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)

A recent study by C.F. Burant et al. (13) demonstrates that GLUT5 is a high-affinity fructose transporter with a much lower capacity to transport glucose. To characterize the potential role of GLUT5 in fructose and glucose transport in insulin-sensitive tissues, we investigated the distribution and insulin-stimulated translocation of the GLUT5 protein in human tissues by immunoblotting with an antibody to the COOH-terminus of the human GLUT5 sequence. GLUT5 was detected in postnuclear membranes from the small intestine, kidney, heart, four different skeletal muscle groups, and the brain, and in plasma membranes from adipocytes. Cytochalasin-B photolabeled a 53,000-M(r) protein in small intestine membranes that was immunoprecipitated by the GLUT5 antibody; labeling was inhibited by D- but not L-glucose. N-glycanase treatment resulted in a band of 45,000 M(r) in all tissues. Plasma membranes were prepared from isolated adipocytes from 5 nonobese and 4 obese subjects. Incubation of adipocytes from either group with 7 nM insulin did not recruit GLUT5 to the plasma membrane, in spite of a 54% insulin-stimulated increase in GLUT4 in nonobese subjects. Thus, GLUT5 appears to be a constitutive sugar transporter that is expressed in many tissues. Further studies are needed to define its overall contribution to fructose and glucose transport in insulin-responsive tissues and brain.
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PMID:Human small intestine facilitative fructose/glucose transporter (GLUT5) is also present in insulin-responsive tissues and brain. Investigation of biochemical characteristics and translocation. 139 12

Enzymological studies have implicated two Ca(2+)-dependent endopeptidases in the conversion of proinsulin to insulin; a type 1 activity which cleaves on the C-terminal side of Arg31-Arg32 and a type 2 activity which cleaves C-terminally to Lys64-Arg65 in the proinsulin sequence. The possibility that these enzymes are related to the recently discovered family of mammalian subtilisin-like gene products (furin, PC2, and PC3) and the yeast propheromone-converting enzyme (KEX-2), was investigated. Degenerate oligonucleotide primers flanking the putative catalytic domain within this gene family were used in a polymerase chain reaction to amplify related sequences from rat insulinoma cDNA. One major product of 700 base pairs was obtained which was greater than 99% identical to the corresponding rat PC2 sequence. This cDNA was subcloned into the bacterial expression vector pGEX-3X to generate a recombinant protein for antibody production. Western blot analysis showed the immunoreactivity was prominent in neuroendocrine tissues as a 65-kDa protein. It was concentrated in secretory granule-enriched fractions of insulinoma tissue, where it was present as a readily solubilized monomeric protein. Deglycosylation studies using endoglycosidase H and N-glycanase showed that the 65-kDa protein was comprised of approximately 9% carbohydrate, consistent with the presence of three consensus sequences for N-linked glycosylation in rat PC2. The immunoreactivity co-eluted with the type 2 proinsulin endopeptidase on gel filtration and ion-exchange chromatography and the antisera specifically immunoprecipitated type 2 activity from insulin granule extracts. N-terminal sequence analysis of the immunoreactive protein gave two sequences which corresponded to residues 109-112 and 112-119 of rat PC2. This indicated that posttranslational processing of PC2 itself occurs C-terminally to basic amino acids to produce the mature enzyme. It is concluded that PC2 is the type 2 endopeptidase involved in proinsulin conversion. Localization of PC2 immunoreactivity to other tissues of the diffuse neuroendocrine system suggests that the type 2 endopeptidase also functions in the processing of precursor forms of other prohormones and polypeptide neurotransmitters.
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PMID:Identification of the type 2 proinsulin processing endopeptidase as PC2, a member of the eukaryote subtilisin family. 163 53

Breast tumor cell lines have been shown to secrete several distinct polypeptide growth factors, although conflicting results exist for the insulin-like growth factors (IGFs). In contrast a limited number of breast tumor cell lines have definitely been shown to secrete the high affinity IGF binding proteins (IGFBPs) that modify IGF actions. To characterize the types of IGFBPs that are secreted by breast tumor cell lines, conditioned medium was collected from seven separate tumor cell lines, three of which were estrogen receptor (ER) negative, and four of which were ER positive. All three of the ER negative cell lines, MDA-231, MDA-330, and HS578T, secreted binding proteins of 49,000 and 43,000 Mr (IGFBP-3) as well as 29,000 (IGFBP-1) and 24,000 Mr. In contrast, all four ER positive cell lines secreted 34,000 (IGFBP-2) or 24,000 Mr forms, and none secreted the 49,000 and 43,000 or 29,000 Mr forms. BT-20, a cell line that is positive for ER messenger RNA (mRNA) but negative for ER protein, secreted predominantly a 34,000 Mr protein. The amount of total IGFBP activity released in 24 h ranged between 0.4 and 5.6 nM equivalents of IGFBP-1, and there was no significant difference between the ER positive and negative cell lines. The MCF-7 cells that produced predominantly 34,000 and 24,000 Mr forms showed a 1.8-fold increase in IGFBP secretion after estrogen stimulation. Immunoblotting and a specific RIA for IGFBP-1 showed that only the ER negative lines MDA-330, MDA-231, and HS578T secreted this form. Northern blotting analysis for the mRNA encoding this protein showed that both MDA-330 and MDA-231 contained a single 1.6 kilobase mRNA species that hybridized with an IGFBP-1 complementary DNA (cDNA) probe. Immunoblotting analysis of the other cell lines showed that only the 34,000 Mr form secreted by the ER positive cell lines reacted with IGFBP-2 antisera. Exposure of the conditioned media from the three ER negative cell lines to N-glycanase revealed that the 49,000 and 43,000 Mr forms of IGFBP were glycosylated and therefore probably represent IGFBP-3. We conclude that ER negative cell lines secrete three forms of IGFBPs, IGFBP-1, IGFBP-3, and a 24,000 Mr form. In contrast, the ER positive cell lines secrete predominantly IGFBP-2 and the 24,000 Mr form but do not secrete IGFBP-3 or 1.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Insulin-like growth factor binding protein secretion by breast carcinoma cell lines: correlation with estrogen receptor status. 170 Nov 24

Tryptase was purified 13,000-fold to apparent homogeneity from rat skin. The two-step procedure involved ammonium sulfate fractionation of the initial extract followed by combined sequential affinity chromatography on agarose-glycyl-glycyl-p-aminobenzamidine and concanavalin A-agarose. The purified enzyme had a specific activity toward N-benzoylarginine ethyl ester (BzArgOEt) of 170 mumol/min mg-1 and was obtained in a yield of 28% as determined by the specific substrate, H-D-Ile-Pro-Arg-p-nitroanilide. Rat skin tryptase was thermal labile, losing 50% of its activity when preincubated for 30 min at 30 degrees C. The presence of NaCl (1 M) improved thermal stability and was necessary for long-term storage. Heparin did not stabilize the enzyme against thermal denaturation, and heparin-agarose failed to bind the enzyme. Rat skin tryptase was inhibited by diisopropylphosphofluoridate, antipain, leupeptin, and aprotinin but not by alpha 1-antitrypsin, ovomucoid, or soybean or lima bean trypsin inhibitors. Substrate specificity studies using a series of tri- and tetrapeptidyl-p-nitroanilide and peptidyl-7-amino-4-methylcoumarin substrates demonstrated the existence of an extended substrate binding site. Rat skin tryptase hydrolyzed [Arg8]vasopressin, neurotensin, and the oxidized B-chain of insulin at the -Arg8-Gly9-NH2, -Arg8-Arg9-, and -Arg22-Gly23-bonds, respectively. No general proteinase activity was observed toward casein, hemoglobin, or azocoll. Rat skin tryptase had a Mr of 145,000 by gel filtration. The subunit Mr was either 34,000 or 30,000 depending on the electrophoretic technique used. Treatment of the enzyme with peptide N-glycosidase F (N-glycanase) decreased the subunit Mr by 4000. The enzyme exhibited multiple isoelectric forms (pI's of 4.5-4.9). Rat skin tryptase was found to be related statistically to other tryptases on the basis of amino acid composition. The N-terminal amino acid sequence was Ile1-Val2-Gly3-Gly4-Gln5-Glu6-Ala7-+ ++Ser8-Gly9-Asn10-Lys11-Trp12-Pro13- Trp14- Gln15-Val16-Ser17-Leu18-Arg19-Val20- --21-Asp-22Thr23-Tyr24-Typ25-, with a putative glycosylation site at residue 21. This sequence was 72-80% homologous with the N-terminus of other tryptases but only 40% homologous with that of bovine trypsin.
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PMID:Tryptase from rat skin: purification and properties. 203 67

In the human circulation, the insulin-like growth factors (IGFs) circulate as part of a growth hormone-dependent 125- to 150-kDa complex. This complex has been postulated to contain, in addition to IGFs and one or more IGF-binding proteins, an acid-labile subunit (ALS) which does not itself bind IGFs. In this study, the ALS has been purified 1600-fold from human serum, and its binding properties have been examined. Fresh serum was fractionated on DEAE-Sephadex, and active fractions (determined by radioimmunoassay) were purified by affinity chromatography on an IGF-agarose column saturated with the plasma IGF-binding protein BP-53. After further high performance anion exchange chromatography, an ALS preparation was obtained which contained only an 84-86-kDa protein doublet, converting to a single 70-kDa band on N-glycanase treatment, and having an amino-terminal sequence unrelated to IGF-binding proteins or receptors. Pure ALS formed a complex with BP-53 (Ka approximately 5 x 10(8) M-1), immunoprecipitable by anti-BP-53 antiserum, only in the presence of IGF-I or IGF-II. This complex appeared at approximately 150 kDa on high performance gel chromatography. Pure ALS had no intrinsic IGF-binding activity and no effect on the binding of IGF-I or IGF-II to BP-53. These studies suggest that formation of the high molecular weight IGF-binding protein complex requires ALS, BP-53, and IGF.
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PMID:High molecular weight insulin-like growth factor binding protein complex. Purification and properties of the acid-labile subunit from human serum. 247 65

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

Freshly isolated rat islets and cultured hamster insulinoma cells (HIT T15) were incubated with a membrane-permeable octanoyl tripeptide (N-octanoyl-ASN-TYR-THR-NH2), which contains an acceptor sequence for ASN-linked glycosylation. Labeled octanoyltripeptide (125[I]TYR) was glycosylated by both islets and HIT cells. The carbohydrate moiety of this glycotripeptide was removed by N-glycanase indicating that glycotripeptide was formed in the lumen of endoplasmic reticulum and, subsequently was secreted via the route for secretory protein. Secretion of glycotripeptide began more rapidly than that of insulin newly synthesized from 3[H]leucine. At 30 min glycotripeptide secretion was already significant but, over a 3-h period, it never represented more than 21% of glycotripeptide produced. Glycotripeptide secretion was not affected by compounds shown to regulate insulin secretion (glucose, forskolin, EGTA and streptozotocin). Thus in beta cells, it appears that glycotripeptide secretion is unregulated and that its cellular secretory pathway is different from that for insulin.
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PMID:Unregulated secretion of an exogenous glycotripeptide by rat islets and HIT cells. 284 81

We have characterized receptors for the insulin-like growth factor (IGF-I) on the mouse neuroblastoma cell line N18 as well as NG108, the hybrid cell line of N18 and rat glioma (C6). In this cell-free system, IGF-I and insulin stimulated the phosphorylation of 95-kDa and 105-kDa proteins. Using appropriate antibodies we were able to demonstrate that the IGF-I receptor beta subunit has two subtypes of 95 kDa and 105 kDa. On the other hand, insulin receptor beta subunit is a separate single 95-kDa protein. Enzymatic digestion of IGF-I receptor beta subunit subtypes by glycopeptidase F resulted in similar molecular masses (84 kDa and 86 kDa) on SDS-PAGE, which suggests that the difference in molecular masses between two subtypes is attributable to the differences in N-linked complex-type carbohydrate chains on the extracellular domain of beta subunits. This conclusion is further supported by peptides of similar molecular mass following staphylococcal V8 protease digestion. Analysis of IGF-I receptor beta subunit subtypes in these cells may provide insights into the mechanism of action of IGF-I on neural tissues.
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PMID:Insulin-like growth factor I receptors on mouse neuroblastoma cells. Two beta subunits are derived from differences in glycosylation. 296 5

In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10(6) receptors per cell. The cell line with the highest insulin binding (NIH 3T3 HIR3.5) had 6 X 10(6) receptors with a Kd of 10(-9) M. This level was not dependent on exposure to metals but could be increased further to 2 X 10(7) receptors per cell by addition of sodium butyrate to the culture medium. The alpha and beta subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the alpha and beta subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis.
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PMID:High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells. 329 82

Transforming growth factor-beta 1 (TGF-beta 1) stimulated growth and glucose uptake in Swiss mouse fibroblasts. DNA synthesis was increased 2-3-fold after 48 h incubation of growing 3T3 cells with TGF-beta 1 in calf serum-containing medium. Glucose transport activity in the cells was increased within 3 h after addition of TGF-beta 1 and this stimulation continued during incubation for 48 h. TGF-beta 1 also increased the levels of a brain type-glucose transporter (GLUT1) mRNA and the GLUT1 protein (55 kDa) in the membranes, consistent with the increase in glucose uptake. Furthermore, a longer exposure of TGF-beta 1 for 24-48 h induced a marked increase in the 65 kDa GLUT1 in 3T3 cell membranes. Other growth factors such as epidermal growth factor, fibroblast growth factor, transforming growth factor-alpha, and insulin did not elevate glucose uptake and the levels of 55 and 65 kDa GLUT1 proteins. Adding tunicamycin or deoxymannojirimycin to the TGF-beta 1-treated and untreated cells caused these 55 and 65 kDa glucose transporters to migrate as one band at 40-43 kDa. In addition, treating membrane proteins with glycopeptidase F, which removes N-linked oligosaccharides, also generated a glucose transporter of 40 kDa, suggesting that the 55 and 65 kDa GLUT1 proteins have a similar or identical core polypeptide but with different N-linked oligosaccharides. These results indicate that TGF-beta 1 modulates the synthesis of GLUT1 protein as well as its glycosylation in Swiss 3T3 cells, and that these changes may contribute to the control of cell proliferation by TGF-beta 1.
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PMID:Modulation of the synthesis and glycosylation of the glucose transporter protein by transforming growth factor-beta 1 in Swiss 3T3 fibroblasts. 843 54


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