Purification and Properties of UDP-GlcNAc:Dolichyl-Pyrophosphoryl-GlcNAc GlcNAc Transferase from Mung Bean Seedling

Kaushal, Gur P.; Elbein, Alan D.

doi:10.1104/pp.81.4.1086

Cited by 22 publications

(4 citation statements)

References 32 publications

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“…The pH optima and kinetic characteristics of the maize endosperm glycosyltransferases were similar to those described for these enzymes from other plant sources (11,18,19,30). The antibiotic tunicamycin, which resembles a transition-state reaction intermediate, inhibits the first enzyme of the dolichol pathway blocking the formation of GlcNAc-PP-dolichol (19).…”

Section: Discussionsupporting

confidence: 52%

Glycoprotein Synthesis in Maize Endosperm Cells

Riedell

Miernyk²

1988

Plant Physiol.

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Microsomal membrane preparations from maize (Zea mays L., inbred A636) endosperm cultures contained enzymes that transferred sugar moieties from uridine diphosphate-N-acetylglucosamine, guanosine diphosphate-mannose, and uridine diphosphate-glucose to dolichol-phosphate. These enzyme activities were characterized with respect to detergent and pH optima, substrate kinetic constants, and product and antibiotic inhibition constants. It was demonstrated by mild acid hydrolysis and high performance liquid chromatography that the products of the N-acetylglucosamine transferases were N-acetylglucosamine-pyrophosphoryl-dolichol and N,N'-diacetyl-chitobiosyl-pyrophosphoryl-dolichol and that the product of the mannose transferase was mannosyl-phosphoryl-dolichol. A large proportion of the products of the glucose transferase activity was stable to mild acid hydrolysis. However, the proportion that was labile was identified as glucosyl-phosphoryl-dolichol. Rate zonal sedimentation and isopycnic banding in linear sucrose density gradients in the presence of 1 millimolar ethylenediaminetetraacetic acid indicated that the glycosyltransferase activities were located in the endoplasmic reticulum. The glycosyltransferases were not solubilized by 500 millimolar KCI or by sequential washes with tris-(hydroxymethyl)aminomethane and water, treatments that release peripheral membrane proteins. Solubilization was achieved with low concentrations of Triton X-100. When sealed microsomal vesicles were incubated with trypsin for 30 minutes in absence of detergent, the activity of N-acetylglucosaminyl-transferase was substantially reduced, while the activity of the glucosyl-transferase was somewhat reduced. Activity of the mannosyl-transferase was resistant to inactivation by incubation with trypsin unless Triton was present.

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Section: Discussionsupporting

confidence: 52%

Glycoprotein Synthesis in Maize Endosperm Cells

Riedell

Miernyk²

1988

Plant Physiol.

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“…At the end of this time, the mixture was adjusted to a CHC13-C-H30H-H20 mixture of 1:1:1 by the addition of water, and the lower layer and interface were removed, concentrated to dryness, and redissolved in CHC13-CH30H-H20 (10:10:3). To this fraction, a small amount of radioactive (20000 cpm) dolichyl-PP-(GlcNAc)2, prepared as previously described (Kaushal & Elbein, 1986b), was added as a marker. The mixture (in 10:10:3) was applied to a DEAE-cellulose (in acetate) column that had been previously equilibrated with CHCl3-CH3OH-H20 (10:10:3).…”

Section: Methodsmentioning

confidence: 99%

Partial purification and characterization of .beta.-mannosyltransferase from suspension-cultured soybean cells

Kaushal¹,

Elbein²

1987

Biochemistry

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The beta-mannosyltransferase that catalyzes the synthesis of Man-beta-GlcNAc-GlcNAc-PP-dolichol from GDP-mannose and dolichyl-PP-GlcNAc-GlcNAc was solubilized from microsomes of suspension-cultured soybean cells by treatment with 1.5% Triton X-100 and was purified about 700-fold by chromatography on DEAE-cellulose, hydroxylapatite, and a GDP affinity column. The purified enzyme was reasonably stable in the presence of 20% glycerol and 0.5 mM dithiothreitol. The enzyme required either detergent (Triton X-100 or NP-40) or phospholipid for maximum activity, but the effects of these two were not additive. Thus, either phosphatidylcholine or Triton X-100 could give maximum stimulation. In terms of phospholipid stimulation, both the head group and the acyl chain appeared to be important since phosphatidylcholines with 18-carbon unsaturated fatty acids were most effective. The purified enzyme had a sharp pH optimum of 6.9-7.0 and required a divalent cation. Mg2+ was the best metal ion with optimum activity occurring at 6 mM, but Mn2+ was reasonably effective while Ca2+ was slightly stimulatory. The Km for GDP-mannose was calculated to be 1.7 X 10(-6) M and that for dolichyl-PP-GlcNAc-GlcNAc about 9 X 10(-6) M. The enzyme was inhibited by a number of guanosine nucleotides such as GDP-glucose, GDP, GMP, and GTP, but various uridine and adenosine nucleotides were without effect. The purified enzyme was apparently free of alpha-1,3-mannosyltransferase (and perhaps other mannosyltransferases) and dolichyl-P-mannose synthase since the only product seen from dolichyl-PP-GlcNAc-GlcNAc and GDP-mannose was Man-beta-GlcNAc-GlcNAc-PP-dolichol.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…However, tunicamycin had no effect on other GlcNAc transferases such as the enzyme that adds the next GlcNAc to dolichyl-PP-GlcNAc (111), the enzymes that add terminal GlcNAc residues to the N-linked oligosaccharide core (107), the phosphotransferase that adds GlcNAc-1-P to high-mannose chains on lysosomal enzymes (112), and the trans ferase that adds GlcNAc to serine residues on nuclear proteins (113). It also had no effect on the formation of undecaprenyl-PP-galactose or undecaprenyl-PP-GalNAc (114), but at high concentrations it did inhibit formation of dolichyl-P-glucose (115).…”

Section: A Tunicamycinmentioning

confidence: 97%