Lipid phase, water concentration change

Conflicting hypotheses were also put forward with respect to the mechanism of interfacial activation. Desnuelle et al. (1960) were the first to suggest that a conformational change in the enzyme could be responsible for the enhancement of activity at the oil-water interface. There were also other hypotheses. For example. Wells (1974) suggested that the apparent activation of lipases is due to the orientation of the scissile ester bond on the surface of micelles Brockerhoff (1968), on the other hand, pointed to the possibility of differences in solvation of the ester bond in solution versus a lipid phase, whereas Brockman et al. (1973) postulated that a steep substrate concentration gradient at the interface may provide an explanation. 

Interpretation of the Calorimetric Results. There is little doubt that the transition observed in M. laidlawii membranes arises from the lipids since it occurs at the same temperature in both intact membranes and in water dispersions of membrane lipids. It is reasonable to conclude that in both membranes and membrane lipids the lipid hydrocarbon chains have the same conformation. The lamellar bilayer is well established for phospholipids in water (I, 20, 29) at the concentration of lipids used in these experiments. In the phase change the hydrocarbon core of the bilayer undergoes melting from a crystalline to a liquid-like state. Such a transition, like the melting of bulk paraffins, involves association between hydrocarbon chains and would vanish or be greatly perturbed if the lipids were apolarly bound to protein. We can reasonably conclude that most of the lipids in M. laidlawii membranes are not apolarly bound to protein. 

The muscles were freed by gross dissection of extraneous tissue which was mainly fat and peripheral nerves, and then stored at -40°C. For an experiment, approximately 1 kg tissue was macerated by a meat grinder and homogenized in ten volumes of tetrahydrofuran 0.01 M KC1 (4 1, v/v), stirred for 3 hours, and filtered through a Buchner funnel. The extraction was repeated twice and the filtrates then combined and concentrated in a rotary evaporator. One liter of chloroform-methanol (2 1, v/v) was added to the lipid extract which has the appearance and consistency of syrup. Gangliosides were partitioned into the upper layer by the addition of 200 ml of water (11) and the lower layer extracted two additional times with theoretical upper phase containing 0.027% KC1. The combined upper layers were then concentrated and dialyzed exhaustively at 4°C with five changes of distilled water. 

An aliphatic ketone (9-heptadecanone) and two keto derivatives of stearic acid (as potassium salts) containing a ketone functionality either at position 5 or 12 were incorporated into bilayers of the phospholipid l,2-dihexadecyl-sn-glycero-3-phosphocholine. Infrared spectra of these mixtures were measured as a function of temperature and amount of added cholesterol. It was found that the presence of cholesterol in these bilayers induces changes in the location of the guest ketone and that these changes are dependent on both temperature and cholesterol concentration. It is also demonstrated that, in the gel phase, the presence of cholesterol induces larger intersheadgroup separations and, therefore, water penetrates deeper into the lipid bilayer. 

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