average of 1.1 pM translocated enzyme [31]. Note that the D9-NBD trials
occurred for a shorter interval and lower concentration of carrier and
cargo, thus we surmise that D9-NBD is a more potent carrier than
Pep-1. We used 800 nM D9-NBD for these experiments because higher
concentrations easily ruptured the DIB membrane. We also examined
the concentration dependence of D9-NBD HRP transport. A four-fold dilution
of the D9-NBD HRP mixture or absence of carrier greatly reduced
translocation. As a control, the 800 nM D9-NBD 40 nM HRP trial was repeated
but with one important change. The droplets were contacted
only briefly and then separated (no DIB formed) and the source droplet
was analyzed for enzyme activity. In 10 trials, no enzyme activity was
detected, showing that translocation occurs only across the DIB membrane
and not by other means. Pep-1-mediated transport is facilitated
by the application of a transmembrane potential or lipid charge
asymmetry [31]. We were curious to see if D9-NBD translocation also
required similar conditions. In contrast to Pep-1, we found that approximately
the same amount of HRP is carried across the DIB by D9-NBD
regardless of the applied potential polarity (Fig. 3). In addition, while
Pep-1 could not transport HRP across a pure PC DIB at 0 mV applied potential,
D9-NBD spontaneously carried HRP across a neutrally charged
membrane with no voltage (Fig. 3). Since membrane charge asymmetry
can drive Pep-1 mediated translocation in the absence of voltage [31],
we also examined the effect of lipid asymmetry on D9-NBD mediated
average of 1.1 pM translocated enzyme [31]. Note that the D9-NBD trialsoccurred for a shorter interval and lower concentration of carrier andcargo, thus we surmise that D9-NBD is a more potent carrier thanPep-1. We used 800 nM D9-NBD for these experiments because higherconcentrations easily ruptured the DIB membrane. We also examinedthe concentration dependence of D9-NBD HRP transport. A four-fold dilutionof the D9-NBD HRP mixture or absence of carrier greatly reducedtranslocation. As a control, the 800 nM D9-NBD 40 nM HRP trial was repeatedbut with one important change. The droplets were contactedonly briefly and then separated (no DIB formed) and the source dropletwas analyzed for enzyme activity. In 10 trials, no enzyme activity wasdetected, showing that translocation occurs only across the DIB membraneand not by other means. Pep-1-mediated transport is facilitatedby the application of a transmembrane potential or lipid chargeasymmetry [31]. We were curious to see if D9-NBD translocation alsorequired similar conditions. In contrast to Pep-1, we found that approximatelythe same amount of HRP is carried across the DIB by D9-NBDregardless of the applied potential polarity (Fig. 3). In addition, whilePep-1 could not transport HRP across a pure PC DIB at 0 mV applied potential,D9-NBD spontaneously carried HRP across a neutrally chargedmembrane with no voltage (Fig. 3). Since membrane charge asymmetrycan drive Pep-1 mediated translocation in the absence of voltage [31],
we also examined the effect of lipid asymmetry on D9-NBD mediated
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