cDNA cloning, expression and characterization of nitric-oxide synthase from the salivary glands of the blood-sucking insect Rhodnius prolixus.
Rhodnius prolixus, a blood-sucking bug, is a unique insect that is known to produce nitric oxide (NO) in the salivary glands to use as a vasodilator for blood sucking. We report here the cloning of the NO synthase (NOS) cDNA from these salivary glands and its expression in a baculovirus system. This cDNA encodes a protein of 1174 amino acids with a calculated molecular mass of 132,331 Da. The primary structures of mammalian NOS, including the putative cofactor-recognition sites for heme, tetrahydrobiopterin (BH4), calmodulin. FMN, FAD and NADPH are all conserved in salivary-gland NOS. Recombinant salivary-gland NOS differed from nerve NOS and endothelial NOS in that it lacked a large N-terminal domain and an N-terminal myristylation sequence, respectively. Salivary-gland NOS produced in a baculovirus system showed NOS activity and demonstrated that salivary-gland NOS was soluble and was Ca2+ and calmodulin dependent, similarly to mammalian constitutive NOS isoforms. Recombinant salivary-gland NOS was purified to near homogeneity and migrated at 130 kDa on SDS/PAGE.
Yuda
M
M
Department of Medical Zoology, School of Medicine, Mie University, Tsu, Japan.
Hirai
M
M
Miura
K
K
Matsumura
H
H
Ando
K
K
Chinzei
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D017924:Nucleopolyhedroviruses; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012239:Rhodnius / Q000201:enzymology*; D012469:Salivary Glands / Q000201:enzymology; D016415:Sequence Alignment
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019001:Nitric Oxide Synthase