scholarly journals The action of glycosylases on dopachrome (2-carboxy-2,3-dihydroindole-5,6-quinone) tautomerase

1992 ◽  
Vol 284 (1) ◽  
pp. 109-113 ◽  
Author(s):  
P Aroca ◽  
J H Martinez-Liarte ◽  
F Solano ◽  
J C García-Borrón ◽  
J A Lozano
Keyword(s):  
Concanavalin A ◽  
Enzymic Activity ◽  
Coated Vesicles ◽  
Carbohydrate Moiety ◽  
Native Enzyme ◽  
Enzyme Treatment ◽  
Strong Affinity ◽  
The Stability ◽  
Beta Galactosidase

It is shown that dopachrome (2-carboxy-2,3-dihydroindole-5,6-quinone) tautomerase (DCT) is a glycoprotein containing N-linked oligosaccharides. The enzymic activity can be stimulated by partial deglycosylation with a number of glycosylases such as neuraminidase, beta-mannosidase and beta-galactosidase. However, the stability of the enzyme after the hydrolytic treatment becomes lower. Thus total deglycosylation with peptide N-glycosidase F directly provokes an inactivation of DCT. The native enzyme also shows a strong affinity for concanavalin A-Sepharose. This affinity decreases after treatment with neuraminidase and/or beta-mannosidase. The DCT associated with coated vesicles seems to be mostly glycosylated, since the action of glycosylases on the enzyme obtained from these vesicles produced a similar stimulation to that with the melanosomal enzyme. Treatment of cultured melanocytes with tunicamycin elicited a decrease in the amount of active DCT inside the cells. All data suggest that the structure of the carbohydrate moiety of DCT should be very similar to, if not identical with, the structure proposed for tyrosinase by Ohkura, Yamashita, Mishima & Kobata (1984) Arch. Biochem. Biophys. 235, 63-77.

scholarly journals Synthesis and properties of oligonucleotides modified with an N-methylguanidine-bridged nucleic acid (GuNA[Me]) bearing adenine, guanine, or 5-methylcytosine nucleobases

2021 ◽  
Vol 17 ◽  
pp. 622-629
Author(s):  
Naohiro Horie ◽  
Takao Yamaguchi ◽  
Shinji Kumagai ◽  
Satoshi Obika
Keyword(s):  
Nucleic Acid ◽  
Binding Affinity ◽  
Antisense Oligonucleotides ◽  
Modified Oligonucleotides ◽  
Biophysical Properties ◽  
Chemical Modifications ◽  
Antisense Technology ◽  
Strong Affinity ◽  
The Stability

Chemical modifications have been extensively used for therapeutic oligonucleotides because they strongly enhance the stability against nucleases, binding affinity to the targets, and efficacy. We previously reported that oligonucleotides modified with an N-methylguanidine-bridged nucleic acid (GuNA[Me]) bearing the thymine (T) nucleobase show excellent biophysical properties for applications in antisense technology. In this paper, we describe the synthesis of GuNA[Me] phosphoramidites bearing other typical nucleobases including adenine (A), guanine (G), and 5-methylcytosine (mC). The phosphoramidites were successfully incorporated into oligonucleotides following the method previously developed for the GuNA[Me]-T-modified oligonucleotides. The binding affinity of the oligonucleotides modified with GuNA[Me]-A, -G, or -mC toward the complementary single-stranded DNAs or RNAs was systematically evaluated. All of the GuNA[Me]-modified oligonucleotides were found to have a strong affinity for RNAs. These data indicate that GuNA[Me] could be a useful modification for therapeutic antisense oligonucleotides.

Purification and characterization of rat epididymal neutral β-galactosidase and its changes during in vivo development

1993 ◽  
Vol 71 (1-2) ◽  
pp. 22-26 ◽  
Author(s):  
Pratima Dutta ◽  
Gopal C. Majumder
Keyword(s):  
Concanavalin A ◽  
Sexual Maturity ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Tissue Homogenate ◽  
Affinity Column ◽  
Ph Optimum

A neutral β-D-galactosidase has been partially purified from rat epididymis and characterized. The enzyme having molecular mass of approximately 50 kilodaltons has been purified 400-fold by using calcium phosphate gel adsorption, DEAE-cellulose chromatography, Sephadex G-100 gel filtration, and concanavalin A - agarose affinity chromatography. Although the neutral enzyme binds to the concanavalin A affinity column, the activity could be eluted with α-methyl mannoside only if the buffer contained salt (NaCl) at a concentration as high as 0.3 M. The enzyme was of cytosolic origin, since 90% of the total enzymic activity of the tissue homogenate was recovered in the soluble fraction of these cells. The neutral β-galactosidase was not dependent on metal ions for its activity and it had a pH optimum of 7.0. Zn2+, p-chloromercuribenzoate, Hg2+, and Pb2+ served as potent inhibitors of the enzyme. There was a marked increase (approximately fourfold) in the specific activity of the neutral β-galactosidase during sexual maturity of epididymis in vivo.Key words: neutral β-galactosidase, rat epididymal, cytosolic, developmental, sexual maturity.

scholarly journals Characterization of human liver α-D-mannosidase purified by affinity chromatography

1976 ◽  
Vol 153 (3) ◽  
pp. 579-587 ◽  
Author(s):  
N C Phillips ◽  
D Robinson ◽  
B G Winchester
Keyword(s):  
Affinity Chromatography ◽  
Concanavalin A ◽  
Human Liver ◽  
Early Stage ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Exchange Chromatography ◽  
Lysosomal Storage ◽  
Sepharose 4B

Human liver acidic α-D-mannosidase was purified 1400-fold by a relatively short procedure incorporating chromatography on concanavalin A-Sepharose and affinity chromatography on Sepharose 4B-epsilon-aminohexanoylmannosylamine. In contrast with the acidic enzymic activity the neutral α-mannosidase did not bind to the concanavalin A-Sepharose so the two types of α-mannosidase could be separated at an early stage in the purification. The only significant glycosidase contaminant after affinity chromatography on the mannosylamine ligand was α-L-fucosidase, which was selectively removed by affinity chromatography on the corresponding fucosylamine ligand. The final preparation was free of other glycosidase activities. The pI of the purified enzyme was increased from 6.0 to 6.45 on treatment with neuraminidase. Although the pI and the mol.wt. (220 000) suggested that α-mannosidase A had been purified selectively, ion-exchange chromatography on DEAE-cellulose indicated that the preparation consisted predominantly of α-mannosidase B. This discrepancy is discussed in relation to the basis of the multiple forms of human α-mannosidase. The purified enzyme completely removed the α-linked non-reducing terminal mannose from a trisaccharide isolated from the urine of a patient with mannosidosis. A comparison of the activity of the pure enzyme towards the natural substrate and synthetic substrates suggests that the same enzymic activity is responsible for hydrolysing all the substrates. These results validate the use of synthetic substrates for determining the mannosidosis genotype. They are also further evidence that mannosidosis is a lysosomal storage disease resulting from a deficiency of acidic α-mannosidase.

scholarly journals A non-glycosylated extracellular superoxide dismutase variant

1992 ◽  
Vol 288 (2) ◽  
pp. 451-456 ◽  
Author(s):  
A Edlund ◽  
T Edlund ◽  
K Hjalmarsson ◽  
S L Marklund ◽  
J Sandström ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Culture Media ◽  
Periodic Acid ◽  
Chinese Hamster ◽  
Glycosylation Site ◽  
Enzymic Activity ◽  
Carbohydrate Moiety ◽  
Size Exclusion ◽  
Extracellular Superoxide Dismutase ◽  
Periodic Acid Schiff

The secretory tetrameric extracellular superoxide dismutase (EC-SOD) is the only glycosylated SOD isoenzyme. The importance of the carbohydrate moiety for the properties of the enzyme is unknown. An expression vector defining nonglycosylated EC-SOD (ngEC-SOD) was constructed by mutagenesis of the codon for Asn-89 into a codon for Gln. The vector was transfected into Chinese hamster ovary DXB-11 cells and ngEC-SOD was isolated to 70% purity from the culture media of selected clones. The absence of glycosylation was established by the lack of affinity for various lectins, the absence of staining with the periodic acid-Schiff reagent, the change in mobility and composition of the tryptic peptide containing the mutated glycosylation site, and the reduction in apparent molecular mass upon SDS/PAGE and size-exclusion chromatography. The tetrameric state was retained. The heparin affinity, a fundamental and distinguishing property of EC-SOD, was found to be slightly increased. The enzymic activity was essentially retained. The major difference from native glycosylated enzyme in physical properties was a marked reduction in solubility. Like glycosylated EC-SOD, ngEC-SOD was, after intravenous injection into rabbits, rapidly sequestered by the vessel endothelium, and was promptly released into plasma after injection of heparin. The only difference from glycosylated EC-SOD in this behaviour, was a slightly more rapid elimination of the mutant enzyme from the vasculature. It is concluded that no specific biological role for the EC-SOD carbohydrate moiety could be revealed.

N-glycosylated catalytic unit meets O-glycosylated propeptide: complex protein architecture in a fungal hexosaminidase

2004 ◽  
Vol 32 (5) ◽  
pp. 764-765 ◽  
Author(s):  
O. Plíhal ◽  
J. Sklenář ◽  
J. Kmoníčková ◽  
P. Man ◽  
P. Pompach ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Enzymic Activity ◽  
Enzyme Activation ◽  
Protein Architecture ◽  
Fungal Enzyme ◽  
Hexa Gene ◽  
Catalytic Unit ◽  
Complex Protein ◽  
The Stability ◽  
Inhibition Studies

β-N-Acetylhexosaminidase from a filamentous fungus Aspergillus oryzae is a secreted enzyme known to be an important component of the binary chitinolytic system. Cloning of the hexA gene and sequencing of the enzyme revealed its unique preproprotein structure. While the enzyme's zincin-like and catalytic domain had significant similarities with members of the glycohydrolase 20 family, the propeptide was unique for the fungal enzyme. Detailed pulse–chase and inhibition studies revealed that propeptide was processed during the biosynthesis of the enzyme. Moreover, the presence of propeptide was necessary for enzyme activation, dimerization and secretion. The catalytic unit was N-glycosylated, and the propeptide was O-glycosylated, both in their C-terminal parts. Deglycosylation experiments revealed that the N-glycosylation increased the stability and solubility of the enzyme. In contrast, O-glycosylated propeptide was necessary to attain the full enzymic activity.

Interactions of Concanavalin A with the Membrane of Influenza Virus Infected Cells and with Envelope Components of the Virus Particle

1972 ◽  
Vol 27 (3) ◽  
pp. 227-233 ◽  
Author(s):  
R. Rott ◽  
H. Becht ◽  
H.-D. Klenk ◽  
C. Scholtissek
Keyword(s):  
Influenza Virus ◽  
Virus Particle ◽  
Concanavalin A ◽  
Early Phase ◽  
Carbohydrate Moiety ◽  
Host Cells ◽  
Con A ◽  
Bhk Cells ◽  
Infected Cells

Treatment of host cells with Concanavalin A prevents the assembly or release of fowl plague virus from chick fibroblasts and inhibits fusion of BHK cells by the paramyxovirus SV5. Metabolic events of the host cells are not greatly impaired during the early phase of infection. There is evidence that the carbohydrate moiety of the Con A receptor of fowl plague virus is associated with the viral neuraminidase.

scholarly journals Substitutions for Glu-537 of β-galactosidase from Escherichia coli cause large decreases in catalytic activity

1994 ◽  
Vol 299 (2) ◽  
pp. 527-531 ◽  
Author(s):  
J Yuan ◽  
M Martinez-Bilbao ◽  
R E Huber
Keyword(s):  
Wild Type ◽  
Inhibitory Effects ◽  
Wild Type Enzyme ◽  
Enzyme Preparations ◽  
Transition State Analogues ◽  
Normal Enzyme ◽  
Enzyme Molecules ◽  
The Stability ◽  
Beta Galactosidase ◽  
Synthetic Oligonucleotides

Glu-537 of beta-galactosidase (EC 3.2.1.23) was replaced by Asp, Gln and Val using synthetic oligonucleotides. The kcat values of the purified enzyme mixtures were reduced by about 100-fold for the Asp mutant, 30,000-60,000-fold for the Val mutant and 160,000-300,000-fold for the Gln mutant. The greatest differences in properties from the wild-type enzyme were found for the Asp-substituted enzyme: the Km values increased (from 0.12 to 0.42 mM for o-nitrophenyl beta-D-galactopyranoside), and from 0.04 to 0.37 mM for p-nitrophenyl beta-D-galactopyranoside), the Ki value for isopropyl beta-D-galactopyranoside increased (from 0.11 to 0.30 mM), the stability to heat decreased and methanol did not act as an acceptor. The enzymes with the other two substitutions had properties similar to those of the wild-type. For all three substituted enzymes, the inhibitory effects of the transition-state analogues (2-deoxy-2-amino-D-galactose and L-ribose) and the Mg2+ effects were similar to those of the normal enzyme. As all of the properties (except the kcat values) of the Gln- and Val-substituted enzyme preparations were similar to those of the wild-type enzyme, the activities in those preparations were probably due to the presence of a few wild-type enzyme molecules (formed from misreads) among the substituted enzymes. The enzymes with Gln and Val substitutions appear to be totally inactive. The results obtained support a recent suggestion that Glu-537 is an important catalytic residue of beta-galactosidase.

scholarly journals Hydrolysis of dietary flavonoid glycosides by strains of intestinal Bacteroides from humans

1987 ◽  
Vol 248 (3) ◽  
pp. 953-956 ◽  
Author(s):  
V D Bokkenheuser ◽  
C H Shackleton ◽  
J Winter
Keyword(s):  
Healthy Subjects ◽  
Enzymic Activity ◽  
Flavonoid Glycosides ◽  
Faecal Flora ◽  
A Cell ◽  
Bacteroides Ovatus ◽  
Dietary Flavonoid ◽  
Beta Glucosidase ◽  
Hydrolysis Of ◽  
Beta Galactosidase

Rutin and quercitrin are hydrolysed to quercetin, and robinin is hydrolysed to kaempferol, by faecal flora from healthy subjects. The enzymes required for these hydrolyses, namely alpha-rhamnosidase and beta-galactosidase, were produced by some strains of Bacteroides distasonis; other strains, however, synthesized beta-glucosidase. The last-named enzyme was also elaborated by Bacteroides uniformis and Bacteroides ovatus. All the enzymes were produced constitutively. A cell-free extract of B. distasonis containing beta-glucosidase displayed an enzymic activity of 1 mumol/10 min per 10 mg of protein.

scholarly journals Post-secretional modification of exo-1,3-β-d-glucanases from Saccharomyces cerevisiae

1983 ◽  
Vol 215 (3) ◽  
pp. 471-474 ◽  
Author(s):  
A Sánchez ◽  
A R Nebreda ◽  
J R Villanueva ◽  
T G Villa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Concanavalin A ◽  
Carbohydrate Moiety ◽  
Enzyme Form

Exo-1,3-beta-D-glucanase secreted by Saccharomyces cerevisiae undergoes extracellular modifications in its carbohydrate moiety that change the affinity towards the lectin concanavalin A. The transition of negatively reacting enzyme form into positively reacting one depends on temperature. Results from experiments with glucono-delta-lactone and from treatments in vitro with hydrolases suggest a glycosidase-mediated mechanism.

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