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:3.5.1.52 (PNGase F)
1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Affinity-purified human testosterone-binding globulin (hTeBG) is composed of two subunits [mol wt (Mr), 52,200 and 48,600], as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretic transfer, and immunochemical localization with a monoclonal antibody raised against rat androgen-binding protein. Fluorography of SDS-PAGE gels on which photoaffinity-labeled hTeBG was analyzed yielded essentially identical results. Enzymatic deglycosylation of hTeBG with neuraminidase to remove sialic acid led to the production of two subunits of 50,800 and 47,300 Mr when assessed by SDS-PAGE. Treatment of hTeBG with an optimal concentration of N-glycanase to remove Asn-linked oligosaccharides produced a single subunit of 44,100 Mr. When hTeBG was treated with neuraminidase and O-glycanase to remove O-linked oligosaccharides, three subunits were seen, two of which had Mr not clearly different from those obtained with neuraminidase treatment alone plus a subunit of 40,900 Mr. Treatment of hTeBG with a combination of all three enzymes produced a single subunit of 42,900 Mr. Chemical deglycosylation with trifluoromethane-sulfonic acid produced a single subunit with a Mr identical to that produced by treatment with all three enzymes. We concluded that this is the Mr of completely deglycosylated hTeBG. Based on this Mr, carbohydrates contribute 18% and 12% to the apparent Mr of the heavy and light subunits of hTeBG, respectively. Two-dimensional PAGE analysis of hTeBG with its oligosaccharides intact indicated that the heavy subunit was composed of seven isoelectric variants with pI values of 5.87-6.55, while the light subunit was composed of four charge variants with pI values of 6.14-6.55. Treatment of hTeBG with the enzymes resulted in a shift in the pH values to a more basic pH range, indicating that carbohydrate removal also removed charged species from the protein. The greatest cathodal shift occurred when hTeBG was treated with a combination of the three enzymes (pI 7.33-7.77) or when it was chemically deglycosylated (pI 6.37-7.02). Despite the apparent removal of all carbohydrates, the single subunit was still composed of multiple isoforms. This finding suggests that other charged species remain on the hTeBG molecule.
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PMID:Human testosterone-binding globulin is a dimer composed of two identical protomers that are differentially glycosylated. 272 45

In the course of characterizing polyclonal antibodies to beta nerve growth factor (NGF) on immunoblot replicas of sodium dodecyl sulfate gels, we observed a protein (designated C protein) migrating as two bands (14.0 and 13.5 kDa) that copurifies with NGF and reacts strongly with its antibodies. The molecule is detectable in the 7 S, beta, and 2.5 S forms of NGF, accounting in the latter two for approximately 2% of total protein. The C protein can be separated from the A and B chains of beta-NGF on acetic acid-urea gels and on two-dimensional gels but not by isoelectric focusing alone. The molecule has been isolated to near purity on reversed-phase high performance liquid chromatography. Amino acid analyses and sequencing through 49 Edman cycles revealed that the protein preparation is composed of the intact and desoctapeptide (des-(1-8] polypeptide chains and suggested a glycosylation site at Asn-45. Following digestion with N-glycanase, the chains migrated on sodium dodecyl sulfate gels identically with the A and B chains of beta-NGF. Although this was accompanied by some degree of proteolytic degradation, the presence of glucosamine (approximately 4 mol/mol of single chain) was confirmed in acid hydrolysates on the amino acid analyzer. No amino sugars were detected in hydrolysates of the A chain nor was galactosamine recovered in either preparation. Glycosylated NGF promotes neuronal growth and survival in a manner indistinguishable from native 2.5 S NGF when tested in the chick sensory ganglion assay and with rat postnatal sympathetic neurons in a dissociated culture cell survival assay or in a compartmentalized culture growth assay. These studies reveal that NGF can be modified by glycosylation in a manner that does not reduce its biological activity.
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PMID:Isolation and characterization of a glycosylated form of beta nerve growth factor in mouse submandibular glands. 274 57

The biosynthesis and turnover of lipoprotein lipase (LPL) have been investigated in adipose 3T3-F442A cells labeled with [35S]methionine. Pulse-chase experiments, endo-beta-N-acetylglucosaminidase H treatment, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis have indicated that LPL is synthesized in the endoplasmic reticulum as a glycoprotein of Mr = 55,500 bearing two N-oligosaccharide side chains of the high mannose-type. This precursor form of LPL is transported within 10 min to the Golgi apparatus, and this event is accompanied by the formation of a mature species of Mr = 58,000. Treatment of the Mr = 58,000 species with glycopeptidase F yielded a Mr = 51,000 protein similar to that observed after treatment of the Mr = 55,500 precursor form or after inhibition of N-glycosylation in tunicamycin-treated cells. The precursor form of LPL of Mr = 55,500 does not accumulate in the cells since, after a labeling period of 2 h, only the Mr = 58,000 species is detected. It is shown that only 20% of the newly synthesized molecules of Mr = 58,000 are constitutively secreted, whereas 80% are degraded, most likely in lysosomes, as indicated by the inhibitory effect of leupeptin upon the degradation process. Under heparin stimulation, quantitative secretion of the mature form of LPL takes place whereas the intracellular degradation is arrested. Heparin is able to mobilize intracellular LPL without changing the rate of LPL export from the endoplasmic reticulum to the cell surface. Sucrose gradient centrifugation of the material from intracellular cisternae shows that the Mr = 55,500 precursor form is present as a monomer (s = 4.1 S), whereas the Mr = 58,000 mature form is present as a homodimer (s = 6.8 S) to which LPL activity is associated. The results are interpreted as LPL being transiently stored under a dimeric form before its degradation. A sorting process of LPL in the Golgi apparatus, followed by its entry either mainly in a regulated pathway or in a constitutive pathway, is proposed.
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PMID:Biosynthesis of lipoprotein lipase in cultured mouse adipocytes. II. Processing, subunit assembly, and intracellular transport. 275 12

Lysosomal enzymes from Dictyostelium discoideum contain unusual sulfated N-linked oligosaccharides, whose synthesis has been well studied in vivo. However, little is known about the properties of the pertinent sulfotransferases. To study these transferases, we have prepared a cell-free system which transfers 35SO4 from 3'-phosphoadenosine 5'-phosphosulfate to either endogenous or exogenous acceptors. We found that the 35SO4 was released from macromolecules by protein N-glycanase F to yield a mixture of anionic oligosaccharides with 1-6 negative charges. Some of the labeled molecules contained acid-stable methyl phosphodiesters but none contained phosphomoesters or acid-labile diesters. The sulfate was found in molecules with the acid stability characteristic of esters of primary alcohols. In all these ways, the products resembled those generated in vivo. We also demonstrated that a membrane-associated form of beta-hexosaminidase and the precursor of alpha-mannosidase were among the products. In addition, glycoproteins prepared from a sulfation-deficient mutant strain could act as exogenous acceptors in permeabilized vesicles.
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PMID:Characteristics of the sulfation of N-linked oligosaccharides in vesicles from Dictyostelium discoideum: in vitro sulfation of lysosomal enzymes. 277 65

A high affinity (1-2 nM) radioiodinated, photoaffinity probe for the dopamine transporter, 1-(2-[bis-(4-fluorophenyl)-methoxylethyl)-4-(2-[4-azido-3- [125I]iodophenyl]ethyl)piperazine ([125I]FAPP) has been synthesized. Upon photolysis, [125I]FAPP incorporates into a striatal polypeptide of apparent Mr 62,000 as visualized by autoradiography following sodium dodecyl sulfate-PAGE. Photoincorporation of [125I]FAPP into the Mr 62,000 polypeptide was stereoselectively inhibited by various dopamine uptake agents with a potency order typical of the dopamine transporter. The glycoprotein nature of the apparent Mr 62,000 polypeptide was assessed following specific exo- and endoglycosidase treatment. The dopamine transporter appears to be associated with complex-type oligosaccharides as indexed by its susceptibility to neuraminidase but not alpha-mannosidase digestion. Complete N-linked deglycosylation of the neuronal dopamine transporter with the endoglycosidase, glycopeptidase-F, increased the electrophoretic mobility of the 62 kDa polypeptide to apparent Mr 48,000. [125I]FAPP should prove to be a useful probe for the molecular characterization of the dopamine uptake site in various tissues and under certain pathophysiological states.
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PMID:Photoaffinity labeling of the mammalian dopamine transporter. 280 48

The ligand-binding subunit of the porcine striatal dopamine D2 receptor was identified by photoaffinity labeling with [125I]N-azidophenethylspiperone ([125I]NAPS). Upon photolysis, [125I]NAPS covalently incorporated into a broad band of apparent Mr congruent 140,000 with an appropriate pharmacological profile for D2 receptors as assessed by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Smaller subunits of apparent Mr congruent 94,000 and 34,000 were specifically labeled by [125I]NAPS with an appropriate D2 receptor profile and were similar to the major ligand-binding subunits of photoaffinity-labeled canine striatal D2 receptors. Photoaffinity labeling in the absence or presence of multiple protease inhibitors did not alter the migration pattern of the Mr congruent to 140,000/94,000 subunits upon denaturing electrophoresis in either the absence or presence of thiol-reducing/alkylating reagents. In order to investigate the possible basis for the existence of these high molecular weight forms of the D2 receptor, we assessed the carbohydrate nature of photolabeled D2 ligand-binding subunits by the use of lectin affinity chromatography and specific exo- and endoglycosidase treatments. Both photoaffinity-labeled D2 receptor proteins from porcine striatum (Mr congruent to 140,000 and 94,000) were glycoproteins as indexed by their absorption and specific elution from wheat germ agglutinin lectin resins. The exoglycosidase neuraminidase altered the electrophoretic mobility of both the Mr congruent to 140,000 and 94,000 labeled subunits to a single band of apparent Mr congruent to 51,000. Prior removal of sialic acid residues did not alter the reversible binding characteristics of [3H]spiperone to D2 receptors. Complete removal of receptor-associated N-linked carbohydrate by the endoglycosidase glycopeptidase F (peptide-N4[N-acetyl-beta-glucosaminyl]asparagine amidase) produced a further increase in the mobility of the Mr congruent to 51,000 subunit to apparent Mr congruent to 44,000. The porcine Mr congruent to 34,000 photolabeled peptide is an N-linked glycoprotein as assessed by lectin affinity chromatography and susceptibility to digestion by glycopeptidase F to a peptide of apparent Mr congruent to 23,000.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Dopamine D2 receptor binding subunits of Mr congruent to 140,000 and 94,000 in brain: deglycosylation yields a common unit of Mr congruent to 44,000. 297 May 86

We describe the isolation of human fibronectin receptors (integrins) from two nonadherent promonocytic cell lines and from peripheral blood monocytes. Integrins purified from U-937 and THP-1 cells exhibited identical electrophoretic migrations on sodium dodecyl sulfate gels run under reducing (approximately Mr 150,000) and nonreducing (alpha, Mr 160,000; beta, Mr 130,000) conditions. Treatment of U-937 or THP-1 cells with phorbol esters induced these cells to express different integrins with electrophoretic mobilities (alpha, Mr 140,000; beta, Mr 115,000, nonreduced) identical to those from normal human peripheral blood monocytes. Receptors isolated from uninduced, nonadherent promonocytic leukemia cells (U-937 and THP-1) were distinct from glycoproteins IIb and IIIa and from leukocyte adhesion molecules (p150/95). However, receptors isolated here did react with an antibody known to block cell adhesion to fibronectin. The differences observed in apparent molecular masses of fibronectin receptors from uninduced and induced U-937 or THP-1 cells are removed by treatment of purified integrins with endoglycosidase F or N-glycanase. In summary, the data presented here demonstrate the purification of integrins by fibronectin affinity chromatography from human leukemia cells and normal peripheral blood monocytes. Our results suggest that these receptors differ in immature and mature monocytic cells, and are altered by glycosylation in the course of cellular maturation.
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PMID:Alteration of fibronectin receptors (integrins) in phorbol ester-treated human promonocytic leukemia cells. 297 31

The Asn-linked oligosaccharides of the pituitary hormone lutropin (LH) contain both sulfate and GalNAc. Bovine pituitary explants incorporate [3H]glucosamine, [3H]mannose, [3H]fucose, and [35S]sulfate into the Asn-linked oligosaccharides of LH. Endoglycosidase F or N-glycanase releases the [3H]glucosamine- and [3H]mannose-labeled oligosaccharides from the protein, which resolve on anion-exchange high pressure liquid chromatography as neutral (S-0), mono- (S-1), and disulfated (S-2) species. Based on sequential enzyme digestion, methylation, periodate oxidation, and nuclear magnetic resonance studies, the proposed structure for S-2 is as follows: formula see text. Sulfate is confined to position 3 or 4 of GalNAc based on periodate and methylation data and can be removed by methanolysis. The presence of beta-linked GalNAc at a position typically occupied by Gal has not previously been observed.
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PMID:Structural elucidation of the disulfated oligosaccharide from bovine lutropin. 299 25

The bombesin receptor present on the surface of murine and human cells was identified using 125I-labeled gastrin-releasing peptide as a probe, the cross-linking agent disuccinimidyl suberate, and sodium dodecyl sulfate gels. A clone of NIH-3T3 cells which possesses approximately 80,000 bombesin receptors/cell with a single binding constant of approximately 1.9 X 10(-9) M was used in these studies. In addition, we used Swiss 3T3 cells and a human glioma cell line which possesses approximately 100,000 and approximately 55,000 bombesin receptors/cell, respectively. Under conditions found optimal for binding, it is demonstrated that 125I-labeled gastrin-releasing peptide can be cross-linked specifically to a glycoprotein of apparent molecular mass of 65,000 daltons on the surface of the NIH-3T3 cells. Similar results were obtained when the cross-linked product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or non-reducing conditions. Moreover, the cross-linking reaction is specific and saturable and the 65,000-dalton polypeptide is not observed when the cross-linking experiments were performed with a NIH-3T3 cell line which is devoid of bombesin receptors. Interestingly, glycoproteins with apparent molecular weights of 75,000 were labeled specifically by 125I-labeled gastrin-releasing peptide when similar experiments were performed with Swiss 3T3 cells and with human glioma cell line GM-340. These different molecular weights may indicate differential glycosylation as treatment with the enzyme N-glycanase reduced the apparent molecular weight of the cross-linked polypeptide to 45,000. On the basis of these results it is concluded that the cross-linked polypeptides represent the bombesin receptor or the ligand-binding subunit of a putative larger bombesin receptor expressed on the surface of these cells.
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PMID:Identification of the bombesin receptor on murine and human cells by cross-linking experiments. 303 12

A commercially available endoglycosidase (N-glycanase, Genzyme, Boston, Mass.) purified from Flavobacterium meningosepticum with a specificity for cleaving asparagine-linked carbohydrate moieties in glycoproteins was tested on several pituitary and chorionic gonadotropins as substrates. All intact hormones tested were resistant to the action of the enzyme as were all beta subunits from the respective gonadotropins. All alpha subunits, however, were susceptible to the enzyme as evidenced by a decrease in molecular size when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Preparative experiments with ovine luteinizing hormone subunit (oLH alpha) indicated that only 35-40% of the carbohydrate was removed after N-glycanase treatment, suggesting that perhaps only one of the two carbohydrate moieties was cleavable under the conditions employed. The enzyme-modified subunit (DG-oLH alpha) was able to recombine with untreated oLH beta. An in vitro steroidogenic bioassay (rat Leydig cell) showed that the recombinant (DG-oLH alpha-oLH beta) was about 22% as potent as the native oLH, but in a testicular membrane binding assay for LH, it was equal in potency to the native hormone in competing with the radioligand.
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PMID:Deglycosylation of gonadotropins with an endoglycosidase. 308 Jul 56


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