with molecular weights ranging from about 0.9 to 1.3 x 10(6). This suggests that in the native state epitectin exists as aggregates of three or four monomer units of 350-400 kDa. Epitectin was found to have significant affinity to hexyl-, octyl- or phenyl agarose indicating that hydrophobic interactions and possibly carbohydrate-carbohydrate interactions may be responsible for the self-association. Chemical and enzymic deglycosylation of [125I]-labeled urine epitectin and metabolically labeled H.Ep.2 epitectin resulted in extremely polydisperse products. The buoyant densities of epitectin purified from urine and H.Ep.2 cells were found to be 1.39-1.40 g ml(-1), suggesting that the total carbohydrate content of these preparations is not significantly different. The O-linked saccharides of epitectin were fractionated by HPLC and analyzed by permethylation and FAB-MS. The neutral saccharides from both sources contain three common structures, namely Gal1 --> 3GalNAc, GlcNAc1 --> 6 (Gal1 --> 3) GalNAc and Gal1 --> 4GlcNAc --> 6 (Gal1 --> 3)GalNAc. The sialic acid of urine epitectin consisted entirely of N-acetylneuraminic acid. The two sources of epitectin, in vitro labeled on sialic acid, were found to have the same sialyl oligosaccharides but in different proportions. Metabolic labeling and N-glycanase susceptibility experiments firmly established the presence of N-linked saccharides in epitectin as minor components. The remarkable similarities in the total carbohydrate content, the carbohydrate composition and structures of saccharides between epitectin from urine, a non-malignant source, and H.Ep.2 cells is surprising in view of the prevailing view that MUC1 glycoproteins of cancer cells are underglycosylated compared to those produced by non-malignant cells.
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PMID:Purification and characterization of the MUC1 mucin-type glycoprotein, epitectin, from human urine: structures of the major oligosaccharide alditols. 953 Sep 55

We studied the interaction of n-octyl-beta-d-glucopyranoside-solubilized VIP receptors (VIPR) with wheat germ agglutinin and found that the addition of the lectin to the detergent extract led to the formation of aggregates that could be pelleted by high speed centrifugation. Resuspension of the pellet in the presence of the competing trisaccharide, N,N', N"-triacetylchitotriose (TAC), dissociated the lectin from the complex without altering the precipitability of VIPR. The final pellet (referred to as TAC pellet) contained an average of 12% of total protein and 96% of total VIP binding activity with a typical rank order of potency for VIP-related peptides. Lipid analysis and electron microscopic examination indicated that the precipitated material was composed of lipid vesicles. VIPR molecules were shown to be integrally inserted in the liposomes because they could not be dissociated from the vesicles at pH 11 or with high salt concentration. By comparing the liposome-associated VIP binding activity in the presence and absence of detergent and by showing accessibility of VIPR to PNGase F, it was concluded that VIP binding sites were not simply trapped within the reconstituted vesicles but likely exposed at the external surface of the liposomes.
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PMID:Functional reconstitution of membrane glycoproteins into lipid vesicles using lectin precipitation. Application to the VIP receptor. 967 72