The effect of phospholipid fatty acid composition in membranous enzymes in Escherichia coli.

Abstract The shape of an Arrhenius plot of glycerol 3-phosphate acyltransferase activity of an unsaturated fatty acid auxotroph of Escherichia coli is identical in membranes containing cis-vaccenic, oleic, linoleic, or linolenic acid as sole unsaturated fatty acid. The curve is linear at low temperatures with a continuous decrease in slope above 15°. In membranes containing trans-unsaturated fatty acids as the sole unsaturated fatty acid, the decrease in slope occurs at 20°. Membranes from cells grown in the presence of oleic acid and then grown for one generation in the absence of unsaturated fatty acids contained 79% saturated fatty acids and 21% oleic acid. Temperature dependence of the enzyme activity in these membranes was intermediate between that observed in membranes containing trans-unsaturated fatty acids and those containing normal amounts of cis-unsaturated fatty acids. 1-Acylglycerol 3-phosphate acyltransferase activity exhibited a linear Arrhenius plot identical in slope in membranes containing cis-unsaturated fatty acids of varying degrees of unsaturation. Membranes from cells deprived of unsaturated fatty acids for one generation or those containing trans-unsaturated fatty acids exhibited a steeper slope. Membranous glycerol 3-phosphate dehydrogenase activity appeared to be independent of membrane fatty acid composition. Linear Arrhenius plots of identical slope were observed in all membrane preparations described above. The difference in the dependence of the temperature characteristics of various membranous enzymes on membrane fatty acid composition suggests a heterogeneity in the relationship between membranous enzymes and membrane phospholipids.

activity of an unsaturated fatty acid auxotroph of Escherichia coli is identical in membranes containing cis-vaccenic, oleic, linoleic, or linolenic acid as sole unsaturated fatty acid. The curve is linear at low temperatures with a continuous decrease in slope above 15". In membranes containing trans-unsaturated fatty acids as the sole unsaturated fatty acid, the decrease in slope occurs at 20". Membranes from cells grown in the presence of oleic acid and then grown for one generation in the absence of unsaturated fatty acids contained 79 % saturated fatty acids and 21% oleic acid. Temperature dependence of the enzyme activity in these membranes was intermediate between that observed in membranes containing trans-unsaturated fatty acids and those containing normal amounts of cis-unsaturated fatty acids.
1-Acylglycerol 3-phosphate acyltransferase activity exhibited a linear Arrhenius plot identical in slope in membranes containing cis-unsaturated fatty acids of varying degrees of unsaturation.
Membranes from cells deprived of unsaturated fatty acids for one generation or those containing transunsaturated fatty acids exhibited a steeper slope. Membranous glycerol 3 -phosphate dehydrogenase activity appeared to be independent of membrane fatty acid composition.
Linear Arrhenius plots of identical slope were observed in all membrane preparations described above. The difference in the dependence of the temperature characteristics of various membranous enzymes on membrane fatty acid composition suggests a heterogeneity in the relationship between membranous enzymes and membrane phospholipids.
The involvement of lipids in membrane function has been investigated in recent years by the use of unsaturated fatty acid auxotrophs of Eschericlzia co& first isolated in this laboratory *  (1). The critical function of the unsaturated fatty acids of phospholipids was illustrated by the isolation of auxotrophs. More important, the availability of these auxotrophs has made possible the manipulation of the membrane phospholipids since the phospholipid fatty acid composition reflected those fatty acids provided in the culture medium (2). Thus the effects of blocking phospholipid synthesis by deprivation of the required unsaturated fatty acid has been studied in order to determine whether simultaneous phospholipid synthesis is required during the synthesis of a functional P-galactoside transport system (3,4). Another type of membrane study utilizing the unsaturated fatty acid auxotrophs took advantage of the fact that the unsaturated fatty acids of the phospholipids could be manipulated by varying the fatty acid supplement added to the growth medium.
Invcstigations of the temperature characteristics of glycoside transport (5)(6)(7)(8) as well as respiration and growth rate (7) indicated that Arrhcnius plots for all these processes were biphasic, the slopes extrapolating to intersections at unique transition temperatures. The transition temperatures for all of these biological processes varied lvith the degree of unsaturation of the fatty acid supplement, being highest for trans-monoenoic, intermediate with cismonoenoic, and lowest for cis-polyenoic acids. Similar transition temperatures were observed in studies of the isolated phospholipids in monolayers at an air-water interface (7), suggesting that the transition temperatures observed in the biological processes refiected transitions in the lipid portion of the membrane.
A similar dependence of the temperature of phase transitions on fatty acid composition has been observed in lipid bilayers and biological membranes by calorimetric and x-ray diffraction tcchniques (9, 10). These studies together with studies using spectroscopic methods (11) suggest that a large portion of lipid in biological membranes is arranged in a bilayer structure, t,he phase properties of which are unperturbed by the protein present in the membrane.
It thus becomes of interest to determine whether all membrane functions are dependent on the phase properties of membrane lipids.
One of the more important functions of the cell membrane is phospholipid biosynthesis. The initial step of phospholipid biosynthesis, acylation of glycerol-3-P to phosphatidic acid, has been studied previously in this laboratory (12,13). Evidence has been obtained which suggests that a single membranous enzyme in E. coli catalyzes the acylation of glycerol-3-P with either palmityl-or oleyl-CoA to form monoacylglycerol-3-l'.
Issue of February 10, 1972 R. D. JIavis a& P. R. Vagelos 653 acid distribution in naturally occurring phospholipids. A separate membranous enzyme appears responsible for the ncylntion of 1-acylglycerol-3-P to form phosphatidic acid, and this enzyme also displays specificity in regard to the thioester substrates.
In the present study we selected these two membranous enzymes and the membranous glycerol-3-P dehydrogenase for investigation of temperature characteristics because sensitive optical assays are aT-nilable.
The effects of varying degrees of unsaturation of membrane fatty acids on the hrrhenius plots of these activities were determined in membranes of an E. coli unsaturated fatty acid ausotroph.

MATERIALS
A?;D METHODS

Materials
Unsat'uratcd fatty acids and fatty acid methyl esters were purchased from the Hormel Institute, Austin, h'linnesota.

Methods
Growth of Bacteria and Preparation oj Membrane Fraction-E. coli strain K-1060, generously proyidecl by Peter Ovcrath, has marker.5 identical with strain K-1059 (7) and is unable to synthesize or degrade unsaturated fatty acids. K-1060 was grown in Medium E (15) supplemented with 0.5% glycerol, 0.3% casamino acids (Difco, vitamin free), 2% Brij 35, and 0.02% fatty acids. One-liter cultures of cells were grown to late exponential phase (200 Klett units, 54 filter) harvested, washed once wit,h cold 0.02 &I potassium phosphate buffer, pI-I 7.0, containing 1% Brij 35, washed two additional times with the same buffer minus Brij 35, resuspended in the same buffer, and frozen. The suspension was thawed and the cells were broken i n a French pressure cell previously cooled in ice. The cell extract was made 2.5 rn3I in MgCl~ and a few crystals of dcoxyribonuclcase were added. The extract was incubated on ice for 30 min and then centrifuged 10 mill nt 6,000 x g to remol-e unbroken cells. The membrane fraction was then collected by ccntrifugation for 45 min at' 50,000 x g. hrter washing twice by resuspension and homogenization in cold 0.02 RI potassium phosphate buffer, pH 7.0, the membrane fraction was resuspended in the same buffer at a protein concentration of 10 mg per ml and stored frozen. Protein was determined by a microbiuret procedure (16).
Fatty Acid Analysis of Jle?nbrane Fraction-Lipids were cxtracted from 0.4 ml of membrane fraction according to the method of Bligh and Dyer (17). The chloroform phase was evaporated to dryness under a stream of nitrogen, dissolved in 1 ml of 2 7" H2S04 in methanol, and heated at 70" for 1 hour. One milliliter of water was then added to the acidic methanol solution and the fatty acid methyl esters were extracted with diethyl ether. The other extract was washed first with dilute sodium bicarbonate and then with water before analysis by gas liquid chromatography on a Varian model 2100 using a 4-foot glass column of 3% Silicone SE-30 on Varaport 30. Temperature was programmed from 140-180" at a rate of 4" per min with helium as carrier gas. Peak size was quantitated with an Infotronics model CRS 100 digital integrator.
Fatty acids were identified by comparison of retention times with those of standard methyl esters. Identities were verified by chromatography on a column of 10% diethylene glycol adipate on Chromasorb W.
Enzymic Assays-The spectrophotometric assays for both the glycerol-3-P acyltransfcrase and the I-acylglycerol-3-P acyltransferase have been described previously (13). Since these assays contain Tris buffer which is known to be affected by temperature, the pH of a typical reaction mixture was measured at 0" and 37", the extreme limits of the temperatures used here. A change of 0.5 pH units, which would not significantly affect the activity of these enzymes, was observed in changing the temperature from 0" to 37O. Glycerol-3-P dehydrogenase was assayed according to Lin et al. (18) by measuring the rate of reduction of the tetrazolium dye, 3-(4,5-dimethyl thiazolyl-2) 2,5-diphenyl tetrazolium bromide, to its formazan, which absorbs at 550 nm. Temperature of the sample compartment of the spectrophotometer was controlled by a circulating constant temperature bath. Reaction temperatures were measured by insertion of a thermocouple directly into the cuvettes.
The initial rates of reactions were linear for several minutes and proportional to the amount of membrane fraction used in all measurements reported.

Attempted Solubilization
of Glycerol-S-P Acyltransferase-Treatment of the particulate fraction of E. coli with sodium dodecyl sulfate, Triton X-100, Triton X-165, sodium deoxycholate, Cutscum, 2-methyl-1-propanol, or 1-pentanol inactivated the glycerol 3-phosphate acyltransferase activity. Treatment with 1 y0 Triton X-305 in the absence of buffer did not affect the acyltransferase activity, and differential centrifugation suggested the activity was associated with a light fraction of the Triton X-305treated membranes.
Partial resolution of the activity from a large portion of the membrane material was achieved by sucrose density centrifugation in the presence of 1 y0 Triton X-305. One milliliter of membrane suspension in 1 y0 Triton X-305 was layered on the top of a discontinuous gradient consisting of 1.5 ml of 70°10 sucrose, 1 ml of 40% sucrose, and 1 ml of 20% sucrose with 1% Triton X-305 throughout in a 4.5-ml centrifuge tube and centrifuged at 34,000 rpm for 2 hours in a Spinco SW 56 swinging bucket rotor.
Two visible bands of turbidity resulted. The lighter band, which was located at the interface between 20 and 40% sucrose, contained approximately 65% of the recovered glycerol-3-P acyltransferase activity and roughly 15y0 of the turbidity measured at 600 nm (Table I). The heavier band, located at the 40 to SOY, interface apparently contained the major portion (fO%) of material applied to the gradient, judging from the absorbance at 600 nm. Only 9% of the recovered glycerol-3-Y acryltransferase activity was located in this heavier band. The glycerol-3-P acyltransferase activity of the upper band could be pelleted by addition of 0.02 M potassium phosphate, pH 7.0, and centrifugation at 50,000 x g for 15 min. This procedure yielded a lo-to 1Bfold purification of the glycerol-3-P acyltransferase.
The activity was rapidly lost upon removal from the sucrose mixture, however, and attempts to further purify the enzyme failed. Due to the instability of this The gradient is described under "Results." Glycerol-3-P acyltransferase activity is presented in nanomoles per min. supplied in the growth media, was found in the membranes and these results are similar to those of Overath, Schairer, and Stoffel (7). The exception was supplementation with cis-vaccenic acid, which is synthesized in wild type E. coli; cis-vaccenic acid was partially converted to its cyclopropane derivative, cis-11 ,12methylene octadecanoic acid, in the mutant. The effect of supplementation with various unsaturated fatty acids on chain length and percentage of saturated fatty acids in the membrane is generally consistent with the observations of this and other laboratories (2,7,(19)(20)(21) and suggests that fatty acid composition of the membrane of E. coli is controlled with respect to the over-all physical properties of the hydrocarbon side chains. Linolenic acid (18 : 3) was incorporated into membrane to a lesser extent than other &-unsaturated fatty acids, and its incorporation was not increased when the mutant was grown at a lower temperature.
The amount of t?ans-unsaturated fatty acids incorporated was greater than the cis-analogues, and the largest amounts of shorter chain saturated fatty acids were found in membranes of the mutant grown in the presence of elaidic acid. Growth of the mutant for one generation in the absence of unsaturated fatty acids after prior growth in oleic acid-supplemented medium yielded membranes containing 79 y. saturated fatty acids consisting of equal amounts of myristic and palmitic   shown in Table III, typical specific for unsaturated fatty acids for one generation had a specific activities of glycerol-3-P acyltransferase at 25" ranged from 1.0 activity of 0.9 on the lower end of the normal range of specific to 2.2 nmoles per min per mg of protein with palmityl-CoA as activities. acyl donor in membranes prepared from cells supplemented with indistinguishable.
Using palmityl-CoA as the acyl donor (Fig.  1, A and B), the curves shown are identical in shape for membranes containing either cis-vaccenic acid or linolenic acid as the Issue of February 10, 1972 R. D. Mavis and P. R. Vagelos 657 as membranes of wild type cells (data for the latter three not shown), gave identical Arrhenius plots of glycerol-3-P acyltransferase activity.
"Starved" membranes exhibited a curve nearly identical with "normal" curves, in contrast to the observations with palmityl-Cob as acyl donor. The shape of "normal" curves with oleyl-CoA as acyl donor is unlike "normal" curves with palmityl-CoA as acyl donor.
Comparison in Fig. 3 shows that glycerol-3-P acyltransferase activity decreased less upon lowering temperature with oleyl-CoA than with palmityl-CoA as acyl donor.
The ratio of palmityl-CoA activity to oleyl-CoA activity at 25" is 40% higher than the ratio at 10". On the other hand, Arrhenius plots of glycerol-3-P acyltransferase activity in trans-fatty acid-containing membranes are very similar in shape with palmityl-CoA and oleyl-CoA as acyl donor at temperatures over 12". The low specific activities of the membranes containing truns-fatty acids made measurements at lower temperatures difficult. E$ect of Phospholipid Fatty Acid Composition on I-Acylglycerol-S-P Acyltransferase Activity-The effect of temperature on l-acylglycerol-3-P acyltransferase activity was also investigated in these membrane preparations of varying fatty acid composition. As shown in Fig. 4, Arrhenius plots of this activity are linear with all membrane preparations studied over the temperature range O-37". The curves with membranes containing oleic or linolenic as well as linoleic (not shown) acids are virtually identical in slope. Membrane preparations from Iruns-fatty acid-supplemented cells, however, exhibit a higher slope which is very similar to that observed with "starved" membranes.
In contrast to the data obtained with glycerol-3-P acyltransferase, the effect of temperature on 1-acylglycerol-3-P acyltransferase was identical when palmityl-CoA or oleyl-CoA was used as acyl donor. Dehydrogenase Activity-A third membra.nous enzyme, glycerol a-phosphate dehydrogenase, was also studied in these membrane preparations.
Bs shown in Fig. 5, linear hrrhenius plots of virtually identical slope were obtained with membrane preparations containing oleic, linolenic, trans.vaccenic, or elaidic acid as well as "starved" membrane preparations.

DISCUSSIOX
The procedure utilized in this work in an attempt to solubilize the E. co& glycerol-3-P acyltransferase is similar to a procedure recently reported by Schnaitman (22) in which he separated two discrete fractions of the cell envelope by isopycnic centrifugation. The more dense fraction was shown by him to be enriched in components of the cell wall; the lighter fraction contained the components of the cytoplasmic membrane.
Enrichment of the glycerol-3-P a,cyltransferase in the lighter fraction obtained here suggests that this enzyme is localized in the inner, cytoplasmic membrane of the E. coli envelope.
Our failure to solubilize this enzyme by using many of the procedures that have succeeded in solubilizing other membranous enzymes emphasizes the fact that new methods are required for studies of membranous enzymes. The finding that glycerol-3-P acyltransferase was inactivated by several delipidating or membrane solubilizing agents suggests that this enzyme has a requirement for some component of intact membrane structure.
Work now in progress indicates that the enzyme is inactivated by pure phospholipase C,' suggesting a phospholipid requirement. However, attempts to reactivate a delipidated, inactive preparation by the addit'ion of phospholipids have not succeeded thus far.
Earlier studies of the temperature characteristics of glycoside transport, with a similar unsaturated fatty acid auxotroph of E. coli have indicated that transport, as well as respiration and growth, reflected the degree of unsaturation of the fatty acid supplied in the growth medium; these biological processes were all characterized by distinct transition temperatures that were determined by the temperatures of the phase transitions of the bulk membrane phospholipids (4-S). Neither dependence of temperature characteristics on the degree of unsaturation of the phospholipid fatty acids nor distinct transition temperatures were observed in studies of the three membrane enzymes investigated in this study. Thus membrane enzymes may or may not reflect the physical properties of the membrane phospholipids, and the differences disclosed by studies of temperature characteristics suggest a heterogeneity among membrane enzymes with regard to dependence upon the membrane phospholipide.
The similarity of slope of the lincar portions of all Arrhenius plots shown for glycerol-3-P acyltransferase suggest,s that at lower temperatures the enzyme exists in the same form indcpendent of the membrane fatty acid composition.
The temperature of departure from lincsrity was increased, however, by the presence of tram-unsaturated fatt'y acids in the membrane, implicating lipid-protein interactions in the action of this enzyme, and suggesting an increase in the phase transition temperature of lipids affecting this enzyme.
The identical shape of the curves reported here for membranes containing c&mono-, cis-di-, or cis-triun-

6%
Effect of Fatty Acids on Membranous Enzymes Vol. 247, X-o. 3 saturated fatty acids, however, indicates that the glycerol-3-P acyltransferase activity is not dependent on the over-all phase properties of membrane lipids, in contrast to the dependencies reported for various transport systems (4-S). A possible explanation for this could be selective association of this enzyme with lipids of appropriate fatty acid composition to maintain its normal temperature profile.
Alternatively, the protein could interact with lipids around it in a way which affects their phase properties.
A role for glycerol-3-l' acyltransferase in the temperature regulation of the membrane fatty acid composition has been suggested by the work of Sinensky (24). A relative increase in glycerol-3-P acyltransferasc activity with unsaturated as opposed to saturated fatty acid thioesters at lower temperatures was shown to correlate with an increased proportion of unsaturated fatty acids found in phospholipids of JY. coli grown at lower temperatures. The decrease in the ratio of enzyme activity with palmityl-CoA to that with oleyl-CoA at lower temperatures as reported here is also consistent with such a role. It would thus be of importance to maintain the temperature profile of this enzmye. Maintenance of a normal arrhenius plot (Fig. 2B) of glycerol-3-P acgltransfcrase activity with oleyl-CoA as acyl donor in membranes of cells starved for unsaturated fatty acid, and therefore containing only 21% c&unsaturated fatty acid, supports the hypothesis of selective association of this enzyme with lipids whose physical properties permit a normal temperature profile independent of the over-all phase properties of the membrane. Enzyme activity with palmityl-Cob as acyl donor (Fig. lB), in contrast, exhibits an Arrhenius plot in membranes from the cells starved for unsaturated fatty acid which is similar to that observed with membranes containing trans.unsaturated fatty acids, consistent with expected similarities in the physical properties of the two membrane preparations.
This contrasting behavior betweet activities with oleyl-Co-4 and palmityl-Coil suggests that the physical properties of the lipids associated with this enzyme in "starved" membranes are intermediate between those of the lipids normally associated with the enzyme and those containing only lrans-unsaturated fatty acids. The abnormal Arrhenius plots obtained with membranes containing Irans-unsaturated fatty acids suggests that maintenance of the normal temperaturedependent substrate specificities depends on the presence of cisunsaturated fatty acids in the lipids associated with this enzyme. The similarity of these plots (Fig. 3) when either palmityl or oleyl-Cob was used as acyl donor in trans.-unsaturated fatty acidcontaining membranes suggests that the ability to distinguish between saturated and unsaturated acyl donors is lost in the presence of only trans-unsaturated fatty acids. An involvement of the unsaturated fatty acid hydrocarbon side chains in the recognition of substrate by the active site of this enzyme is thus suggested.
The effect of temperature on I-acylglycerol 3-phosphate acyltransferase activity as a function of membrane fatty acid composition is different from that observed with glycerol-3-P acyltransferase. A linear Arrhenius plot was obtained which is identical for membranes containing &mono-, cis-di-, or cis-triunsaturated fatty acids but has a steeper slope in membranes from cells starved for unsaturated fatty acids or containing trans-unsaturated fatty acids (Fig. 4). This suggests that this enzyme exists in a different form in the presence of Irans-unsaturated or a higher proportion of saturated fatty acids, in contrast to the glycerol-3-1' acyltransferaae, which exhibits a similar slope in all membrane preparations at lower temperatures. No evidence of a change in slope or phase transition is observed with the l-acylglycerol-3-P acyltransferase, yet interaction of lipid and protein is suggested by the steeper slope of the Arrhenius plot in membranes containing trans.unsaturated or membranes from cells starved for unsaturated fatty acids. Thus a highly specific association of lipids with this enzyme may be postulated in which the phase transitional properties of the lipids are not expressed. Only the over-all temperature characteristic of the lipoprotein complex is affected by the physical properties of the lipid hydrocarbon side chains. The higher slope of the plots with membranes containing trans.unsaturated or predominantly saturated fatty acids suggests that the enzyme is a less effective catalyst when &-unsaturated fatty acids are not available. The possibility that the temperature characteristics observed in studies of the two acyltransferases were influenced by temperature effects on the substrates acyl-CoA or I-acylglycerol-3-P cannot be entirely discounted. However, substrate saturation curves of acyl-CoA were similar at high and low temperatures; thus no obvious differences were detected at this level. Moreover, since major differences in temperature characteristics were noted between enzyme preparation made from membranes containing different fatty acids, although the assays were carried out under identical conditions, it is likely that the temperature profiles largely refiected the properties of the membranous enzymes.
Glycerol-3-P dehydrogenase activity, in contrast to the acyltransferase activities, appears to be independent of membrane fatty acid composition.
If lipid-protein interaction is essential to the action of this enzyme, the requirement is either nonspecific with respect to the over-all physical properties of hydrocarbon side chains or exclusive for saturated fatty acids. A third, more obvious possibility is a total lack of lipid-protein interaction in the action of this membranous enzyme.
The unique response of each of three membranous enzyme activities to variations in membrane fatty acid composition indicates a heterogeneity of membrane proteins with respect to lipid dependence.
In addition to transport systems, which exhibit changes in temperature characteristics and distinct breaks in Arrhenius plots at temperatures corresponding to temperatures of over-all phase transition of the bulk of membrane lipid (MS), at least three additional classes of membrane proteins appear to exist. Lack of response of these enzymes to changes in over-all phase properties of the membrane effected by increasing the degree of unsaturation in membrane fatty acids suggests that lipids associated with these proteins have phase properties distinct from the bulk of membrane lipid. This could be due either to specific association of the protein with lipids of fatty acid composition unlike the average composition of the whole membrane, or to a perturbation of the phase properties of the lipids in its immediate environment by the protein itself. In either case, a microheterogeneity of membrane lipids with respect to phase properties is indicated.
Temperature studies of other membranous enzymes have been recently reported. Raison,Lyons,and Thomson (25) found that disruption of mitochondria from rat liver and sweet potato by sonication, hypotonic swelling, and freezing and thawing did not alter the discontinuity in the Arrhenius plot exhibited by mitochondrial respiratory enzymes, including the succinate oxidase system, succinate dehydrogenase, and cytochrome c oxidase. Thus intact membrane structures are not required for the demonstration of transition temperatures. Disruption of the mito-Issue of February 10, 1972 R. D. Mavis and P. R. Vagelos 659 chondrial membranes with detergent brought about a change in the temperature characteristics of all three enzyme systems with a loss of the discontinuity in the i\~henius plot. The authors interpreted these results as suggesting that the temperature-induced change in activation energy of these membranous enzymes was associated with a phase change in the lipid component of membranes.
On the other hand Zeylemaker et al. (26) noted a breakpoint in the Arrhenius plot with both particulate and soluble succinate dehydrogenase of heart muscle which they interpret'ed as indicating temperature-dependent conformational changes in the enzyme.