Localization of lysophosphatidylcholine in bovine chromaffin granules.

One of the unique features of the chromaffin granule membrane is the presence of about 17 mol% lysophosphatidylcholine. Lysophosphatidylcholine isolated from the granules could be degraded by approx. 94% by lysophospholipase. This result is consistent with chemical analyses data showing that about 9% of this lysophospholipid is 1'-alkenyl glycerophosphocholine. The localization of the acylglycerophosphocholine in the chromaffin granule membrane was studied by using pure bovine liver lysophospholipases. In intact granules only about 10% of the total lysophosphatidylcholine was directly available for enzymic hydrolysis. In contrast, when granule membranes (ghosts) were treated with lysophospholipases approx. 60% of the lysophosphatidylcholine was deacylated. These values did not increase after pre-treatment of intact granules or ghosts with trypsin. Added 1-[1-14C]palmitoyl-sn-glycero-3-phosphocholine did not mix with the endogenous lysophosphatidylcholine pool(s) and remained completely accessible to added lysophospholipases.

Availability of lysophosphatidylcholine in single bilayer vesicles for hydrolysis by lysophospholipase.

Single bilayer vesicles were prepared from total rat liver microsomal lipids to which 5 mol% lysophosphatidylcholine had been added. The availability of lysophosphatidylcholine for enzymatic hydrolysis by lysophospholipase (EC 3.1.1.5) was found to be higher in vesicles prepared by the cholate dispersion technique when compared with sonicated vesicles. Sepharose 4 B chromatography showed that the vesicles prepared by the cholate technique were smaller than those prepared by sonication. This is in contrast to previous observations for egg phosphatidylcholine vesicles. Total rat liver microsomal extracts were found to contain proteolipid, which could be removed by ether precipitation. Cholate vesicles prepared from proteolipid-free extracts were still smaller than sonicated vesicle from this extract. Experiments with [14C] dextran entrapped in the vesicles indicate that there is no loss of the permeability barrier of the vesicles for high molecular weight solutes during vesicle treatment with lysophospholipase. The high availability of lysophosphatidylcholine in cholate vesicles of total rat liver microsomal lipids is discussed in terms of a highly asymmetric distribution of lysophosphatidylcholine over the inner and outer monolayer of the bilayer.

Distribution of lysophosphatidylcholine in single bilayer vesicles prepared without sonication.

The cholate method originally introduced by Kagawa et al. (J. Biol. Chem. (1973) 248, 676-684) and further developed by Brunner et al. (Biochim. Biophys. Acta (1976) 455, 322-331) has been used to prepare single bilayer vesicles containing 5 mol% lysophosphatidylcholine embedded in a matrix of phosphatidylcholine. The distribution of lysophosphatidylcholine over outer and inner monolayer was found to be highly asymmetric (ratio 9:1), as determined by lysophospholipase treatment of the vesicles. This distribution is similar to the value found in sonicated vesicles. Up to 20 mol% cholesterol could be incorporated in the vesicles by the cholate method. The method was succesfully used also for the preparation of single bilayer vesicles from total rat liver microsomal lipids, to which 5 mol% of 1-[1-14C]palmitoyl lysophosphatidylcholine had been added. Surprisingly, almost 100% of lysophosphatidylcholine in the latter vesicles was directly available for hydrolysis by sophospholipase. In contrast, only 70% of the lysophosphatidylcholine is sonicated vesicles of similar composition could be hydrolyzed by lysophospholipase.