Expression, reconstitution and characterization of prolixin-S as a vasodilator--a salivary gland nitric-oxide-binding hemoprotein of Rhodnius prolixus.
Prolixin-S, an anticoagulant from the salivary gland of the blood-sucking insect Rhodnius prolixus is also one of the members of salivary gland hemoproteins. We produced recombinant protein using a baculovirus-insect cell expression system, reconstituted the hemoprotein and made some characterization of it as a nitric oxide carrier. The reconstituted protein exhibited the absorption spectrum of a high-spin ferric hemoprotein with a Soret absorption peak at 400 nm. By binding nitric oxide (NO-prolixin-S), the Soret band shifted from 400 nm to 420 nm and two sharp bands (Q bands, at 535 nm and 565 nm) also appeared in the visible region. In a bioassay with aortic smooth muscle, NO-prolixin-S showed strong relaxation activity in a dose-dependent manner, which demonstrated that prolixin-S really acts as an NO carrier. A Soret absorption change also indicated that nitric oxide was gradually released under these conditions (pH 7.4 and 37 degrees C). However, at low temperature (20 degrees C) and/or low pH (pH 6), which mimic those in the insect's salivary glands, the releasing became very slow. These different NO-binding properties would enable prolixin-S to reserve nitric oxide in the salivary glands and release it in the host's blood vessels.
Yuda
M
M
Department of Medical Zoology, School of Medicine, Mie University, Tsu, Japan.
Higuchi
K
K
Sun
J
J
Kureishi
Y
Y
Ito
M
M
Chinzei
Y
Y
D000818:Animals; D000925:Anticoagulants / Q000302:isolation & purification / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D006420:Hemeproteins / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D066298:In Vitro Techniques; D019476:Insect Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects; D009569:Nitric Oxide / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D012239:Rhodnius; D012471:Salivary Proteins and Peptides / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D013053:Spectrophotometry; D013696:Temperature; D014665:Vasodilator Agents / Q000302:isolation & purification* / Q000494:pharmacology
D000925:Anticoagulants; D006420:Hemeproteins; D019476:Insect Proteins; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; D014665:Vasodilator Agents; C090560:nitrophorin; D009569:Nitric Oxide