Ispitivanje elemenata respiratornog lanca gljive Phycomyces blakesleeanus burgeff: veza sa metabolizmom fosfatnih jedinjenja

Abstract

U ovom radu je ispitivano postojanje i aktivnost alternativnih komponenata elektron transportnog lanca kod gljive Phycomyces blakesleeanus, sa akcentom na enzim alternativna oksidaza (AOX), kao i ponašanje respiratornog sistema u uslovima smanjene koncentracije kiseonika. Odgovor respiratornog sistema na ovaj stres je upoređen sa odgovorom druge komponente energetskog metabolizma, polifosfata (PPn), sa namerom da se utvrdi da li su, i na koji način, ova dva metabolička sistema povezana. Ispitivanjima izvedenim pomoću kiseonične elektrode tipa Klark je utvrđeno postojanje aktivnosti alternativnih elemenata respiratornog lanca kod P. blakesleeanus, a za zabeleženu aktivnost su odgovorni AOX, zatim enzim ili enzimski kompleks koji se indukuje u uslovima dugotrajne inhibicije Kompleksa III i nazvan je Kompleks IIIPAR, a vrlo je verovatno da postoji i spoljašnja alternativna NADH:dehidrogenaza (NDE). Utvrđeno je da se AOX sintetiše u citoplazmi nakon čega se enzim unosi u mitohondriju energetski zavisnim transportom. U uslovima smanjene koncentracije kiseonika kapacitet AOX se ne menja, ali učešće enzima u respiraciji značajno raste zbog inhibicije citohrom c oksidaze (COX) u ovim eksperimentalnim uslovima. Ovakav odgovor respiratornog sistema P. blakesleeanus se može pripisati potrebi za brzom odbranom od nastanka reaktivnih kiseoničnih vrsta (ROS) prilikom reoksigenacije koja dovodi do velikog porasta protoka elektrona kroz citohromski respiratorni put. 31P NMR spektroskopija je pokazala da u istim eksperimentalnim uslovima odnos intenziteta centralnog signala PPn i unutarćelijskog neorganskog fosfata (PPc/Pi), koji je dobar pokazatelj energetskog stanja ćelije, opada, ali iznenađujuće, dolazi do rasta nivoa ATP-a. Analiza glavnih komponenti (PCA), je pokazala snažnu negativnu korelaciju između ova dva parametra, ali i negativnu korelaciju između odnosa PPc/Pi i učešća alternativne u ukupnoj respiraciji. Osim toga, dodatak azida, snažnog inhibitora COX, nije dodatno smanjio odnos PPc/Pi u uzorcima micelijuma koji su bili izloženi tretmanu smanjene koncentracije kiseonika. Ovi rezultati ukazuju na povezanost ove dve komponente energetskog metabolizma, odnosno na mogućnost da se hidrolizom PPn nadoknadi manjak ATP-a u ćelijama izazvan inhibicijom citohromskog lanca...

The existence and activity of the alternative components of electron transport chain in fungus Phycomyces blakesleeanus, especially alternative oxidase (AOX), as well as respiratory chain behavior in the conditions of oxygen deprivation, was investigated in this work. Response of respiratory system to these stress conditions was compared to response of another component of energy metabolism, polyphosphates (PPn), with the intention of determining whether, and in what way, are these two metabolic systems connected. Activity of the alternative elements of respiratory chain in P. blakesleeanus was recorded by means of Clark type oxygen electrode, and the elements responsible for this activity were AOX and an enzyme or enzymatic complex induced by long-term inhibition of Complex III which was named Complex IIIPAR. Also, there is a strong possibility of external alternative NADH:dehydrogenase (NDE) existence. It was established that synthesis of AOX takes place in the cytosol, and the enzyme is then imported into the mitochondrion by energy dependent transport. In the conditions of oxygen deprivation AOX capacity is not affected, but its engagement in total respiration increases significantly due to the cytochrome c oxidase (COX) inhibition. This type of respiratory chain response in P. blakesleeanus can be attributed to a need for fast defense from reactive oxygen species (ROS) formed during reoxygenation, which leads to the increase in electron flow through the cytochrome respiratory path. 31P NMR spectroscopy revealed that the intensity ratio of core PPn to intracellular inorganic phosphate signal (PPc/Pi), which is a good indicator of overall cellular energy metabolism, decreases in these experimental conditions, but surprisingly, ATP level raises. Principal component analysis (PCA), has shown strong negative correlation between these two parameters, and also between PPc/Pi ratio and participation of alternative in total respiration. Apart from that, addition of azide, a strong COX inhibitor, did not additionally decrease PPc/Pi ratio in mycelium samples exposed to oxygen deprivation. These results indicate that there is a connection between these two components of energy metabolism, i.e., there is a possibility that the decrease in cellular ATP content caused by cytochrome chain inhibition can be compensated by PPn hydrolysis...