11

Biochem. J. (1979) 180, 11-24 Printed in Great Britain

The Isolation and Partial Characterization of the Plasma Membrane from

Trypanosoma brucei By H. PAUL VOORHEIS,* JEAN S. GALE, MICHAEL J. OWEN and W. EDWARDS Department ofBiochemistry, University of Cambridge, Cambridge CB2 1 Q W, U.K. (Received 2 October 1978)

Whole sheets of plasma membrane, each with their attached flagellum, were purified from Trypanosoma brucei. The method devised for their isolation included a new technique of cell breakage that used a combination of osmotic stress followed by mechanical sheer and avoided the problem of extreme vesiculation as well as the trapping of organelles in cell 'ghosts'. The purified membranes all contained the pellicular microtubular array. The antigenic surface coat was completely released from the plasma membrane during the isolation procedure. The membranes had a very high cholesterol/phospholipid ratio (1.54). A large proportion (42%) of the cellular DNA was recovered in the plasma-membrane fraction unless a step involving deoxyribonuclease treatment, which decreased the DNA content to less than 13%, was included before sucrose-density-gradient centrifugation. This step also aided the separation of plasma membranes from other cellular components. The ouabain-sensitive Na++K+-stimulated adenosine triphosphatase and adenylate cyclase co-purified with the plasma membranes. Although 5'-nucleotidase was thought to be a plasma-membrane component, it was easily detached from the membrane. The purified membranes were essentially free of L-alanine-ax-oxoglutarate aminotransferase, L-asparte-a-oxoglutarate aminotransferase, malate dehydrogenase, oligomycin-sensitive adenosine triphosphatase, glucose 6-phosphatase, Mg2+-stimulated p-nitrophenyl phosphatase and catalase. The isolation of the plasma membrane from a wide variety of types of cells has received considerable attention during the last decade and has been reviewed critically by Wallach & Lin (1973). An increasing number of important cellular events are being recognized as involving the active participation of the plasma membrane, particularly in regulatory events. Prominent among these are mechanisms of transport, cell-cell recognition and the immune response.

The plasma membrane of the salivarian trypanosomes is of particular fascination to biologists because

it provides the attachment sites for the antigenic surface coat in bloodstream forms and the assembly template for a complex and regular array of microtubules. The ability of these invasive parasitic cells to change their surface coat repeatedly and so defeat the host's immune defence mechanism (Massaglia, 1907; Ritz, 1914; Broom & Brown, 1940; Gray, 1965; Vickerman & Luckins, 1969; Vickerman, 1969; Cross, 1975) provides additional interest. More recently the selective inhibition of the Na+-independent Nl-system amino acid-transport carrier in the plasma membrane of Trypanosoma brucei has Abbreviations used: DNAase, deoxyribonuclease; Tes, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulphonic acid; ATPase, adenosine triphosphatase. * Present address: Department of Biochemistry, Trinity College, Dublin 2, Ireland. Vol. 180

been reported (Owen & Voorheis, 1976), which may make it possible in further studies to label, isolate and characterize an active-transport carrier from the plasma membrane of this eukaryote. Further progress in each of these investigations would be helped by a simple, reliable and well-characterized procedure for the isolation of the plasma membrane with its associated supporting cytoskeleton. Many of the techniques that are used for the isolation and characterization of plasma membranes have been reviewed by DePierre & Karnovsky (1973) and have been collected together in convenient format by Fleischer & Packer (1974a,b). In the present paper a new technique of breaking cells is described that allows the subsequent isolation of whole plasma membranes. The morphologically distinct microtubular network is used as one of several markers for the unequivocal identification of the isolated membrane material as being derived exclusively from the plasma membrane of Trypanosoma brucei. Materials Distilled and deionized water was used in all experiments. Purified DNAase I (EC 3.1.4.5) of the highest grade available was from Worthington Biochemical Corporation, Freehold, NJ 07728, U.S.A. [3H]DNA from the lambdoid phage 080 and [32P]RNA from Escherichia coli were gifts from

H. P. VOORHEIS, J. S. GALE, M. J. OWEN AND W. EDWARDS

12

Dr. M. Blundel, and yeast tRNA was a gift from Dr. T. Hunt. All biochemicals and enzyme substrates were from either Sigma (London) Chemical Co., Kingston upon Thames KT2 7BH, U.K., or Boehringer Corp. (London), London W.5, U.K. Other reagents were of analytical grade and were from BDH Chemicals, Poole, Dorset, U.K. Bloodstream forms of Trypanosoma brucei (427-12/ICI-060) were originally isolated from the blood of a sheep in 1960 at Tororo in Uganda (Cunningham & Vickerman, 1962) and maintained by serial syringe passage at The Lister Institute, London, U.K. It is now a monomorphic strain and was cloned by Dr. R. W. F. Le Page and recloned by Dr. G. A. M. Cross, both at the University of Cambridge. The organisms were stored in liquid N2 until used. Methods

Growth, harvesting and preparation of cells The techniques for obtaining cells have been described previously (Owen & Voorheis, 1976; Voorheis, 1977). Bloodstream forms of T. brucei were

harvested from rats 3 days after intraperitoneal inoculation. Organisms were separated from blood elements by using the cell chromatography method of Lanham (1968) on short columns of DEAE-cellulose at 5°C. Cells were washed by centrifuging at 600g for 10min (5°C) and resuspending in the appropriate medium (5°C). The entire harvesting, purification and washing procedure requires less than 60min to perform. Cells were used immediately and were actively motile.

Procedure for the preparation of sheets of plasma membrane with their associated flagella and microtubular networks Swelling and rupture. This procedure is summarized in Scheme 1. A fresh preparation of 3 x 1010 bloodstream forms of T. brucei (Owen & Voorheis, 1976) was centrifuged at 650g for 10min (4°C) and the pellet resuspended in Tes buffer (lOmI) containing 2mM-Tes, l5mM-KCI, 1 mM-EDTA, 1 mM-2-mercaptoethanol and 0.1 mM-phenylmethanesulphonyl fluoride adjusted to pH 7.5 with 1 M-NaOH and prepared fresh each day. Usually three successive 10ml

T. brucei (427-12/ICI-060) 3 x 1010 bloodstream forms in pellet (I) Suspend in lOml of Tes buffer at 0°C containing 0.1 mM-phenylmethanesulphonyl fluoride (complete buffer composition given in the Methods section). (2) Add deionized water (3 x IO ml) containing 0.1 mM-phenylmethanesulphonyl fluoride slowly with stirring at 0°C. (3) Homogenize by one to three strokes of a very tight-fitting Dounce homogenizer. (4) Immediately add 2 ml of 3M-KCI and mix. (5) Centrifuge at 7500g,, for 10s.

I

Supernatant (SI

Pellet (PI) (I) (2) (3) (4)

Supernatant (S2)

Suspend in 10 ml of Tes buffer without EDTA and with S mM-MgCI2 (20°C). Add purified DNAase (0.1 mg, 240 units); incubate for 5min (20°C). Terminate reaction by adding SOml of Tes buffer with I mM-EDTA (0°C). Centrifuge at 7500g., for lOs.

1

Pellet (P2) (I) Suspend in 40% (w/v) sucrose in Tes buffer. (2) Layer on linear 40-60% sucrose gradient in Tes buffer. (3) Centr fuge at 70 000g8 for 3 h.

Remainder of gradient including pellet of free flagella

Most prominent dense band (I) Dilute with 50ml of Tes buffer. (2) Centrifuge at 7500g,v. for 2min.

Pellet (1) Suspend in SOmI of Tes buffer. (2) Centrifuge at 7500gm, for 10s. (3) Repeat (I) and (2) twice.

Plasma membranes

Scheme 1. Preparation ofplasma membranes from bloodstream forms of T. brucei

1979

PLASMA MEMBRANE FROM T. BRUCEI

portions of distilled and deionized water (0°C) were added with vigorous stirring. It proved important to follow the progress of cell swelling with phase-contrast microscopy by examining samples after each addition of water because the susceptibility to swelling of different preparations of cells varied. Microscopic control allowed the addition of water to be stopped early or even continued further than usual until the preparation had the appearance illustrated in Plate 1(c). The swollen cells were ruptured by the use of a very tight-fitting glass Dounce homogenizer with a spherical Teflon pestle machined so that entry of the pestle into the sleeve was possible only below a temperature of 5°C. A very close clearance was achieved by exploiting the difference between the coefficients of thermal expansion of Teflon and glass. The average clearance was estimated to be 10-20um on the radius at O-5°C. After about 9 months of use an unacceptable looseness occurred exclusively on the glass portion; this was easily remedied by replacing the sleeve. Between one and three complete strokes were required for 95 % rupture as assessed microscopically. Cells that were not swollen did not rupture with this procedure, but their free flagella were severed at the point where they first attached to the main body of the cell. Immediately after rupturing of the cells the ionic strength of the homogenate was raised by the addition of 2 ml of 3 M-KCl for each 40ml of homogenate. This manoeuvre eliminates contamination from hexokinase (results not shown), and therefore presumably elutes this enzyme from its attachment to the plasma membrane that can occur as an artifact under these breakage conditions or else prevents rupture of the glycosomes altogether. Differential centrifugation and DNAase treatment. The homogenate was centrifuged in an MSE-18 centrifuge in an 8 x 50ml head by accelerating to 7500gav, maintaining this speed for lOs and then decelerating with the brake on; this procedure produces pellets of membrane in good yield with a minimum of damage to the bilaminar structure. The pellet was resuspended in Tes buffer (lOmI) without EDTA and containing 5mM-MgCl2 and then warmed to 20°C. Highly purified DNAase (240 units) was added and the suspension incubated (20°C) for 5 min. The reaction was terminated by adding ice-cold Tes buffer (50mI), mixing and centrifuging at 7500g2,. (4°C) for IOs. The pellet was resuspended in 40 % (w/v) sucrose in Tes buffer (4ml for each 1010 cells starting material). Continuous density-gradient centrifugation. The suspension (4ml/gradient) was layered on top of a linear 40-60% (w/v) sucrose gradient (25 ml) with a 60% (w/v) sucrose bottom cushion (2ml) and was centrifuged at 70000gav. for 3 h at 4°C. The gradient and the cushion were prepared in Tes buffer. Gently disturbing the boundary between the suspension and Vol. 180

13 the gradient with a stirring rod before centrifuging aided the celiular material to enter the gradient smoothly and completely. Two constant and prominent bands as well as several more variable smaller bands resulted from the centrifugation procedure adopted. Plasma-membrane sheets comprised the largest and most prominent dense band at 51.3 % sucrose (w/v) and had a density of 1.243g/l (5°C). Final wash and storage. The band of plasma membranes was removed with a Pasteur pipette after aspiration of the overlying gradient. The membranes in this fraction were diluted to 50 ml by the addition of Tes buffer and centrifuged at 7500gav. for 2min. The pellet was resuspended in Tes buffer (5ml/10'0 cells starting material) and portions were used immediately or stored frozen at either -20°C or -196°C. Assay of enzymic activities All enzymic assays were conducted at 30°C. Continuous assays were performed in a Unicam SP. 500 recording spectrophotometer. Stopped assays were incubated for 30min and terminated by addition of 2ml of 15 % (w/v) trichloroacetic acid unless otherwise indicated. The release of Pi was measured by the method of Fiske & SubbaRow (1925). The ouabain-sensitive Na+ + K+-dependent ATPase (EC 3.6.1.3) was assayed by measuring Pi release by using the method of Post & Sen (1967). The activity of the enzyme was taken to be the difference between that measured in the presence of 100mM-NaCI and 20mMKCI and that measured in the presence of 0.17mmouabain. Adenylate cyclase (EC 4.6.1.1) was assayed by the method of Salomon et al. (1974) with [a-32P]ATP as substrate synthesized by the method of Martin & Voorheis (1977). The activity of 5'-nucleotidase (EC 3.1.3.5) was measured as described by Michell & Hawthorne (1965). The y-glutamyl transpeptidase (EC 2.3.2.2) was assayed by the method of Orlowski et al. (1969) with L-y-glutamate p-nitroanilide as substrate. The hydrolytic activity of glucose 6-phosphatase (EC 3.1.3.9) was measured by the method of Nordlie & Arion (1966). Malate dehydrogenase (EC 1.1.1.37) was assayed with the method ofWolfe & Nielands (1956) in the direction of NAD+ reduction with L-malate as substrate. The mitochondrial F1 oligomycin-sensitive ATPase (EC 3.6.1.3) was assayed under the control conditions for Mg2+-dependent ATPase described by Post & Sen (1967) and the activity of the enzyme was taken as the difference between that measured in the presence and absence of 0.67,ug of oligomycin/ml. Citrate synthase (EC 4.1.3.7) was measured by the method of Srere et al. (1963). Fumarase (EC 4.2.1.2) was assayed by the method of Hill & Bradshaw (1969). Catalase was assayed polarographically (02 production) with a Rank oxygen electrode (Rank Brothers, Bottisham,

-14

H. P. VOORHEIS, J. S. GALE, M. J. OWEN AND W. EDWARDS

Cambridge, U.K.) under the conditions described by Chance & Maehly (1955) for particulate samples and crude homogenates. All samples were sonicated for 15s at maximum intensity in a bath-type sonicator (model KS-101, Kerry Ultrasonic, Hitchin, Herts., U.K.) before assay. The medium was deoxygenated by bubbling with filtered N2 and the small blank rate Of 02 diffusion into the closed electrode vessel determined before the addition of enzyme. Glycollate oxidase (EC 1.1.3.1) and urate oxidase (EC 1.7.3.3) were assayed polarographically under conditions described by Baker & Tolbert (1966) with either 40mM-glycollate or 0.2mM-urate as substrate. The activity of p-nitrophenyl phosphatase was assayed in a medium (pH 6.5) containing 0.075 % (w/v) Triton X-100, 40mM-citrate, 14mM-MgCI2 and 11.5mM-pnitrophenyl phosphate. Incubations (1.3 ml) were for 15 min at 30°C, and the reaction was stopped and the colour developed by adding 2 ml of 0.1 M-NaOH. A AA435 of 1.0 was taken to be equivalent to 54.1 nmol of p-nitrophenol produced/ml. The L-alanine-aoxoglutarate aminotransferase (EC 2.6.1.2) was assayed by the method of Wro6blewski & La Due (1956) and L-aspartate-a-oxoglutarate aminotransferase (EC 2.6.1.1) by the method of Karmen et al. (1955).

Analytical methods Protein was determined by the method of Lowry et al. (1951) with bovine serum albumin as standard. The DNA and RNA of cells and membranes were extracted and separated as described by Schneider (1957). DNA was determined by the method of Burton (1956) with purified calf thymus DNA as standard, and RNA was assayed by the method of Schneider (1957) with purified yeast tRNA as standard. For the determination of total lipid a known weight of dried cells or membranes was extracted twice with 10ml of chloroform/methanol (2:1, v/v) at room temperature by shaking for 30min. The combined extracts were washed once with 10ml of 0.58% (w/v) NaCl. The organic phase was evaporated to dryness at 37°C in a current of filtered N2 and then overnight in vacuo over P205. The weight of the residue was taken to be the amount of total lipid. Cholesterol was measured on extracted lipid by the procedure of Maclntyre & Ralston (1954). Total phospholipid was measured after hydrolysis by measuring phosphate release by using a modification (J. M. Stein, personal communication) of the method of Bartlett (1959). Dry samples (1-5pg of P) of extracted lipid were digested for 30min at 180°C in a marble-covered boiling tube containing 50,ul of I % (w/v) ammonium molybdate and 0.2 ml of 9: 1 (v/v) solution of conc. (98 0, w/v) H2SO4/conc. (72%, w/v) HC104. After the mixture had cooled 2.8ml of deionized water was carefully added followed by 0.3ml of 5% (w/v) ammonium

molybdate and 0.2ml of a solution of 1-amino-2naphthol-4-sulphonic acid containing NaHSO3 and Na2SO3 prepared as described by Bartlett (1959). Samples were then boiled for 7min in a marblecovered boiling tube, cooled for 20min at room temperature and their absorbance was measured at 830nm. Calculations were based on the assumption that the average molecular weight of the phospholipids present corresponded to that of dipalmitoyl phosphatidylcholine. Carbohydrate was measured by the method of Devor (1950) with glucose as standard. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was carried out by the procedure of Fairbanks et al. (1971). Molecular weights were estimated as described by Weber & Osborn (1969) with phosphorylase b (125000), bovine serum albumin (68000), glyceraldehyde 3-phosphate dehydrogenase (36000) and lysozyme (14400) as standards. The periodic acid/Schiff procedure (Zacharius et al., 1969) was used for staining carbohydrate and Coomassie Blue for protein (Fairbanks et al., 1971). Microscopy All fractions were examined by phase-contrast microscopy during each stage of the purification procedure. For electron microscopy samples of whole cells, isolated membranes and material at various stages of purification were fixed by addition of an equal volume of 4 % (w/v) glutaraldehyde prepared in the same medium in which the samples were suspended. After a period of 20min at 4°C the samples were centrifuged for 1 h at 700g also at 4°C. The pellets of material were cut into sectors and washed overnight in 0.05 M-NaH2PO4 buffer, adjusted to pH7.0 with 0.05M-NaOH, and containing 0.295Msucrose. After leaving for 3 h in 20% (w/v) OS04 in phosphate-buffered sucrose, the sectors were rinsed free of OS04 with phosphate-buffered sucrose and were dehydrated with ethanol and embedded in the medium of Spurr (1969) or after passage through propylene oxide embedded in Epon (Luft, 1961). Thin sections were stained with 10 % (w/v) uranyl acetate in methanol (Stempach & Ward, 1964) followed by lead citrate (Reynolds, 1963) and then viewed in an AEI 801 electron microscope. Negatively stained specimens were prepared by the drop method discussed by Haschenmeyer & Myers (1972) without fixation and with 0.1 0% (w/v) sodium phosphotungstate (pH 7.5).

Results Preparation ofplasma membranes Disruption of cells. The most critical part of the preparative procedure was found to be the homogenization step. Various methods of disrupting cells 1979

15

PLASMA MEMBRANE FROM T. BRUCE1 were tried and no one type of method proved satisfactory. For example osmotic lysis yielded resealed 'ghosts' (Plate Id) with many trapped organelles that made further fractionation difficult, and even mild sonication gave rise to small vesicles that lacked the characteristic morphological feature (microtubular array) of plasma membranes in trypanoson.es. The procedure finally developed (see the Methods section) used a combination of two different techniques, osmotic stress and a shear force, that for success relied on the peculiar structure of the trypanosomal pellicle. The pellicular microtubules in trypanosomes consist of twisted closed loops of tubules that spiral around the length of the cell from one end to the other and then return in the same way (Anderson & Ellis, 1965). These microtubules were observed to unwind progressively when the cells were subjected to increasing osmotic pressure, distorting the shape of the cell (Plate lb and Ic) until all of the microtubular loops came to lie in parallel circular planes. The twisting of the cell during swelling was best demonstrated by the manner in which the flagellum became wrapped around the cell up to three times in fully swollen cells (Plate Ic). Electron microscopy of thin sections of glutaraldehyde-fixed swollen cells revealed a generalized decrease in the average electron density of the cytoplasm (Plate 2b). The majority of cells observed at this stage in thin section were found to have intact plasma membranes. Most of the cell lysis that was observed in these sections was thought to have oc-

curred after fixation and to have been an artifact of preparation for electron microscopy for three reasons: first, intracellular organelles were only rarely found outside the plasma membrane profiles; secondly, where breakage was observed the ends of the plasma membrane were not rolled in the fashion to be described later when breakage unequivocally occurred before fixation; thirdly, less than 5% cell lysis was observed by phase-contrast microscopy at the optimum osmotic strength for swelling before rupture in the Dounce homogenizer (compare Plates I c and Id). Under these swollen conditions the shear forces, generated by streamlined solvent flow through the narrow gap of a tight-fitting Dounce homogenizer, sliced swollen cells lengthwise in a cleavage plane stabilized by the now parallel circular microtubules. Whole sheets of plasma membrane with their unique arrays of microtubules still attached were produced. The release of osmotic pressure after rupture of the cells allowed some recovery of the microtubular twisting so that many of the plasma-membrane sheets partly rolled up like loose scrolls (Plate 2c). This appearance was maintained throughout the purification procedure (Plates 3, Sb and 6b). The nucleus and the mitochondrion do not survive this procedure for rupturing cells as intact organelles (Plate 2c); as a result, markers for these components appear in the first supernatant (48 % of the cellular DNA and 92% of the oligomycin-sensitive ATPase found in the first supernatant). Careful attention to three critical details of the

Table 1. Removal of DNA and RNA from the plasma-membrane fraction prepared as outlined in Schenle 1 Plasma-membrane fractions were prepared as outlined in Scheme 1, but with omission of the addition of KCI to the homogenate and the DNAase treatment, and were washed once in Tes buffer after removal from the sucrose gradient. Membranes derived from 1010 cells were treated as indicated in the Table, washed three times with Tes buffer, resuspended in 2 ml of 400% (w/v) sucrose in Tes buffer and layered on top of a 40-60, (w/v) sucrose gradient (25 ml) and centrifuged at 70000g0s. for 3 h. The plasma-membrane band was collected and washed three times with Tes buffer and analysed for residual nucleic acid. Each value represents the mean+S.E.M. The number of separate determinations (different preparations of membranes) is given in parentheses. Residual nucleic acid in Membranes Cells

(jug/10'0 cells) DNA Control, no treatment Treated with DNAase (500,ug), MgSO4 (IOmM) for 5min at 20°C; 2ml final vol. Washed with KCI (0.5M) at 0°C RNA Control, no treatment Washed with KCI (0.5 M) at 0°C Treated with RNAase (200,ug), MgSO4 (10mM) for 30min at 20°C; 1 ml final vol. Treated with puromycin (5 mM), MgSO4 (5 mM) for 30min at 20°C

Vol. 180

746 + 69 (7)

(jug/membranes from 1010 cells)

317± 35 (11) 94 + 8 (3) 264 ± 31 (3)

6870 ± 490 (4)

1050 ± 120 (10) 1220± 87 (3) 193 + 28 (3)

147 ± 21 (3)

16

H. P. VOORHEIS, J. S. GALE, M. J. OWEN AND W. EDWARDS

procedure is required for best results: first, the inclusion of the proteinase inhibitor phenylmethanesulphonyl fluoride at all stages and particularly during cell breakage, together with the use of proteinase-free DNAase; secondly, adjustment of the osmotic strength of the medium so that no more than three and preferably only one stroke of the homogenizer is required for adequate cell breakage; thirdly, centrifugation of membranes into pellets at the lowest possible gravitational field strengths and for the shortest times so that resuspension can be easily and quickly accomplished without repeated excessive shear force being applied to the membrane leaflets. Failure to observe these points leads to the selective loss of membrane material, resulting in preparations containing little else other than the microtubular array and some flagella. Membrane-bound DNA: characteristics and re-

moval to facilitate further fractionation. The conditions for cell breakage that were optimal for the recovery of whole plasma membranes also led to disruption of nuclei with the consequent appearance of broken nuclear profiles in electron-microscopic sections (Plate 2c). The DNA in trypanosotnes is thought to lack histone proteins (Beck & Walker, 1964), and when released from broken nuclei quickly associated with the plasma membranes and caused them to aggregate and to become intractable to adequate resuspension. The extent of this problem may be assessed by reference to Table 1, which shows that about half of the cellular DNA was found in the final purified membrane fraction even after washing in 0.5 M-KCI unless special steps were taken. The ability of trypanosomal DNA to stick to plasma membranes was not shared by double-stranded 3H-labelled DNA from the lambdoid phage 080, which also lacks

Table 2. Recovery of [3H]DNA and [32P]RNA during the purification ofplasma membranes [3H]DNA (5.54x 105d.p.m., Expt. 1; 1.39x 106d.p.m., Expt. 2) and [32P]RNA (5.02x I16d.p.m., Expt. 3; 4.92x

106d.p.m., Expt. 4) were added to 1010 whole cells in separate experiments. Cells were homogenized and plasma membranes were prepared according to Scheme 1. Samples were removed at each stage and their radioactivities determined by liquid-scintillation counting. For sources of labelled nucleic acids see the Materials section. [32P]RNA recovered [3H]DNA recovered

(%) Fraction Supematant from homogenate First wash Second wash Sucrose gradient Final membranes Total recovery

Expt. 1 66.2 20.3 2.8

Expt. 2 57.4 34.0 7.7

Expt. 3 68.5

0.14 89.3

0.27 99.4

2.6 71.1

Expt. 4 86.6 5.1 1.4 1.3 0.7 95.1

_

Table 3. Comparison of the density distributtion of DNA extractedfrom whole cells with that extractedfrom the purified plasmamembrane fraction Plasma membranes were purified according to Scheme 1, but omitting the addition of KCI to the homogenate and DNAase treatment step. Samples of membrane pellets containing 20/ig of DNA were extracted for 2h at 37°C with 2ml of 1°Y (w/v) sodium dodecyl sulphate in a medium containing 0.02M-Tris/HCI (pF 8.5), 0.1 M-EDTA and 0.15MNaCI. The resulting suspensions were incubated with 1 mg of Pronase/ml for 2h at 37°C, dialysed and then subjected to analytical CsCI-density-gradient ultracentrifugation until equilibrium was reached. The area under each peak in the curves of u.v. absorbance (260nm) versus distance along the gradient was obtained by integration (peak height x width at half height) of microdensitometer tracings of in-flight photographs of the gradient, and the results were compared with those obtained with DNA extracted from whole cells after integration of the experimental curves reported by Newton & Burnett (1972). The relationship between buoyant density and distance along the gradient was determined by the use of an added marker DNA. Whole cellst Plasma membranes Buoyant density Buoyant density Y. of total 1.707 54.5 1.708 1.702 28.1 (37.8*) 1.702 1.695 9.7 1.691 7.5 1.692 * Value represents the sum of components at buoyant densities of 1.702 and 1.695. t Values calculated for comparison from the data of Newton & Burnett (1972).

%, of total 51.9 34.6 13.4

1979

17

PLASMA MEMBRANE FROM T. BRUCEI histones, or by 32P-labelled rRNA from E. cali. These nucleic acids were removed easily from plasma membranes by washing in 0.5 M-KCI (Table 2). This result makes it unlikely that the trypanosomal DNA simply acted like a polyanion with exposed functional groups that bound to the membrane by an electrostatic interaction. It seemed unlikely that the DNA was found in the membrane fraction because of physical trapping in closed membrane vesicles for two reasons: first, vesicular profiles were almost completely absent from electron-microscopic sections of purified membrane sheets (Plate 3c), and, secondly, the ready accessibility of this DNA to degradation by highly purified DNAase (Table 1). The possibility was examined that the plasma membrane had selected certain unique species of trypanosomal DNA or a particular population of species that were tightly and specifically bound. Accordingly the DNA associated with the purified plasma membranes was extracted with 1.0 % (w/v) sodium dodecyl sulphate, treated with 1 mg of Pronase/ml (2 h at 37°C), dialysed and then subjected to CsCl-densitygradient centrifugation (Newton & Burnett, 1972). The results (Table 3) were compared with a published report of the density distribution of whole-cell DNA from this strain of T. brucei (Newton & Burnett, 1972)..There was no significant selection of any particular density band of DNA. Further purification of plasma membranes was facilitated by removal of most of the membraneassociated DNA by treatment with highly purified DNAase. Impure or partially purified preparations of DNAase were found to contain substantial proteinase activity. The time course of removal of DNA shows this process to be complete in 5min (Fig. lb). Under these conditions the content of RNA is not affected (Fig. Ib). The protein content of the membrane fraction was also monitored during DNAase treatment (Fig. Ia). The control treatment (buffer only) in the absence of DNAase shows that protein is not lost as a result of the incubation conditions or a proteinase activity of the membranes themselves that had been previously treated with 0.1 mM-phenylmethanesulphonyl fluoride. After treatment with DNAase the protein content of the membrane fraction depended on whether or not the membranes were washed before assaying for protein. Without washing the protein content was unchanged regardless of the length of DNAase treatment, which indicated a lack of proteinase activity in the purified DNAase. With washing there was a progressive loss of protein from the membrane fraction during DNAase treatment. This result was interpreted to mean that membranebound DNA could bind non-membrane proteins to the DNA-membrane complex and that their removal was effected by removal of the DNA. This conclusion was corroborated by the finding that DNAase treatment increased the specific activity of an unequivocal Vol. 180

120r

o

1201

0

5

10

20

30

Time (min) Fig. 1. DNA, RNA and protein contents of the membrane fraiction before and after treatment with purified DNAase I Pellets (Pl) from the first stage of the plasma-membrane purification procedure (see Scheme 1) were resuspended in Tes buffer without EDTA and containing 5mM-MgCI2. They were incubated (6ml; 5mg of PI fraction protein/ml) for 20min in the presence of purified DNAase I (0.05 mg, 120 units). Samples (1 .2ml) were withdrawn for assay at the times indicated, placed in 5 ml of 15% trichloroacetic acid and centrifuged at 600gav. for 5 min. The pellets were assayed for protein and their DNA and RNA separated and assayed (see the Methods section). (a) Protein: U, control without DNAase; o, with DNAase; *, with DNAase, but samples washed once with Tes buffer by centrifuging and resuspending before adding to trichloroacetic acid solution. (b) DNA and RNA: U, control without DNAase and analysed for DNA; e, analysed for RNA; El, samples incubated with DNAase and analysed for DNA; o, analysed for RNA. Values are the means+S.E.M. for three separate determinations.

plasma-membrane marker, the ouabain-sensitive Na++K+-stimulated ATPase (Table 4). Purification by density-gradient centrifugation. Further purification was achieved by centrifuging the DNAase-treated and washed membrane fraction for 3h to equilibrium on a continuous 40-60% sucrose density gradient. The profile across the gradient of a typical run is shown in Fig. 2. The exact position of the major band, which consisted of sheets of plasma

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H. P. VOORHEIS, J. S. GALE, M. J. OWEN AND W. EDWARDS

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