INT . J . RAIIAT . BIOL .,
1975,
VOL .
28,
NO .
3, 267-272
CORRESPONDENCE Reactivity of a free radical probe with erythrocyte membranes ROGER H . BISBY and MICHAEL R . PRICE Int J Radiat Biol Downloaded from informahealthcare.com by York University Libraries on 12/29/14 For personal use only.
Cancer Research Campaign Laboratories, University Park, Nottingham NG7 2RD, England .
ROBERT B . CUNDALL Chemistry Department, University Park, Nottingham NG7 2RD, England
PETER WARDMAN Gray Laboratory of the Cancer Research Campaign, Mount Vernon Hospital, Northwood, Middlesex HA6 2RN, England (Received 30 yune 1975 ; accepted 18 August 1975)
1.
Introduction
On structure of bio-membranes, there is a major controversy concerning the way in which protein molecules are disposed with respect to the lipid bilayer (Cook and Stoddart 1973, Hendler 1974) . The early Danielli-Davson model (Danielli and Davson 1934) showed the membrane protein associated with the polar head groups of the lipid and hence coating the lipid bilayer at the aqueous interfaces . More recently, much work, mainly with erythrocyte membranes, has indicated that protein is inserted into, or in some instances, spans the lipid bilayer (Steck, Fairbanks and Wallach 1971, Singer and Nicolson 1972, Marchesi, Tillack, Jackson, Segrest and Scott 1972, Juliano 1973, Steck 1974, Morris, Meuller and Huber 1974) . Here we show how a radiolytically-generated free radical, which reacts selectively with aromatic amino acids, may be used as a novel probe to investigate the accessibility of protein in both native and enzymetreated erythrocyte ghost membranes . The Br2 radical anion is generated in irradiated aqueous solutions containing bromide (Sutton, Adams, Boag and Michael 1965) H2O ,,V- -> OH, H, e aq OH - + Br - Br- + OH Br' + Br Br2 Using pulse radiolysis (Dorfman 1974), it is possible to follow the reaction of Br2 with a solute in the microsecond time region . At neutral pH, Br2 has been found to react selectively with amino acids, oxidizing only tryptophan tyrosine, histidine and cysteine at measurable (k > 10 6 dm' M -1 s-1) rates (Adams, Aldrich, Bisby, Cundall, Redpath and Willson 1972) . We have previously used this selectively to show that such amino acids are not readily accessible at the erythrocyte membrane surface, and to follow protein solubilization from the membrane by dodecyl sulphate (Bisby, Cundall and Wardman 1975).
26 8
Correspondence
Int J Radiat Biol Downloaded from informahealthcare.com by York University Libraries on 12/29/14 For personal use only.
2.
Materials and methods Haemoglobin-free erythrocyte ghosts were prepared from defibrinated sheep's blood by the method of Dodge, Mitchell and Hanahan (1963) . Fractions of the resulting suspension in 5 mM phosphate buffer (pH 7 . 9) were incubated at 37 ° C for 1 hour in the presence of either a-chymotrypsin (0 . 1 mg per mg of membrane protein), papain (0 .1 mg per mg of membrane protein, activated with 5 mM L-cysteine) or neuraminidase (16 I .U . per mg of membrane protein) . After incubation, the suspension was washed twice by centrifugation (78 000 gav for 20 min), and the supternatant fluids were collected and immediately frozen and stored. The resulting pellets were washed once more, and KBr was added to the supernatant fluids and resuspended pellets (in 5 mM, pH 7 . 9, phosphate buffer) to a final concentration of 50 mM . A sample of the ghost suspension was solubilized by treatment with dodecyl sulphate . Using pulse radiolysis, the rates of reaction of Br 2- with enzyme-treated ghosts, untreated controls, solubilized ghosts and supernatant fluids were measured by following the decay of the Br, - absorption at 380 nm (Sutton, Adams, Boag and Michael 1965) . By subtracting the rate of Br 2 - decay in the supernatant fluid from that in the ghost suspension, the effect of any soluble material that remained after the three washings was eliminated . Protein concentrations were measured by the method of Lowry, Rosebrough, Farr and Randall (1951), and neuraminic acid was assayed with thiobarbituric acid (Warren 1959) . Molecular-weight distributions of proteins in the membrane pellets and supernatant fluids were analysed by electrophoresis on 10 per cent polyacrylamide-SDS gels (Weber and Osborn 1969), and visualized by staining with Coomassie Blue . 3.
Results and discussion The table shows the percentages of protein and neuraminic acid liberated by the various enzyme treatments from erythrocyte ghosts . Figure 1 shows
Treatment
Per cent proteint solubilization
Per cent neuraminic acid solubilization
None Neuraminidase§ a-Chymotrypsin 11 Papainll
1.3 5 21 . 1 48 . 6
1 .7 74 . 2 6.3 56 . 7
Second-order decay$ of Br2(x 10-3 sec1 mg-1 ml 1)
1.3 0. 5 10 . 6 12 . 8
Second-order decay of Br, after dodecyl sulphate treatment (x 10 -3 sec 1 mg 1 ml-1) 16 . 9 23 . 5 31 . 9 31 . 8
t Corrected for added enzyme . $ Corrected for soluble material in supernatant fluid . § Neuraminidase (from Vibrio cholerae), Behringwerke . 11 a-Chymotrypsin and papain were supplied by the Sigma Chemical Co . (Kingstonon-Thames). Reactivity of Br2- with enzyme-treated and control untreated erythrocyte membranes measured by pulse radiolysis.
Correspondence
269
Int J Radiat Biol Downloaded from informahealthcare.com by York University Libraries on 12/29/14 For personal use only.
densitometric scans of gels run for the supernatant fluids from the first two washings after enzyme treatment (containing the liberated material) and for the pellets obtained after the final centrifugation . Neuraminidase releases about 75 per cent of the neuraminic acid from the membrane but only a negligible amount of protein . Gel electrophoresis shows that the molecular-weight distribution of the membrane proteins is not significantly altered by treating the membranes with neuraminidase . In contrast, the proteolytic enzymes, A-chymotrypsin and papain, release substantial amounts of protein from the ghosts (table), and gel electrophoresis shows that the membranes then contain
Ip+ -O
O
O `~
Al
Bi
A2
C U 0
n 0 n N
A3
B3
a
i
I
A4
B4
i
4
6 8 0 2 Migration distance (cm)
6
8
Figure 1 . Molecular-weight distributions of proteins and polypeptides present in enzymetreated erythrocyte membranes (A) and corresponding solubilized fractions, (B) determined by electrophoresis on 10 per cent polyacrylamide dodecyl sulphate gels (Weber and Osborn, 1969) . Electrophoresis was continued until the leading edge of the bromophenol blue tracking dye migrated to the end of the gel . Protein bands were detected by densitometric scans of the gels stained with Coomassie Blue . Analyses were performed on untreated erythrocyte membranes (1) and membranes treated with neuraminidase (2), «-chymotrypsin (3) and papain (4) . Supernatant fluids containing solubilized material were lyophilized and reconstituted in volumes sufficient to allow visualization of stained protein on the gels . Arrows denote stained bands due to the presence of the enzyme. R .B .
U
2 70
Correspondence
Int J Radiat Biol Downloaded from informahealthcare.com by York University Libraries on 12/29/14 For personal use only.
many more smaller-molecular-weight polypeptides . Lower-molecular-weight fragments also appear in the supernatant fluids after treatment with these enzymes . In the case of papain, digestion is so extensive that all protein appears as small-molecular-weight molecules (10 000-30 000 daltons) . The rates of reaction of Br2 with treated and untreated membranes are included in the table . Neuraminidase treatment slightly lowers the reactivity of Br, - , whereas treatments with a-chymotrypsin and with papain both increase the rate of reaction, by almost a factor of 10 in the case of papain . As was noted previously (Bisby et al. 1975) solubilization of the untreated membrane by dodecyl sulphate causes a pronounced increase in the reactivity of Br, - . This was proposed to be due to exposure of reactive sites, which are not available for reaction with Br2 in the native membrane . Br2 reacts selectively with tryptophan, tyrosine, and to a lesser extent, histidine (Adams et al . 1972) (and also with cysteine, although amino-acid analysis shows little of this in erythrocyte membrane proteins (Fuller, Boughter and Morazzani 1974) . It was therefore suggested (Bisby et al . 1975) that in the erythrocyte membrane, these residues, which are amongst the most hydrophobic amino acids (Tanford 1962, Nozaki and Tanford 1971), interact with the non-polar region of the lipid bilayer and must therefore be located inside the membrane structure . There, they would be protected from oxidative attack by Br 2 - by the remaining unreactive protein, and possibly also by the negatively-charged carbohydrate groups (such as N-acetyl-glucosamine and N-acetyl neuraminic acid which are both unreactive (k < 5 x 10 6 dm 3 M -1 s -1 ) towards Br2 (R . H . Bisby and P . Wardman, unpublished observations)) . These are attached to the regions of protein molecules known to be predominantly exposed to the extracellular environment (Marchesi et al . 1972, Juliano 1973, Steck 1974, Segrest, Kahane, Jackson and Marchesi 1973) . That neuraminidase treatment does not significantly affect the reactivity of
24F 0
16
la d
7 2
m
0 z
8
Y
0 / I 1 1 1
0
10 4
10 3
[Sodium dodecyl
1072
10 1
sulphate M
Figure 2 . Effect of dodecyl sulphate concentration on the second-order rate for reaction of Br, - with bovine serum albumin (1 mg/ml) (Sigma) measured by pulse radiolysis in N2 0-saturated solutions containing 40 mM KBr at pH 7 . 9 .
Int J Radiat Biol Downloaded from informahealthcare.com by York University Libraries on 12/29/14 For personal use only.
Correspondence
27 1
Br2 indicates that this masking effect is not due solely to the negatively-charged neuraminic acid residues . The removal of polypeptides and glycopeptides by the proteolytic enzymes does, however, facilitate the reaction of Br2 with the membrane protein (table) . A further increase in reactivity of Br2 is observed when the membrane is solubilized by dodecyl sulphate . A model experiment with bovine serum albumin (figure 2) shows that the interaction of dodecyl sulphate with a protein molecule causes a decrease in the rate of Br2 reaction with the protein ; this is probably due to the negative charge of the adsorbed dodecyl sulphate molecules. The observed rates of Br 2 - reaction in the enzyme-treated membranes are therefore still much slower than if the protein existed in free solution . The reactivity of Br2 (calculated in units of ml mg -1 s-1 ) is higher for dodecyl-sulphate-solubilized, enzyme-treated membranes than the dodecylsulphate-solubilized, untreated membranes . This indicates that the removal of membrane protein and glycoprotein by proteolytic enzymes leaves the membrane-bound protein residue with a higher number of reactive sites (i .e . tryptophan and tyrosine residues) per unit weight . In effect, the reactive sites are concentrated in the remaining protein . Our results are compatible with the view that a relatively large amount of membrane-associated protein spans, or is inserted into, the membrane, and that the hydrophobic amino acids are concentrated into the central region of the lipid bilayer, where hydrophobic interactions with the non-polar hydrocarbon chains of the lipid would add stability to the structure . ACKNOWLEDGMENT
These studies were supported by grants from the Cancer Research Campaign .
REFERENCES ADAMS, G . E ., ALDRICH, J . E ., BISBY, R . H ., CUNDALL, R . B ., REDPATH, J . L ., and WILLSON, R . L ., 1972, Radiat . Res ., 49, 278 . BISBY, R . H ., CUNDALL, R . B ., and WARDMAN, P ., 1975, Biochim. biophys, Acta ., 389, 137 . COOK, G . M . W ., and STODDART, R. W., 1973, Surface Carbohydrates of the Eukaryotic Cell (London, New York : Academic Press), Chap. 1 . DANIELLI, J . F ., and DAvsoN, H ., 1934, J. cell. Comp. Physiol., 5, 495 . DODGE, J . T ., MITCHELL, C ., and HANAHAN, D . J ., 1963, Archs Biochem . Biophys., 100, 119 . DORFMAN, L. M ., 1974, Investigation of Rates and Mechanisms of Reactions, Part II, 3rd edition, edited by G . Hammes (New York : Wiley-Interscience), pp . 463-519 . FULLER, G . M ., BOUGHTER, J . M ., and MORAZZANI, M ., 1974, Biochemistry, 13, 3036 . HENDLER, R. W ., 1974, Biomembranes, Vol . 5, edited by L. Manson (New York : Plenum Press), pp . 251-274 . JULIANO, R . L., 1973, Biochim . biophys . Acta, 300, 341 . LowRY, 0 . H., ROSEBROUGH, N . J ., FARR, A . L ., and RANDALL, R . J ., 1951, Y. biol . Chem ., 193, 265 . MARCHESI, V . T ., TILLACK, T . W ., JACKSON, R. L., SEGREST, J . P ., and SCOTT, R . E ., 1972, Proc . natn . Acad . Sci . U.S. A ., 69, 1445 . MORRIS, M ., MEULLER, J . J ., and HUBER, C . T ., 1974, Y. biol. Chem ., 249, 2658 . NozAKI, Y ., and TANFORD, C ., 1971, Y. biol . Chem ., 246, 2211 . SEGREST, J . P ., KAHANE, I ., JACKSON, R. L ., and MARCHESI, V . T ., 1973, Archs Biochem . Biophys ., 155, 167 . SINGER, S . J ., and NICOLSON, G . L ., 1972, Science, N.Y., 175, 720 . STECK, T . L ., 1974, Y. Cell Biol ., 62, 1 .
272
Correspondence
Int J Radiat Biol Downloaded from informahealthcare.com by York University Libraries on 12/29/14 For personal use only.
STECK, T . L ., FAIRBANKS, G ., and WALLACH, D . F. H ., 1971, Biochemistry, 10, 2617 . SUTTON, H . C ., ADAMS, G . E ., BOAG, J . W ., and MICHAEL, B . D ., 1965, Pulse Radiolysis, edited by M . Ebert, J . P . Keene, A . J . Swallow and J . H . Baxendale (London, New York : Academic Press), pp . 61-81 . TANFORD, C ., 1962, J . Am. chem. Soc ., 84, 4240 . WARREN, L ., 1959, J. biol . Chem ., 234, 1971 . WEBER, K ., and OSBORN, M ., 1969, J. biol. Chem ., 244, 4406 .
1975,
VOL .
28,
NO .
3, 267-272
CORRESPONDENCE Reactivity of a free radical probe with erythrocyte membranes ROGER H . BISBY and MICHAEL R . PRICE Int J Radiat Biol Downloaded from informahealthcare.com by York University Libraries on 12/29/14 For personal use only.
Cancer Research Campaign Laboratories, University Park, Nottingham NG7 2RD, England .
ROBERT B . CUNDALL Chemistry Department, University Park, Nottingham NG7 2RD, England
PETER WARDMAN Gray Laboratory of the Cancer Research Campaign, Mount Vernon Hospital, Northwood, Middlesex HA6 2RN, England (Received 30 yune 1975 ; accepted 18 August 1975)
1.
Introduction
On structure of bio-membranes, there is a major controversy concerning the way in which protein molecules are disposed with respect to the lipid bilayer (Cook and Stoddart 1973, Hendler 1974) . The early Danielli-Davson model (Danielli and Davson 1934) showed the membrane protein associated with the polar head groups of the lipid and hence coating the lipid bilayer at the aqueous interfaces . More recently, much work, mainly with erythrocyte membranes, has indicated that protein is inserted into, or in some instances, spans the lipid bilayer (Steck, Fairbanks and Wallach 1971, Singer and Nicolson 1972, Marchesi, Tillack, Jackson, Segrest and Scott 1972, Juliano 1973, Steck 1974, Morris, Meuller and Huber 1974) . Here we show how a radiolytically-generated free radical, which reacts selectively with aromatic amino acids, may be used as a novel probe to investigate the accessibility of protein in both native and enzymetreated erythrocyte ghost membranes . The Br2 radical anion is generated in irradiated aqueous solutions containing bromide (Sutton, Adams, Boag and Michael 1965) H2O ,,V- -> OH, H, e aq OH - + Br - Br- + OH Br' + Br Br2 Using pulse radiolysis (Dorfman 1974), it is possible to follow the reaction of Br2 with a solute in the microsecond time region . At neutral pH, Br2 has been found to react selectively with amino acids, oxidizing only tryptophan tyrosine, histidine and cysteine at measurable (k > 10 6 dm' M -1 s-1) rates (Adams, Aldrich, Bisby, Cundall, Redpath and Willson 1972) . We have previously used this selectively to show that such amino acids are not readily accessible at the erythrocyte membrane surface, and to follow protein solubilization from the membrane by dodecyl sulphate (Bisby, Cundall and Wardman 1975).
26 8
Correspondence
Int J Radiat Biol Downloaded from informahealthcare.com by York University Libraries on 12/29/14 For personal use only.
2.
Materials and methods Haemoglobin-free erythrocyte ghosts were prepared from defibrinated sheep's blood by the method of Dodge, Mitchell and Hanahan (1963) . Fractions of the resulting suspension in 5 mM phosphate buffer (pH 7 . 9) were incubated at 37 ° C for 1 hour in the presence of either a-chymotrypsin (0 . 1 mg per mg of membrane protein), papain (0 .1 mg per mg of membrane protein, activated with 5 mM L-cysteine) or neuraminidase (16 I .U . per mg of membrane protein) . After incubation, the suspension was washed twice by centrifugation (78 000 gav for 20 min), and the supternatant fluids were collected and immediately frozen and stored. The resulting pellets were washed once more, and KBr was added to the supernatant fluids and resuspended pellets (in 5 mM, pH 7 . 9, phosphate buffer) to a final concentration of 50 mM . A sample of the ghost suspension was solubilized by treatment with dodecyl sulphate . Using pulse radiolysis, the rates of reaction of Br 2- with enzyme-treated ghosts, untreated controls, solubilized ghosts and supernatant fluids were measured by following the decay of the Br, - absorption at 380 nm (Sutton, Adams, Boag and Michael 1965) . By subtracting the rate of Br 2 - decay in the supernatant fluid from that in the ghost suspension, the effect of any soluble material that remained after the three washings was eliminated . Protein concentrations were measured by the method of Lowry, Rosebrough, Farr and Randall (1951), and neuraminic acid was assayed with thiobarbituric acid (Warren 1959) . Molecular-weight distributions of proteins in the membrane pellets and supernatant fluids were analysed by electrophoresis on 10 per cent polyacrylamide-SDS gels (Weber and Osborn 1969), and visualized by staining with Coomassie Blue . 3.
Results and discussion The table shows the percentages of protein and neuraminic acid liberated by the various enzyme treatments from erythrocyte ghosts . Figure 1 shows
Treatment
Per cent proteint solubilization
Per cent neuraminic acid solubilization
None Neuraminidase§ a-Chymotrypsin 11 Papainll
1.3 5 21 . 1 48 . 6
1 .7 74 . 2 6.3 56 . 7
Second-order decay$ of Br2(x 10-3 sec1 mg-1 ml 1)
1.3 0. 5 10 . 6 12 . 8
Second-order decay of Br, after dodecyl sulphate treatment (x 10 -3 sec 1 mg 1 ml-1) 16 . 9 23 . 5 31 . 9 31 . 8
t Corrected for added enzyme . $ Corrected for soluble material in supernatant fluid . § Neuraminidase (from Vibrio cholerae), Behringwerke . 11 a-Chymotrypsin and papain were supplied by the Sigma Chemical Co . (Kingstonon-Thames). Reactivity of Br2- with enzyme-treated and control untreated erythrocyte membranes measured by pulse radiolysis.
Correspondence
269
Int J Radiat Biol Downloaded from informahealthcare.com by York University Libraries on 12/29/14 For personal use only.
densitometric scans of gels run for the supernatant fluids from the first two washings after enzyme treatment (containing the liberated material) and for the pellets obtained after the final centrifugation . Neuraminidase releases about 75 per cent of the neuraminic acid from the membrane but only a negligible amount of protein . Gel electrophoresis shows that the molecular-weight distribution of the membrane proteins is not significantly altered by treating the membranes with neuraminidase . In contrast, the proteolytic enzymes, A-chymotrypsin and papain, release substantial amounts of protein from the ghosts (table), and gel electrophoresis shows that the membranes then contain
Ip+ -O
O
O `~
Al
Bi
A2
C U 0
n 0 n N
A3
B3
a
i
I
A4
B4
i
4
6 8 0 2 Migration distance (cm)
6
8
Figure 1 . Molecular-weight distributions of proteins and polypeptides present in enzymetreated erythrocyte membranes (A) and corresponding solubilized fractions, (B) determined by electrophoresis on 10 per cent polyacrylamide dodecyl sulphate gels (Weber and Osborn, 1969) . Electrophoresis was continued until the leading edge of the bromophenol blue tracking dye migrated to the end of the gel . Protein bands were detected by densitometric scans of the gels stained with Coomassie Blue . Analyses were performed on untreated erythrocyte membranes (1) and membranes treated with neuraminidase (2), «-chymotrypsin (3) and papain (4) . Supernatant fluids containing solubilized material were lyophilized and reconstituted in volumes sufficient to allow visualization of stained protein on the gels . Arrows denote stained bands due to the presence of the enzyme. R .B .
U
2 70
Correspondence
Int J Radiat Biol Downloaded from informahealthcare.com by York University Libraries on 12/29/14 For personal use only.
many more smaller-molecular-weight polypeptides . Lower-molecular-weight fragments also appear in the supernatant fluids after treatment with these enzymes . In the case of papain, digestion is so extensive that all protein appears as small-molecular-weight molecules (10 000-30 000 daltons) . The rates of reaction of Br2 with treated and untreated membranes are included in the table . Neuraminidase treatment slightly lowers the reactivity of Br, - , whereas treatments with a-chymotrypsin and with papain both increase the rate of reaction, by almost a factor of 10 in the case of papain . As was noted previously (Bisby et al. 1975) solubilization of the untreated membrane by dodecyl sulphate causes a pronounced increase in the reactivity of Br, - . This was proposed to be due to exposure of reactive sites, which are not available for reaction with Br2 in the native membrane . Br2 reacts selectively with tryptophan, tyrosine, and to a lesser extent, histidine (Adams et al . 1972) (and also with cysteine, although amino-acid analysis shows little of this in erythrocyte membrane proteins (Fuller, Boughter and Morazzani 1974) . It was therefore suggested (Bisby et al . 1975) that in the erythrocyte membrane, these residues, which are amongst the most hydrophobic amino acids (Tanford 1962, Nozaki and Tanford 1971), interact with the non-polar region of the lipid bilayer and must therefore be located inside the membrane structure . There, they would be protected from oxidative attack by Br 2 - by the remaining unreactive protein, and possibly also by the negatively-charged carbohydrate groups (such as N-acetyl-glucosamine and N-acetyl neuraminic acid which are both unreactive (k < 5 x 10 6 dm 3 M -1 s -1 ) towards Br2 (R . H . Bisby and P . Wardman, unpublished observations)) . These are attached to the regions of protein molecules known to be predominantly exposed to the extracellular environment (Marchesi et al . 1972, Juliano 1973, Steck 1974, Segrest, Kahane, Jackson and Marchesi 1973) . That neuraminidase treatment does not significantly affect the reactivity of
24F 0
16
la d
7 2
m
0 z
8
Y
0 / I 1 1 1
0
10 4
10 3
[Sodium dodecyl
1072
10 1
sulphate M
Figure 2 . Effect of dodecyl sulphate concentration on the second-order rate for reaction of Br, - with bovine serum albumin (1 mg/ml) (Sigma) measured by pulse radiolysis in N2 0-saturated solutions containing 40 mM KBr at pH 7 . 9 .
Int J Radiat Biol Downloaded from informahealthcare.com by York University Libraries on 12/29/14 For personal use only.
Correspondence
27 1
Br2 indicates that this masking effect is not due solely to the negatively-charged neuraminic acid residues . The removal of polypeptides and glycopeptides by the proteolytic enzymes does, however, facilitate the reaction of Br2 with the membrane protein (table) . A further increase in reactivity of Br2 is observed when the membrane is solubilized by dodecyl sulphate . A model experiment with bovine serum albumin (figure 2) shows that the interaction of dodecyl sulphate with a protein molecule causes a decrease in the rate of Br2 reaction with the protein ; this is probably due to the negative charge of the adsorbed dodecyl sulphate molecules. The observed rates of Br 2 - reaction in the enzyme-treated membranes are therefore still much slower than if the protein existed in free solution . The reactivity of Br2 (calculated in units of ml mg -1 s-1 ) is higher for dodecyl-sulphate-solubilized, enzyme-treated membranes than the dodecylsulphate-solubilized, untreated membranes . This indicates that the removal of membrane protein and glycoprotein by proteolytic enzymes leaves the membrane-bound protein residue with a higher number of reactive sites (i .e . tryptophan and tyrosine residues) per unit weight . In effect, the reactive sites are concentrated in the remaining protein . Our results are compatible with the view that a relatively large amount of membrane-associated protein spans, or is inserted into, the membrane, and that the hydrophobic amino acids are concentrated into the central region of the lipid bilayer, where hydrophobic interactions with the non-polar hydrocarbon chains of the lipid would add stability to the structure . ACKNOWLEDGMENT
These studies were supported by grants from the Cancer Research Campaign .
REFERENCES ADAMS, G . E ., ALDRICH, J . E ., BISBY, R . H ., CUNDALL, R . B ., REDPATH, J . L ., and WILLSON, R . L ., 1972, Radiat . Res ., 49, 278 . BISBY, R . H ., CUNDALL, R . B ., and WARDMAN, P ., 1975, Biochim. biophys, Acta ., 389, 137 . COOK, G . M . W ., and STODDART, R. W., 1973, Surface Carbohydrates of the Eukaryotic Cell (London, New York : Academic Press), Chap. 1 . DANIELLI, J . F ., and DAvsoN, H ., 1934, J. cell. Comp. Physiol., 5, 495 . DODGE, J . T ., MITCHELL, C ., and HANAHAN, D . J ., 1963, Archs Biochem . Biophys., 100, 119 . DORFMAN, L. M ., 1974, Investigation of Rates and Mechanisms of Reactions, Part II, 3rd edition, edited by G . Hammes (New York : Wiley-Interscience), pp . 463-519 . FULLER, G . M ., BOUGHTER, J . M ., and MORAZZANI, M ., 1974, Biochemistry, 13, 3036 . HENDLER, R. W ., 1974, Biomembranes, Vol . 5, edited by L. Manson (New York : Plenum Press), pp . 251-274 . JULIANO, R . L., 1973, Biochim . biophys . Acta, 300, 341 . LowRY, 0 . H., ROSEBROUGH, N . J ., FARR, A . L ., and RANDALL, R . J ., 1951, Y. biol . Chem ., 193, 265 . MARCHESI, V . T ., TILLACK, T . W ., JACKSON, R. L., SEGREST, J . P ., and SCOTT, R . E ., 1972, Proc . natn . Acad . Sci . U.S. A ., 69, 1445 . MORRIS, M ., MEULLER, J . J ., and HUBER, C . T ., 1974, Y. biol. Chem ., 249, 2658 . NozAKI, Y ., and TANFORD, C ., 1971, Y. biol . Chem ., 246, 2211 . SEGREST, J . P ., KAHANE, I ., JACKSON, R. L ., and MARCHESI, V . T ., 1973, Archs Biochem . Biophys ., 155, 167 . SINGER, S . J ., and NICOLSON, G . L ., 1972, Science, N.Y., 175, 720 . STECK, T . L ., 1974, Y. Cell Biol ., 62, 1 .
272
Correspondence
Int J Radiat Biol Downloaded from informahealthcare.com by York University Libraries on 12/29/14 For personal use only.
STECK, T . L ., FAIRBANKS, G ., and WALLACH, D . F. H ., 1971, Biochemistry, 10, 2617 . SUTTON, H . C ., ADAMS, G . E ., BOAG, J . W ., and MICHAEL, B . D ., 1965, Pulse Radiolysis, edited by M . Ebert, J . P . Keene, A . J . Swallow and J . H . Baxendale (London, New York : Academic Press), pp . 61-81 . TANFORD, C ., 1962, J . Am. chem. Soc ., 84, 4240 . WARREN, L ., 1959, J. biol . Chem ., 234, 1971 . WEBER, K ., and OSBORN, M ., 1969, J. biol. Chem ., 244, 4406 .
