Biomedical Engineering Reference
In-Depth Information
2.4.2
Lipid Membrane Stabilization
It was [
117
,
118
] observed by Rubinsky and coworkers that the external cell
membrane integrity was enhanced during cooling, and protection against rupture
by AFGP and AFP occurred [
119
]. They suggested that the enhancement of the
membrane integrity was due to the blocking of ion fluxes across the membrane
by AFGP. Biological antifreezes were then inferred to be directly interacting
with membrane-bound ion channels. However, the suggestion has met with some
disagreement [
119
-
121
]. It was confirmed a few years later by Hayes et al. [
122
]
that AFGP/AFP enhanced membrane integrity during cooling, but they suggest an
alternate mechanism consistent with a nonspecific interaction with the liposome
membrane. In this work, four different types of phospholipids which were all
phosphatidylcholine derived that contained carboxyfluorescein (CF) probes were
used to prepare liposomes. Liposomes were observed to leak up to 50% of the
trapped marker as they were cooled through the thermotropic phase transition
in the absence of AFGP. During this transition state (typically between 12 and
41
ı
C), both gel and liquid crystalline states of the lipid membrane coexist [
122
-
124
], provoking a mismatch of the hydrocarbon chains and ultimately facilitating
leakage of the lipid membrane. In the presence of less than 1 mg/mL of AFGP,
no leakage was observed during cooling or warming through the thermotropic
phase transition. Other proteins were also shown to inhibit leakage. The study by
Hayes et al. was further expanded by Wu and Fletcher [
125
] and they examined
interactions of AFGP, AFP type 1-3, and albumin interactions with liposomes
as model cell membranes. Because most cell membranes are more complex
than a phosphatidylcholine (PC) liposome, they prepared liposomes derived from
dielaidoylphosphatidylethanolamine and dielaidoylphosphatidylglycerol. Complex
AFGP was found to be extremely effective at preventing leakage from the liposomes
as each was cooled through its respective thermotropic phase transition. These
researchers concluded that AFGP may interact with the lipid bilayer in one of
two ways. NMR studies have suggested that AFGP adopts a threefold left-handed
helical conformation. The carbohydrate moieties line up on one side of the helix
while the hydrophobic alanine residues line up on the other [
126
,
127
]. With this
given arrangement, the hydrophilic carbohydrate moieties might interact with the
polar head groups of the lipid bilayer, while the hydrophobic backbone of AFGP
was hypothesized to be partially immersed in the lipid bilayer and that this may be
sufficient to prevent disruption of the bilayer.
The only AFP I that has been studied with liposomes is the naturally occurring
protein TTTT. A concentration of 5 mg/mL of TTTT was found to be able to
completely inhibit the leakage of the fluorophore carboxyfluorescein from the
center of the liposomes as the liposomes were chilled through their thermotropic
phase transition temperature. In contrast, this protection against leakage was not
observed when the same experiments were performed with low-molecular-weight
fractions of AFGPs. Independent experiments indicated that significant amounts
of TTTT remained associated with the lipid portions of the bilayer even after
