Development of Antibody to Human GM3 Synthase and Immunodetection of the Enzyme in Human Tissues

N. K. Golovanova^1*, N. N. Samovilova¹, E. V. Gracheva¹, M. M. Peklo¹, T. N. Vlasik¹, A. Yu. Sobolev², Yu. V. Jurchenko², and N. V. Prokazova¹

¹Institute of Experimental Cardiology, Cardiology Research Center, Russian Ministry of Health, 3-ya Cherepkovskaya ul. 15A, Moscow 121552, Russia; fax: (7-095) 149-0559; E-mail: golov@cardio.ru

²Institute of Molecular Genetics, Russian Academy of Sciences, pl. Akademika Kurchatova 2, Moscow 123182, Russia; fax: (7-095) 196-0221; E-mail: alex_sobolev@aport.ru

^* To whom correspondence should be addressed.

Received July 18, 2003

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Polyclonal antibody was raised to a cloned fragment of human GM3 synthase. Affinity purified R27C1 antibody to the tagged recombinant protein inhibited GM3 synthase activity in human liver and HL-60 cells in a dose-dependent manner. However, the R27C1 antibody did not affect liver sialyltransferase activity towards asialofetuin. We are the first to measure GM3 synthase activity in human liver (194 ± 60 pmol NeuAc/h per mg protein), which was about 10-fold lower than in phorbol myristate acetate-stimulated HL-60 cells (1353 ± 573 pmol NeuAc/h per mg protein). On immunoblotting the R27C1 antibody recognized a common protein band in a number of human tissues (liver, brain, atherosclerotic aortic intima, HL-60 cells) with molecular mass of about 60 kD, which is similar to that of the purified GM3 synthase from rat liver. In human liver and aortic intima, the 60-kD band was almost a single band, which makes possible the use of the R27C1 antibody for immunohistochemical studies in these tissues.

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KEY WORDS: human GM3 synthase activity, antibody to human GM3 synthase, human liver, HL-60 cells

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