PMID: 2125471Sep 25, 1990Paper

Purification and characterization of dolichyl-P-mannose:Man5(GlcNAc)2-PP-dolichol mannosyltransferase

Biochemistry
C B SharmaA D Elbein

Abstract

The dolichyl-P-mannose:dolichyl-PP-heptasaccharide alpha-mannosyltransferase (2.4.1.130), which catalyzes the transfer of mannose from dolichyl-P-mannose to the Man5(GlcNAc)2-PP-dolichol acceptor glycolipid, was solubilized from pig aorta microsomes with 0.5% NP-40 and purified 985-fold by a variety of conventional methods. The partially purified enzyme had a pH optimum of 6.5 and required Ca2+, at an optimum concentration of 8-10 mM, for activity. Mn2+ was only 20% as effective as Ca2+, and Mg2+ was inhibitory. The mannosyltransferase activity was also inhibited by the addition of EDTA to the enzyme, but this inhibition was fully reversible by the addition of Ca2+. The enzyme was quite specific for dolichyl-P-mannose as the mannosyl donor and Man5(GlcNAc)2-PP-dolichol as the mannosyl acceptor. The Km values for dolichyl-P-mannose and the acceptor lipid Man5(GlcNAc)2-PP-dolichol were 1.8 and 1.6 microM. On Bio-Gel P-4 columns and by HPLC, the radiolabeled oligosaccharide formed during incubation of dolichyl-P-[14C]mannose and unlabeled Man5(GlcNAc)2-PP-dolichol with the purified enzyme behaved like Man6(GlcNAc)2. This octasaccharide was susceptible to digestion by endoglucosaminidase H, indicating that the newly added mannose w...Continue Reading

References

Jan 1, 1987·Annual Review of Biochemistry·A D Elbein
Jan 1, 1987·Annual Review of Biochemistry·C B Hirschberg, M D Snider
Jan 1, 1987·Annual Review of Biochemistry·M A KukuruzinskaB J Jackson
Apr 27, 1987·Biochimica Et Biophysica Acta·W Tanner, L Lehle
May 1, 1987·Analytical Biochemistry·S HiraniJ R Rasmussen
Jan 1, 1985·Annual Review of Biochemistry·R Kornfeld, S Kornfeld
Jan 1, 1981·Annual Review of Biochemistry·S C Hubbard, R J Ivatt

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Citations

Dec 21, 1993·Biochimica Et Biophysica Acta·R Kleene, E G Berger
Nov 24, 1998·Critical Reviews in Oral Biology and Medicine : an Official Publication of the American Association of Oral Biologists·M A Kukuruzinska, K Lennon

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