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Chinese hamster ovary cells with reduced hexokinase activity maintain normal GDP‐mannose levels
Author(s) -
O'Rear Jessica L.,
Scocca Jane R.,
Walker Brian K.,
Kaiden Adina,
Krag Sharon S.
Publication year - 1999
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/(sici)1097-4644(19990101)72:1<56::aid-jcb7>3.0.co;2-h
Subject(s) - mannose , chinese hamster ovary cell , hexokinase , glycoprotein , biochemistry , biology , dolichol , microbiology and biotechnology , metabolism , enzyme , biosynthesis , glycolysis , receptor
Parental Chinese hamster ovary (CHO) cells were mutagenized and subjected first to a mannose suicide selection technique and second to a screen of individual colonies grown on polyester discs for reduced mannose incorporation into protein. The incorporation of radioactivity for the selection and the screen was conducted at 41.5°C instead of the normal growth temperature of 34°C in order to allow for the isolation of temperature‐sensitive lesions. This selection/screening procedure resulted in the isolation of MI5–4 cells, which had three‐ to five‐fold lower incorporation of [2‐ 3 H]mannose into mannose 6‐phosphate, mannose 1‐phosphate, GDP‐mannose, oligosaccharide‐lipid, and glycoprotein at 41.5°C. We detected no difference in the qualitative pattern of mannose‐labeled lipid‐linked oligosaccharides compared to parental cells. MI5–4 cells synthesized dolichol. The defect of MI5–4 cells was determined to be in hexokinase activity; crude cytosolic extracts were eight‐ to nine‐fold lower in hexokinase activity in MI5–4 cells compared to parental cells. As a result of this defect, incorporation of labeled mannose from the medium was significantly decreased. However, the level of GDP‐mannose in MI5–4 cells was 70% of normal. The phenotype of MI5–4 was a lower specific activity of labeled GDP‐mannose, not a substantial reduction in the level of GDP‐mannose. Consistent with these results, no alterations in the glycosylation of a model glycoprotein, G protein of vesicular stomatitis virus, were observed. These cells grew slower than parental cells, especially in low‐glucose medium. J. Cell. Biochem. 72:56–66, 1999. © 1999 Wiley‐Liss, Inc.