Molecular characterization of NAD:arginine ADP-ribosyltransferase from rabbit skeletal muscle.
Mono-ADP-ribosylation is a reversible modification of proteins, with NAD:arginine ADP-ribosyltransferases (EC 2.4.2.31) and ADP-ribosylarginine hydrolases (EC 3.2.2.19) catalyzing the opposing reactions in an ADP-ribosylation cycle. A membrane-associated arginine-specific (mono)-ADP-ribosyltransferase was purified 215,000-fold from rabbit skeletal muscle. On the basis of the amino acid sequences of HPLC-purified tryptic peptides, degenerate oligonucleotide primers were synthesized and used in a polymerase chain reaction (PCR)-based procedure to generate cDNA. A specific probe, based on PCR-generated sequence, was used to screen a rabbit skeletal muscle cDNA library. A composite cDNA sequence, obtained from library screening and rapid amplification of the 5' end of the cDNA, contained a 981-base-pair open reading frame, encoding a 36,134-Da protein. The deduced amino acid sequence contained the sequences of the tryptic peptides, hydrophobic amino and carboxyl termini, and two potential sites for N-linked glycosylation. Escherichia coli cells transformed with an expression vector containing transferase-specific sequence expressed ADP-ribosyltransferase activity. A transferase-specific oligonucleotide probe recognized a 4-kilobase mRNA expressed primarily in rabbit skeletal and cardiac muscle. There was no extended similarity in deduced amino acid sequences of the muscle transferase and several bacterial ADP-ribosylating toxins. The hydrophobic amino and carboxyl termini may represent a signal peptide and a site for a glycosyl-phosphatidylinositol anchor, respectively.
Zolkiewska
A
A
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Nightingale
M S
MS
Moss
J
J
D036002:ADP Ribose Transferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins; D012995:Solubility
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D036002:ADP Ribose Transferases