ISOLATION AND CHARACTERIZATION OF A NITRIC OXIDE SYNTHASE (NOS)-LIKE PROTEIN OF PEA (PISUM SATIVUM L.)

Abstract

Nitric oxide synthase (NOS) activity based on citrulline formation assay, which was used in mammalian system, was detected in Pisum Sativum L. (pea) extracts. The pea NOS-like protein was most efficiently extracted with the addition of protease inhibitors (ethylene bis (oxyethylenenitrilo)tetraacetic acid (EGTA) and leupeptin) in the extraction buffer and under alkaline condition (pH 8.5–9.0) as compared to neutral condition in mammalian system. The precipitation of this protein with various concentrations of ammonium sulfate, sodium citrate and sodium chloride caused rapid loss of NOS activity, in contrast to that in the mammalian system, and the protein was not precipitated by organic solvents (acetone or polyethylene glycol, PEG). The pea NOS-like protein was successfully isolated using ion-exchange column, but did not bind to ?-nicotinamide adenine dinucleotide phosphate (NADPH) and calmodulin affinity columns suggesting that it lacked binding sites for the cofactors NADPH and calmodulin that were required for NOS activity in mammalian cells. The results indicated that the pea NOS like protein was significantly different in structure from mammalian NOS.

Aktiviti nitrik oksida sintase (NOS) berdasarkan pembentukan asal cerakin sitrulina yang digunakan dalam sistem mamalia, dapat dikesan dalam ekstrak Pisum Sativum L. (kacang manis). Protein seakan-NOS dalam kacang manis diekstrak paling efisien menggunakan penimbal ekstrak yang ditambah penghalang protease [ethylene bis (oxyethylenenitrilo)tetraacetic acid (EGTA) dan leupeptin)] dan dalam keadaan alkali (pH 8.5–9.0) berbanding keadaan neutral dalam sistem mamalia. Pemendakan protein menggunakan pelbagai kepekatan garam ammonium sulfat, natrium sitrat dan natrium klorida menyebabkan kehilangan aktiviti NOS, berbeza dengan sistem mamalia, dan ia tidak dimendakkan oleh pelarut organik (aseton atau polietilena glikol, PEG). Protein seakan-NOS dalam kacang manis telah diasingkan dengan jayanya menggunakan turus pertukaran-ion, tetapi ia tidak terikat kepada turus ?-nicotinamide adenine dinucleotide phosphate (NADPH) dan kalmodulin yang menunjukkan kekurangan tapak untuk mengikat kofaktor NADPH dan kalmodulin yang diperlukan oleh aktiviti NOS dalam sel mamalia. Keputusan ini menunjukkan bahawa protein seakan-NOS dalam kacang manis adalah berbeza daripada NOS mamalia dari segi struktur.