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)

Insulin-like growth factor-I (IGF-I), the principal IGF in adult rat serum, occurs complexed to specific binding proteins. After fractionation of serum on Sephadex G-200 at neutral pH, 62% of the immunoreactive IGF-I is recovered in the 150K region, 38% in the 40K region, and none is present as free 7.5K IGF-I. Adult rat serum also contains unoccupied binding sites for IGFs that also are predominantly (77%) located in the 150K region and have preferential binding affinity for IGF-II. IGF-binding protein components in the 150K and 40K regions were evaluated by affinity cross-linking to 125I-labeled IGFs and by ligand blotting (i.e. incubation of nitrocellulose blots of sodium dodecyl sulfate (SDS)-gels with [125I]IGFs). Affinity cross-linking of the 150K region revealed a major 43K binding protein complex and several minor covalent complexes of 97-210K that are formed during the cross-linking reaction. The 40K region of the gel filtration column contains a predominant 32K complex and smaller amounts of the 43K complex. Ligand blotting of the 150K region identifies a predominant cluster of binding components of about 40K and a smaller 29K protein. The apparent molecular masses of the 40K and 29K proteins are decreased by incubation with N-glycanase, indicating that they contain N-linked oligosaccharides. These glycoprotein components, designated gp40 and gp29, presumably combine with an acid-labile nonbinding subunit of about 100K to generate the 150K complex. The gp40 cluster represents glycosylation variants of a 34K protein; gp29 has been shown to correspond to an amino-terminal fragment of gp40. Ligand blotting of the 40K region indicates that it contains smaller amounts of gp40 and gp29, possibly representing free subunits not combined with the nonbinding subunit, as well as two proteins of apparent molecular mass 24K and 30K (p24 and p30) that are not glycosylated. Although p30 is similar in size to the binding protein from BRL-3A cells (BP-3A) that is present in fetal rat serum, immunoprecipitation and immunoblotting of whole and fractionated adult serum with an antiserum to BP-3A indicate that p30 in adult rat serum is an antigenically distinct protein. Serum levels of gp40 and gp29 are decreased by hypophysectomy and are restored by GH treatment; p24 and p30 show similar but smaller changes.
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PMID:Structure, specificity, and regulation of the insulin-like growth factor-binding proteins in adult rat serum. 254 90

A receptor for acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) was isolated from 7-day embryonic chick. Chromatography of solubilized membrane proteins on wheat germ agglutininagarose and aFGF-Sepharose yielded three major polypeptides migrating at 150, 70, and 45 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides were eluted from aFGF-Sepharose with either 1.0 M NaCl or 100 micrograms/ml heparin, but were not retained on underivatized Sepharose. Cross-linking of 125I-aFGF or 125I-bFGF to either crude membrane preparations or to purified fractions yielded a 165-kDa complex, suggesting the existence of a 150-kDa FGF receptor after subtraction of approximately 15 kDa for 125I-FGF. Addition of excess aFGF or bFGF competed for binding of either 125I-aFGF or 125I-bFGF to FGF receptor preparations. Purified FGF receptor fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to Immobilon membranes, and incubated with 125I-aFGF or 125I-bFGF in order to identify FGF-binding polypeptides. Bound 125I-aFGF and 125I-bFGF were displaced by aFGF and bFGF, but not epidermal growth factor, consistent with the identification of the 150-kDa polypeptide as a receptor for acidic and basic FGF. Treatment of purified FGF receptor fractions with N-glycanase demonstrated that the 150-kDa polypeptide contained approximately 10 kDa of N-linked oligosaccharide. The apparent molecular mass of the 150-kDa polypeptide was unaffected by treatment with heparitinase, indicating that the 150-kDa polypeptide is not a heparan sulfate proteoglycan. Together, these data suggest that the 150-kDa polypeptide is a FGF receptor that may mediate the biological activities of aFGF and bFGF.
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PMID:Isolation of a receptor for acidic and basic fibroblast growth factor from embryonic chick. 255 17

A monoclonal antibody (C219) that recognizes the P-glycoprotein (Mr = 170,000) in plasma membranes of multidrug-resistant Chinese hamster ovary (CHO) cell lines was used to assay renal brush border membrane (BBM) and basolateral membrane (BLM) fractions for the presence of a cross-reactive polypeptide. The C219 antibody bound to a 155,000 dalton protein in immunoblots of rat BBM but not BLM proteins resolved by sodium dodecyl sulfate gel electrophoresis. The corresponding human kidney BBM and dog kidney BBM proteins had molecular weights of 170,000 and 160,000 respectively. The glycoprotein nature of the renal protein was shown by its sensitivity to N-glycanase treatment which reduced the apparent molecular weight of the dog protein to 120,000. In addition, dog P-glycoprotein could be bound to and eluted from immobilized wheat germ agglutinin. The molecular weight, antibody crossreactivity, glycosidase sensitivity and lectin binding show that this protein is a normal kidney analogue of the P-glycoprotein induced in multidrug resistant cell lines.
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PMID:Identification of P-glycoprotein in renal brush border membranes. 256 33

The human fibrinogen B beta chain was expressed in Escherichia coli to study the functions of fibrinogen associated with this subunit. Recombinant B beta chains were expressed at 100 ng/mL in an IPTG-dependent manner. A first cistron sequence, inserted into the expression vector 5' to the B beta chain cDNA, was required to express the protein. Recombinant B beta chains were expressed within five minutes after induction with IPTG and were soluble in physiologic buffers. The recombinant B beta chains migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a rate identical to B beta chains from fibrinogen treated with N-glycanase. Recombinant B beta chains were cleaved by thrombin, as demonstrated by the loss of cross-reactivity with a monoclonal antibody (MoAb) specific for the undigested B beta 1-42 fragment. The levels of expression of the B beta chain were much lower than those reported previously for the gamma chain of fibrinogen expressed in a similar vector in E coli. However, these levels are sufficient to allow further characterization of this fibrinogen subunit.
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PMID:Expression in Escherichia coli of the human fibrinogen B beta chain and its cleavage by thrombin. 264 71

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 order to evaluate the structural identification of various recombinant human interferon-beta 1s, the recombinant proteins were produced in four different mammalian cells (human PC12 and PC8 lung adenocarcinoma cells, Chinese hamster ovary cells and mouse C127 cells) and characterized. Each mammalian-cell-derived recombinant human interferon-beta 1 represented a single band of 23 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, the same molecular mass as fibroblast-derived natural human interferon-beta 1. Specific activities, amino acid compositions, amino-terminal sequences, peptide maps on C18 reversed-phase high-performance liquid chromatography and circular dichroic spectra of recombinant proteins were in good agreement with natural ones. On the other hand, the patterns of isoelectric focusing were different between mammalian-cell-derived recombinant human interferon-beta 1s and natural human interferon-beta 1. Sugar composition analysis revealed that the recombinant protein from Chinese hamster ovary cells has a similar sugar composition to that of natural protein and the other recombinant proteins have increased amounts of galactose and glucosamine in comparison to the natural protein. Furthermore, there is no galactosamine in the natural protein, while small amounts of galactosamine were detected in the oligosaccharides released from PC8- and C127-derived recombinant proteins by N-glycanase. These results indicate that mammalian-cell-derived recombinant human interferon-beta 1s have identical polypeptides to those of natural human interferon-beta 1 but their carbohydrate moieties, including unusual N-linked oligosaccharides, are individually different from natural ones and depend on the host cell.
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PMID:Characterization of four different mammalian-cell-derived recombinant human interferon-beta 1s. Identical polypeptides and non-identical carbohydrate moieties compared to natural ones. 273 37

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

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

In order to determine whether the human insulin receptor ectodomain can be expressed as a functional protein, the coding regions for the transmembrane and cytoplasmic domain of a full-length human insulin receptor cDNA were deleted by site-directed mutagenesis, and the resultant construct was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH3T3 cells, a cell line secreting an insulin binding protein was isolated. The insulin binding alpha subunit had an Mr of 138,000 and a beta subunit of Mr 48,000 (compared to 147,000 and 105,000 for the full-length human insulin receptor expressed in NIH3T3 cells). This difference in size of the alpha subunit was due to a difference in glycosylation as N-glycanase digestion reduced the apparent size of the alpha subunits of secreted and normal membrane-bound receptors to identical values. The secreted receptor formed disulfide-linked heterotetrameric structures with an Mr of 280,000. It was synthesized as an Mr 160,000 precursor which was cleaved into mature subunits with a t1/2 of 3 h. Increasing expression of the cDNA by induction with sodium butyrate lead to the appearance of an Mr 180,000 protein in the medium as well as the mature alpha and beta subunits. A Scatchard plot of insulin binding to the secreted receptor was curvilinear with a Kd of 7 X 10(-10) M for the high affinity sites and 10(-7) M for the low affinity site (compared to Kd values of 1.1 X 10(-9) M and 10(-7) M, respectively, for human insulin receptors expressed in these cells.
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PMID:Secretion of soluble functional insulin receptors by transfected NIH3T3 cells. 283 Feb 71


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