Erythrocyte membranes — Some effects of sonication

Chem. Phys. Lipids 8 (1972) 341-346 © North-Holland Publishing Company

E R Y T H R O C Y T E M E M B R A N E S - S O M E EFFECTS OF SONICATION

v. B. KAMAT, A. J. WYATT* and M.A.F. DAVIS Biophysics Division, Unilever Research Laboratory, Colworth/ Welwyn, England I. Introduction

In recent years, increasing use is being made of ultrasonic irradiation to clarify erythrocyte membrane suspensions in order to render then suitable for certain experiments 1-8). It is therefore important to know the effects of sonication on the structure and function of isolated membranes. Available evidence suggests that sonication reduces the size of membrane particles 2, a) and alters the activities of some membrane-bound enzymes S, 7). However, the basic markers for membrane identification, viz. the thickness (as judged by electron microscopy) a) and X-ray diffraction patterns 9) are reported to be similar for unsonicated and sonicated membranes. The purpose of this communication is to further explore the nature of membrane dispersions obtained under conditions used in the preparation of samples for the proton magnetic resonance (pmr) studies 1, 2). II. Experimental

Haemoglobin-free erythrocyte ghosts were prepared 9) from human blood (group A, Rh positive), washed with distilled and deionised water and stored frozen (around - 1 5 ° C ) in small aliquots until used. Each aliquot was thawed and prespun at 50,000 rpm for l0 min in a preparative ultracentrifuge to remove any free or loosely bound protein. The pellets were suspended in 2 mM Tris buffer (pH 8), or Tris containing 2 mM CaCI 2. The suspension was adjusted to a known turbidity (1.4 at 400 m/~) or a known protein concentration (0.8 ~ 1.2 mg/ml). It was placed in a lusteroid tube surrounded by icecold water and subjected to sonic oscillations at 20 KHz using a Dawe Instruments Soniprobe type 1130 fitted with a microtip. The tip was l cm below the upper level of suspension. The power setting was adjusted to record an amperage input of 2. Sonication was interruped at l min interval * Present address: The Bohringer Corporation (London) Ltd. 341

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V.B. KAMAT, A . J . W Y A T T AND M . A . F . DAVIS

for measuring the turbidity of the suspension at 400 m/~. It was stopped when the decrease in turbiduty reached a plateau (5 min). For some experiments, sonication was continued for up to 15 min. Unless otherwise stated, a 5 rain sonicated dispersion and the unsonicated control at identical protein concentrations were examined as follows: (1) Electron microscopy - fixation with glutaraldehyde and osmium tetroxide followed by staining 10). (2) Electron spin resonance (esr) spectrum of the membrane labelled with the protein specific nitroxide-maleimide label ~). (3) Derivative proton magnetic resonance (pmr) spectrum. (4) Differential centrifugation - at 30,000, 105,000 and 300,000 times gravity for 1 hr. The supernatants were examined for proteinlZ) and lipid phosphorous la). (5) lsopycnic density gradient centrifugation in continuous preformed buffered (pH 8-2 mM Tris) sucrose gradients. Gradients were analysed for the distribution of lipid-phosphorous and protein. (6) Alterations in the activities of phosphoglycerate kinase ( P G K ) and Na-K-ATPase. (7) Antibody absorptions - Anti-A-serum (Dade Chemicals) or a solution of phytohaemagglutinin (PHA-Burroughs Welcome Co.) were incubated with sonicated and unsonicated membranes. The incubates were centrifuged in high ionic strength media at 300,000xg for 1½ hr. Supernatants and control antibody solutions were checked for haemagglutination titres against intact (A, Rh positive) red cells in an isotonic medium at pH 7.4. I11. Results and discussion

An electron micrograph of unsonicated membranes reveals large membranous films, consisting of layers approximately 80 ,~ thick; whereas that of the sonic dispersion reveals a large population of closed vesicles ( < 6 0 0 ,~, in diameter) bounded by unit membrane of about 80 A thickness. Occasionally some large vesicles about 1000,--2000 A in diameter can be seen. The results indicate that sonication produces membrane fragments. They are in agreement with the observations of other investigators a, 6). The thickness of lipid vesicles (e.g., lecithin-cholesterol-water) has, however, been reported to be approximately 44 ,~ 1~). The esr spectrum of the membrane, after 1 min of sonication, shows an increase (about 25~o) in intensity of the narrow component. The broad component remains unaltered. Continued sonication up to 10 min causes no further spectral changes. Similarly, the derivative pmr spectrum of intact membranes shows at least a 109/o reduction in the intensity of the broad

E R Y T H R O C Y T E MEMBRANES - SOME EFFECTS OF S O N I C A T I O N

343

component upon sonication, and a marked increase in the narrow chemically shifted components. These results are consistent with a reduction in particle size and possible alteration of the molecular packing of the cell surface. All the lipid and protein of the unsonicated membranes sediment at 30,000 x g in 1 hr. The proportions of non-sedimentable lipid and protein after differential centrifugation of the sonicated dispersion are shown in fig. 1. About 70% of the protein and 80% of the lipid can be sedimented at o Phospholipid} Sonicated ~n • Protein 2rnM Tris ~ Phospholipid} 2 mM Tris Protein +2raM Ca"'

7O mE cm60

X

30,000

105,000 300,000 Average relative centrifugal force (g) Fig.

1.

300,000 x g in 1 hr. The lipid to protein ratio of the supernatants at various speeds is between 0.6-0.75. In the presence of 2mM Ca + +, all the lipid and 90% of the protein becomes sedimentable at 300,000 x g in I hr. The marked effect of Ca + + ions appears to be consistent with the reported aggregation of sonic dispersions into membranous films 15). The distribution of lipid-phosphorous and protein during isopycnic centrifugation of the unsonicated and sonicated membranes in sucrose gradients is shown in fig. 2. In both these cases, the lipid distribution follows the protein distribution faithfully, suggesting strongly the presence of lipoprotein structures. There is no evidence of free lipid. In general, the sonic dispersions distribute over lower density regions compared with the unsonicated control. This may be due to greater osmotic shrinkage of the unsonicated membranes which have a larger internal compartment than the sonicated vesicles, lsopycnic centrifugation in buffered (pH 8, 2mM Tris) sodium bromide gradients shows that the median densities of unsonicated

344

V.B. KAMAT, A. J. WYATT AND M. A. F. DAVIS

50= ~ Protein o-...... o Lipid 40 /

'

I

el1 .>_

0-,*" 20

Sonicated

' ~,'ll" ° m e m' ',b, ~r a n e s

i

~

~- Un sonicated membranes

ii /q' /~ ",'JI /

1

8 n

1C

1.02 1.04 1.06 1.08

:.10 1.12 1.14 1:16 Density

138 1.20

F i g . 2.

and sonicated membranes, as judged by protein distribution, are respectively 1.168, 1.143. The protein distribution profiles for both these membranes overlap between the density region 1.12-1.28. There is no detectable free lipid or free protein existing in these systems. The P G K activity of the membrane (2.1 x 10 -3 #moles/mg protein/min) increases by about four-fold (8 x 10 -3/~moles/mg protein/min) after 1 min of sonication and remains unaltered up to 10 min of sonication. The Na-K ATPase activity (in the presence of Mg ÷ +) is lost after 5 min of sonication. There is no change in the Mg ÷+ (plus either Na ÷ or K +) ATPase activity under these conditions. These results are in agreement with those of other workers 5, 7, is). Haemagglutination tests show that both the unsonicated and sonicated membranes absorb out anti-A serum or PHA to the same degree, and upon absorption the supernatant antibody solutions exhibit the same decrease in haemagglutination titres compared with control antibody titres. Since a large population of dispersed membranes are closed vesicles, the results indicate that these vesicles retain their original surface orientations.

ERYTHROCYTE MEMBRANES - SOME EFFECTS OF SONICATION

345

A t t e m p t s have also been m a d e to study changes in protein c o n f o r m a t i o n u p o n sonication o f the m e m b r a n e s . The I R film spectrum does not show a n y change in the c o n t o u r o f the A m i d e l - b a n d after sonication 15). Sonication has been used to reduce scattering and obtain circular d i c h r o i s m spectra 4) devoid o f a n o m a l i e s observed in the spectra o f unsonicated m e m b r a n e s . C l a i m s have been m a d e for an i m p r o v e d estimate o f s e c o n d a r y structure o f m e m b r a n e protein. However, m a j o r changes in m e m b r a n e protein conf o r m a t i o n by sonication have been observed by other w o r k e r s 17). The foregoing results thus confirm the i n t e r p r e t a t i o n that sonication u n d e r the c o n d i t i o n s described reduces the isolated m e m b r a n e s to small closed vesicles which retain their original surface orientation*, the layered thickness, and at least some enzymatic a n d antigenic determinants. W e suggest that sonicated m e m b r a n e dispersions provide useful e x p e r i m e n t a l systems for u n d e r s t a n d i n g lipid-protein interactions in m e m b r a n e s . It must however be b o r n e in mind that even the isolated ghosts m a y not necessarily represent the true native state o f the m e m b r a n e and that recently differences in structural and functional b e h a v i o u r have been r e p o r t e d between h a e m o globin-free and h a e m o g l o b i n - c o n t a i n i n g ghosts is, 29, 20).

Acknowledgement W e wish to a c k n o w l e d g e the assistance o f Mr. R.A. L a m b a n d Miss G . A . Lawrence.

References 1) 2) 3) 4) 5) 6) 7) 8) 9) I0) I 1) 12) 13)

D. Chapman, V. B. Kamat, J. de Gier and S. A. Penkett, J. Mol. Biol. 31 (1968) 101 V. B. Kamat and D. Chapman, Biochim. Biophys. Acta 163 (1968) 411 S. A. Rosenberg and J. R. Mclntosh, Biochim. Biophys. Acta 163 (1968) 285 A. S. Schneider, M. T. Schneider and K. Rosenbeek, Proc. Natl. Acad. Sci. U.S. 66 (1970) 793 A. Askari and J. C. Fratantoni, Biochim. Biophys. Acta 92 (1964) 132 V. T. Marchesi and G. E. Palade, J. Cell. Biol. 35 (1967) 385 S. L. Schrier, Am. J. Physiol. 210 (1) (1966) 139 J. B. Finean, R. Coleman, W. G. Green and A. R. Limbrick, J. Cell Sci. I (1966) 28 J. T. Dodge, C. Mitchell and D. J. Hanahan, Arch. Biochem. Biophys. 100 (1963) ! 19 D. D. Sabatini, K. Bensch and R. J. Barnett, J. Cell. Biol. 17 (1963) 19 D. Chapman, M. D. Barratt and V. B. Kamat, Biochim. Biophys. Acta 173 (1969) 154 O. H. Lowry, N. R. Roberts, K. Y. Leiner, M. L. Wu and A. L. Farr, J. Biol. Chem. 207 (1954) 1 O. H. Lowry, N. J. Rosebrough, A. L. Farr and R.J. Randall, J. Biol. Chem. 193 (1951) 265

* Neuraminidase treatment of sonicated dispersion releases as much sialic acid as digestion with 0.1 N H2SO4. Sonication p e r se does not release any detectable sialic acid from the vesicles.

346 14) 15) 16) 17) 18) 19) 20)

V. B. K A M A T , A. J. W Y A T F A N D M. A. F. D A V I S

A. D. Bangham and R. W. Horne, J. Mol. Biol. 8 (1964) 660 R. G. Kirk, Proc. Natl. Acad. Sci. U.S. 60 (1968) 614 D. Chapman, V. B. Kamat and R. J. Levene, Science 160 (1968) 314 D. F. H. Wallach, Discussion, this conference S. Knutton, J. B. Finean, R. Coleman and A. R. Limbrick, J. Cell. Sci. 7 (1970) 357 T. A. Bramley, R. Coleman and J. B. Finean, Biochim. Biophys. Acta 241 (1971) 752 L. L. M. van Deenen, Paper presented at this conference