Polymorphic forms of human ADP-ribosyltransferase-1 differences in their catalytic activities revealed by labeling of membrane-associated substrates.
Full length cDNA encoding ADP-ribosyltransferase-1 (ART1) was generated from human skeletal muscle. A single base variation from the published sequence was observed (C770-->T), and was established as a polymorphism by the screening of a population of 50 Caucasians. The base variation predicted a nonconservative substitution of Leu for Pro at codon 257. Cell lines with stable and doxycycline-inducible expression of the two polymorphic forms of ART1 were generated from Chinese hamster V79 cells, and exploited in studies to compare the activities of ART1-Pro257 and ART1-Leu257. The results revealed no differences in the capacity of phosphoinositide-specific phospholipase C to cleave the two ART1 isoforms from the plasma membrane. Furthermore, the capacities of ART1-Pro257 and ART1-Leu257 to ADP-ribosylate agmatine or fibroblast growth factor-2 were similar. Differences in the catalytic activities of ART1-Pro257 and ART1-Leu257 were however, identified when measurements were made of their capacities to ADP-ribosylate membrane-associated proteins on the surface of V79 cells. Protein(s) of molecular mass 80-110 kDa were more extensively ADP-ribosylated by ART1-Pro257 than ART1-Leu257, in accordance with the Vmax (59.5 +/- 5.5 and 37.0 +/- 3.0) and Km values (12.5 +/- 4.5 and 5.0 +/- 1. 9) for ART1-Pro257 and ART1-Leu257, respectively.
Yadollahi-Farsani
M
M
Section on Clinical Pharmacology, Division of Medicine, Imperial College School of Medicine, London, UK.
Kefalas
P
P
Saxty
B A
BA
MacDermot
J
J
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D012150:Polymorphism, Restriction Fragment Length; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D017931:DNA Primers; D016222:Fibroblast Growth Factor 2; D011065:Poly(ADP-ribose) Polymerases; D010738:Type C Phospholipases; D043265:Phosphatidylinositol Diacylglycerol-Lyase