Development of antibody to human GM3 synthase and immunodetection of the enzyme in human tissues.
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.
Golovanova
N K
NK
Institute of Experimental Cardiology, Cardiology Research Center, Russian Ministry of Health, Moscow 121552, Russia. golov@cardio.ru
Samovilova
N N
NN
Gracheva
E V
EV
Peklo
M M
MM
Vlasik
T N
TN
Sobolev
A Yu
AY
Jurchenko
Yu V
YV
Prokazova
N V
NV
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D001011:Aorta / Q000201:enzymology* / Q000276:immunology / Q000473:pathology; D001161:Arteriosclerosis / Q000201:enzymology / Q000276:immunology / Q000473:pathology; D001212:Asialoglycoproteins / Q000737:chemistry; D001921:Brain / Q000201:enzymology* / Q000276:immunology; D060748:Fetuins; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology* / Q000276:immunology; D008970:Molecular Weight; D009928:Organ Specificity / Q000276:immunology; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012799:Sialyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D017539:Tunica Intima / Q000201:enzymology* / Q000276:immunology / Q000473:pathology; D000509:alpha-Fetoproteins / Q000737:chemistry
D000906:Antibodies; D001212:Asialoglycoproteins; D060748:Fetuins; D011994:Recombinant Proteins; D000509:alpha-Fetoproteins; C017029:asialofetuin; D012799:Sialyltransferases; C021343:haematoside synthetase