POSTERS RELATED T O BIOLOGICAL OXIDATIONS
THU 2 A P R I L
Thu-J-40B
Thu-J-41B
CHANGES OF MEMBRANE POTENTIAL DURING NITRATE
RESPIRATION BY ESCHERICHIA COLI K 32 VESICLES
ANOMALOUS REDUCTION OF CYT b IN HIGHLY
PURIFIED COMPLEX I11 FROM BAKER'S YEAST.
F.F. de la Rosa and G. Palmer". Dpt. of
Biochemistry,University o f Sevilla,Spain.
* h t . of Biochemistry. Rice University.
Hobston, USA.
Tapani Huttunen
Department of Biochemistry, University of Turku,
SF-20500 Turku 50, Finland
The extents of the steady-state nlenibrane potentials (dy),generated by the E_. coli membrane vesicles during the oxidation of formate with the acceptors nitrate, duroquinone or oxygen, were compared to those obtained by inhibiting the electron
transfer at the levels of formate ciehycirogenase
(FDH), cytochronie b_ or terminal oxidases. The d
increased by about two t o three-fold up to a value
of about 250 nlV (negative inside), which is substantially larger than reported previously, upon
inhibition of the activity of FDH by hypophosphite.
This inhibitor decreased the initial specific activities of about 2-3 pmoles/min/nig prot. by 75 to
90%. Since no corresponding increase of b y was
observed in the presence of the other inhibitors,
it is suggested that the extent of the steadystate d y is controlled by the redox state of the
electron transfer chain below the level of cytochrome(s) b_.
In highly purified bc -complex frombaker's
yeast,the reduction o * cyt 5 and partial
reduction o f cyt
is obtainkd by catalytic
amount o f succinate dehydrogenase and succinate in the presence o f 7pM antimycin.
After the addition of ferricyanide the s,
is re-oxidized and a increase in thereduction of
is observed.Using stopped-flow
we established that the oxidation o f 5 by
ferricyanide proceeds as a pseudo-firs$
order reaction and the reduction of
is
faster and with two phases.0ur observation
suggests that these two processes are not
directly interconected and that other corn
ponent than 2, must be the "control factor"
in the anomalous reduction of cyt b_.
Thu-J-42B
Thu-J-43B
INSIDE-OUT SUBMITOCHONDRIAL PARTICLES (SMP) FROM
PLAN" MITOCHONDRIA
CROSS LINKING CYTOCHROME C T O YEAST
CYTOCHROME C PEROXIDASE
ett
ew.G.W. and Seilman,S.
F e t . E t ,Dept .of Biochemistry,Royal (Dick)
School o f Vet .S tudies ,Summerhall,
-
.
'
,
Bergman, A.2 Mdller, I.M.I, Gardestrdm, P
Ericson, I.2'and Palmer, J.M.I
'Dept. Bot. , Imperial College, Prince Consort Rd.,
London SW7 2BB, England
*Dept.Biochem., Umea Univ. , 901 87 Umei., Sweden
SMP were produced from Amunmculatwn mitochondria
by sonication. The SMP were about 90% inside-out
as measured by the oxidation of reduced cytochrome c -+ Triton X-100. l%o peaks were found by
analyzing the particles by counter-current distribution in a dextran-PEG two-phase system. The
main peak was about 9 5 % inside-out and the minor
about 85%. Purification of the main peak could
also be performed by extracting the bottom phase
three times with top phase. The properties of
NRDH oxidation by these inside-out SMP w i l l be
presented and discussed in relation to the known
characteristics of malate oxidation by plant
mitochondria.
Edinburgh,UK
Cytochrome c (horse heart) was
covalently linked to yeast cytochrome c
peroxidase using the cleavable reagent
dithiobis (succinimidyl propionate) a t pH 7
The cross linking is inhibited by polyglutamate and reversed by mercaptoethanol.
The cross linked species c a n be isolated
in 5% yield after DEAE cellulose
chromatography and contains 1 cytochrome
and 1 peroxidase. The ability of the
complex to accept electrons and carry out
the reduction of peroxide has been studied
T h e relative orientation of the two
proteins can be assessed b y investigation
of cross linked peptides
'
.
Thu-J-44B
Thu-J-45B
E f f e c t s of 4 - k e t o a c i d s on peroxisonal
f -oxidation,
H.Osmundsen, I n s t i t u t e of Medical Biochemistry, University of Oslo, Oslo,
Norway.
Peroxisomalp-oxidation i s i n r a t l i v e r s
induced by hypolipidaemic drugs and by
high € a t d i e t s . I t i s n o t clear how t h i s
process i s i n t e g r a t e d i n t o c e l l u l a r metabolism. This p o i n t i s r e l e v a n t a s r e g a r d s
d i s p o s a l of NADH from peroxisomal netabol i s m . I t i s shown t h a t f - o x i d a t i o n by i s o l a t e d peroxisomal f r a c t i o n s i s s t i m u l a t e d
by more than 1 0 0 % by addedd-keto a c i d s ,
i n p a r t i c u l a r o x a l o a c e t a t e and pyruvate.
I n h i b i t i o n by added NADII i s p a r t i a l l y rel i e v e d by added&-keto a c i d s . A l s o extent
of f a t t y a c i d o x i d a t i o n is markedly influeneced by added<-keto a c i d s . I t i s
suggested t h a t re-oxidation of N M U can
be r a t e - l i m i t i n g for peroxisomal
isod a t i o n . R a t l i v e r peroxisomes w e e-oxil a t e d i n a self-generated P e r c o l l gradient.
O R I G I N OF PHOSPHORYLATION COUPLED TO THE OXIDATION OF EXTRAMITOCHONDRIAL NADH
L. Ernster. B.S. Bharaj*, and K. Nordenbrand
Dept. o f Biochemistry, Arrhenius Laboratory.
Univ. o f Stockholm. S-106 91 Stockholm. Sweden
Demonstration o f ATP synthesis coupled t o t h e oxid a t i o n o f extramitochondrial NADH c o n s t i t u t e s important evidence f o r t h e occurrence o f phosphoryl a t i o n i n t h e r e s p i r a t o r y chain. This o x i d a t i o n
i s dependent on added c y t - c , i s i n s e n s i t i v e t o
amytal. rotenone and antimycin. and probably proceeds v i a t h e outer-membrane NADH-cyt.bg reductase-cyt.bg pathway and cyt.c oxidase. A t low (< 2
mM) NADH. t h e PI0 r a t i o i s < 1. and t h e phosphor y l a t i o n o r i g i n a t e s mostly from S i t e 111. A t high
( > 5 mM) NADH. o r i n t h e presence o f f l u o r i d e s t h e
o x i d a t i o n i s s t i l l cyt.c dependent and i n s e n s i t i ve t o t h e above i n h i b i t o r s . but t h e PI0 r a t i o i s
b 2 . and t h e phosphorylation i s l a r g e l y abolished
by amytal. rotenone and antimycin, i n d i c a t i n g
t h a t i t involves t h e o x i d a t i o n o f intramitochondr i a l NADH and or: i n a t e s mainly f r o m S i t e s I and
SAREC! Sw den! o n - l e ve from
11. - *Fellqw
t h e Dept.of Blochem., Univ.07 N a i r o b i , Wenya.
B
07
319P