M. Tihova et al., ELECTRON-MICROSCOPY OF CYTOCHROME-C OXIDASE-CONTAINING PROTEOLIPOSOMES - IMAGING ANALYSIS OF PROTEIN ORIENTATION AND MONOMER-DIMER BEHAVIOR, Biochemical journal, 292, 1993, pp. 933-946
1. Cytochrome c oxidase-containing vesicles were prepared by cholate d
ialysis using bovine heart cytochrome c oxidase with egg and sphatidyl
choline/dioleoylphosphatidylethanolamines (1: 1, w/w) at two ratios of
phospholipid to protein (25 mg/mg and 10 mg/mg). With each mixture, o
ne or two (FII, FIII) fractions with mostly outward-facing cytochrome
aa3 were separated from a fraction (FI) containing mostly inward-facin
g enzyme and protein-free liposomes by DEAE-Sephacel chromatography. 2
. FII and FIII fractions from egg phospholipid mixtures had 60-80% out
ward-facing enzyme; FII and FIII fractions from dioleoyl phospholipids
showed 50-70% outward-facing enzyme. Egg and dioleoyl phospholipid mi
xtures maintained good respiratory control ratios (8-13) only at the h
igher lipid/protein ratios. 3. Platinum/carbon replicas of freeze-frac
tured vesicle surfaces were subjected to image analysis. The results s
howed two types of membrane projection with average heights of 7.5 nm
and 3.5 nm from the fracture plane. The former were more numerous on t
he convex faces. Calculated areas of the projections indicated the pro
bable presence of both enzyme dimers and higher aggregates. Oxidase di
mers may have membrane areas of 70 80 nm2 at the high (7.5 nm) side an
d 40-50 nM2 on the low (3.5 tim) side. 4. Proteoliposomes prepared wit
h enzyme depleted of subunit III contained predominantly much smaller
projecting areas. These probably represent monomers with high side are
as of 35-40 nm2 and low side areas of 20-25 nm2. Electron microscopy t
hus directly confirms the predicted change of aggregation state result
ing from subunit depletion. 5. The results are compared with those fro
m two-dimensional crystals. Assuming that the high and low projections
are two sides of one family of transmembrane molecules, a total lengt
h of 11 nm matches 11-12 nm lengths obtained by crystallography. Our m
embrane areas match the areas obtained in earlier 'crystal' studies be
tter than the small areas obtained recently by electron cryomicroscopy
.