Detergent Properties of Water-soluble Choline Phosphatides SELECTIVE SOLUBILIZATION OF ACYL-CoA:LYSOLECITHIN ACYLTRANSFERASE FROM THYMOCYTE PLASMA MEMBRANES*

Several analogs of lysolecithin were found to solubilize human erythrocyte ghosts comparably or even better than other detergents. Derivatives with aliphatic chains of 12 to 14 carbons were most effective. The phosphorylcholine detergents apparently possess low protein-denaturing properties, since they, for the first time, allowed the solubilization of enzymatically active acyl-CoA:lysolecithin acyltransferase from thymocyte plasma membranes. The solubilized enzyme was not sedimented at 177,000 x g for 60 min and penetrated into Sepharose 6B gels. Low detergent concentration resulted in a selective extraction of the acyltransferase (about 70%) as compared to alkaline phosphatase, nucleotide pyrophosphatase, gamma-glutamyltransferase or Mg2+-ATPase (30 to 40%). The selectivity was reflected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of soluble and sedimentable membrane fractions; three bands of approximately 53, 84, and 94 x 10(3) daltons were enriched in the supernatants, whereas one band of about 68 x 10(3) daltons was concentrated in the pellet. The preferential extraction of acyltransferase may be related to particularly high affinity of lysolecithin analogs for this enzyme, which at higher concentrations was competitively inhibited by these detergents. The inhibitor constants ranged from 1400 micron for the C10 analog (ET-10-H) to 80 micron for the compound with 16 carbons (ET-16-H) per aliphatic chain.

one band of about 68 x lo" daltons was concentrated in the pellet. The preferential extraction of acyltransferase may be related to particularly high affinity of lysolecithin analogs for this enzyme, which at higher concentrations was competitively inhibited by these detergents.
The inhibitor constants ranged from 1400 PM for the Cl0 analog (ET-lo-H) to 80 PM for the compound with 16 carbons (ET-16-H) per aliphatic chain.

Solubilization
of membrane-bound enzymes without loss of activity has in the past been achieved by use of nondenaturing detergents such as Triton X-100. Lipid-metabolizing acyltransferases, however, which transfer long chain fatty acids to lipid precursors, are usually completely inactivated by these detergents.
One possible explanation for such inactivation would be that the detergents replace membrane phospholipids, the association of which with the enzyme might be essential for enzymatic activity. It has therefore been suggested by Tanford and Reynolds (1) that natural detergents of phospholipid structure, such as lysophospholipids, might possess distinct advantages for the isolation of particularly labile membrane components.  (11) and suspended in Hepes buffer, pH 7.2, at 2 mg protein/ml. Samples (0.1 ml) of these suspensions were mixed with increasing amounts of the various phosphatides, diluted to a total volume of 0.6 ml, and kept for 20 min at 0°C and the extinction of the solution was determined at 514 nm. In the absence of detergent, this extinction was approximately 0.4/cm (control). We have found that the degree of solubilization under these conditions is primarily determined by the ratio of detergent to membrane protein and not so much by the absolute detergent concentration.
Therefore, in Table I we have compiled the magnitude of this ratio necessary for a 50% decrease in turbidity.
The data reveal that a considerable number of compounds, particularly those with Cl2 and Cl4 aliphatic chains, are comparable to or even more active in this test than Triton X-100 or SDS. Shorter as well as longer hydrophobic chains result in less potent detergents. In fact, all saturated Cl8 compounds (with the noteworthy exception of ET-K+ OCH3) increase rather than decrease the turbidity.
The importance of relatively small structural changes for the detergent properties of these phospholipids is emphasized by the finding that an exchange of saturated for unsaturated Cl8 aliphatic chains leads to rather potent long chain detergents. Moreover, the benzylation of the hydroxyl function in ET-12-OH resulted in the most active membrane solubilizer of our series (ET-12-OCH&Hri).
A more detailed impression of the solubilization properties of a selection of compounds is given in Fig. la.
Since turbidity decrease may not necessarily coincide with true solubilization of membrane material, we have, for some of the compounds employed, compared these data with the percentage of membrane protein remaining in ultracentrifugation supernatants (last column of Table I, and Fig. lb). The data reveal that, in general, good agreement exists between the two methods but that some quantitative variations will have to be considered. Regarding the importance of the length of the hydrophobic chain for the solubilization power of phosphorylcholine detergents (Fig. la), it is interesting to note that the poorest detergents in this assay (ET-l&H, ET-16-H) are the hemolytically most active substances of the series (19,20).
SDS-polyacrylamide gel electrophoresis (data not shown) of 100,000 X g supernatants and pellets after solubilization with ET-12-H and ET-16-H showed that all membrane proteins can be extracted by this procedure.
No principal differences between the action of the long and short chain derivative have been observed. Inhibition of Acyl-CoA:LysoZecithin

Acyltransferase by Lysolecithin
Analogs-To test the proposed "mildness" of phosphorylcholine detergents for sensitive membrane components, we planned to extract acyl-CoA:lysolecithin acyltransferase from the plasma membrane of calf thymocytes. Since all phosphatides used here were substrate analogs for the acyltransferase, it was reasonable to assume that the analogs might be inhibitors of the enzyme. Fig. 2 shows for the series of alkyl-deoxy lysophosphatides (ET-n-H) that this is, in fact, the case. The degree of inhibition increased with increasing chain length up to an optimal length of 16 carbon atoms. ET-18-H exhibited a significantly lower inhibition than ET-16-H. To investigate the mechanism of this inhibition, we used the most active compound ET-16-H. Fig. 3 shows the Lineweaver-Burk plot (21) of a kinetic experiment using palmitoyl lysolecithin as substrate and ET-16-H as inhibitor. The results from these studies are consistent with a competitive mechanism.
The dissociation constants determined were K, = 6.5 pM for palmitoyl lysolecithin and K, = 100.5 pM for ET-16-H.
In another series of experiments, we determined the inhibitor constants from Dixon plots (22) using increasing concentrations of the inhibitor. These studies also revealed that ET-16-H exhibited the highest and ET-IO-H    the lowest inhibition of the acyl-CoA:lysolecithin acyltransferase (Table II).
However, it should be kept in mind that the system used is not a homogeneous one since most of the added substrates and detergents is adsorbed to the membrane (20). Therefore, the capacity of the detergent to inhibit the enzyme and to solubihze membrane proteins depends within the fist approximation not on the concentration but on the ratio of detergent to the membrane mass. This will be demonstrated also in the series of experiments using ET-12-H as detergent to solubihze thymocyte plasma membranes. trations which were so low that only about 20% of the membrane was solubtied and which caused at least a 90% inhibition of the acyltransferase: 1% cholate, 0.2% glycocholate, 0.25% deoxycholate, 0.05% T&on X-100,0.25% Nonidet P-40, and 0.25% Berol-043 (MoDo Kemi AB, Stenungsund, Sweden).
From the results described above, we knew that ET-12-H at a suitable concentration exhibits a relatively high capacity to solubilixe membranes without inhibition of the lysolecithin acyltransferase.
Thus, the inhibition studies showed that a concentration of 20 to 40 rmrol of ET-12-H/20 pg of membrane protein inhibited the acyltransferase only slightly (1 to 3% inhibition).
In the following experiments, we therefore used pmol/mg of membrane protein (corresponding to 0.2 to 1.0 mg of ET-12-H/mg of membrane protein). Table III shows that ET-12-H solubilixes the acyltransferase without loss of activity, which is documented by a recovery of 90 to 100%. The activities of the alkaline phosphatase, nucleotide pyrophosphatase, and the y-glutamyltransferase were also not impaired whereas, surprisingly, the Mg2+-ATPase was inhibited to an extent of 50 to 70%. Washing of plasma membranes with buffer (Hepes, 10 mM, pH 7.4) removed appreciable amounts of protein (16%). However, these proteins did not contain any activity of the membrane-bound enzymes tested. It is of particular interest that ET-12-H extracts the acyltransferase with preference since optimal concentrations of   cd i 333 about 0.5 mg of ET-12-H/mg of membrane protein solubilizes 70% of the total acyltransferase activity while only 30 to 40% of all other enzyme activities were removed. If 60% of the membrane is solubilized by treatment with 0.9 mg of ET-12-H/mg of membrane protein (not shown in Table III), this  selectivity is lost and all enzymes are extracted to a similar extent (60 to 70%). Fig.  4 shows the SDS-gel electrophoresis of plasma membranes solubilized with ET-12-H and the residual pelleted membrane. This extract was derived from an experiment where 0.5 mg of ET-12-H/mg of membrane protein was added resulting in a solubilization of 41%. As can be seen, most bands occurring in the control membranes are extracted, with three bands (94,000, 84,000, and 53,000 daltons) being more prominent in the supernatant.

SDS-Polyacrylamide Gel Ekctrophoresis--
One other band (68,000 daltons) is enriched in the pellet. DISCUSSION Lysophosphatidylcholine has occasionally been used as a detergent, and particularly with mitochondrial membranes, a certain selectivity in the solubilization of membrane components has been reported (23)(24)(25)(26)(27)(28). These studies have led to the notion that lysolecithin may combine the solubilizing properties of detergents with enzyme stabilization or even activation properties of membrane lipids (25,29). We have taken advantage of the availability of the great variety of physicochemically characterized synthetic derivatives of lysophosphatidylcholine (6,19,20) and have screened these compounds for their membrane solubilizing properties. As has been demonstrated in this study, a fair number of these analogs compare favorably with established detergents such as Triton X-100 or SDS. In general, our data reveal that lysolipids with aliphatic chains of 12 to 14 carbons or unsaturated long chain compounds are the most potent solubilizers (Table I, Fig. 1). This is in remarkable contrast to the cytolytic activities of these substances. In this respect, the saturated CIG/Cls lysophosphatides are by far the most active ones (20). As mentioned above, the qualities of a detergent are, however, not simply determined by its solubilization power but more so by its ability to protect natural protein conformation in mice&r solutions, i.e. to leave enzymatic activities unimpaired. Only this property will enable the investigator to use it for purification and characterization of membrane-bound enzymes. With regard to lysophosphatides, we have demonstrated that, at least for the case of acyl-CoA:lysolecithin acyltransferase, these surfactants possess distinct advantages over all other detergents tested.
Only a few successful solubilizations of long chain acyltransferases have been reported in the literature (30)(31)(32). In all cases, however, the enzyme (glycerophosphate acyltransferase of various sources) was inevitably inactivated by the detergents (Triton X-100 or cholate) and could only be determined upon reactivation by a large excess of membrane lipids. Moreover, all reports consistently show that these preparations exhibit no activity for the acylation of monoacyl-sn-glycero-3phosphate. The latter compound was the only product during the acylation of glycerophosphate. This failure can be explained either by an irreversible inactivation of the lysophosphatidate acyltransferase by the detergent treatment or by the fact that the lipid-soluble acceptor molecule (lysophosphatidate) becomes inaccessible because it is partitioned into the excess of liposomal lipid. Thus, to our knowledge, the extraction of membranes with analogs of lysolecithin is the first procedure which leads to an active and soluble lysophosphatide acyltransferase.
We have defined effective membrane solubilization as re-leasing membrane components not sedimented at 177,090 x g., for 60 min. Moreover, we tested extracts from thymocyte plasma membranes by chromatography on Sepharose 6B in ET-12-H containing buffer. These experiments revealed that the acyl-CoA:lysolecithin acyltransferase activity was included into the gel and eluted significantly later than the void volume.
Our results further showed that the relatively low lysophosphatide concentrations used did not impair biochemical analysis and, hence, did not have to be removed. An additional advantage of using short chain deoxy-alkyl analogs of lysolecithin comes from the fact that they do not interfere with the natural substrate of acyl-CoA:lysolecithin acyltransferase and that they may be separated by thin layer chromatography from natural lysolecithin.
Moreover, these detergents apparently exhibit a selectivity for the extraction of the acyltransferase, leading, at low detergent concentrations, to about a 2fold enrichment in the extracts as compared to the original membrane material. This selectivity seems to be rather restricted, since an electrophoretic comparison of extracts and pellets (Fig. 4) revealed only few differences. Three bands in the SDS-polyacrylamide gel electrophoresis, however, appear to be enriched in the extracts: two of 80,000 to 90,000 daltons and one of 50,000 to 55,009 daltons.
The results should not be discussed without recalling the rather special conditions of our system. The detergent is a substrate analog of the enzyme to be isolated and may, at higher concentrations, act as a competitive inhibitor. The inhibitor properties of the various analogs are apparently linked directly to the membrane affinities of these substances (20). Like those, inhibition is most pronounced for the Cle derivative and decreases with shorter as well as with longer aliphatic residues (Fig. 2, Table II). The discrepancy with a report of Wittels and Hulbert (33) who found that ES-NO-H did not inhibit acyl-CoA:lysolecithin acyltransferase may be resolved by the notion that these authors (a) used a different enzyme source, (b) employed an acyl-instead of an alkyldeoxy lysolipid, and (c) used maximally 40 pM concentrations, whereas we found 50% inhibition for approximately 200 pM concentration (Fig. 2). The effective solubilization of thymocyte plasma membranes by ET-12-H contrasts with the findings of Peterson and Deamer (34) who observed that sarcoplasmic reticulum membranes only can be solubilized by lysolecithins of a chain length of 16 to 18 carbon atoms but not by those carrying Cl2 chains. Thus, this difference can only be explained by the fact that we used a lysolecithin analog with an ether linkage (position 1) and a deoxy group (position 2) or, more likely, one has to assume that the thymocyte plasma membrane is more susceptible to solubilization by these detergents than sarcoplasmic reticulum membranes. Solubilization of membrane-bound enzymes by natural lysolecithins obviously has some advantages. Rydstrom et al. (35) reported that mitochondrial nicotinamide nucleotide transhydrogenase together with succinate dehydrogenase could be preferentially solubilized with lysolecithin, thus leading to preparations of higher specific activities of these enzymes compared with Triton X-190 extracts. In contrast to Triton X-100, even an excess of lysolecithin did not inhibit the transhydrogenase (35). A similar property of this detergent was observed in studies on the Ca*'-ATPase of sarcoplasmic reticulum. Peterson and Deamer (34) found that low concentrations of lysolecithin inhibited this enzyme up to 70% but it was possible to reactivate the ATPase by 3 to 6 times higher concentrations of lysolecithin. However, in these studies, the ATPase was not solubilized but remained enriched in the membranous pellet (25). The surprising selectivity in the extraction of acyltransfer-ase with ET-12-H might possibly be related to the aforementioned enzyme/inhibitor relation. In fact, this correlation may be encouraging for further attempts to use "custom-made" detergents for special solubilization problems. The use of other lyso compounds of appropriate hydrophobicity such as analogs of lysophosphatidylethanolamine or lysophosphatidylserine, to mention only some possibilities, may be rewarding for solubilization studies of certain lipid-requiring enzymes or surface antigens.