Characterization and Composition of the Purple and Red Membrane from Halobacterium cutiru&ruml
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S. C . KUSHWAHA, M. KATES,AND W. G. MARTIN Department of Biochemistry, University o f Ottawa, and Division of BioCogical Sciences, National Research Council, Bttalva, Canada K I N 6N5 Received August 29, 1974 Kushwaha, %. C., Kates, M. & Martin, W. G. (1975) Characterization and Composition cutirubrrsm. Can. J. Biochcm. 53, sf the Purple and Red Membranes from HaEobacte~~iunz 284-292 The purple membrane (bacteriorhsdopsin) isolated from cells of Halobacters'urn ctltirubrum grown anaerobically in the light was shown to contain 77% protein and 20% lipids by weight. The protein component consisted of a single protein mseity, having a molecular weight of (19.6 iz 0.8) x 10" complexed with retinal in mole ratio of 2: 1, respectively. The protein moeity is not glycosylated but may be phosphorylated (ca. 2 mol of phosphate per mole of protein). The red membrane contains 56% protein and 38% lipids, including bacterioruberins. Several polypeptide components ape present including some which may be glycosylated and/or phosphorylated. The lipids of both membranes contained phosphatidyl glycerophosphate (52% ) and phosphatidyl glycerol (3-4% ) but the sulfated lipid components, glycolipid sulfate and phosphatidyl glycerosulfate, were present excltlsive?y in the purple membrane, the red membrane containing instead two unidentified glycolipids. Neutral lipids (squalenes, vitamin MK-8, etc.) were present in both membranes to the extent of 7-9%. Kushwaha, S. C., Kates, M. & Martin, W. G. (1975) Characterization and Composition of the Purple and Red Membranes from NaPobacteriu~ncutirubrirm. Can. J. Biocher~a.53. 284-292 Ea membrane pourpre (bacttriorhodopsine) isolte des cellules de Halsbacteriuttz cutirubrum cultivt en anatrobiose B la lumi2re contient 77% de prottine et 20% de lipides en poids. Cette teneur prottique consiste en une seule prottine de poids molCculaire &gal (19.6 iz 0.8) x lo3 et formant un complexe avec le rttinal dans un rapport molaire de 2: 1. La fraction prottique n9est pas glycosylte mais elle peut Stre phosphsrylte (ca. 2 mol de phosphate par mole de prottine). La membrane rouge contient 56% de prottine et 38% de lipides, y compris les bacttriorubtrines; plusieurs composts polypeptidiques sont prtsents parmi lesquels certains peuvent &re glycosylts et/ou phosphorylts. Les lipides des deux membranes contiennent de phosphatidyl glyctrophosphate (52%) et de phosphatidyl glyctrol (3-4% ) . Ees composts lipidiques sulfatds, glycolipide sulfate et phosphatidyl glyctrosulfate, sont trouvts exclusivement dans la membrane pourpre, la membrane rouge contenant plutbt deux glycolipides non identifits. kes deux membranes contiennent jusqaa987 4 % de lipides neutres (squalbnes, vitamine MK-8, etc. ) . [Traduit par le journal]
Intrsductisn Pigmented extremely halophilic bacteria grown aerobically produce largely a red membrane in which the red pigments consist of CBo carotenoids called ""bcterioruberins" ( 1, 2). When the cells are grown anaerobically in light, the membrane is largely purple, the chromophore consisting of a retinal-protein complex called ""bcteriorhodspsin" (3-8). This purple 'A preliminary report has been presented at the 17th annual meeting of the Canadian Federation of Biological Societies, Hamilton, Ontario, Canada, June 25-28, 1974. (Proc. Can. Fed. Biol. Soc. 17, Abstr. No. 447 4 1974)).
membrane complex was first found in Halobacteriurn halobium (3-8), but has since been shown to occur in Halobacterium cutirubru~n (2, 9 ) and in several other pigmented extremely halophilic bacteria ( 10). Under anaerobic conditions in light, the purple membrane appears to function as a light-driven proton pump and the cells utilize the resulting cherniosrnotic gradient for ATP synthesis (7, 8 ) . Preliminary studies indicated that the purple membrane H. cutirubrum (9) . , had a-so&what difierent composition than that from H. "'lobiurn 6). the lipid ponents of the purple membrane had not yet
fronn.
(59
KUSHWAHA ET AL.: HALOBACTERIUM CUTIRUBRUM MEMBRANES
285
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been examined in detail. The work presented purple membrane") was suspended in distilled water here deals with further analysis and character- (18 ml) and further purified by centrifugation on a discontinuous sucrose gradient, prepared by layering ization of the protein mseity and the individual 6 ml of 1.3 M sucrose solution on top of 3 ml of 1.5 M lipid components of the purple membrane from sucrose solution. These operations were carried out in H . cutirubrum. For comparison, the charac- a cold room at 4 " C . Centrifugation was carried out terization of lipids and proteins sf the red mem- at 40 000 r.p.m. (260 000 x g ) for 18 h in an SW41Ti swinging-bucket rotor in an L2C 65B Beckman ultrabrane is also presented. centrifuge at 5 "C. Materials and Methods
Materials Deoxyribonuclease (BN-100, from beef pancreas) and vitamin K1were purchased from Sigma Chemical Co. and General Biochemicals, respectively. Silica gel H for thin-layer chromatography was obtained from Brinkmann Instruments (Canada) Ltd., and some of the silica gel G plates (500 pm thickness) used here were supplied by Mandel Scientific Co. Ltd., Montreal. Hexadecyltrimethylammonium bromide (cetyltrimethylammonium bromide, technical grade) was supplied by 3. T. Baker Chemical Co. Authentic samples of all trans-retinal was a gift from Dr. 0. Isler, Hoffmann-LaRoche and Co. Ltd., Basle, Switzerland. All solvents used here were either spectral grade or were purified according to Vogel ( 11) . Calibration proteins for gel electrophoresis were obtained from the following sources: bovine plasma albumin, crystalline, Pentex Brand (Miles Research Laboratories) ; human hemoglobin (blann Research Laboratory) ; ovalbumin (Sigma Chemical Co.); trypsin and a-chymotrypsin (Worthington Biochemical Corp.) . Preparation of Purple Mentbrarae The purple membrane was prepared essentially by the procedure of Stoeckenius and co-workers (3-5) from cells grown anaerobically in the light (7, 8 ) as follows: cells of H . cutirubrmrm were first grown aerobically at 37 "C in 1.5-1 batches of the standard complex medium for halophiles (12, 13), in 4-1 shaker flasks under fluorescent light ( a bank of eight 20411. ( 5 1-cm) "cool white" fluorescent tubes), in an incubator shaker (New Brunswick Scientific Co., Inc.), at a shaking rate of 120 r.p.m. for 8 h. Aeration was then reduced by lowering the rate of shaking to 100 r.p.m. for the remainder of the 4-day incubation period, other conditions remaining the same. Cells from six 1.5-1 batches were harvested by centrifugation at 10 000 x g for 20 min, washed twice with basal salt solution (NaC1, 250 g/l; MgSO,, 9.8 g/l; KC1, 2 g/l; pH 6.5), resuspended in 20 ml of basal salt solution and treated with about 10 mg of deoxyribonuclease for 30 min at room temperature with stirring. The cell suspension was then dialyzed at 4 "C against 6 1 of distilled water for 6 h with two changes of water at 2-h intervals. The dialysate was centrifuged at 10 000 x g for 20min to remove cell debris, and the supernatant was centriflaged at 50 000 x g for 1.5 h. The pellet was suspended in approximately 2QOml of distilled water and centrifuged at 10 000 x g for 20 min; the pellet was discarded and the supernatant was recentrifuged at 50 000 x g for 1.5 h. The 50 000 x g pellet ("crude
The purple membrane band appearing at the interface of the 1.5 M and 1.3 M sucrose layers, and the red band at the top of the 1.3 M sucrose layer, were displaced from the centrifuge tubes, collected, dialyzed against distilled water to remove sucrose, and recentrifuged in a similar sucrose gradient as described above. The purple membrane, which now formed a single sharp band, was removed and stored in sucrose solution at 4 "C; it was dialyzed against deionized distilled water just before use. The yield of pure purple membrane was about 2 mg/l of anaerobic culture (weight ratio of purp1e:red membranes in these cells was ccs. 311). Preparatiorl of Red Mertlbrane ( 3 , 4 ) For the preparation of large amounts of red membrane, ceIls of H. cutirubrum were grown aerobically in the light for 4 days at 37 "C in 1-1 batches of complex medium for halophiles at a shaking rate of 120-140 r.p.m. Cells were harvested and washed as described above. The procedure for the preparation of the red tnembrane was similar to that for the purple membrane described above, except that the second densitygradient centrifugation was done on a discontinuous sucrose gradient prepared by layering 5 ml of 1 M sucrose soIution on top of 3 ml of 1.3 M sucrose. After centrifugation for 18 h at 40 000 r.p.m., the red membrane formed a single sharp band at the junction of the 1.3 M and 1 M sucrose layers. It was recovered and stored as described for the purple membrane; the yield of red membrane was 20 mg/l of aerobic culture. Extraction of Lipids frortz Purple or Red Rlenzbrunes Total lipids were extracted from the sucrose-free red or purple membranes essentially by the method of Bligh and Dyer (14). To 5 ml of a suspension of the membrane (10-20 mg) was added 12.5 ml of methanol and 6.25 ml of chloroform, and the contents were thoroughly mixed. After 5-10 min equal volumes (6.25 ml each) of chloroform and water were added and the mixture was centrifuged. The chloroform phase was separated, diluted with benzene, and evaporated to dryness under a gentle stream of nitrogen; the residue of total lipids was dissolved to a known volume in chloroform, and aliquots were taken for chemical analyses. Fractionation of the lipids by acetone precipitation to separate polar and non-polar lipids was carried out as described previously ( 12). All operations were carried out under nitrogen and in semidarkness. Extraction of Retinal from the Purple Membrane To 400 pl of the sucrose-free dialvzed purple-membrane suspension (equivalent to 239 pg of Lowry protein) was added, with mixing, BOO pl of 0.08 M cetyltrimethylammoniurn bromide (pH 8) and 300 pl
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of 0.5 M hydroxylamine base (freshly prepared by mixing 10 ml of 1 M hydroxylamine hydrochloride with 5 ml of 2 N NaOH and adjusting the pH to 8 in a final volume of 20 ml). After 1-4 h at room temperature in the dark under nitrogen, the mixture was extracted by adding 2.0 ml methanol and 1.8 ml chIoroform, followed after several minutes by the addition of 1.0 ml each of chloroform and water. After centrifugation, the chloroform phase was separated, diluted with benzene, and evaporated to dryness under a gentle stream of nitrogen. The residue was dissolved in ethanol and the amount of retinyloxime was determined spectrophotometrically using the extinction coefficient E'%,,, at 355 m of 2020 (15). The protein remaining after the above extraction procedure was dissolved in 10% formic acid and its spectrum measured in the visible-IN range.
A r?-mino-AcidAnalysis Whole membrane or lipid-free membrane protein samples were hydrolyzed for 24 h or 48 h in 6 N HCl at 110 "C in evacuated sealed tubes. The acid was removed on a rotary evaporator at 37 "C and the residue finally dried over KOH pellets in vacuo. Amino acid composition was determined on a Beckman model I21 analyzer using the single-column procedure (Beckman methodology brochure, A-TB-059A). Hydrolyses were also carried out at 110 ' C for 4 and 6 h in 4 N HGl in sealed evacuated tubes for estimation of hexosamines. Tryptophane was estimated spectrophotometrically (25) in 0.1 N NaOH in a Cary spectrophotometer. Acrg~lanaide-GelEBeetrophoresis Electrophoresis and staining of whole membrane and lipid-free membrane protein was carried out according to the methods of Weber and Osborn (26) Thin-Layer Claromatography und ddentificatisrz of on 7 and 10% polyacrylamide gels except that the Lipid Components samples were dissolved in 2% (w/v) sodium dodecyl The polar and non-polar lipid components from sulfate. 0.02 M dithiothreitol, 8.0 M urea, and 0.01 M purple and red membranes were separated by thin- phosphate buffer at pH 9.0 and placed in boiling water layer chromatography on silica gel G or silica gel H for 2 min before layering on the gels. Background stain (500 pm-thick layers) using the following solvent (Coomassie blue) was removed by washing in 7% systems: A, 0.3% ethyl ether in hexane, for the separa- acetic acid for several days. tion of squalene, dihydrosqualene. tetrahydrosqualene, and @-carotene; B, 6% ethyl ether in hexane for the Sedimentation Anakysis Sedimentation experiments were carried out in 6 N separation of vitamin MK-8; C, 1% ethyl ether in chloroform for the separation of retinal and bisan- guanidine hydrochloride, 0.01 M dithisthreitol, 0.05 M hydrobacterioruberin; D, 7% methanol in chloroform Tris buffer, pH7.8, in a synthetic boundary cell at for the separation of monoanhydrobacterioruberins 20 "C and 60 000 r.p.m. Sedimentation rates were and bacterioruberins. Phospholipids and glycolipids calculated according to Schachman (28), and molecwere separated ( 16, 17) by two-dimensionaI thin-layer ular weight was estimated from the sedimentation rate chromatography using solvent E, chloroform - and the amino-acid analyses using the formula sf methanol - concentrated NHDM (6%:3 5 :5, v/v/v) in Tanford (29 ) . the first direction; and solvent F, chloroform - 90% Dry -Weight Deterplainations of the Membranes acetic acid - methanol (30 :20 14, v/v/v) in the second Dry weights of the membranes were determined by direction. Solvent systems A. B. and C were used only drying suitabk aliquots of dialyzed membrane suswith silica gel G plates while D, E, and F were used pensions in a weighing bottle over KQH in a vacuum with silica gel H plates. Before use. thin-layer chroma- desiccator (ea. 1 mm Hg (133 N/m2)) at room temtographic plates were washed with chloroformperature. methanol (1 :I, v/v), air dried, and activated at I00 "C Mensurernent of Spectra and Quantitative for 12 h. Determination sf Hsoprenoid Compounds The separated components were detected by their Visible and ultraviolet spectra of purple-membrane visual colors (retinal, C 4 0 and Cmpigments), by iodine vapor (squalene, cHi- and tetrahydrosqualenes m d and red-membrane suspensions and of MK-8, retinal, vitamin MK-8), by phosphorus spray reagent ( 18, 19) retinyloxime, and red pigments in suitable organic for phospholipids or by the a-naphthol spray reagent solvents were recorded with a Coleman-Hitachi-PerkinElmer model 124 spectrophstsmeter. The amount of ( 18) for glycolipids. The isoprenoid neutral lipids (9, 10, 12) and the phospholipids ( 16, 20) and glycolipids each isoprenoid compound was calculated by the (17, 20) were identified as described previously. The formula given in our previous communication (10) , values: MK-$, 268 at 248 phospholipid and glycolipid components were quanti- using the following El%'.,, tated by scraping each spot directly into digestion tubes nm in petroleum ether; retinal, 1510 at 383 nrn in and determining the phosphorus or sugar content, ethanol; retinyloxime, 2028 at 355 nm in ethanol; red pigments, 2540 at 4490 nm in acetone. respectively.
Chemical Analyses Phosphorus was determined by a modification (18) of Allen's procedure (21) or by the micromethod of Bartlett (22). Total hexose content was determined by the phenol - sulfuric acid procedure of Dubois er al. (23). Protein determinations on the dialyzed membrane suspensions were carried out by the method of Lowry et ai. (24).
Results General e&earacteri~ation 04 Fractions purple ~~~b~~~~ The purified preparation of purple membrane from H.cutirubrum showed absosp-
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KUSHWAHA ET AL. : HALOBACTERIUM CUTITPUBRUM MEMBRANES
TABLE I . Molecular-weight determination by SDS gel electrophoresis Molecular weight X 10-3
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Protein
7% gel*
10YQgel?
Purple membrane Y
Average, 19.6+0.8 Red membrane$ Band 1 2 3
I
4 I
'K)O
I
'
400
_
I
SW
t
l
600
WAVELENGTH, nm
5
6 *Results for two separate runs, each in duplicate. ?Results for one run in quadruplicate. $Bands are numbered from bottom to top of gel (Fig. 2B).
108
400
WAVELENGTH, nm
FIG. I. (A) Absorption spectrum of the pure purple membrane from H. clltirlrbrlrm in water ; concentration, 153 pg/ml; (B) absorption spectrum of the pure red membrane from H . cmsrr'rubrirna in water; concentration, 3.1 mg/ml.
tion maxima at 565 nm and 275 nm (Fig. 1A). The ratio of absorbance at 565 nm and 275 nm is 1 :2, and the molar extinction at 565 nm is 4.8 X 10% essentially as was reported for H. halobium purple membrane (5 ) .On disc-gel electrophoresis on 10% polyacrylamide gels, various preparations of the purple membrane consistently migrated as a single sharp band (Fig. 2A) ; similar results were obtained on 7 % gels (Fig. 2B). The lipid-free protein also gave a single band on disc-gel electrophoresis. From the plot of migration distance against log molecular weight for the membrane and the calibration proteins, a molecular weight of (19.6 -t- 0.8) X lo3was obtained (Fig. 3, Table 1 ) . The molecular weight calculated from the amino-acid composition (Table 2 ) was
18.5 X 10" assuming 1 mol of histidine per mole of protein. The sedimentation rate rneasured in guanidinium hydrochloride was 0.47 S. However, the protein was not stable in the solvent and gradually precipitated. Aggregates were also observed in the ultracentrifugal schlieren patterns. The partial specific volume of the protein was calculated from the amino-acid analyses (Table 2 ) according to Cohn and Edsall (30) and then corrected (28) by subtracting 0.81 to compensate for the type of solvent used. From this calculated value (0.736) and the sedimentation rate, the molecular weight was calculated (28) to be 19 X lo3. These results thus provide consistent evidence that the molecular weight of the purple membrane of H. cutirubrum is close to 20 000, a value significantly lower than that reported ( 5 ) for H.halsbium purple membrane (26 000). Amino-acid compositional data (Table 2) show that the mole ratios for most amino acids in the purple membrane from H. cutirubrum are generally similar to those reported for the H . halobium membrane ( 5 ) . The proportion of polar amino-acid residues (40% ) was also similar to that in the H. halsbium membrane. No significant amount of hexosamines was detected in the purple membrane, indicating that it is not a glycoprotein. Extraction by the Bligh-Dyer procedure alone (9) gave a value of 0.33% retinal corresponding to a retinal:protein mole ratio of 1 : 3.2. However, extraction in the presence of hexa-
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CAN. J. BIOCHEM. VOL. 53, 1955
FIG.2. Disc-gel electrophoresis on poiyacrylamide of purple and red membranes from H . curirubr~mmin sodium dodecyl sulfate. (A) 18$; polyacrylamide: (a) calibration mixture of bovine plasma albumin, ovalbumin, a-ehyrnotrypsin; (b) mixture of bovine plasma albumin, ovalbumin, purple membrane (preparation I); ( c ) purple membrane (preparation I); (d) calibration mixture of bovine plasma albumin, ovalbumin, trypsin, hemoglobin; (e) purple membrane (preparation 11); (f) mixture of bovine plasma albumin, ovalbumin. purple membrane (preparation 11). (B) 7yi polyacrylamide: (m) calibration mixture of bovine plasma albumin (ditner and monomer), ovalbumin, trypsin, hemoglobin; (n) purple membrane (preparation 111); (o) red membrane; (p) red membrane overloaded.
45 ) a retha1:protein mole ratio for H . Ir~celobiurn purple membrane of 1:1. The purple membrane of H. cutirubrurn had 10 % gel 20% total lipids by weight and 77% protein (Lowry et al. (24)) (Table 3 ) . The phosphorus content of the purple membrane was found to be 1.03 %, of which only 79% was found in lipids (Table 3 ) , the remainder, presuinably, being bound to the protein (ca. 1.8 atoms per mole protein). The hexose content of the purple membrane was found to be 2.6% , a11 of which was accounted for in the lipids (Table 3 ) . This finding supports the above conclusion that the MIGRATION DISTANCE, cm membrane is not a glycoprotein. FIG.3. Plot of migration distance vs. logarithm of Weal Membrane molecular weights of the purple-membrane and calibraThe purified red-membrane preparation tion proteins after electrsphoresis on 7 and 10yOpolyshowed absorption maxima at 535, 500, and acrylamide gels in sodium dodecyl sulfate. 478 nm (Fig. IB), typical of bacterioruberins decyltrimethyl ammonium bromide and hy- ( 1, 2, 10). On disc-gel electrophoresis (Fig. droxylamine as described in the methods section 2B) it showed the presence of at least six bands; gave a higher retinal content corresponding to a the lipid-free membrane preparation showed the retinal: protein mole ratio of 1 : 2.2 (Table 3 ) . same six bands. There was considerable backIn contiast, Oesterhelt and Stoeckenius reported ground staining between bands but the top and
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KUSHWAHA EF AL.: HALOBACTERIUM CUFIRUBRUM MEMBRANES
TABLE2. Amino-acid composition of purple- and red-membrane preparations Purple membrane
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Amino acid Asp Thr Ser Clu Pro G~Y Ala Val Met I le Leu TY~ Phe His =JY~ Arg Try 3 cys
rno1/100 mol
Red membrane mole ratiosa
H . cutbritbr~irnb H. halobirlnzC
H. cutirrlbriinz
H . hnlobium*
mol/ 100 mol H . ciirinthrume
H . hadohiumC
8.6 5.7 7.8, 6.8 5.6, 5 .O 7.4 6.5 4.3 4.6 10.6 9.6 10.9 11.8 8.4 7.3 2.7 4.1 5.6 5.6 12.2 13.2 3.6 3.7 4.2 4.6 0.6 8.6 2.4 5 .O 2.7 5.9 2.30 (2.3) 0.6 (0.1) Total amino-acid residues
Welative to histidine. hAverage of six determinations on two separate preparations. cData from Stoeckenius and Kunau (4) and Oesterhelt and Stoeckenius ( 5 ) . *Data from Oesterhelt and Stoeckenius (5). eAverage of four determinations on one preparation. fExtrapoled to zero time of hydrolysis. UDetermined spkctrophstometrically (25). hCalcutated molecular weight, 18 500. fCalculated molecular weight. 26 000.
TABLE 3. Overall composition of membrane preparations Purple
Density, g/ml (5 "C)
c, 5%
H, 56 N, 57% Protein, %, Lipid, O/, kipid:protein, weight ratio Retinal," % Retinal :protein, mole ratio Total P, ';, Total lipid P, % Total hexose, 'r, Total lipid hexose, % *Extracted in presence of hydesxylamine (5.
15).
Red
37-38% by weight and the protein (Lowry et al. (24)) accounted for 55-56% of the weight (Table 3 ) . The phosphorus content was 1.896, of which 82% was found in the lipids (Table 3 ) , indicating the presence of some phosphoproteins in the membrane. The hexose and hexosamine content of the red membrane was found to be 6.9%, of which 66% was accounted for in the lipids. This would suggest that the red membrane contains glycoproteins. The amino-acid composition was very similar (Table 2) to that reported for H. halobium (4). Only traces of hexosamines were present in samples hydrolyzed for 12 h.
Lipid Components of Purple and Red bottom of the gels were clear. The major bands Membranes (Fig. 2B) had molecular weights given in Table Thin-layer chromatography of the total lipids 1; there was no band in the region of the single from the purple membrane showed that the polar protein band from the purple membrane in lipids were phosphatidyl glycerophosphate, neighboring gels. triglycosyl diether, glycolipid sulfate, phosphaThe lipid content of the red membrane was tidy1 glycerosulfate, and phosphatidyl glycerol
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CAN. J. BIQCHEM. VOL. 53, 1975
FIG.4. Two-dimensional thin-layer chromaaograms on silica gel H of total lipids of: (A) pure purple membrane; (B) red membrane, from H. cratir~lbnlm.Identity of spots: GLS, glycolipid sulfate; TGD, triglycosyli diether; PGP, phosphatidyl glycerophosphate; PGS, phosphatidyl glycerosulfate; PG, phosphatidyl glycerol; X I and X2, unidentified glycolipids; PA, phosphatidic acid; NL, neutral lipids. Solvent systems: (1) chloroform - methanol - concentrated NHIOH (65:35:5, v//v); (2) chloroform - 90% acetic acid - methanol (30:20:4, v/v). TABLE 4. Lipid composition of membrane preparations*
Total iipids, Lipid component Neutral lipids Squalene, dihydrosqualene, tetrahydrosqualene @-Carotene MK-8 Retinal C60 red pigments Phospholipids Phosphatidyl glycerol Phosphatidyl glycerophosphate Phosphatidyl glycerosulfate Glyccdlipids
x x
1
Triglyccdsyl diether 2
Glycolipid sulfate *See Figs. 4 and 5. ?The amount of XI
Purple membrane 4.5 Trace 1.6 2.5 -
Traces 69.3 Traces 10.3
7;by weight Red membrane 5 Traces 1.2 0.4
t t
Traces Traces
+ Xa was about 32-3570 by weight.
(diphytanyl glycerol ether analogues) (Fig. placed by the two unidentified glycolipids in the 4A, Table 4). In contrast, no sulfated polar red membrane (Table 4). lipids (glycolipid sulfate and phosphatidyi glyIn regard to the neutral lipids, these amounted cerosulfate were detected in the red membrane to 7 4 % of the total lipids in both membranes (Fig. 4B, Table 4), the main polar lipids being and consisted mostly ~f squalene, dihydro- and phosphatidyl glycerophosphate, phosphatidyl tetrahydrosqualenes, vitamin MK-8, diphytanyl glycerol, and two unidentified glycolipids (XI glycerol ether, and pigments; retinal was only in and X2). It is of interest that the contents of the the purple membrane and C50bacteriomberins major and minor phospholipids, phosphatidyl were only in the red membrane (Fig. 5 A,B ) . It glycerophosphate and phosphatidyl glycerol, re- is of interest that the contents of squalenes were spectively, are essentially the s m e in both mem- in the order, squalene < dihydrosqardene < branes, and that the glycolipid sulfate and trigly- tetrahydrosqualene in both membranes obtained cosy1 diether in the purple membrane are re- from anaerobic cells; this order was reversed in
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KUSHWAHA ET AL.: HALOBACTERIUM CUFIRUBRUM MEMBRANES
FIG.5. Thin-layer chromatogram on silica gel 6 of neutral (acetone-soluble) lipids of the purple and red membranes from H. cubirubruna. (A) Chromatogram of squalenes and carotenes, developed in solvent A, ethyl ether - hexane (0.3 : 99.7, v!v). (1) Acetone-soluble lipids from whole cells of H. clitirr4brrrm; (2) mixture of squalenes; (3, 4) lipids of the purple and red membrane, respectively, from anaerobically grown cells; (5) lipids of red membrane from cells grown aerobically. (B) Chromatogram of vitamin K and retinal developed in solvent B, ethyl ether - hexane (6:94, v!v). (1) Squalenes; (2) acetone-soluble lipids from whole cells of H. cutinibnrm; (3) vitamin Kr; 4, all-trans-retinal; (5,6) lipids of purple and red membranes, respectively, from cells grown anaerobically; 4, lipids of the red membrane from cells grown aerobically. Identity of spots: (a) @-carotene,(b) squalene, (c) dihydrosqualene, (d) tetrahydrosqualene, (e) saturated hydrocarbons, (f) vitamin K1, (g, h) cis- and trans-retinal. Dotted lines indicate that the spots were seen by their visual colors.
the membranes from cells grown aerobically (Fig. 5A).
Discussion As mentioned in our previous report (9), the purple membrane from H . cutirubrum appears to be generally similar to that of H . halobiunz (5), but differs in several respects. The present results show that in the H. cutirubrtim purple membrane the molecular weight of the protein moeity is CQ. 20 000 and the mole ratio of retina1:protein is 1: 2, compared to a protein molecular weight of 26 000 and a retinal : protein mole ratio of 1:1 reported for the H. halobiunz membrane ( 5 ) . It should be noted that the nlolecular weight of 20 000 in the case of H. cutirubrum was consistently obtained by three independent methods and is probably reliable to within t4% and thus is significantly lower than that for theH. halobium protein. However, it would be desirable to measure the molecular weight by equilibrium centrifugation in a suitable solvent, using a reliable partial specific volume. On the other hand, the amino-acid compositional data for the purple-membrane preparations are very similar, although significant differences in mole ratios of aspartic acid, methionine, arginine, and lysine were observed (Table 2). Also, the lipid to protein
weight ratio of the two preparations was similar ( 1:3 (Ref. 5 ) vs. 1:4 (Table 3)). The analytical data for the H. cutirubrum membrane (Table 3 ) show clearly that the protein is not glycosylated but may be phosphorylated (about 2 mol phosphate per mole protein). In regard to the retinal: protein ratio of 1:2 obtained for the W.cutirubrum preparation, it is possible that not all of the retinal was extracted, although the procedure used (ethyltrimethylammonium bromide (CTAB) plus hydroxylamine) was essentially the same as that used by Oesterhelt and Stoeckenius ( 5 ) . Control experiments using N-retinylidene hexylamine showed that free retinal could not be extracted from either the protonated or the unprotonated SchiR base by the Bligh-Dyer procedure or by the CTAB plus hydroxylamine procedure described in the methods section. Thus the retinal that was extracted must have been in the extractable free aldehyde or hydrated aldehyde form. The possibility that some retinal may be covalently bound to the protein in an unextractable form was eliminated by the finding that the protein remaining after extraction of the membrane, as described in the methods section was colorless and showed no absorption in the region 300500 nm characteristic of the protonated or unprotonated retinal Schiff base.
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CAN. J.
BIOGHEM.
The striking feature of the purple membrane, relative to the red membrane, is the exclusive presence of sulfated lipids (glycolipid sulfate and ghosphatidyl glycerosdfate) in the former membrane and their complete absence from the latter (Fig. 4 A,B). It may be noted that the glycolipid sulfate was found to be essential for formation of stable bilayers of H. cutirubrum Hipids ( 3 1 ) and that squalenes were considered to play a role in the salt-dependence of the halophile membranes (32). The purple and red membranes were also quite distinct with regard to their protein components, the single purplemembrane protein with molecular weight 20 080 being entirely absent from the red-membrane proteins, which consisted of about six components ranging in molecular weight from 10 000 to 60 008. Furthermore, the purplemembrane protein is phosphorylated but not glycosylated, but the red membrane contains both phosphorylated and glycosylated proteins. The findings presented here raise the question whether species differences occur in the purple membranes from different extremely halophilic bacteria. The present results suggest that the purple membrane of M. cutirubrum differs from that of the H.halobium, but direct comparison of the two membrane preparations would be desirable. This work was supported by a grant from the Medical Research Council of Canada (MA-4103). We are indebted to Dr. 0. Isler, Hoffmann-LaRoche Inc., Basle, Switzerland, for supplying several authentic samples, and to Mr. P. Pilon, Mr. J. Giroux, and Mrs. P. Fejer for technical assistance. 1. Kelly, M., Norgard, S, & Liaaen-Jensen, S. (1970) Aeta Chem. Scarzd 24, 2169-2182 2. Gocknauer, M. B., Kushwaha, S. C . , Kates, M. & Kuskner, D, J. (1972) Arkiv. Mikrobisl. 84, 339-349 3. Stoeckenius, W. & Rswen, R. (1967) J. Cell Biol. 34, 365-393 4. Stmkenius, W. & Kunau, W. H. (8968) J. Cell Biol. 38, 337-357 5. Oesterhelt, D. & Stoeckenius, W. (1971) Rrat. New Biol. 233, 149-152
VOL. 53, 1975
6. Blaanrock, A, E. & Stoeckenius, W. (1971) Nat. New Biol. 233, 152-154 7. Oesterhelt, D. & Stoeckenius, W. (8973) Proc. Rraal. Acad. Sci. U.S. 70, 2853-2857 8. Danon, A. & Stoeckenius, W. (1974) Proc. Rratl. Acad. Sci. U.S. 71, 1234-1238 9. Kushwaha, S. C . & Kates, M. (8973) Biochirn. Bioph-9s. Actca 316, 235-243 18. Kushwaha, S. C . , Gochnauer, M. B., Kushner, D, J. & Kates, M. (1974) Cat?.J. Microbial. 20, 241-245 81. Vogel, A. I. (1956) Practical Orgartie Chemistry, pp. 161-175, Longmans Green & Co. Ltd., London 12. Kushwaha, S. C., Pugh, E. L., Kramer, J. K. G. & Kates, M. (1972) Biochim. Bioplz~s.Acfa 260,492-506 13. Sehgal, S. N., Kates, M. & Gibbons, N. E. (1962) Can. J . Biochem. Plydol. 40, 69-8 1 14. Bligh, E. G. & Dyer, W. J. (1959) Carz. J. Biochem. Physiol. 37, 911-917 15. Wald, G. & Brown, P. K. (1953,/54) J . Gerz. Plysiol. 37, 189-200 16. Hancock, A. J. & Kates, M. (1973) J. Lipid Res. 14, 422-429 17. Kates, M. & Deroo, P. W. (1973) J . Lipid Res. 14, 438-445 18. Kates, M. (1972) Techniqnres of Lipidology, pp. 354359, North-Holland Publishing Co., Amsterdam and London 19. Vaskovsky, V. E. & Kostetsky, E. Y . (1968) J. Lipid Res. 9, 396 20. Kates, M. (1972) in Ether Lipids, Clzemistr~)arzd Biology (Snyder, F., ed.), pp. 351-398, Academic Press Inc., New York, N.Y. 21. Allen, R. J. L. (1948) Biochem. J. 34, 858-865 22. Bartlett, G. R. (1959) J. Biol, Clzern. 234, 466-468 23. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. (1956) Anal. Chem. 28, 350-354 24. Eowry, 0. H., Rosebroangh, N. J., Farr, A. L. & Randall: R.J. (1951) J. Biol. Chrtm. 193, 265-275 25. Bencze, W. L. & Schrnid, K. (1957) Anal. Clrena. 29, 1193-1196 26. Weber, K. & Osborn, M. (1969) J. Br'ol. Chetm. 244, 4406-44 12 27. Schwert, G. W. & Kaufman, S. (1951) J . Ba'ol. Chem. 198, 807-816 28. Schachman, H. K. (1957) kfeth. Etazymol. 4, 32-103 29. Tanford, C., Kawahara, K. & Lopanje, S. (1967) J. Am. Clzent. Ssc. 89, 729-736 30. Cohn, E. J. & Edsall, J. T. (1943) in Proteins, Amino Acids and Peptides (Cohn, E . J. & Edsall, J. T., eds), pp. 378-381, Reinhold Publishing Corp., New York, N.Y. 31. Chen, J. S., Barton, P. G., Brown, D. & Kates, M. (1 974) Biochim. Biophys. Acaa 352, 202-217 32. Eanyi, J. K., Plachy, W. Z . & Kates, M. (1974) Biochemistry, 13, 4914-4920
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S. C . KUSHWAHA, M. KATES,AND W. G. MARTIN Department of Biochemistry, University o f Ottawa, and Division of BioCogical Sciences, National Research Council, Bttalva, Canada K I N 6N5 Received August 29, 1974 Kushwaha, %. C., Kates, M. & Martin, W. G. (1975) Characterization and Composition cutirubrrsm. Can. J. Biochcm. 53, sf the Purple and Red Membranes from HaEobacte~~iunz 284-292 The purple membrane (bacteriorhsdopsin) isolated from cells of Halobacters'urn ctltirubrum grown anaerobically in the light was shown to contain 77% protein and 20% lipids by weight. The protein component consisted of a single protein mseity, having a molecular weight of (19.6 iz 0.8) x 10" complexed with retinal in mole ratio of 2: 1, respectively. The protein moeity is not glycosylated but may be phosphorylated (ca. 2 mol of phosphate per mole of protein). The red membrane contains 56% protein and 38% lipids, including bacterioruberins. Several polypeptide components ape present including some which may be glycosylated and/or phosphorylated. The lipids of both membranes contained phosphatidyl glycerophosphate (52% ) and phosphatidyl glycerol (3-4% ) but the sulfated lipid components, glycolipid sulfate and phosphatidyl glycerosulfate, were present excltlsive?y in the purple membrane, the red membrane containing instead two unidentified glycolipids. Neutral lipids (squalenes, vitamin MK-8, etc.) were present in both membranes to the extent of 7-9%. Kushwaha, S. C., Kates, M. & Martin, W. G. (1975) Characterization and Composition of the Purple and Red Membranes from NaPobacteriu~ncutirubrirm. Can. J. Biocher~a.53. 284-292 Ea membrane pourpre (bacttriorhodopsine) isolte des cellules de Halsbacteriuttz cutirubrum cultivt en anatrobiose B la lumi2re contient 77% de prottine et 20% de lipides en poids. Cette teneur prottique consiste en une seule prottine de poids molCculaire &gal (19.6 iz 0.8) x lo3 et formant un complexe avec le rttinal dans un rapport molaire de 2: 1. La fraction prottique n9est pas glycosylte mais elle peut Stre phosphsrylte (ca. 2 mol de phosphate par mole de prottine). La membrane rouge contient 56% de prottine et 38% de lipides, y compris les bacttriorubtrines; plusieurs composts polypeptidiques sont prtsents parmi lesquels certains peuvent &re glycosylts et/ou phosphorylts. Les lipides des deux membranes contiennent de phosphatidyl glyctrophosphate (52%) et de phosphatidyl glyctrol (3-4% ) . Ees composts lipidiques sulfatds, glycolipide sulfate et phosphatidyl glyctrosulfate, sont trouvts exclusivement dans la membrane pourpre, la membrane rouge contenant plutbt deux glycolipides non identifits. kes deux membranes contiennent jusqaa987 4 % de lipides neutres (squalbnes, vitamine MK-8, etc. ) . [Traduit par le journal]
Intrsductisn Pigmented extremely halophilic bacteria grown aerobically produce largely a red membrane in which the red pigments consist of CBo carotenoids called ""bcterioruberins" ( 1, 2). When the cells are grown anaerobically in light, the membrane is largely purple, the chromophore consisting of a retinal-protein complex called ""bcteriorhodspsin" (3-8). This purple 'A preliminary report has been presented at the 17th annual meeting of the Canadian Federation of Biological Societies, Hamilton, Ontario, Canada, June 25-28, 1974. (Proc. Can. Fed. Biol. Soc. 17, Abstr. No. 447 4 1974)).
membrane complex was first found in Halobacteriurn halobium (3-8), but has since been shown to occur in Halobacterium cutirubru~n (2, 9 ) and in several other pigmented extremely halophilic bacteria ( 10). Under anaerobic conditions in light, the purple membrane appears to function as a light-driven proton pump and the cells utilize the resulting cherniosrnotic gradient for ATP synthesis (7, 8 ) . Preliminary studies indicated that the purple membrane H. cutirubrum (9) . , had a-so&what difierent composition than that from H. "'lobiurn 6). the lipid ponents of the purple membrane had not yet
fronn.
(59
KUSHWAHA ET AL.: HALOBACTERIUM CUTIRUBRUM MEMBRANES
285
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been examined in detail. The work presented purple membrane") was suspended in distilled water here deals with further analysis and character- (18 ml) and further purified by centrifugation on a discontinuous sucrose gradient, prepared by layering ization of the protein mseity and the individual 6 ml of 1.3 M sucrose solution on top of 3 ml of 1.5 M lipid components of the purple membrane from sucrose solution. These operations were carried out in H . cutirubrum. For comparison, the charac- a cold room at 4 " C . Centrifugation was carried out terization of lipids and proteins sf the red mem- at 40 000 r.p.m. (260 000 x g ) for 18 h in an SW41Ti swinging-bucket rotor in an L2C 65B Beckman ultrabrane is also presented. centrifuge at 5 "C. Materials and Methods
Materials Deoxyribonuclease (BN-100, from beef pancreas) and vitamin K1were purchased from Sigma Chemical Co. and General Biochemicals, respectively. Silica gel H for thin-layer chromatography was obtained from Brinkmann Instruments (Canada) Ltd., and some of the silica gel G plates (500 pm thickness) used here were supplied by Mandel Scientific Co. Ltd., Montreal. Hexadecyltrimethylammonium bromide (cetyltrimethylammonium bromide, technical grade) was supplied by 3. T. Baker Chemical Co. Authentic samples of all trans-retinal was a gift from Dr. 0. Isler, Hoffmann-LaRoche and Co. Ltd., Basle, Switzerland. All solvents used here were either spectral grade or were purified according to Vogel ( 11) . Calibration proteins for gel electrophoresis were obtained from the following sources: bovine plasma albumin, crystalline, Pentex Brand (Miles Research Laboratories) ; human hemoglobin (blann Research Laboratory) ; ovalbumin (Sigma Chemical Co.); trypsin and a-chymotrypsin (Worthington Biochemical Corp.) . Preparation of Purple Mentbrarae The purple membrane was prepared essentially by the procedure of Stoeckenius and co-workers (3-5) from cells grown anaerobically in the light (7, 8 ) as follows: cells of H . cutirubrmrm were first grown aerobically at 37 "C in 1.5-1 batches of the standard complex medium for halophiles (12, 13), in 4-1 shaker flasks under fluorescent light ( a bank of eight 20411. ( 5 1-cm) "cool white" fluorescent tubes), in an incubator shaker (New Brunswick Scientific Co., Inc.), at a shaking rate of 120 r.p.m. for 8 h. Aeration was then reduced by lowering the rate of shaking to 100 r.p.m. for the remainder of the 4-day incubation period, other conditions remaining the same. Cells from six 1.5-1 batches were harvested by centrifugation at 10 000 x g for 20 min, washed twice with basal salt solution (NaC1, 250 g/l; MgSO,, 9.8 g/l; KC1, 2 g/l; pH 6.5), resuspended in 20 ml of basal salt solution and treated with about 10 mg of deoxyribonuclease for 30 min at room temperature with stirring. The cell suspension was then dialyzed at 4 "C against 6 1 of distilled water for 6 h with two changes of water at 2-h intervals. The dialysate was centrifuged at 10 000 x g for 20min to remove cell debris, and the supernatant was centriflaged at 50 000 x g for 1.5 h. The pellet was suspended in approximately 2QOml of distilled water and centrifuged at 10 000 x g for 20 min; the pellet was discarded and the supernatant was recentrifuged at 50 000 x g for 1.5 h. The 50 000 x g pellet ("crude
The purple membrane band appearing at the interface of the 1.5 M and 1.3 M sucrose layers, and the red band at the top of the 1.3 M sucrose layer, were displaced from the centrifuge tubes, collected, dialyzed against distilled water to remove sucrose, and recentrifuged in a similar sucrose gradient as described above. The purple membrane, which now formed a single sharp band, was removed and stored in sucrose solution at 4 "C; it was dialyzed against deionized distilled water just before use. The yield of pure purple membrane was about 2 mg/l of anaerobic culture (weight ratio of purp1e:red membranes in these cells was ccs. 311). Preparatiorl of Red Mertlbrane ( 3 , 4 ) For the preparation of large amounts of red membrane, ceIls of H. cutirubrum were grown aerobically in the light for 4 days at 37 "C in 1-1 batches of complex medium for halophiles at a shaking rate of 120-140 r.p.m. Cells were harvested and washed as described above. The procedure for the preparation of the red tnembrane was similar to that for the purple membrane described above, except that the second densitygradient centrifugation was done on a discontinuous sucrose gradient prepared by layering 5 ml of 1 M sucrose soIution on top of 3 ml of 1.3 M sucrose. After centrifugation for 18 h at 40 000 r.p.m., the red membrane formed a single sharp band at the junction of the 1.3 M and 1 M sucrose layers. It was recovered and stored as described for the purple membrane; the yield of red membrane was 20 mg/l of aerobic culture. Extraction of Lipids frortz Purple or Red Rlenzbrunes Total lipids were extracted from the sucrose-free red or purple membranes essentially by the method of Bligh and Dyer (14). To 5 ml of a suspension of the membrane (10-20 mg) was added 12.5 ml of methanol and 6.25 ml of chloroform, and the contents were thoroughly mixed. After 5-10 min equal volumes (6.25 ml each) of chloroform and water were added and the mixture was centrifuged. The chloroform phase was separated, diluted with benzene, and evaporated to dryness under a gentle stream of nitrogen; the residue of total lipids was dissolved to a known volume in chloroform, and aliquots were taken for chemical analyses. Fractionation of the lipids by acetone precipitation to separate polar and non-polar lipids was carried out as described previously ( 12). All operations were carried out under nitrogen and in semidarkness. Extraction of Retinal from the Purple Membrane To 400 pl of the sucrose-free dialvzed purple-membrane suspension (equivalent to 239 pg of Lowry protein) was added, with mixing, BOO pl of 0.08 M cetyltrimethylammoniurn bromide (pH 8) and 300 pl
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CAN. J. BPOCWEM. VOL. 53, 1975
of 0.5 M hydroxylamine base (freshly prepared by mixing 10 ml of 1 M hydroxylamine hydrochloride with 5 ml of 2 N NaOH and adjusting the pH to 8 in a final volume of 20 ml). After 1-4 h at room temperature in the dark under nitrogen, the mixture was extracted by adding 2.0 ml methanol and 1.8 ml chIoroform, followed after several minutes by the addition of 1.0 ml each of chloroform and water. After centrifugation, the chloroform phase was separated, diluted with benzene, and evaporated to dryness under a gentle stream of nitrogen. The residue was dissolved in ethanol and the amount of retinyloxime was determined spectrophotometrically using the extinction coefficient E'%,,, at 355 m of 2020 (15). The protein remaining after the above extraction procedure was dissolved in 10% formic acid and its spectrum measured in the visible-IN range.
A r?-mino-AcidAnalysis Whole membrane or lipid-free membrane protein samples were hydrolyzed for 24 h or 48 h in 6 N HCl at 110 "C in evacuated sealed tubes. The acid was removed on a rotary evaporator at 37 "C and the residue finally dried over KOH pellets in vacuo. Amino acid composition was determined on a Beckman model I21 analyzer using the single-column procedure (Beckman methodology brochure, A-TB-059A). Hydrolyses were also carried out at 110 ' C for 4 and 6 h in 4 N HGl in sealed evacuated tubes for estimation of hexosamines. Tryptophane was estimated spectrophotometrically (25) in 0.1 N NaOH in a Cary spectrophotometer. Acrg~lanaide-GelEBeetrophoresis Electrophoresis and staining of whole membrane and lipid-free membrane protein was carried out according to the methods of Weber and Osborn (26) Thin-Layer Claromatography und ddentificatisrz of on 7 and 10% polyacrylamide gels except that the Lipid Components samples were dissolved in 2% (w/v) sodium dodecyl The polar and non-polar lipid components from sulfate. 0.02 M dithiothreitol, 8.0 M urea, and 0.01 M purple and red membranes were separated by thin- phosphate buffer at pH 9.0 and placed in boiling water layer chromatography on silica gel G or silica gel H for 2 min before layering on the gels. Background stain (500 pm-thick layers) using the following solvent (Coomassie blue) was removed by washing in 7% systems: A, 0.3% ethyl ether in hexane, for the separa- acetic acid for several days. tion of squalene, dihydrosqualene. tetrahydrosqualene, and @-carotene; B, 6% ethyl ether in hexane for the Sedimentation Anakysis Sedimentation experiments were carried out in 6 N separation of vitamin MK-8; C, 1% ethyl ether in chloroform for the separation of retinal and bisan- guanidine hydrochloride, 0.01 M dithisthreitol, 0.05 M hydrobacterioruberin; D, 7% methanol in chloroform Tris buffer, pH7.8, in a synthetic boundary cell at for the separation of monoanhydrobacterioruberins 20 "C and 60 000 r.p.m. Sedimentation rates were and bacterioruberins. Phospholipids and glycolipids calculated according to Schachman (28), and molecwere separated ( 16, 17) by two-dimensionaI thin-layer ular weight was estimated from the sedimentation rate chromatography using solvent E, chloroform - and the amino-acid analyses using the formula sf methanol - concentrated NHDM (6%:3 5 :5, v/v/v) in Tanford (29 ) . the first direction; and solvent F, chloroform - 90% Dry -Weight Deterplainations of the Membranes acetic acid - methanol (30 :20 14, v/v/v) in the second Dry weights of the membranes were determined by direction. Solvent systems A. B. and C were used only drying suitabk aliquots of dialyzed membrane suswith silica gel G plates while D, E, and F were used pensions in a weighing bottle over KQH in a vacuum with silica gel H plates. Before use. thin-layer chroma- desiccator (ea. 1 mm Hg (133 N/m2)) at room temtographic plates were washed with chloroformperature. methanol (1 :I, v/v), air dried, and activated at I00 "C Mensurernent of Spectra and Quantitative for 12 h. Determination sf Hsoprenoid Compounds The separated components were detected by their Visible and ultraviolet spectra of purple-membrane visual colors (retinal, C 4 0 and Cmpigments), by iodine vapor (squalene, cHi- and tetrahydrosqualenes m d and red-membrane suspensions and of MK-8, retinal, vitamin MK-8), by phosphorus spray reagent ( 18, 19) retinyloxime, and red pigments in suitable organic for phospholipids or by the a-naphthol spray reagent solvents were recorded with a Coleman-Hitachi-PerkinElmer model 124 spectrophstsmeter. The amount of ( 18) for glycolipids. The isoprenoid neutral lipids (9, 10, 12) and the phospholipids ( 16, 20) and glycolipids each isoprenoid compound was calculated by the (17, 20) were identified as described previously. The formula given in our previous communication (10) , values: MK-$, 268 at 248 phospholipid and glycolipid components were quanti- using the following El%'.,, tated by scraping each spot directly into digestion tubes nm in petroleum ether; retinal, 1510 at 383 nrn in and determining the phosphorus or sugar content, ethanol; retinyloxime, 2028 at 355 nm in ethanol; red pigments, 2540 at 4490 nm in acetone. respectively.
Chemical Analyses Phosphorus was determined by a modification (18) of Allen's procedure (21) or by the micromethod of Bartlett (22). Total hexose content was determined by the phenol - sulfuric acid procedure of Dubois er al. (23). Protein determinations on the dialyzed membrane suspensions were carried out by the method of Lowry et ai. (24).
Results General e&earacteri~ation 04 Fractions purple ~~~b~~~~ The purified preparation of purple membrane from H.cutirubrum showed absosp-
287
KUSHWAHA ET AL. : HALOBACTERIUM CUTITPUBRUM MEMBRANES
TABLE I . Molecular-weight determination by SDS gel electrophoresis Molecular weight X 10-3
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Protein
7% gel*
10YQgel?
Purple membrane Y
Average, 19.6+0.8 Red membrane$ Band 1 2 3
I
4 I
'K)O
I
'
400
_
I
SW
t
l
600
WAVELENGTH, nm
5
6 *Results for two separate runs, each in duplicate. ?Results for one run in quadruplicate. $Bands are numbered from bottom to top of gel (Fig. 2B).
108
400
WAVELENGTH, nm
FIG. I. (A) Absorption spectrum of the pure purple membrane from H. clltirlrbrlrm in water ; concentration, 153 pg/ml; (B) absorption spectrum of the pure red membrane from H . cmsrr'rubrirna in water; concentration, 3.1 mg/ml.
tion maxima at 565 nm and 275 nm (Fig. 1A). The ratio of absorbance at 565 nm and 275 nm is 1 :2, and the molar extinction at 565 nm is 4.8 X 10% essentially as was reported for H. halobium purple membrane (5 ) .On disc-gel electrophoresis on 10% polyacrylamide gels, various preparations of the purple membrane consistently migrated as a single sharp band (Fig. 2A) ; similar results were obtained on 7 % gels (Fig. 2B). The lipid-free protein also gave a single band on disc-gel electrophoresis. From the plot of migration distance against log molecular weight for the membrane and the calibration proteins, a molecular weight of (19.6 -t- 0.8) X lo3was obtained (Fig. 3, Table 1 ) . The molecular weight calculated from the amino-acid composition (Table 2 ) was
18.5 X 10" assuming 1 mol of histidine per mole of protein. The sedimentation rate rneasured in guanidinium hydrochloride was 0.47 S. However, the protein was not stable in the solvent and gradually precipitated. Aggregates were also observed in the ultracentrifugal schlieren patterns. The partial specific volume of the protein was calculated from the amino-acid analyses (Table 2 ) according to Cohn and Edsall (30) and then corrected (28) by subtracting 0.81 to compensate for the type of solvent used. From this calculated value (0.736) and the sedimentation rate, the molecular weight was calculated (28) to be 19 X lo3. These results thus provide consistent evidence that the molecular weight of the purple membrane of H. cutirubrum is close to 20 000, a value significantly lower than that reported ( 5 ) for H.halsbium purple membrane (26 000). Amino-acid compositional data (Table 2) show that the mole ratios for most amino acids in the purple membrane from H. cutirubrum are generally similar to those reported for the H . halobium membrane ( 5 ) . The proportion of polar amino-acid residues (40% ) was also similar to that in the H. halsbium membrane. No significant amount of hexosamines was detected in the purple membrane, indicating that it is not a glycoprotein. Extraction by the Bligh-Dyer procedure alone (9) gave a value of 0.33% retinal corresponding to a retinal:protein mole ratio of 1 : 3.2. However, extraction in the presence of hexa-
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CAN. J. BIOCHEM. VOL. 53, 1955
FIG.2. Disc-gel electrophoresis on poiyacrylamide of purple and red membranes from H . curirubr~mmin sodium dodecyl sulfate. (A) 18$; polyacrylamide: (a) calibration mixture of bovine plasma albumin, ovalbumin, a-ehyrnotrypsin; (b) mixture of bovine plasma albumin, ovalbumin, purple membrane (preparation I); ( c ) purple membrane (preparation I); (d) calibration mixture of bovine plasma albumin, ovalbumin, trypsin, hemoglobin; (e) purple membrane (preparation 11); (f) mixture of bovine plasma albumin, ovalbumin. purple membrane (preparation 11). (B) 7yi polyacrylamide: (m) calibration mixture of bovine plasma albumin (ditner and monomer), ovalbumin, trypsin, hemoglobin; (n) purple membrane (preparation 111); (o) red membrane; (p) red membrane overloaded.
45 ) a retha1:protein mole ratio for H . Ir~celobiurn purple membrane of 1:1. The purple membrane of H. cutirubrurn had 10 % gel 20% total lipids by weight and 77% protein (Lowry et al. (24)) (Table 3 ) . The phosphorus content of the purple membrane was found to be 1.03 %, of which only 79% was found in lipids (Table 3 ) , the remainder, presuinably, being bound to the protein (ca. 1.8 atoms per mole protein). The hexose content of the purple membrane was found to be 2.6% , a11 of which was accounted for in the lipids (Table 3 ) . This finding supports the above conclusion that the MIGRATION DISTANCE, cm membrane is not a glycoprotein. FIG.3. Plot of migration distance vs. logarithm of Weal Membrane molecular weights of the purple-membrane and calibraThe purified red-membrane preparation tion proteins after electrsphoresis on 7 and 10yOpolyshowed absorption maxima at 535, 500, and acrylamide gels in sodium dodecyl sulfate. 478 nm (Fig. IB), typical of bacterioruberins decyltrimethyl ammonium bromide and hy- ( 1, 2, 10). On disc-gel electrophoresis (Fig. droxylamine as described in the methods section 2B) it showed the presence of at least six bands; gave a higher retinal content corresponding to a the lipid-free membrane preparation showed the retinal: protein mole ratio of 1 : 2.2 (Table 3 ) . same six bands. There was considerable backIn contiast, Oesterhelt and Stoeckenius reported ground staining between bands but the top and
289
KUSHWAHA EF AL.: HALOBACTERIUM CUFIRUBRUM MEMBRANES
TABLE2. Amino-acid composition of purple- and red-membrane preparations Purple membrane
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Amino acid Asp Thr Ser Clu Pro G~Y Ala Val Met I le Leu TY~ Phe His =JY~ Arg Try 3 cys
rno1/100 mol
Red membrane mole ratiosa
H . cutbritbr~irnb H. halobirlnzC
H. cutirrlbriinz
H . hnlobium*
mol/ 100 mol H . ciirinthrume
H . hadohiumC
8.6 5.7 7.8, 6.8 5.6, 5 .O 7.4 6.5 4.3 4.6 10.6 9.6 10.9 11.8 8.4 7.3 2.7 4.1 5.6 5.6 12.2 13.2 3.6 3.7 4.2 4.6 0.6 8.6 2.4 5 .O 2.7 5.9 2.30 (2.3) 0.6 (0.1) Total amino-acid residues
Welative to histidine. hAverage of six determinations on two separate preparations. cData from Stoeckenius and Kunau (4) and Oesterhelt and Stoeckenius ( 5 ) . *Data from Oesterhelt and Stoeckenius (5). eAverage of four determinations on one preparation. fExtrapoled to zero time of hydrolysis. UDetermined spkctrophstometrically (25). hCalcutated molecular weight, 18 500. fCalculated molecular weight. 26 000.
TABLE 3. Overall composition of membrane preparations Purple
Density, g/ml (5 "C)
c, 5%
H, 56 N, 57% Protein, %, Lipid, O/, kipid:protein, weight ratio Retinal," % Retinal :protein, mole ratio Total P, ';, Total lipid P, % Total hexose, 'r, Total lipid hexose, % *Extracted in presence of hydesxylamine (5.
15).
Red
37-38% by weight and the protein (Lowry et al. (24)) accounted for 55-56% of the weight (Table 3 ) . The phosphorus content was 1.896, of which 82% was found in the lipids (Table 3 ) , indicating the presence of some phosphoproteins in the membrane. The hexose and hexosamine content of the red membrane was found to be 6.9%, of which 66% was accounted for in the lipids. This would suggest that the red membrane contains glycoproteins. The amino-acid composition was very similar (Table 2) to that reported for H. halobium (4). Only traces of hexosamines were present in samples hydrolyzed for 12 h.
Lipid Components of Purple and Red bottom of the gels were clear. The major bands Membranes (Fig. 2B) had molecular weights given in Table Thin-layer chromatography of the total lipids 1; there was no band in the region of the single from the purple membrane showed that the polar protein band from the purple membrane in lipids were phosphatidyl glycerophosphate, neighboring gels. triglycosyl diether, glycolipid sulfate, phosphaThe lipid content of the red membrane was tidy1 glycerosulfate, and phosphatidyl glycerol
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CAN. J. BIQCHEM. VOL. 53, 1975
FIG.4. Two-dimensional thin-layer chromaaograms on silica gel H of total lipids of: (A) pure purple membrane; (B) red membrane, from H. cratir~lbnlm.Identity of spots: GLS, glycolipid sulfate; TGD, triglycosyli diether; PGP, phosphatidyl glycerophosphate; PGS, phosphatidyl glycerosulfate; PG, phosphatidyl glycerol; X I and X2, unidentified glycolipids; PA, phosphatidic acid; NL, neutral lipids. Solvent systems: (1) chloroform - methanol - concentrated NHIOH (65:35:5, v//v); (2) chloroform - 90% acetic acid - methanol (30:20:4, v/v). TABLE 4. Lipid composition of membrane preparations*
Total iipids, Lipid component Neutral lipids Squalene, dihydrosqualene, tetrahydrosqualene @-Carotene MK-8 Retinal C60 red pigments Phospholipids Phosphatidyl glycerol Phosphatidyl glycerophosphate Phosphatidyl glycerosulfate Glyccdlipids
x x
1
Triglyccdsyl diether 2
Glycolipid sulfate *See Figs. 4 and 5. ?The amount of XI
Purple membrane 4.5 Trace 1.6 2.5 -
Traces 69.3 Traces 10.3
7;by weight Red membrane 5 Traces 1.2 0.4
t t
Traces Traces
+ Xa was about 32-3570 by weight.
(diphytanyl glycerol ether analogues) (Fig. placed by the two unidentified glycolipids in the 4A, Table 4). In contrast, no sulfated polar red membrane (Table 4). lipids (glycolipid sulfate and phosphatidyi glyIn regard to the neutral lipids, these amounted cerosulfate were detected in the red membrane to 7 4 % of the total lipids in both membranes (Fig. 4B, Table 4), the main polar lipids being and consisted mostly ~f squalene, dihydro- and phosphatidyl glycerophosphate, phosphatidyl tetrahydrosqualenes, vitamin MK-8, diphytanyl glycerol, and two unidentified glycolipids (XI glycerol ether, and pigments; retinal was only in and X2). It is of interest that the contents of the the purple membrane and C50bacteriomberins major and minor phospholipids, phosphatidyl were only in the red membrane (Fig. 5 A,B ) . It glycerophosphate and phosphatidyl glycerol, re- is of interest that the contents of squalenes were spectively, are essentially the s m e in both mem- in the order, squalene < dihydrosqardene < branes, and that the glycolipid sulfate and trigly- tetrahydrosqualene in both membranes obtained cosy1 diether in the purple membrane are re- from anaerobic cells; this order was reversed in
Can. J. Biochem. 1975.53:284-292. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 02/08/15. For personal use only.
KUSHWAHA ET AL.: HALOBACTERIUM CUFIRUBRUM MEMBRANES
FIG.5. Thin-layer chromatogram on silica gel 6 of neutral (acetone-soluble) lipids of the purple and red membranes from H. cubirubruna. (A) Chromatogram of squalenes and carotenes, developed in solvent A, ethyl ether - hexane (0.3 : 99.7, v!v). (1) Acetone-soluble lipids from whole cells of H. clitirr4brrrm; (2) mixture of squalenes; (3, 4) lipids of the purple and red membrane, respectively, from anaerobically grown cells; (5) lipids of red membrane from cells grown aerobically. (B) Chromatogram of vitamin K and retinal developed in solvent B, ethyl ether - hexane (6:94, v!v). (1) Squalenes; (2) acetone-soluble lipids from whole cells of H. cutinibnrm; (3) vitamin Kr; 4, all-trans-retinal; (5,6) lipids of purple and red membranes, respectively, from cells grown anaerobically; 4, lipids of the red membrane from cells grown aerobically. Identity of spots: (a) @-carotene,(b) squalene, (c) dihydrosqualene, (d) tetrahydrosqualene, (e) saturated hydrocarbons, (f) vitamin K1, (g, h) cis- and trans-retinal. Dotted lines indicate that the spots were seen by their visual colors.
the membranes from cells grown aerobically (Fig. 5A).
Discussion As mentioned in our previous report (9), the purple membrane from H . cutirubrum appears to be generally similar to that of H . halobiunz (5), but differs in several respects. The present results show that in the H. cutirubrtim purple membrane the molecular weight of the protein moeity is CQ. 20 000 and the mole ratio of retina1:protein is 1: 2, compared to a protein molecular weight of 26 000 and a retinal : protein mole ratio of 1:1 reported for the H. halobiunz membrane ( 5 ) . It should be noted that the nlolecular weight of 20 000 in the case of H. cutirubrum was consistently obtained by three independent methods and is probably reliable to within t4% and thus is significantly lower than that for theH. halobium protein. However, it would be desirable to measure the molecular weight by equilibrium centrifugation in a suitable solvent, using a reliable partial specific volume. On the other hand, the amino-acid compositional data for the purple-membrane preparations are very similar, although significant differences in mole ratios of aspartic acid, methionine, arginine, and lysine were observed (Table 2). Also, the lipid to protein
weight ratio of the two preparations was similar ( 1:3 (Ref. 5 ) vs. 1:4 (Table 3)). The analytical data for the H. cutirubrum membrane (Table 3 ) show clearly that the protein is not glycosylated but may be phosphorylated (about 2 mol phosphate per mole protein). In regard to the retinal: protein ratio of 1:2 obtained for the W.cutirubrum preparation, it is possible that not all of the retinal was extracted, although the procedure used (ethyltrimethylammonium bromide (CTAB) plus hydroxylamine) was essentially the same as that used by Oesterhelt and Stoeckenius ( 5 ) . Control experiments using N-retinylidene hexylamine showed that free retinal could not be extracted from either the protonated or the unprotonated SchiR base by the Bligh-Dyer procedure or by the CTAB plus hydroxylamine procedure described in the methods section. Thus the retinal that was extracted must have been in the extractable free aldehyde or hydrated aldehyde form. The possibility that some retinal may be covalently bound to the protein in an unextractable form was eliminated by the finding that the protein remaining after extraction of the membrane, as described in the methods section was colorless and showed no absorption in the region 300500 nm characteristic of the protonated or unprotonated retinal Schiff base.
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292
CAN. J.
BIOGHEM.
The striking feature of the purple membrane, relative to the red membrane, is the exclusive presence of sulfated lipids (glycolipid sulfate and ghosphatidyl glycerosdfate) in the former membrane and their complete absence from the latter (Fig. 4 A,B). It may be noted that the glycolipid sulfate was found to be essential for formation of stable bilayers of H. cutirubrum Hipids ( 3 1 ) and that squalenes were considered to play a role in the salt-dependence of the halophile membranes (32). The purple and red membranes were also quite distinct with regard to their protein components, the single purplemembrane protein with molecular weight 20 080 being entirely absent from the red-membrane proteins, which consisted of about six components ranging in molecular weight from 10 000 to 60 008. Furthermore, the purplemembrane protein is phosphorylated but not glycosylated, but the red membrane contains both phosphorylated and glycosylated proteins. The findings presented here raise the question whether species differences occur in the purple membranes from different extremely halophilic bacteria. The present results suggest that the purple membrane of M. cutirubrum differs from that of the H.halobium, but direct comparison of the two membrane preparations would be desirable. This work was supported by a grant from the Medical Research Council of Canada (MA-4103). We are indebted to Dr. 0. Isler, Hoffmann-LaRoche Inc., Basle, Switzerland, for supplying several authentic samples, and to Mr. P. Pilon, Mr. J. Giroux, and Mrs. P. Fejer for technical assistance. 1. Kelly, M., Norgard, S, & Liaaen-Jensen, S. (1970) Aeta Chem. Scarzd 24, 2169-2182 2. Gocknauer, M. B., Kushwaha, S. C . , Kates, M. & Kuskner, D, J. (1972) Arkiv. Mikrobisl. 84, 339-349 3. Stoeckenius, W. & Rswen, R. (1967) J. Cell Biol. 34, 365-393 4. Stmkenius, W. & Kunau, W. H. (8968) J. Cell Biol. 38, 337-357 5. Oesterhelt, D. & Stoeckenius, W. (1971) Rrat. New Biol. 233, 149-152
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6. Blaanrock, A, E. & Stoeckenius, W. (1971) Nat. New Biol. 233, 152-154 7. Oesterhelt, D. & Stoeckenius, W. (8973) Proc. Rraal. Acad. Sci. U.S. 70, 2853-2857 8. Danon, A. & Stoeckenius, W. (1974) Proc. Rratl. Acad. Sci. U.S. 71, 1234-1238 9. Kushwaha, S. C . & Kates, M. (8973) Biochirn. Bioph-9s. Actca 316, 235-243 18. Kushwaha, S. C . , Gochnauer, M. B., Kushner, D, J. & Kates, M. (1974) Cat?.J. Microbial. 20, 241-245 81. Vogel, A. I. (1956) Practical Orgartie Chemistry, pp. 161-175, Longmans Green & Co. Ltd., London 12. Kushwaha, S. C., Pugh, E. L., Kramer, J. K. G. & Kates, M. (1972) Biochim. Bioplz~s.Acfa 260,492-506 13. Sehgal, S. N., Kates, M. & Gibbons, N. E. (1962) Can. J . Biochem. Plydol. 40, 69-8 1 14. Bligh, E. G. & Dyer, W. J. (1959) Carz. J. Biochem. Physiol. 37, 911-917 15. Wald, G. & Brown, P. K. (1953,/54) J . Gerz. Plysiol. 37, 189-200 16. Hancock, A. J. & Kates, M. (1973) J. Lipid Res. 14, 422-429 17. Kates, M. & Deroo, P. W. (1973) J . Lipid Res. 14, 438-445 18. Kates, M. (1972) Techniqnres of Lipidology, pp. 354359, North-Holland Publishing Co., Amsterdam and London 19. Vaskovsky, V. E. & Kostetsky, E. Y . (1968) J. Lipid Res. 9, 396 20. Kates, M. (1972) in Ether Lipids, Clzemistr~)arzd Biology (Snyder, F., ed.), pp. 351-398, Academic Press Inc., New York, N.Y. 21. Allen, R. J. L. (1948) Biochem. J. 34, 858-865 22. Bartlett, G. R. (1959) J. Biol, Clzern. 234, 466-468 23. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. (1956) Anal. Chem. 28, 350-354 24. Eowry, 0. H., Rosebroangh, N. J., Farr, A. L. & Randall: R.J. (1951) J. Biol. Chrtm. 193, 265-275 25. Bencze, W. L. & Schrnid, K. (1957) Anal. Clrena. 29, 1193-1196 26. Weber, K. & Osborn, M. (1969) J. Br'ol. Chetm. 244, 4406-44 12 27. Schwert, G. W. & Kaufman, S. (1951) J . Ba'ol. Chem. 198, 807-816 28. Schachman, H. K. (1957) kfeth. Etazymol. 4, 32-103 29. Tanford, C., Kawahara, K. & Lopanje, S. (1967) J. Am. Clzent. Ssc. 89, 729-736 30. Cohn, E. J. & Edsall, J. T. (1943) in Proteins, Amino Acids and Peptides (Cohn, E . J. & Edsall, J. T., eds), pp. 378-381, Reinhold Publishing Corp., New York, N.Y. 31. Chen, J. S., Barton, P. G., Brown, D. & Kates, M. (1 974) Biochim. Biophys. Acaa 352, 202-217 32. Eanyi, J. K., Plachy, W. Z . & Kates, M. (1974) Biochemistry, 13, 4914-4920
