To test the hypothesis that the activation of protein kinase C (PKC) is influenced by lateral heterogeneities of the components of the lipid bilayer, the thermotropic phase behavior of dimyristoylphosphatidylcholine (DMPC)/dimyristoylphosphatidylserine (DMPS)/dioleoylglycerol (DO) vesicles was compared with the activation of PKC by this system. Differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to monitor the main transition (i.e., the gel-to-fluid phase transition) as a function of mole fraction DO (chi(DO)) in DMPC/DO, DMPS/DO, and [DMPC/DMPS (1:1, mol/mol)]/DO multilamellar vesicles (MLVs). In each case, when chi(DO) < or approximately 0.3, DO significantly broadened the main transition and shifted it to lower temperatures; but when chi(DO) > approximately 0.3, the main transition became highly cooperative, i.e., narrow, again. The coexistence of overlapping narrow and broad transitions was clearly evident in DSC thermograms from chi(DO) approximately 0.1 to chi(DO) approximately 0.3, with the more cooperative transition growing at the expense of the broader one as chi(DO) increased. FTIR spectroscopy, using analogs of DMPC and DMPS with perdeuterated acyl chains, showed that the melting profiles of all three lipid components in [DMPC/DMPS (1:1, mol/mol)]/DO MLVs virtually overlay when chi(DO) = 0.33, suggesting that a new type of phase, with a phospholipid/DO mole ratio near 2:1, is formed in this system. Collectively, the results are consistent with the coexistence of DO-poor and DO-rich domains throughout the compositions chi(DO) approximately 0.1 to chi(DO) approximately 0.3, even at temperatures above the main transition. Comparison of the phase behavior of the binary mixtures with that of the ternary mixtures suggests that DMPS/DO interactions may be more favorable than DMPC/DO interactions in the ternary system, especially in the gel state. PKC activity was measured using [DMPC/DMPS (1:1, mol/mol)]/DO MLVs as the lipid activator. At 35 degrees C (a temperature above the main transition of the lipids), PKC activity increased gradually with increasing chi(DO) from chi(DO) approximately 0.1 to chi(DO) approximately 0.4, and activity remained high at higher DO contents. In contrast, at 2 degrees C (a temperature below the main transition), PKC activity exhibited a maximum between chi(DO) approximately 0.1 and chi(DO) approximately 0.3, and at higher DO contents activity was essentially constant at 20-25% of the activity at the maximum. We infer from these results that the formation of DO-rich domains is related to PKC activation, and when the lipid is in the gel state, the coexistence of DO-poor and DO-rich phases also contributes to PKC activation.

The mixing of phosphatidylserine (PS) and phosphatidylcholine (PC) in fluid bilayer model membranes was studied by measuring binding of aqueous Ca2+ ions. The measured [Ca2+]aq was used to derive the activity coefficient for PS, gamma PS, in the lipid mixture. For (16:0, 18:1) PS in binary mixtures with either (16:0, 18:1)PC, (14:1, 14:1)PC, or (18:1, 18:1)PC, gamma PS > 1; i.e., mixing is nonideal, with PS and PC clustered rather than randomly distributed, despite the electrostatic repulsion between PS headgroups. To understand better this mixing behavior, Monte Carlo simulations of the PS/PC distributions were performed, using Kawasaki relaxation. The excess energy was divided into an electrostatic term Uel and one adjustable term including all other nonideal energy contributions, delta Em. Uel was calculated using a discrete charge theory. Kirkwood's coupling parameter method was used to calculate the excess free energy of mixing, delta GEmix, hence In gamma PS,calc. The values of In gamma PS,calc were equalized by adjusting delta Em in order to find the simulated PS/PC distribution that corresponded to the experimental results. We were thus able to compare the smeared charge calculation of [Ca2+]surf with a calculation ("masked evaluation method") that recognized clustering of the negatively charged PS: clustering was found to have a modest effect on [Ca2+]surf, relative to the smeared charge model. Even though both PS and PC tend to cluster, the long-range nature of the electrostatic repulsion reduces the extent of PS clustering at low PS mole fraction compared to PC clustering at an equivalent low PC mole fraction.

The action of phospholipase A2 (PLA2) toward zwitterionic bilayers is modulated by lateral phase separation of reaction products and substrate. The experiments here address the mechanism of this modulation. PLA2 is particularly active toward lipid dispersions containing reaction products and substrates in which lateral phase separation has occurred. Here, we study PLA2 activity in two related model systems: first in a system in which lateral phase separation can be produced a priori, and second in a system in which the action of PLA2 produces sufficient reaction product in situ such that lateral phase separation occurs. The dispersions in which lateral phase separation occurs a priori form either disk micelles or disk vesicles, not canonical vesicles. When lateral phase separation occurs due to in situ PLA2 activity, there is an abrupt change in vesicle structure and a simultaneous profound increase in catalytic rate. This observation is surprising in light of several reports that vesicles remain intact even when the entire outer monolayer has been hydrolyzed. Membrane curvature and the associated structural defects and dynamic fluctuations in membrane structure have been proposed to modulate PLA2 activity. The mechanism by which lateral phase separation modulates PLA2 activity has been unclear. The data presented here indicate that lateral phase separation affects PLA2 activity by altering membrane curvature and/or inducing defects in the membrane structure.

To test the hypothesis that activation of protein kinase C (PKC) is related to the interface between coexisting diacylglycerol- (DAG-) enriched and DAG-poor phases, the thermotropic phase behavior of the ternary mixtures dimyristoylphosphatidylcholine (DMPC)/dimyristoylphosphatidylserine (DMPS)/dioleoylglycerol (DO), DMPC/DMPS/1-palmitoyl-2-oleoylglycerol (PO), and DMPC/DMPS/dimyristoylglycerol (DM) was analyzed and compared with the ability of the lipid mixtures to support PKC activity. Differential scanning calorimetry (DSC) was used to monitor the gel-to-liquid crystalline phase transition as a function of the mole fraction of DO (chiDO), PO (chiPO), or DM (chiDM) in DMPC/DMPS (1:1) multilamellar vesicles (MLVs) and of chiDO in large unilamellar vesicles (LUVs). The addition of DAG at low mole fractions gave rise to the appearance of two or more overlapping transitions. The phase boundaries of the ternary mixtures deduced from the partial phase diagrams were chiDO = approximately 0.10 and approximately 0.3 for DMPC/DMPS/DO, chiPO = approximately 0.05 and approximately 0.4 for DMPC/DMPS/PO, and chiDM = approximately 0.025 and approximately 0.5-0.6 for DMPC/ DMPS/DM. Above these mole fractions of DAG, the transitions again became very sharp. The ability of the lipid mixtures to support activity of PKC alpha and PKC eta was examined below and above the gel-to-liquid crystalline phase transition. In the gel phase, PKC activity went through a maximum as a function of increasing mole fraction of each DAG and was restricted to lipid compositions in which coexisting phases were observed. Maximal activity decreased with increasing saturation of the DAG. In the fluid state, maximal PKC activity was shifted to higher DO mole fractions and the peak was much broader. Collectively, these data support a role for both the presence and nature of interface between compositionally distinct domains in activation of PKC.

Fruit of tomato landrace Alcobaca, containing the recessive allele alc, ripen more slowly, with a reduced level of ethylene production, and have prolonged keeping qualities. The levels of polyamines in pericarp tissues of alc and 'wild type' Alc (cv Rutgers and Alcobaca-red) fruit were measured by HPLC in relation to ripening. Putrescine was the predominant polyamine with a lower content of spermidine, while spermine was just detectable. The level of putrescine was high at the immature green stage and declined in the mature green stage. In Alc fruit the decline persisted but in alc fruit the putrescine level increased during ripening to a level smilar to that present at the immature green stage. There was no pronounced change or difference in spermidine levels. The enhanced polyamine level in ale fruit may account for their ripening and storage characteristics.member of the polyamine sequence, the diamine PTC,3 is synthesized in most plant systems from arginine via ADC, though in tomato fruit the principal pathway appears to be similar to that in animals and microorganisms, namely from ornithine via ODC (4,5,8,9). The level of ODC is highest shortly after pollination and declines to a low level of maturity (9), while inhibitors of ODC activity inhibit fruit development (4). Actual levels of polyamines have not been recorded through development and ripening, nor have ODC or ADC been followed through ripening in any tomato line. As the conversion of PTC to the other polyamines, SPD and SPM, involves SAM, which is also the precursor of ethylene (7), it follows that the synthesis of polyamines and ethylene are competitive.In view of the reduced ethylene production and delayed senescence of alc fruit, the polyamine content of these fruit was analyzed to determine whether it might vary from normal tomato fruit.Alcobaca is a landrace oftomato in which the fruit ripen more slowly than standard horticultural varieties (60 versus 50 d from anthesis) and have prolonged keeping qualities. If picked ripe, fruit can be kept at 20C for four times longer than standard varieties (33 versus 9 d) (13,15). This characteristic is determined by a single recessive gene, designated alc, whose inheritance and linkage have been described (13,14). The storability of alc fruit is not due to extreme firmness of the harvested fruit, but to an attenuation of the overripening process, including a slower rate of softening possibly because of lower polygalacturonase levels (12; MA Mutschler, in preparation). alc Fruit lack a climacteric pattern of CO2 and ethylene production and produce less than 25% of the ethylene produced by cv Rutgers (15).A spontaneous mutant in Alcobaca has given rise to a plant with normal ripening fruit but with all the other characteristics of the original Alcobaca landrace. The fruit of this line, which has been named Alcobaca-red, ripen to a crimson red as compared to the medium orange color of the original Alcobaca fruits and do not possess prolonged keeping qualities. Genetic analysis has shown that the al...

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