Kinetics of inhibition of purified and mitochondrial cytochrome c oxidase by psychosine (β-galactosylsphingosine)

Abstract

1. Psychosine (beta-galactosylsphingosine) is the toxic agent in Krabbe's disease (globoid cells leukodystrophy). It inhibits purified bovine heart mitochondrial cytochrome c oxidase; there is a rapid phase of inhibition (complete within 10-15 s) and a slower phase (complete within 10-15 min). Both phases are also seen in rat liver mitochondria. IC50 is about 200 microM psychosine in the purified enzyme and less than 20 microM in mitochondria. Psychosine inhibition is due to binding to cytochrome oxidase, not cytochrome c. 2. Bovine heart submitochondrial particles show inhibition similar to rat liver mitochondria. However, although proteoliposomes containing bovine heart cytochrome oxidase show an identical fast phase, they have no noticeable slow phase of inhibition. Addition of phospholipid liposomes to submitochondrial particles relieved the majority of psychosine inhibition, consistent with the removal of those molecules binding in the slow phase. Psychosine can inhibit cytochrome oxidase molecules facing in either direction in proteoliposomes and submitochondrial particles, suggesting that it can rapidly interact with both sides of a membrane when added externally. 3. At high ionic strength, the presence of psychosine decreases the Vmax. of cytochrome oxidase with little effect on the Km for cytochrome c. This non-competitive inhibition suggests that the psychosine-enzyme complex is kinetically inactive and not labile over the time course of the assay. Psychosine does not inhibit the reduction of haem a or haem a3 by artificial electron donors, but does inhibit the reduction of haem a by cytochrome c.

Galactosylsphingosine (psychosine)-induced demyelination is attenuated by sphingosine 1-phosphate signalling

“…Instead, psychosine may induce astrocyte cell death by altering mitochondrial function and electron transport, as determined by increased JC1 levels in the cytosol and decreased NAD(P)H oxidase function measured by MTT, respectively. In agreement with this idea, previous studies have demonstrated that psychosine alters mitochondrial function and electron transfer, probably via a mechanism involving changes in the lipid environment of the membrane (Cooper et al, 1993;Tapasi et al, 1989). Moreover, studies have also reported that pFTY720 can stabilise mitochondrial function, supporting our findings that pFTY720 rescues mitochondrial dysfunction induced by psychosine.…”

Psychosine, the cytotoxic sphingolipid that accumulates in globoid cell leukodystrophy, alters membrane architecture

“…Indeed, several lines of evidence would seem to suggest a membrane-based toxicity mechanism. Psy causes hemolysis ( 32 ), causes the inhibition of cytochrome c oxidase irrespective of the protein's orientation ( 33,34 ), and has never been shown to bind directly to any receptor or signaling protein despite signifi cant investigation. Furthermore, a large number of proteins and cellular systems are affected by psy, making the likelihood that psy directly interacts with every one of them unlikely.…”

Interaction of Peroxynitrite with Mitochondrial Cytochrome Oxidase

“…This indicates that ONOO Ϫ or one of its short-lived breakdown products are responsible for the heme degradation. However, the steady state kinetics are not consistent with simple enzyme inactivation, as, if this were the case, there would be a V max increase with no change in the K m for oxygen (46). A possible clue as to the mechanism for the raised K m can be found in the proteoliposomal studies.…”