Biosynthesis and characterization of lipid-linked sugars and glycoproteins in aorta.

Chambers, J; Elbein, Alan D.

doi:10.1016/s0021-9258(19)41018-1

Cited by 81 publications

(8 citation statements)

References 58 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Each well was washed with another 1 mL of PBS, and this rinse was added to the initial tube. Radioactivity incorporated into lipid-linked monosacchrides and oligosaccharides was determined after extraction with chloroform/methanol/ water (1:1:1) and chloroform/methanol/water (10:10:3), respectively, as previously described (Chambers & Elbein, 1975). For the incorporation of radiolabel into protein, cells were extracted with TCA at a final concentration of 20% in the presence of 500 µg of bovine serum albumin.…”

Section: Methodsmentioning

confidence: 99%

Australine, a pyrrolizidine alkaloid that inhibits amyloglucosidase and glycoprotein processing

Tropea¹,

Molyneux²,

Kaushal³

et al. 1989

Biochemistry

107

View full text Add to dashboard Cite

Australine [(1R,2R,3R,7S,7aR)-3-(hydroxymethyl)-1,2,7-trihydroxypyrrolizid ine] is a polyhydroxylated pyrrolizidine alkaloid that was isolated from the seeds of the Australian tree Castanospermum australe and characterized by NMR and X-ray diffraction analysis [Molyneux et al. (1988) J. Nat. Prod. (in press)]. Since swainsonine and catanospermine are polyhydroxylated indolizidine alkaloids that inhibit specific glycosidases, we tested australine against a variety of exoglycosidases to determine whether it would inhibit any of these enzymes. This alkaloid proved to be a good inhibitor of the alpha-glucosidase amyloglucosidase (50% inhibition at 5.8 microM), but it did not inhibit beta-glucosidase, alpha- or beta-mannosidase, or alpha- or beta-galactosidase. The inhibition of amyloglucosidase was of a competitive nature. Australine also inhibited the glycoprotein processing enzyme glucosidase I, but had only slight activity toward glucosidase II. When incubated with cultured cells, this alkaloid inhibited glycoprotein processing at the glucosidase I step and caused the accumulation of glycoproteins with Glc3Man7-9(GlcNAc)2-oligosaccharides.

show abstract

Section: Methodsmentioning

confidence: 99%

Australine, a pyrrolizidine alkaloid that inhibits amyloglucosidase and glycoprotein processing

Tropea¹,

Molyneux²,

Kaushal³

et al. 1989

Biochemistry

107

View full text Add to dashboard Cite

show abstract

“…After incubation with label, the monolayers were washed well with phosphate-buffered saline, and the cells were removed from the plates by scraping and placed in tubes. For the analysis of lipid-linked saccharides, the cell suspensions were sequentially extracted with CHC13/CH30H/H20 (1:1:1) and then with CHC13/CH30H/H20 (10:10:3) to isolate the lipid-linked monosaccharides and the lipid-linked oligosaccharides (Chambers & Elbein, 1975). For analysis of protein synthesis, cell suspensions were sonicated and then extracted with 20% trichloroacetic acid.…”

Section: Methodsmentioning

confidence: 99%

Mannostatin A, a new glycoprotein-processing inhibitor

Tropea¹,

Kaushal²,

Pastuszak³

et al. 1990

Biochemistry

View full text Add to dashboard Cite

Mannostatin A is a metabolite produced by the microorganism Streptoverticillium verticillus and reported to be a potent competitive inhibitor of rat epididymal alpha-mannosidase. When tested against a number of other arylglycosidases, mannostatin A was inactive toward alpha- and beta-glucosidase and galactosidase as well as beta-mannosidase, but it was a potent inhibitor of jack bean, mung bean, and rat liver lysosomal alpha-mannosidases, with estimated IC50's of 70 nM, 450 nM, and 160 nM, respectively. The type of inhibition was competitive in nature. This compound also proved to be an effective competitive inhibitor of the glycoprotein-processing enzyme mannosidase II (IC50 of about 10-15 nM with p-nitrophenyl alpha-D-mannopyranoside as substrate, and about 90 nM with [3H]mannose-labeled GlcNAc-Man5GlcNAc as substrate). However, it was virtually inactive toward mannosidase I. The N-acetylated derivative of mannostatin A had no inhibitory activity. In cell culture studies, mannostatin A also proved to be a potent inhibitor of glycoprotein processing. Thus, in influenza virus infected Madin Darby canine kidney (MDCK) cells, mannostatin A blocked the normal formation of complex types of oligosaccharides on the viral glycoproteins and caused the accumulation of hybrid types of oligosaccharides. This observation is in keeping with other data which indicate that the site of action of mannostatin A is mannosidase II. Thus, mannostatin A represents the first nonalkaloidal processing inhibitor and adds to the growing list of chemical structures that can have important biological activity.

show abstract

“…Preparation and Assay of Enzyme Fractions. The intimal layer from fresh pig aorta was homogenized, and a particulate © enzyme fraction was isolated as described previously (Chambers & Elbein, 1975). This particulate fraction was solubilized by mixing it with 0.5% Nonidet P-40 (NP-40) and allowing it to stir in an ice bath for several minutes.…”

Section: Methodsmentioning

confidence: 99%

“…Incubations were done at room temperature or at 37 °C for the times indicated, and reactions were terminated by the addition of 2 mL of CHC13/CH30H (1:1) and 0.5 mL of water. After thorough mixing, the phases were separated by centrifugation, and the dolichyl-linked monosaccharides were isolated as previously described (Chambers & Elbein, 1975;Heifetz & Elbein, 1977). The radioactive contents of these lipids were determined by scintillation counting as a measure of their synthesis.…”

Section: Methodsmentioning

confidence: 99%