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)

To allow for structural analysis of the human acetylcholinesterase (hAChE) subunit, a series of eukaryotic vectors was designed for efficient expression. Several eukaryotic multicistronic expression vectors were tested in various mammalian cell lines. All expression vectors contained the selectable neo gene under control of a weak promoter, while the hAChE cDNA was under control of the cytomegalovirus (CMV) immediate-early or Rous sarcoma virus long terminal repeat (RSV LTR) or simian virus 40 (SV40) early promoters. Optimal production and secretion of recombinant hAChE (rehAChE) was achieved in the embryonal kidney 293 cell line transfected either with the RSV-hAChE or with CMV-hAChE expression vectors. Clones expressing and secreting as much as 5-25 pg of enzyme per cell per 24 h were obtained without resorting to coamplification techniques or continuous maintenance of cells under selective pressure. The purified (specific activity of 6000 units per mg protein) homodimer and tetramer enzyme molecules displayed typical AChE biochemical properties: a Km value of 120 microM for acetylthiocholine; a kcat value of 3.9 x 10(5)/min, and selective by AChE-specific inhibitors. Catalytic subunit dimers (130 kDa) exhibit differential N-glycosylation patterns, and upon reduction resolve into 67- and 70-kDa monomeric subunits. These two forms appear as a single discrete 62-kDa band following deglycosylation by N-glycanase. The N-terminal amino acid sequence analysis of the purified mature enzyme suggests the existence of two alternative cleavage sites for the removal of the signal peptide, in which the 'mature' position 1 is either Ala31 or Gly33. Both of these positions conform with the consensus signal peptide recognition sequences and demonstrate bidirected processing of signal peptides on a native molecule.
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PMID:Production and secretion of high levels of recombinant human acetylcholinesterase in cultured cell lines: microheterogeneity of the catalytic subunit. 144 27

FeLV-FAIDS, an immunodeficiency-inducing isolate of feline leukemia virus, is composed of a pathogenic but replication-defective genome (molecular clone 61C) and a replication-competent but non-immunodeficiency-inducing variant genome (molecular clone 61E). The chimeric virus EECC, composed of the 5' gag-pol of 61E fused to the env-3' LTR of 61C, also induces immunodeficiency. The 61C (or EECC) gp80 can be distinguished from that of 61E on the basis of antigenic recognition, size, and rate of posttranslational processing. We found that the nascent precursor polypeptides of the two viruses were the same size; however, the 61E gp80 rapidly shifted to a smaller size and was subsequently cleaved to gp70, whereas EECC gp80 maintained its nascent size and was cleaved to gp70 only after a prolonged time. Endo-beta-N-acetyl glucosaminidase H and N-glycanase digestions of newly formed glycoproteins resulted in a similar banding pattern for both viruses, indicating that both contained the same number of oligosaccharide side chains and that all of these were high mannose sugars. The metabolic inhibitors of glycosylation, castanospermine or N-methyldeoxynojirimycin, prevented both the rapid trimming of 61E gp80 and its cleavage to gp70. Treatment with mannosidase inhibitors, however, did not affect 61E gp80 processing or size, suggesting that retention of glucose residues on EECC was responsible for these distinguishing properties of the glycoprotein. The pathological consequence of aberrant viral glycoprotein processing was evaluated in feline 3201 T lymphocytes, which are infectable by both 61E and EECC but are killed only by EECC. As in fibroblasts, the EECC glycoprotein produced in lymphocytes was larger, antigenically distinct, and processed more slowly than was the glycoprotein of 61E. Castanospermine treatment of 61E-infected 3201 T cells, however, not only abrogated the antigenic differences between the 61E and EECC glycoproteins but also resulted in a cytopathic effect. Our results suggest that (i) intracellular accumulation of EECC envelope glycoprotein may occur consequent to retention of glucose residues on carbohydrate side chains and (ii) a strong correlation exists between delayed glycoprotein processing and cytopathicity in FeLV-FAIDS-infected T lymphocytes.
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PMID:Characterization and significance of delayed processing of the feline leukemia virus FeLV-FAIDS envelope glycoprotein. 216 20

Aminopeptidase W is a newly discovered enzyme of the renal and intestinal brush borders, having been first isolated as a 130 kDa glycoprotein recognized by a monoclonal antibody [Gee & Kenny (1985) Biochem. J. 230, 753-764]. It is particularly effective in the hydrolysis of dipeptides, Glu-Trp (Km 0.57 mM; kcat. 6770 min-1) being a favoured substrate. Dipeptides with tryptophan, phenylalanine or tyrosine in the P1 position were rapidly hydrolysed, but the requirements in respect of the P1 residue were not stringent. The activity of aminopeptidase W is markedly influenced by ionic conditions. The highest activity was observed in 100 mM-Tris/HCl, pH 8; phosphate ions were strongly inhibitory. Activity was also greatly affected by bivalent metal ions, and the magnitude and direction of the effects depended on the nature of the buffer anions and on pH. The most effective inhibitors were amastatin and bestatin. Some thiols also inhibited, but other chelating agents, EDTA and 1,10-phenanthroline, had no effect over the concentration range 1-10 mM. Other group-specific inhibitors, for cysteine, serine or aspartic peptidases, were also ineffective. Some molecular properties were studied. Deglycosylation by treatment with N-glycanase diminished the apparent subunit Mr from 130,000 to 90,000. The enzyme contained zinc, 1.2 atoms/subunit, and in spite of the atypical properties of this enzyme in respect of chelating agents, a zinc-catalysed mechanism is the most probable. Its roles in digestion and in renal function are not yet clear.
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PMID:Proteins of the kidney microvillar membrane. Enzymic and molecular properties of aminopeptidase W. 289 Mar 46

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

Porcine pancreatic alpha-amylase (PPA) and its isoforms (PPA-I and PPA-II) were deglycosylated by peptide-N-glycosidase F (PNGase F) to investigate the role of bound carbohydrate. On deglycosylation, the effect on thermal stability was less pronounced. Deglycosylation resulted in a shift of the mid-point of thermal transition by 1-2 degrees C towards lower temperature. The fluorescence emission maxima of PPA, PPA-I and PPA-II were found to be 340nm indicating the presence of tryptophan residues in a fairly hydrophilic environment. A red shift in emission spectra accompanied by an increase in fluorescence intensity was observed upon deglycosylation.
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PMID:Porcine pancreatic alpha amylase and its isoforms--effect of deglycosylation by peptide-N-glycosidase F. 1848 69