Modulation of cytochrome oxidase kinetics by indirect antibody action

Abstract

Polyclonal antibodies raised against isolated subunit V from beef heart cytochrome oxidase or against the intact enzyme increase its apparent affinity for the substrate cytochrome c at the high-affinity site while diminishing the turnover at that site. At the low-affinity site the major action of both types of antibody is to reduce the apparent affinity for cytochrome c. At high ionic strengths the kinetic effect of anti-subunit V is very small although it still binds to the enzyme. The results are interpreted in terms of a model for the enzyme in which antibodies can modulate cytochrome oxidase kinetics by affecting the binding of cytochrome c, even if the antibody-binding site is on a subunit not directly involved in substrate binding.

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

“…This is different from the more complicated effects seen on addition of other inhibitors to the enzyme (e.g. antibodies [19]). Table 1 Effect of psychosine on cytochrome oxidase kinetic parameters…”

“…It is not possible from this trace to say whether this represents a small population of enzyme molecules that have no psychosine bound or that the psychosine-bound enzyme is 10% active. Similar problems exist with interpreting data from antibodyinhibition studies of cytochrome oxidase [19]. However, the degree of inhibition is similar whether the enzyme is assayed polarographically or spectrophotometrically ( Figure 2), suggesting that psychosine is unlikely to be having specific kinetic effects on the enzyme/substrate interaction [18] and favouring the idea that the enzyme-psychosine complex is inactive.…”

“…Liposomes and cytochrome c-loaded cytochrome oxidase proteoliposomes were prepared [17] using 'asolectin' type (Sigma Type IV-S, 'phosphatidylcholine'). Psychosine was stored as a stock solution (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) mg/ml) in 50 % ethanol or a buffer appropriate for the experiment in question. In the latter case, bath sonication was required to dissolve the psychosine.…”