Effect of long-term cyanide exposure on cyanide-sensitive respiration and phosphate metabolism in the fungus Phycomyces blakesleeanus

Abstract

The effects of long-term exposure (5 h) of Phycomyces blakesleeanus mycelium to 5 mM KCN on respiration and phosphate metabolites were tested. Exposure to cyanide, antimycin A and azide lead to a decrease in the activity of cyanide-sensitive respiration (CSR), and the ratio of core polyphosphates (PPc) and inorganic phosphates (Pi), which is a good indicator of the metabolic state of a cell. After 5 h of incubation, the activity of CSR returned to control values. For this, the recovery of cytochrome c oxidase (COX) was required. In addition, the PPc/Pi ratio started to recover shortly after initiation of COX recovery, but never reached control values. This led us to conclude that the regulation of polyphosphate (PPn) levels in the cell is tightly coupled to respiratory chain functioning. In addition, acutely applied cyanide caused two different responses, observed by 31P NMR spectroscopy, that were probably mediated through the mechanism of glycolytic oscillations, triggered by the effect of cyanide on mitochondria. [Projekat Ministarstva nauke Republike Srbije, br. 173040]

ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore

“…The inhibition of ORIC by pretreatment of CD with azide demonstrates the metabolic sensing role of ORIC, since, as described by our group 57 , sodium-azide is a potent blocker of cellular respiration in P.blakesleeanus in the concentration range used in this study. As a consequence of respiration block by azide, the ratio of core polyphosphates to inorganic phosphate (Pp/Pi) in P.blakesleeanus is reduced, showing the e cient reduction of metabolic activity.…”

“…As a consequence of respiration block by azide, the ratio of core polyphosphates to inorganic phosphate (Pp/Pi) in P.blakesleeanus is reduced, showing the e cient reduction of metabolic activity. Pp/Pi is tightly connected to the oxidative phosphorylation chain and was previously shown to be a good indicator of metabolic state of P.blakesleeanus 57 . Inactivation of ORIC under the conditions of ATP scarcity is likely to have a protective role, since hypoosmotic conditions can signal lack of sugars in the environment.…”

ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore

“…In filamentous fungi, the essential role of the hyperosmotic-response pathway in nutrient sensing and its direct connection to metabolism regulation has been described in Neurospora 62 . The inhibition of ORIC by pretreatment of CD with azide demonstrates the metabolic sensing role of ORIC, since, as described by our group 63 , sodium-azide is a potent blocker of cellular respiration in P. blakesleeanus in the concentration range used in this study . As a consequence of respiration block by azide, the ratio of core polyphosphates to inorganic phosphate (Pp/Pi) in P. blakesleeanus is reduced, showing the efficient reduction of metabolic activity.…”

“…As a consequence of respiration block by azide, the ratio of core polyphosphates to inorganic phosphate (Pp/Pi) in P. blakesleeanus is reduced, showing the efficient reduction of metabolic activity. Pp/Pi is tightly connected to the oxidative phosphorylation chain and was previously shown to be a good indicator of metabolic state of P. blakesleeanus 63 . Inactivation of ORIC under the conditions of ATP scarcity is likely to have a protective role, since hypoosmotic conditions can signal lack of sugars in the environment.…”

In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi

“…The cell wall integrity pathway is involved in the response to osmotic stress 54 , fungal morphogenesis, and fungal pathogenicity. The respiratory inhibitor azide was used throughout the protocol because it is the only respiratory chain inhibitor capable of almost complete inhibition of classical respiration for prolonged periods (unlike cyanide 55 ) without significant induction of alternative respiration (unlike antimycin 55 ); its use resulted in a long-term reduction in total respiration to 20% while maintaining viability. Under conditions of this pronounced metabolic suppression, all intracellular signaling pathways that depended on kinase activity and therefore required ATP, including the cell wall integrity response, were assumed to be suppressed.…”