[Microtechnic for qualitative and quantitative cardiolipin flocculation test].

This study provides evidence of a novel function for mitochondrial creatine kinase (MtCK) and nucleoside diphosphate kinase (NDPK-D). Both are basic peripheral membrane proteins with symmetrical homo-oligomeric structure, which in the case of MtCK was already shown to allow crossbridging of lipid bilayers. Here, different lipid dilution assays clearly demonstrate that both kinases also facilitate lipid transfer from one bilayer to another. Lipid transfer occurs between liposomes mimicking the lipid composition of mitochondrial contact sites, containing 30 mol % cardiolipin, but transfer does not occur when cardiolipin is replaced by phosphatidylglycerol. Ubiquitous MtCK, but not NDPK-D, shows some specificity in the nature of the lipids transferred and it is not active with phosphatidylcholine alone. MtCK can undergo reversible oligomerization between dimeric and octameric forms, but only the octamer can bridge membranes and promote lipid transfer. Cytochrome c, another basic mitochondrial protein known to bind to anionic membranes but not crosslinking them, is also incapable of promoting lipid transfer. The lipid transfer process does not involve vesicle fusion or loss of the internal contents of the liposomes.

Cardiolipins↗

PubMed2006

Voltage gating of VDAC is regulated by nonlamellar lipids of mitochondrial membranes.

Evidence is accumulating that lipids play important roles in permeabilization of the mitochondria outer membrane (MOM) at the early stage of apoptosis. Lamellar phosphatidylcholine (PC) and nonlamellar phosphatidylethanolamine (PE) lipids are the major membrane components of the MOM. Cardiolipin (CL), the characteristic lipid from the mitochondrial inner membrane, is another nonlamellar lipid recently shown to play a role in MOM permeabilization. We investigate the effect of these three key lipids on the gating properties of the voltage-dependent anion channel (VDAC), the major channel in MOM. We find that PE induces voltage asymmetry in VDAC current-voltage characteristics by promoting channel closure at cis negative applied potentials. Significant asymmetry is also induced by CL. The observed differences in VDAC behavior in PC and PE membranes cannot be explained by differences in the insertion orientation of VDAC in these membranes. Rather, it is clear that the two nonlamellar lipids affect VDAC gating. Using gramicidin A channels as a tool to probe bilayer mechanics, we show that VDAC channels are much more sensitive to the presence of CL than could be expected from the experiments with gramicidin channels. We suggest that this is due to the preferential insertion of VDAC into CL-rich domains. We propose that the specific lipid composition of the mitochondria outer membrane and/or of contact sites might influence MOM permeability by regulating VDAC gating.

Cardiolipins↗

PubMed2006

Phospholipid dependence and liposome reconstitution of purified hyaluronan synthase.

Previous radiation inactivation and enzyme characterization studies demonstrated that the Streptococcus equisimilis hyaluronan synthase (seHAS) is phospholipid-dependent and that cardiolipin (CL) is the best phospholipid for enzyme activation. Here we investigated the ability of seHAS, purified in the absence of added lipid, to be activated by synthetic phosphatidic acid (PA), phosphatidylserine, or CL lipids containing fatty acyl chains of different length or different numbers of double bonds. The most effective lipid was tetraoleoyl CL (TO-CL), whereas tetramyristoyl CL (TM-CL) was ineffective. None of the phosphatidylserine species tested gave significant activation. PAs containing C10 to C18 saturated acyl chains were not effective activators, and neither were oleoyl lyso PA, dilinoleoyl PA, or PA containing one oleoyl chain and either a palmitoyl or stearoyl chain. In contrast, dioleoyl PA stimulated seHAS approximately 10-fold, to approximately 20% of the activity observed with TO-CL. The tested acidic lipids such as PA and CL activated the enzyme most efficiently if they contained only oleic acid. Mixing experiments showed that the enzyme interacts preferentially with TO-CL in the presence of TM-CL. Similarly, seHAS incorporated into phosphotidylcholine-based liposomes showed increasing activity with increasing TO-CL, but not TM-CL, content. Inactivation of membrane-bound seHAS by solubilization with Nonidet P-40 was prevented by TO-CL, but not TM-CL. The pH dependence of seHAS in the presence of synthetic or naturally occurring CLs showed the same pattern of lipid preference between pH 6 and 10.5. Unexpectedly, HAS showed lipid-independent activity at pH 11.5. The results suggest that Class I HAS enzymes are lipid-dependent and that assembly of active seHAS-lipid complexes has high specificity for the phospholipid head group and the nature of the fatty acyl chains.

Cardiolipins↗