Biology Reference
In-Depth Information
belong to the large family of enzymes known as P-Type ATPases because they form a phos-
phorylated intermediate during transport. Their job is to pump Ca
2
þ
from inside to outside
the cell using ATP as the energy source. The pumps maintain constant, low intercellular Ca
2
þ
levels that are required for proper cell signaling. In 1972 Efraim Racker
[53]
reported
a successful reconstitution of the sarcoplasmic reticulum Ca
2
þ
ATPase into soybean phos-
pholipid proteoliposomes. Importantly, the reconstituted vesicles pumped Ca
2
þ
at the
expense of ATP and were sensitive to chlorpromazine, a known inhibitor of calciumATPases.
Two years later, Warren et al.
[54]
reconstituted the SERCA into proteoliposomes where
>
99% of the native membrane lipids had been replaced by synthetic dioleoyl phosphatidyl-
choline (DOPC). However, full expression of the capacity to accumulate Ca
2
þ
into the proteo-
liposomes required the presence of internal oxalate (
Figure 13.19
), a Ca
2
þ
chelator. The
explanation of these observations is that a truly functional calcium ATPase had been recon-
stituted into a proteoliposome devoid of native phospholipids but comprised of a synthetic
PC. However, both the right side out and inside out protein conformations were found in the
proteoliposomes. ATP caused Ca
2
þ
to be pumped into the vesicle interior by one conforma-
tion, whereupon the opposite conformation ATPase would pump the Ca
2
þ
back out, thus
limiting net Ca
2
þ
uptake. In the presence of internal oxalate, Ca
2
þ
that was pumped into
the vesicle was chelated as water-insoluble calcium oxalate (the major component of kidney
stones) and so remained sequestered, resulting in a large increase in net Ca
2
þ
accumulation.
Bacterial Proline Transporter
Reconstitution studies with the bacterial proline transporter into proteoliposomes demon-
strate how a variety of biochemical 'membrane tricks' can be used to deduce the transporter's
molecular mechanism
[55,56]
.
PROTEOLIPOSOME PRODUCTION
The bacterial proline transporter was isolated using a 17 amino acid polyhistidine tag
attached to the C-terminal and a nickel-affinity column
[55]
. The purified transporter was
reconstituted into preformed detergent (Triton X-100) destabilized LUVs. Triton X-100 was
removed by polystyrene beads. Proline transport was supported by PE and PG, but not
PC and CL.
ENERGETICS
Experiments with reconstituted proteoliposomes proved the mechanism of action for the
bacterial proline transporter
[56]
. Proline uptake was shown to be an example of active sym-
port (a form of co-transport, see Chapter 14). The bacterial proline transporter was reconsti-
tuted into proteoliposomes in a KCl buffer and diluted into a NaCl buffer. The
proteoliposomes were therefore 'K
þ
-loaded' with a K
þ
gradient that was high inside and
low outside and a Na
þ
gradient that was high outside and low inside. Upon addition of
the K
þ
-ionophore valinomycin, K
þ
moved down its concentration gradient generating
a net negative potential in the proteoliposome interior. The negative interior drove Na
þ
down its gradient from outside to inside, carrying with it proline. This mechanism is active
sym-port. Formation of the trans-membrane electrical gradient was followed by fluorescence
of the lipophilic cationic fluorescent dye carbocyanine. As carbocyanine entered the proteo-
liposome it became concentrated, resulting in fluorescenc self-quenching. The decrease in net
