The effect of growth temperature on the membrane lipid environment of the psychrophilic bacterium Micrococcus cryophilus

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 224, No. 2, July 15, pp. 718-727, 1983

The Effect of Growth Temperature on the Membrane Lipid Environment of the Psychrophilic Bacterium Micrococcus cryophilus MARIAN FOOT,* ROGER JEFFCOAT,? MARTIN D. BARRATT,* AND NICHOLAS J. RUSSELL**’ *Department of Biochemistry, University College, P.O. Box 78, Card$ CFl IXL, South Wales; tBiosciences L3ivision, Unilever Research, Colworth House, Sharnbrook, Be@rrd MK& lLQ, England; and SEnvironmental Sqfetq Laboratory, Unilever Research, Colworth House, Sharnbrook, Be&rd MK.4.4 lLQ, England Received January

19, 1983, and in revised form April

4, 1983

The relationship between the A9-desaturase activity of the psychrophilic bacterium Micrococcus cryophilus grown at different temperatures and the physical state of its membrane lipids as measured by ESR spectroscopy has been studied. Arrhenius plots of desaturase activity were biphasic with a discontinuity at a temperature which depended upon the bacterial growth temperature. Changes in the desaturase activation energy, which increased as the growth temperature was lowered, are discussed in the context of membrane lipid fluidity adaptation to changing environmental temperature. The fluidity of membranes and isolated lipids was measured using nitroxide-labeled fatty acids. The spectra of 2-(l0-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinoxyl in membranes indicated that there were two lipid environments within the membrane whose relative proportions were dependent both on temperature of measurement and on bacterial growth temperature. In contrast, 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinoxyl spectra showed a single lipid environment and plots of log order parameter (S,) vs l/T were biphasic with inflexion temperatures which were closely related to the bacterial growth temperature. As with membranes, plots of log S, vs l/T for total lipids, phosphatidylglycerol and cardiolipin, but not phosphatidylethanolamine, were biphasic and showed inflexions which correlated well with bacterial growth temperature. These results are interpreted as being consistent with a location for the desaturase within the bulk lipid of the membrane rather than in association with specific lipid types. is believed that the maintenance of correct membrane lipid bilayer fluidity is necessary to ensure the proper functioning of membrane-bound enzyme and transport systems (4-7). The fact that desaturase enzymes are membrane bound raises the possibility that they may not only modify fluidity, but may themselves in turn be regulated by their lipid environment. Thompson and Nozawa (8) have pointed out that it may be very difficult to determine whether temperature affects desaturase activity directly or is mediated indirectly through changes in membrane flu-

The ability of procaryotes and eucaryotes to adapt their membrane lipid composition in response to changes in environmental temperature has been well documented (l3). This adaptation usually involves a change in fatty acyl unsaturation, although other changes such as acyl chain length or methyl branching may occur, especially in procaryotes. The changes in unsaturation are mediated by desaturase enzymes or in some bacteria by alterations in the anaerobic pathway of fatty acid biosynthesis. It 1 To whom correspondence

should be addressed.

0003-9861/83 $3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

718

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idity. In Tetrahymenu the activity of the temperature-sensitive palmitoyl-CoA desaturase appears to be regulated by the physical state of the endoplasmic reticulum membrane where the enzyme is located (8, 9), although there may also be changes in the amount of desaturase as well (10). The phospholipid AEGdesaturase of rat liver microsomes appears to be regulated by membrane fluidity (11,12). Microsomes of the yeast Candida lipol&ca contain an oleoyl-CoA and a phospholipid Ala-desaturase, but only the oleoyl-CoA desaturase is activated by a decrease in growth temperature (11). In contrast, in bacilli that contain an inducible A5-desaturase the enzyme is unaffected by changes in membrane fluidity. Instead, there is a temperature-sensitive modulator protein and the turnover of desaturase protein is stimulated by higher temperatures (13, 14). The psychrophilic bacterium lkkrococcu~ cryophilus responds to changes in growth temperature by altering not the unsaturation but the chain length of its lipids (15). Its membrane phospholipids contain, throughout the growth temperature range, acyl chains that are almost entirely monounsaturated (95%), the double bonds being synthesized by a A9-desaturase (16, 17). This microorganism, therefore, provides an opportunity to investigate the effects of temperature and membrane fluidity on desaturase activity in a system in which the physical state of membrane lipids is not regulated through changes in their unsaturation. The relatively simple lipid composition of the organism is a particular advantage when investigating the effects of temperature on membrane function. The present paper describes experiments in which the effect of temperature on desaturase activity has been determined and correlated with the fluidity of membranes and isolated lipids as measured using electron spin resonance spectroscopy. The results are discussed in the context of the thermal adaptation of M. cryophilus to its psychrophilic habitat. MATERIALS

AND METHODS

Chemicals. The nitroxide spin probe 2-(3-carboxypropyl) - 4,4 - dimethyl - 2 - tridecyl - 3 - oxazolidinoxyl

IN BACTERIAL

MEMBRANES

719

(5NS)’ was purchased from Syva Associates, Palo Alto, California, and 2-(lo-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinoxyl(12NS) was prepared by the method of Waggoner et al (18). [1-“C]Palmitic acid (sp act 2.07 GBq mmol-‘) and [1-“Clstearic acid (sp act 2.12 GBq mmol-‘) were obtained from Amersham International PLC, Amersham, Buckinghamshire, England, and stored as solutions (1.85 MBq ml-‘) in ethanokwater (l:l, v/v) at -20°C. Authentic phospholipid and fatty acid standards were obtained from Sigma (London) Chemical Company Ltd., Poole, Dorset, England, or Nu-Chek Prep, Elysian, Minnesota. All other reagents and solvents were of analytical reagent grade. Organism and cultural conditions. Micrococcus ergophilus (ATTC 15174)was grown in a defined casamino acids-salts medium (NM medium) at temperatures between 0 and 21°C in a refrigerated orbital incubator (for details see (19)). Bacterial cultures were grown for at least four generations and two subcultures at the required temperature prior to use in an experiment. Preparatim of me&runes. Bacteria were harvested from cultures in mid exponential growth phase and disrupted using a French pressure cell as described previously (20). Membranes were collected by centrifuging cell lysates at 105,OOOgfor 1 h. The membrane pellet was resuspended in 1-2 ml of NM buffer (i.e., NM medium lacking casamino acids). Protein was measured by a modification of the Lowry method (21) using bovine serum albumin as a standard. If not required immediately, the membrane suspensions were stored at -20°C. Extraction and fraxtimatim of lipids. Bacteria were collected by centrifugation at 10,OOOg for 15 min and the cell pellet was resuspended in a small volume of NM buffer. The lipid was extracted overnight by the addition of 3.75 ml of chloroform:methanol(1:2, v/v) per ml of aqueous suspension and washed according to the procedure of Garbus et al. (22). The total lipid extract was estimated gravimetrically and stored at -20°C as a solution in chloroform:methanol (l:l, v/v). When required, phospholipid fractions were isolated by thin-layer chromatography (TLC) of total lipid (for details see Russell (23)). Phospholipid classes (phosphatidylethanolamine, PE; phosphatidylglycerol, PG; and cardiolipin, CL) were isolated by

’ Abbreviations used: 5NS, 2-(3-carboxypropyl)-4,4dimethyl-2-tridecyl-3-oxazolidinoxyl; 12NS, 2-(10carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxs~ NM medium, casamino acids-salts medium; NM buffer, NM medium lacking casamino acids; PE, phosphatidylethanolamine; PG, phosphatiylglycerol; CL, cardiolipin; TLC, thin-layer chromatography; GLC, gas-liquid chromatography; 16~0,palmitic acid; 130, stearic acid; 161, palmitoleic acid; 131, oleic acid.

720

FOOT ET AL.

preparative TLC on 0.3-mm-thick plates of silica gel H (Merck, Darmstadt, West Germany) using chloroform:methanol:acetic acid:water (35:15:10:3.5,by vol) as the developing solvent. Phospholipids were visualized by spraying with aqueous Rhodamine 6G (0.012%, v/v) and eluted with four aliquots of chloroform:methanol:diethyl ether (l:l:l, by vol). Individual phospholipids were estimated gravimetrically and stored as solutions at -20°C in chloroform:methanol (l:l, v/v). The fatty acyl composition of total lipid or phospholipid was determined by gas-liquid chromatography (GLC) analysis of fatty acid methyl esters prepared by transesterification using 2.5% (v/v) concentrated HzSOI in methanol. Fatty acid methyl esters were separated using a Perkin-Elmer Fll gas chromatograph as described previously (24). hatumse assay. Desaturase activity wae measured by the addition of 37 kBq of [1-i4C]palmitic or stearic acid to 10 ml portions of bacterial culture, which had been preincubated for 2 min at the assay temperature. After 6 min the reaction was terminated by the addition of 37.5 ml of chloroform:methanol (1:2, v/v), the was lipid extracted, and the phospholipid was isolated as described above. Preliminary experiments demonstrated that the incorporation and desaturation of fatty acids was linear for at least 6 min. Fatty acid methyl esters were prepared as described above, except that 4 mg of oleic acid and 2 mg of stearic acid were added to each transmethylation reaction tube. The fatty acid methyl esters were separated by argentation TLC on 0.2-mm-thick plates of silica gel HF (Merck) containing 10% (w/w) AgNOa using 8% (v/v) diethyl ether in light petroleum (bp 40-60°C) as the developing solvent. Fatty acid methyl esters were visualized using uv light and eluted from the gel using three aliquots of diethyl ether. After the solvent was removed, 14 ml of MI-97 scintillation cocktail (Packard Instrument Co. Ltd.) was added and the radioactivity measured using a Phillips Model PW 4510 liquid scintillation analyzer. Enzyme activity was expressed as “percentage desaturation per minute,” and the apparent energy of activation of desaturation was calculated from the slope of Arrhenius plots using the relationship E. = - slope X 2.30 X 8.314 kJ mol-‘. The lines on Arrhenius plots were drawn on the basis of visual inspection and regression analysis of the upper and lower portions of the plots. Although it is difficult to eliminate all subjectivity using this method, we did not have more sophisticated computer analysis available. Preparation of samples for ESR spectroswpg. (a) MEMBRANES: An appropriate amount of spin label dissolved in methanol was added to a dry glass tube and the solvent removed with a stream of nitrogen. A 200-p] aliquot of a suspension of membranes (4-5 mg of protein) in NM buffer (see above) was added and agitated to ensure partition of the spin label (final concentration lo-* M) in the membrane lipid. These

conditions ensured that the molar ratio of membrane phospholipid to spin label was at least 150~1. K,Fe(CN), was added to membrane samples to give a final concentration of 0.1 M in order to prevent biological reduction of the spin label. (b) LIPID SAMPLES: Approximately 5 mg of lipid, dissolved in chloroform:methanol (l:l, v/v), was mixed with spin label (final concentration lo-” M), the solvent evaporated, and the lipid resuspended in 200 ~1 of NM buffer by agitation, so as to give a lipid-to-spin label ratio of approximately 333:l. Purified PE and CL required sonication for 60 s to disperse them fully. Recording and interpretation of ESR spectra ESR spectra were recorded in the form of their first derivative using a Varian E4 X-band spectrometer fitted with an E257-9 variable temperature accessory. Sample temperature was measured using a copper-constantan thermocouple. The order parameter, Ss. was calculated from spectra of 12NS and 5NS as described in the literature (25, 26). RESULTS 1.

Efect of Temperature on Desaturase Activity

The desaturase activity of bacteria grown at 3,7.5,12,16.5, and 21°C was measured at 3” intervals between 3 and 21°C using [l-‘4C]palmitic or stearic acid as the substrate (results from palmitic acid are shown in Table I). Results from experiments using bacteria grown at 16.5”C are presented in the form of Arrhenius plots in Fig. 1. Generally, these plots were clearly biphasic with a discontinuity at a temperature (inflexion temperature) which was a function of the growth temperature, but which was independent of the chain length of the substrate (Fig. 2). However, in a few cases it was more difficult to construct biphasic plots (e.g., the line for stearate desaturation in Fig. l), and indeed it could be argued that the plot should be a curve. Although it is not possible to be categorical about this point (see Materials and Methods), the fact that the inflexion temperatures form a progression with growth temperatures, and that the majority of the plots are clearly biphasic, suggests that this is a valid treatment of the data. The activation energy of the enzyme, calculated from the slope of that part of the Arrhenius plot encompassing the growth temperature, was not constant, but increased as the bacterial growth temper-

TEMPERATURE TABLE

ADAPTATION

IN BACTERIAL

721

MEMBRANES

I

DEPENDENCE OF DESATURASE ACTIVITY ON BACTERIAL GROWTH TEMPERATURE AND ASSAY TEMPERATURE WITH PALMITIC ACID AS SUBSTRATE Bacterial growth temperature (“C) Desaturase assay temperature (“C)

3

7.5

12

16.5

21

3 6 9 12 15 18 21

2.73 3.68 4.53 4.94 -

3.55 4.20 4.48 4.81 5.64 6.20 6.60

3.42 4.78 5.60 5.97 6.45 6.96 6.27

4.31 5.81 7.33 7.87 8.53 9.10 9.74

6.28 6.53 7.16 7.83 8.31 8.68 8.75

Note. All values are the averages of two determinations and are expressed as percentage desaturation/min. Mean variation = 4.16%.

ature was lowered (Fig. 3). The activation energy for stearic acid desaturation showed a greater temperature dependence than that for palmitic acid desaturation; this may reflect a difference in the solubilities of the substrates in the aqueous medium or their transport into the bacteria. The ratios of C&C6 fatty acids from

FIG. 1. Arrhenius plots of desaturase activity in bacteria grown at 16.5”C. Portions (10 ml) of culture were equilibrated at the required temperature for 2 min before starting the assay with 37.4 kBq of [l“C]16:0 (m) or L8:O (0). After a 6-min incubation the reaction was stopped with chloroform:methanol and desaturation measured by argentation TLC as described under Materials and Methods. Each experimental value is the mean of duplicate assays, indicated by the bars.

04 0

/ ’ 5

10 GROWTH

15 TEMP.

10

15

‘C

FIG. 2. Correlation between bacterial growth temperature and the inflexion temperatures from Arrhenius plots of desaturase activity and log S, versus l/T plots of spin-labeled fatty acids in membranes and total lipid extracts. Desaturase activity: C16:0,0, Cls:,,, 0,5NS/memhranes, A; 5NWtotal lipid, 0; 12NS/ total lipid, W. The lines of best fit were determined by linear regression analysis for the desaturase and spin label experiments separately.

the total lipids of M. cryophilus grown at different temperatures are shown in Table II. These results confirm that the bacteria had adapted to lower growth temperatures by synthesizing greater proportions of fatty acids with the shorter acyl chain length (16, 17).

4

II Growth

12 16 Iempcrotura

20 (‘Cc)

FIG. 3. Correlation between bacterial growth temperature and the activation energy of desaturation. The apparent activation energies (E.) of the upper slopes on Arrhenius plots of the desaturation of [l14C]16:0 (W) or 180 (O), obtained from a series of experiments as described in Fig. 1, were calculated using the relationship E, = - slope X 2.303 X 8.314 kJ mol-‘.

722

FOOT TABLE

II

THE DEPENDENCE OF LIPID COMPOSITION OF M. cryqphilus ON GROWTH TEMPERATURE Growth temperature (“C)

of

2

h

Cls/CIG ratio” of phospholipid fatty acyl chains

3 7.5 12 16.5 21 ‘Ratio

ET AL.

1.1 1.7 2.0 2.2 3.3

Cls:of CI~:I&CO + Ci6:i.

2 Fluidity Measurements on Membranes and Isolated Lipids Using Spin Probes ESR spectra of the spin-labeled fatty acids 5NS and 12NS in membranes of MI cy/ophilus grown at 21°C are shown in Fig. 4. Whereas the spectra of 5NS consisted of a single component over the temperature range 3-32”C, those of 12NS contained two components showing that two environments for the spin probe coexisted within the membrane. The T,, values for 5NS and both T,, values for 12NS in the membrane spectra decreased as the temperature was raised, indicating that both spin probes were located in lipid environments. The relative intensities of the two environments for 12NS appeared to change

FIG. 4. ESR spectra (20°C) of (A) 5NS and (B) 12NS in membranes of bacteria grown at 21°C showing the measurement of T,, !&, T;,, a and b.

01 10 20 30 Assay l.mp.rotur~

40 ( ‘c)

50

FIG. 5. Temperature dependence of the ratio a/b from the ESR spectra of 12NS in membranes of bacteria grown at 0 (m) and 21’C (0).

with temperature; the more mobile component (!I’;,) predominating at higher temperatures. The proportion of the two environments for the 12NS spin probe was also dependent on the bacterial growth temperature. This is shown empirically in Fig. 5, where the ratio a/b (indicated in Fig. 4) is plotted against temperature for membranes of bacteria grown at 0 and 21°C. Below 25°C (the upper growth temperature limit of the organism), spectra of membranes from bacteria grown at lower temperatures contained a greater proportion of the more mobile component. The ESR spectra of 5NS and 12NS in aqueous dispersions of the total lipids isolated from M. cryophilus grown at 21°C are shown in Fig. 6. These spectra consisted of a single spectral component indicating one environment for the spin probe, in contrast with spectra of 12NS in membranes. The order parameters for the spin probes in extracted lipids were always lower than those of the corresponding membranes. Plots of log (order parameter, S,) versus l/T were constructed from the spectra of those systems which exhibited a single environment for the spin probe, i.e., 5NS in membranes, and 5NS and 12NS in total lipids. Plots of this type have previously been shown to be biphasic with inflexions attributed to lipid phase transitions (26, 27). Plots of log S, versus l/T for spin probes in membranes and in total lipid dispersions from bacteria grown at differ-

TEMPERATURE

ADAPTATION

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723

same as those obtained with total lipid; PE, however, gave linear plots and showed no inflexions. DISCUSSION

1. Temperature Dependence of

Desaturase Activity

FIG. 6. ESR Spectra (20°C) of (A) 5NS and (B) 12NS in total lipid extracts of bacteria grown at 21°C.

ent temperatures are shown in Fig. ‘7.The majority of these plots were biphasic with an inflexion at a temperature which showed a reasonable correlation with the bacterial growth temperature; these inflexion temperatures are plotted alongside those for the desaturase in Fig. 2. As discussed above for the Arrhenius plots of desaturase activity, it was not always possible to be certain of the inflexion temperature. In several instances the plots might be drawn as curves and some appear to have a second inflexion at higher temperatures; since these second inflexions occur above the upper growth temperature limit they are not considered further. The direction of the inflexion for the plot of 5NS in total lipid (Fig. ‘7B) was opposite to that for 5NS in membranes (Fig. ‘7A) and 12NS in total lipid (Fig. 7C). Although similar differences have been observed elsewhere (29,30), the explanation for this is not clear. Phospholipids constitute 85% of the total lipid of M crgophilus membranes. These consist of PG, PE, and CL which constitute 45, 45, and 10% of the total, respectively. ESR spectra were obtained from 5NS and 12NS in aqueous dispersions of these phospholipids, and plots of log & versus l/T were constructed (not illustrated). PG and CL gave results which were essentially the

The ability of iM cryophilus to alter its membrane lipid acyl composition in response to growth temperature changes, as exemplified by the results in Table II, is well established (15,24). It seems reasonable to suggest, therefore, that the effects of growth temperature on the behavior of the desaturase result from changes in the physical state of the surrounding phospholipids. In studies on other membranebound enzymes or on transport systems, similar inflexions in Arrhenius plots have been attributed either to phase transitions (e.g., gel = liquid crystalline) or to phase separation of phospholipids (31-34), giving rise to conformational changes in the proteins (cf. 35,36). The activity of liver stearoyl-CoA desaturase has been shown to depend on the nature of its lipid environment; inflexions in Arrhenius plots in this case were interpreted as being due to sudden changes in the rate-limiting step of desaturation (37). There is no unified interpretation of the biological significance of inflexions in Arrhenius plots of membrane protein activity. Indeed, not all the enzymes within a particular membrane may show such discontinuities (38), and if they do, the temperatures at which they occur may not be the same (39, 40). It could be argued that the inflexions were due to conformational changes in the enzyme protein induced directly by temperature. However, in the present experiments there are not only concomitant compositional changes in the lipids, but also modifications to their physical properties. The fact that the desaturase activation energy (calculated from the slopes of the Arrhenius plots encompassing the growth temperature) increases with decreasing growth temperature (Fig. 3) probably indicates that the organism did not fully adapt to the lower growth temperature with respect to maintaining a constant

40

30

33

1+x

103

20

35

10

0

OC 37 31

40

30 3.3 l,T x 103

20

--

-.

FIG. 7. Plots of log & versus l/T from ESR spectra of 5NS and 12NS in membranes in membranes, (B) 5NS in total lipid, (C) 12NS in total lipid.

31

10

0

37

"C

and total lipids of bacteria

3.5

grown at different

temperatures;

(A) 5NS

TEMPERATURE

ADAPTATION

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725

demonstrated in AchoLeplaama laidlawii probed with 12NS (47) and in red cell membranes probed with 5NS (48). In each case the proportions of the two lipid environments varied with temperature, although the less mobile environment was less temperature sensitive. The presence of two environments in A. bidlutii mem2 Membrane Fluidity Measurements branes was confirmed by NMR spectrosThe observation of two motional envi- copy, which also ruled out protein-lipid inronments for the spin probe 12NS in iso- teractions as the source of the second lipid lated membranes of M. crgophilus is of environment (49, 50). Thus it seems that considerable interest. The parallel obser- lipid-protein interactions need not necvation of a single environment for 5NS essarily be invoked to account for the preswhich probes the polar region of the mem- ence of two lipid environments in membrane suggests either that the major dif- branes of M. cryophilus. ferences in environment exist in the acyl In contrast to the plots of log S’Sversus chain region of the membrane (rather than 1/T for 5NS in membranes and for 5NS the polar head group region) or that the and 12NS in dispersions of total lipid, PG, ESR spectrum of 12NS is simply more sen- or CL, which all had inflexions showing a sitive to differences of environment than close correlation with the bacterial growth that of 5NS. temperature, no inflexions were observed The identities of the two environments in plots from dispersions of PE. Since there are not clear: they may represent domains were no significant differences in the fatty of different lipid composition or regions of acyl composition of the individual phosphospholipid influenced to different extents pholipids, this difference appears to arise by the presence of membrane proteins (44). from the nature of the phospholipid polar The latter explanation is supported by the head groups. Unsaturated PE is known to observation of a single environment in the form a hexagonal phase rather than a biESR spectra of 12NS in dispersions of total layer in model systems unless it is mixed lipid extracts. The former cannot be ex- with other phospholipids (51, 52). Thus, cluded, however, since the presence of absence of an inflexion in this plot does membrane proteins could influence the not rule out the involvement of PE in the separation of phospholipids into different control of the desaturase. domains. One possibility is that these doThe origin of the inflexions in the order mains may represent lipids of the inner parameter plots is not clear. They are cerand outer membranes of the Gram nega- tainly not due to chain-melting transitions tive cell envelope. Studies on Escherichia since differential scanning calorimetry coli are inconclusive (e.g., cf. (45, 46)). shows that these occur at temperatures The dependence of the ratio of the two well below zero in M. crgophilus (N. J. Rusenvironments for 12NS both on temperasell and P. J. Quinn, unpublished results). ture and on lipid chain length (Fig. 5) fa- Lateral phase separation or formation of vors the existence of an equilibrium be- clusters would seem to be a more likely tween domains of phospholipid, probably explanation, since these properties would of different composition or different phases be dependent on temperature and on C18/ (e.g., lamellar and hexagonal), which is Cl6 ratio which is highly sensitive to the sensitive to changes in acyl chain length bacterial growth temperature (Table II). (i.e., growth temperature) and assay temperature. There is evidence from 31PNMR 3. Relationship between Desaturase that the lipids of M. crgophilus can adopt Activity and Membrane Fluidity hexagonal phases (N. J. Russell, unpubThe inflexions in the Arrhenius plots of lished results). Two lipid environments have also been desaturase activity and those from the orlipid fluidity. Similar incomplete adaptation to lowered temperature has been observed in Streptococczlsfaecalis membranes (41), in the synaptosomal membranes of fish (42), and in the endoplasic reticulum of Tetrahymena (43).

726

FOOT

der parameter/temperature plots show a close correlation with the bacterial growth temperature (Fig. 2). The proportions of the two lipid environments present in the bacterial membrane also show a dependence on temperature and on the bacterial growth temperature (Fig. 5). Similar correspondence between growth temperature and the physical state of phospholipids has been observed by some workers (29,46,5357) but not others (58). M. crgophilus regulates membrane fluidity by changes in acyl chain length and a temperature-dependent elongation mechanism has been proposed (20). The present results suggest that as the growth temperature drops, there is not only an increase in the proportion of ($6 acyl chains, but there is also a preferred desaturation of palmitic acid. This is demonstrated by the differential effect on activation energies for the desaturase acting on palmitic or stearic acid as the substrate (Fig. 3). Thus, as the proportion of C16acyl chains rises, the high degree of unsaturation of the phospholipids is maintained. Given that the spin label reports the motion of the bulk lipid phase, the close relationship between the enzymatic activity of the desaturase and the physical properties of the membrane phospholipids as monitored by such probes suggests that the enzyme is probably located within the bulk lipid of the membrane, rather than in association with specific lipids with properties different from that of the main bilayer regions. REFERENCES 1. FULCO, A. J. (1974) Annu. Rev. B&hem 43,215241. 2. ESSER, A. F., AND SOUZA, K. A. (1976) in Extreme Environments. Mechanisms of Microbial Adaptation (Heinrich, M. R., ed.), pp. 283-294, Academic Press, New York. 3. SMITH, M. W. (1978) in Biochemical Society Symposium No. 41 (Smellie, R. M. S., and Pennock, J. F., eds.), pp. 43-60, The Biochemical Society, London. 4. FARfAs, R. N., BLOJ, B., MORERO, R. D., SIRERIZ, F., AND TRUCCO, R. E. (1975) Biochim Biophys. Acta 415, 231-251. 5. SANDERMANN, H., Jr. (1978) B&him Biqphys. Acta 515, 209-237.

ET AL. 6. MCELHANEY, R. N. (1976) in Extreme Environments. Mechanisms of Microbial Adaptation (Heinrich, M. R., ed.), pp. 255-281, Academic Press, New York. 7. KATES, M., AND KUKSIS, A. (eds.) (1980) Membrane Fluidity; Biophysical Techniques and Cellular Recognition, Humana Press, Clifton, N. J. 8. THOMPSON, G. A., JR., AND NOZAWA, Y. (1977) Biochim Biophys. Acta 472.55-92. 9. FUKUSHIMA, H., NAGAO, S., OKANO, Y., AND NoZAWA, Y. (1977) Biochim. Biophys. Actu 468. 442-453. 10. FUKUSHIMA, H., NAGAO, S., AND NOZAWA, Y. (1979) Biochim Biophys Actu 572.178-182. 11. PUGH, E. L., AND KATES, M. (1979) Lipids 14,159165. 12. PUGH, E. L., KATES, M., AND SZABO, A. G. (1980) Cad J. Biochem 56,952-958. 13. FULCO, A. J. (1972) .I Biol Chem. 247,3511-3519. 14. FUJII, D. K., AND Fu~co, A. J. (1977) .I Biol Chem 252,3660-3670. Biuphys. Acta 231, 15. RUSSEL.L,N. J. (1971) B&him 254-256. 16. RUSSELL, N. J. (1977) Biochem Sot Tram. 5,14921494. 17. RUSSEU, N. J. (1978) Biochim Biqphys. Acta 531. 179-186. 18. WAGGONER, A. S., KINGZETT, T. J., ROI-CSCHAEFER, S., GRIFFITH, 0. H., AND KEITH, A. D. (1969) Chem Phys. Lipids 3.245-253. 80, 21719. RUSSELL, N. J. (1974) J. Gen Microbid 225. 20. SANDERCOCK, S. P., AND RUSSELL, N. J. (1980) Biochem J. 188.585-592. 21. MARKWELL, M. A. K., HAAS, S. M., BIEBER, L. L., AND TOLBERT, N. E. (1978) AnaL Biochem 87, 206-210. 22. GARBUS, J., DE LUCA, H. F., LOOMANS, M. E., AND STRONG, F. M. (1963) J. Bzbl Chem 238.59-63. I&t. 4, 23. RUSSELL, N. J. (1978) FEMS Microbial 335-338. 24. RUSSELL, N. J., AND VOLKMAN, J. K. (1980) J. Gen Microbid 118, 131-141. 25. GAFFNEY, B. J. (1975) Proc. Nat. Acad Sci USA 72, 664-668. 26. BALES, B. L., LESIN, E. S., AND OPPENHEIMER, S. B. (1977) B&him Biophys. Ada 465, 400407. 27. INESI, G., MILLMAN, M., AND ELETR, S. (1973) J. Mol Biol 81,483-504. 28. LAGGNER, P., AND BARRATT, M. D. (1975) Arck B&hem. Biophys. 170, 92-101. Bic29. YANG, L. L., AND HAUG, A. (1979) B&him phys. Ada 573,308-320. 30. BALDASSARE, J. J., RHINEHART, K. B., AND SILBERT, D. F. (1976) Biochemistry 15,2986-2994. 31. WARREN, G. B., BIRDSALL, N. J. M., LEE, A. G., AND METCALFE, J. C. (1974) in Membrane Pro-

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teins in Transport and Phosphorylation (Azzone, G. S., Klingenberg, M. E., Quagliariello, E., and Siliprandt, N., eds.), pp. 1-12, Elsevier, New York. 32. LEE, A. G. (1975) Progr. Biophys. Mol. Biol. 29,

MEMBRANES

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