- Endogenous inhibitors of insect microsomal oxidases are a serious
problem in in-vitro studies of insecticide metabolism.
chemical nature, modes of action, and their effectiveness as inhibitors
In the case of house flies, there may be as many as
The most important of these is known to be heat
stable and to be found mainly in the head of the fly.
was investigated to determine its identity and mode of action.
as assayed by its effect on microsomal aldrin
Fly heads were homogenized with distilled water, heated,
filtered and used as inhibitor preparations.
was used to analyze for dieldrin, the product of the enzyme reaction.
The activities of microsomal NADPH:cytochrome C oxidoreductase and
NADPH:neotetrazolium oxidoreductase were measured spectrophotnmetrically using cytochrome C, 2,6-dichlorophenolindophenol (DCPIP), and neotetrazolium as substrates. The house flies used in these experiments differed genetically in respect to their level and type of insecticide
resistance, their microsomal oxidase level, and their eye color.
The head inhibitor did not affect the stability of the epoxidase
enzyme since the percent inhibition was constant at various times of
Double reciprocal plots of reaction velocity versus
substrate concentration showed that the inhibitor is not a competitor
with aldrin for the same active site on the enzyme.
these methods, the inhibitor does not compete with NADPH for the site
of reaction, but it does reduce the activity of the microsomal electron
The amount of head inhibitor was not dependent on the age, sex,
nature of insecticide resistance, or microsomal oxidase activity of
However, the inhibitor was absent in house fly strains with
the white and ocra eye color mutations and was present at a reduced
level in the flies with carmine colored eyes.
These results showed
that the pigment required for wild type eye coloration is directly
involved with the inhibitor.
This pigment, xanthommatin, a product of
tryptophane metabolism, is absent in strains with genetic blocks at
the third and fifth chromosomes.
Xanthommatin was obtained by synthesis and by isolation from fly
When included at 5 x 10⁻⁶ M and 5 x 10⁻⁷ M in microsome
incubations, xanthommatin inhibited the activity of the epoxidase
enzyme 72.5% and 17.5%, respectively.
The precursor of xanthommatin,
3-hydroxykynurenine, was not inhibitory. Xanthommatin increased the rate of oxidation of NADPH by the
microsomes, as did cytochrome C, DCPIP, and neotetrazolium. Because
dihydroxanthommatin is rapidly air-oxidized, the reduction of xanthommatin by NADPH and microsomes could be detected only under anaerobic
it was concluded that xanthommatin serves as an electron
acceptor for the microsomal electron transport system, limiting the
supply of electrons to the epoxidase system.
The inhibitory effect
of xanthommatin is enhanced by the auto-oxidation of dihydroxanthommatin which provides additional electron acceptor.
A double-reciprocal plot of enzyme reaction velocity versus
substrate concentration showed that xanthommatin inhibits neotetrazolium
The data indicate that the inhibitor
limits the flow of reducing potential to the non-heme iron protein,
Similar experiments with NADPH:cytochrome C oxidoreductase showed a
Xanthommatin appeared to increase the rate of
cytochrome C reduction, but this increase was found to be non-enzymic.
It is suggested that the site of action of xanthommatin is NADPH:
cytochrome C oxidoreductase.
When tested in epoxidase incubations which contained the inhibitor,
bovine serum albumin (BSA) did not affect the percent inhibition,
effect of xanthommatin on the electron transport components was not
altered by the presence of BSA.
However, BSA decreased the reduction
of DCPIP approximately 30% when used at a level of 1 to 2 mg/fly
equivalent of microsomes.
This same concentration of BSA was found to
be optimum for maximum enhancement of microsomal aldrin
epoxidation. The minimum increase of epoxidase activity due to BSA occurred under optimum conditions of incubation. It was concluded that BSA does not counteract the inhibitory effect of xanthommatin, and it is suggested that the enhancing effect of BSA on the microsomal oxidases is due to an influence on the configuration of the microsome.