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An improved spectrophotometric assay of pyruvate dehydrogenase in lactate dehydrogenase contaminated mitochondrial preparations from human skeletal muscle.
[pyruvate dehydrogenase deficiency]
In
mitochondria-enriched
preparations
of
human
skeletal
muscle
,
the
measurement
of
pyruvate
dehydrogenase
activity
,
as
determined
by
conventional
spectrophotometric
assay
of
NADH
accumulation
,
is
underestimated
due
to
the
oxidizing
activity
of
the
contaminating
lactate
dehydrogenase
.
Using
a
model
reaction
system
consisting
of
varying
mixtures
of
purified
lactate
and
pyruvate
dehydrogenases
,
we
found
that
the
presence
of
oxamate
,
a
competitive
inhibitor
of
the
lactate
dehydrogenase
,
allowed
the
measurement
of
a
linear
rate
of
pyruvate
dehydrogenase
activity
without
interference
from
lactate
dehydrogenase
.
In
the
presence
of
25
mM
oxamate
,
this
holds
true
up
to
a
ratio
of
30
:
1
for
lactate
to
pyruvate
dehydrogenases
,
respectively
.
A
similar
result
was
obtained
when
using
human
skeletal
muscle
mitochondria
contaminated
by
lactate
dehydrogenase
.
Rates
of
pyruvate
dehydrogenase
activity
ranging
from
50
to
120
nmol
/
min
/
mg
protein
could
be
routinely
measured
in
such
mitochondrial
fractions
.
We
concluded
that
the
use
of
oxamate
allows
a
spectrophotometric
assay
for
pyruvate
dehydrogenase
activity
to
be
utilized
when
screening
for
pyruvate
dehydrogenase
deficiency
in
mitochondria-enriched
preparations
of
human
skeletal
muscle
.
