Electron spin resonance studies of membrane proteins in erythrocytes in myotonic muscular dystrophy

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

177,

226-234

(1976)

Electron Spin Resonance Studies of Membrane Proteins Erythrocytes in Myotonic Muscular Dystrophy’ D. ALLAN

BUTTERFIELD

,2, 4 ALLEN D. ROSES3 D. B. CHESNUT2

STANLEY

in

H. APPEL,3

AND ‘Paul

M. Gross Chemical Laboratory, Duke Medicine, Division of Neurology,

University, Durham, North Carolina 27706, and Duke University, Durham, North Carolina 27710

Received

March

?Ychool

of

15, 1976

A sulfhydryl group specific spin label has been used to study membrane proteins in erythrocyte ghosts from patients with myotonic muscular dystrophy (MyD). The resulting electron spin resonance spectra of labeled permeable MyD and control ghosts demonstrate two chief populations of sulfhydryl groups, one weakly immobilized, the other strongly immobilized. Approximately one-third of the labeled sulfbydryl groups are in weakly immobilized sites which, based on their nitrogen hyperfine splitting value and their accessibility to both spin label and ascorbate, appear to be located near the membrane surface in a highly polar environment. Strongly immobilized sulfhydryl groups appear to be located both at the membrane surface and deep within the lipid bilayer. While the linewidths of each class of sites are equal in control and MyD membranes, the ratio of the spectral amplitude of the spin label attached to weakly immobilized sites to that of strongly immobilized sites is significantly greater in MyD membranes (P = 0.001). This effect is due to a decreased incorporation of the spin label into the strongly immobilized sulfhydryl group sites and suggests that membrane organizational or protein conformational differences exist between MyD and control erythrocyte membranes.

Myotonic muscular dystrophy (MyDj5 is considered a defect of muscle sarcolemma

based on the persistence of repetitive membrane depolarization after nerve block or neuromuscular blockade (1). Our previous biochemical and biophysical studies have suggested that membrane alterations are present in red blood cells as well as muscle (2-7). A decrease in endogenous membrane-bound protein kinase activity (6, 71, an alteration of the stoichiometry of the sodium pump (41, and an increase in the number of stomatocytes observed by scanning electron microscopy (5) are observed in MyD erythrocytes. Electron spin resonance (esr) studies using stearic acid methyl ester spin labels which probe the lipid bilayer suggest that MyD erythrocyte membranes are more fluid near the membrane surface than those of normal controls (2, 3). In addition, a correlation of increased erythrocyte membrane fluidity with the presence of myotonia has been suggested on the basis of esr studies of red blood cells of MyD, Duchenne muscular

1 This work was supported in part by National Institutes of Health NINDS Grant 1 F22 NSO-136401 to D.A.B., NSF Grant GP-22546, NIH Grant NSO 7872, Multiple Sclerosis Society Grant 558-D-S, NIH Grant NS 12213, a Basil O’Connor Starter Research Grant from the National Foundation March of Dimes, and the Multiple Sclerosis Society Grant 923-A-2. 4 NIH Postdoctoral Fellow, 1974-1975. Present address: Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506. ’ Abbreviations used: esr, electron spin resonance; SH, sulfhydryl; MyD, myotonic muscular dystrophy; RBC, red blood cell; MAL-6, 2,2,6,6tetramethylpiperidine-1-oxyl-4-maleimide; PCMB, parachloromercuribenzoate; NEM, N-ethyl maleimide; PBS, phosphate-buffered saline, containing 5 mM sodium phosphate, 150 mM NaCl, pH 8.0; 5P8, 5 mM sodium phosphate buffer, pH 8.0; SDS, sodium dodecyl sulfate. TCA, trichloroacetic acid; EGTA, ethylene glycobis(aminoethyl)-NJ’-tetraacetic acid; S, strongly immobilized; W, weakly immobilized; and DTNB, 5,5-dithiobis(2-nitrobenzoic acid). 226 Copyright All riehts

0 1976 by Academic Press, Inc. of reuroduction in anv form reserved.

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dystrophy, congenital myotonia, and several nonmyotonic muscular dystrophies (8). This increased fluidity may be a secondary consequence of an altered proteinlipid organization in the membrane, a question potentially amenable to experiment by the use of a sulfhydryl group specific protein spin label. Sulfhydryl (SH) groups of erythrocyte membrane proteins are important in several membrane properties, including sodium and potassium active transport, glucose uptake, and protection against hemolysis (9). The spin labeling technique has been previously used to study SH groups of red blood cell (RBC) membranes (10-15). Using the spin probe 2,2,6,6-tetramethylpiperidine-1-oxyl-4-maleimide (MAL-6), which labels SH groups almost exclusively isome amino group binding may occur), two classes of labeled SH groups were detected: one strongly immobilized, the other weakly immobilized. The effects of various procedures and agents on the relative proportion of each class of sites were monitored by this esr method and one attempt (15) to define the location of labeled SH groups in bovine erythrocytes has been reported. This latter study demonstrated that intact cells and membrane ghosts gave essentially the same kind of esr spect.rum and that some SH groups (not necessarily those labeled by MAL-6) were located at sites where membrane chloride exchange was blocked. However, a clear delineation of the location of the two classes of SH groups labeled by MAL-6 was not demonstrated. Proper interpretation of the spin labeling results requires knowledge of the degree to which gross structural perturbations are caused by MAL-6, the relative proportion of labeled weakly and strongly immobilized sites, and the topology of labeled sulfhydryl groups of membrane proteins. A potential clue as to the SH groups bound by MAL-6 may be found in the action of other sulfhydryl reagents. Parachloromercuribenzoate (PCMB), a commonly used SH agent, has been shown to extract membrane proteins from erythrocytes (16) and is consequently not suitable for our purposes. N-ethyl malemide (NEM), however, does not extract membrane proteins

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from erythrocytes (16, 17); moreover, its structural similarity to MAL-6 offered the possibility that these two reagents might compete for sulfhydryl groups binding. The binding of [14C]NEM to RBC membrane proteins has been previously characterized (18, 19). In the present experiments, competition between NEM and MAL-6 has been used to validate the binding of MALB as descriptive of a covalently bonded reagent, while the esr spectra have been interpreted as descriptive of the spin probe existing in differing locations and environments within the membrane. Ascorbate reduction studies further characterized the topology of labeled sulfhydryl groups in erythrocyte membrane proteins. The present experiments utilize the sulfhydryl group specific and covalently bound spin label MAL-6 to study membrane proteins in erythrocytes in MyD and normal controls. Our studies demonstrate reproducible and statistically significant differences in the esr spectra of normal and MyD erythrocyte membranes labeled with MAL-6 and suggest that membrane organizational alterations are found in MyD. MATERIALS

AND

METHODS

MAL-6 was obtained from Syva, while [“HINEM (230 $Xmmol) and [‘*Clascorbate (5.9 @X/mmol) were purchased from New England Nuclear. The cold NEM (199.9% purity) used was from Aldrich Chemical Company. All other reagents were of highest purity obtainable. The esr spectra were recorded on a standard Varian V-4502-15 system equipped with frequency and power monitoring devices. Modulation and power broadening of spectral lines were avoided by recording all spectra at low modulation amplitude (0.2 G) and low power (20 mW) incident on the rectangular cavity containing the quartz aqueous sample cell. The modulation amplitude and microwave power used in all samples was the same so that the only instrumental variable to affect the esr spectral amplitudes was the spectrometer gain, a quantity determined to be essentially linear for our instrument. Individual peak amplitudes in normal MyD spectra were thus compared by correcting each amplitude both to a single spectrometer gain and to the amount of protein in the sample. Heparinized blood from 17 healthy normal volunteers and 14 MyD patients from eight different families was used in this study. The intact cells were washed four times with 5 rnM sodium phosphate, 150 mM NaCl, pH 8.0 (PBS), and the buffy coats were carefully removed. Permeable ghosts were prepared

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BU’ITERFIELD

by hypotonic lysis in 5 mM sodium phosphate buffer, pH 8.0 (5P8) according to Fairbanks et al. (20), a procedure which results in minimal membrane protein loss; resealed membranes were prepared according to Method II of Steck and Kant (21). Membrane protein content was estimated by the method of Lowry et al. (22), while the method of Sedlak and Lindsay (23) was used to determine the total sulfhydry1 group content. Lipids were extracted by a modification of the Folch procedure (24). The membrane ghosts were spin labeled with MAL-6 by reacting 1 vol of packed permeable ghosts at 4°C overnight with 10 vol of 5P8 buffer containing the spin label in a 1:50 weight ratio to total membrane protein in the sample. The spin labeled ghosts were centrifuged at 27,000g for 10 min and washed in the cold with 5P8 until no further free MAL-6 esr signal could be detected in the supernatant (live washes were sufficient for this purpose). Studies involving the reduction of MAL-6 by ascorbate were performed using 5 mM ascorbate in 5P8. The pH of the ascorbate solution was adjusted to 8.0 prior to reaction. In some experiments, ghosts were spin labeled for only 30 min at 4°C instead of overnight. Short-term labeling was performed by incubating a total of 0.5 ml of ghosts and MAL-6 in 5P8 (1:50, w/ w, spin label to membrane protein) for 30 min, after which time the mixture was rapidly diluted by a factor of 30 with 5P8, centrifuged at 27,OOOg, and washed twice more. No free MAL-6 esr signal was observed in the last supernatant using this procedure. The total time elapsed with this short-term labeling method was less than 1 h, a period in which the rate of NEM uptake is rapid (Fig. 3). Six percent SDS-polyacrylamide gels were prepared and the gel electrophoresis was carried out as previously described (20). Each reaction was run in duplicate mixtures containing 50 mM sodium acetate buffer (pH 6.51, 1.5 mM EGTA, 8 PM to 2 mM NEM, and 200 pg of erythrocyte membrane protein with a total volume of 0.2 ml. In the competition experiments, aqueous MAL-6 solution (final concentration, 0.2 mM) was added at the expense of H,O,

ET AL. keeping the total reaction volume at 0.20 ml while the total NEM concentration was varied. The blank consisted of the usual reagents with 13HINEM replaced by cold NEM (10 mM final concentration). The reaction was run at 4°C overnight to ensure that equilibrium had been reached (maximal t3H]NEM uptake was obtained under these conditions after 8 h, Fig. 3) and was stopped by the sudden addition of a solution containing 10% SDS, 100 mM NEM, and 5 mM EDTA to a final SDS concentration of 1%. The appropriate [3H]NEM solution (8 FM to 2 mM final concentration) was immediately added to blank samples while an equal volume of water was added to the reaction sample. After electrophoresis for 16 h, the gels were fixed in 10% TCA, stained with a Coumassie brilliant blue dye solution, and destained in 7% acetic acid. The individual bands of each gel were cut and solubilized in 100 ~1 of 30% H,O, for 2 h at 100°C and each band was assayed for 3H-radioactivity with a Packard Tri-Carb Liquid Scintillation Spectrometer Model 2405. RESULTS

Figure 1A shows the structural formulas for MAL-6 and NEM. MAL-6 is a nitroxide spin label analog of NEM, labels SH groups almost exclusively, and reacts as shown in Fig. 1B. The protein SH group schematically indicated in Fig. 1B forms a covalent linkage across the double bond of MAL-6 (or NEM). Figure 2A demonstrates a densitometry scan of a 6% SDS-polyacrylamide gel of erythrocyte ghost proteins which have been subjected to electrophoresis. Prior incubation with MAL-6 or NEM produces no change on the electrophoretic profile and does not extract any membrane protein into the supernatant phase. The time course of the uptake of tritiated NEM into erythrocyte membrane pro-

MAL-6

NEM

FIG. reaction

1. (A) Structural of MAL-6 with

formulas a membrane

of MAL-6 protein

and NEM. SH group.

(B) Schematic

representation

of the

ESR

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BAND

OF

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PROTEINS

NUMBER

FIG. 2. (A) A typical SDS-polyacrylamide gel densitometry scan of a human erythrocyte preparation. (B) Bar graph of the incorporation of NEM into the various protein bands of human erythrocyte membrane proteins after an 18-h incubation at 4°C. The height of each rectangle represents the number of picomoles of NEM incorporated into the indicated bands per milligram of total membrane protein. Band nonmenclature is after Steck and Yu (17) and Fairbanks et al. (201.

teins is indicated for band III in Fig. 3. The reaction is nearly half completed after approximately 1 h and the uptake is maximal after 8 h. The incorporation of NEM into other protein bands demonstrates similar kinetic behavior. Membranes were reacted with NEM for 8 h and washed with 5P8 to remove excess reagent. No [3H]NEM could be released by subsequent dialysis over a 24-h period. Figure 2B shows a bar graph of the incorporation of [3H]NEM into the proteins of the human erythrocyte membrane. The greatest incorporation of 13HlNEM is in the large polypeptide bands known as spectrin (I and III), in the higher molecular weight proteins present in lesser amounts, and in peak III. Spectrin, which comprises approximately 25% of the membrane protein, accounts for approximately 30% of the [3HJNEM binding, while the proteins in largest abundance in the membrane, peak III (30%), account for only ll%, suggesting that NEM incorporation is not directly proportional to the amount of the particular protein band present in

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the erythrocyte membrane. Figure 2B also shows that bands IV-VI are not labeled to any great extent by NEM and consequently these protein bands would not be expected to contribute greatly to the overall MAL-6 esr spectra. The relatively large radioactivity peak which occurs just behind the tracking dye is probably due to NEM incorporation into residual hemoglobin that is not completely removed in the preparation of the membranes. No such peak occurs when NEM, isolated membrane proteins, or isolated lipids are individually reacted with [3HlNEM and subjected to electrophoresis. However, considerable radioactivity in the same position on the gel as noted above is observed when purified hemoglobin is reacted with 13HlNEM and the SDS-gel is assayed for NEM incorporation. The esr spectrum of MAL-6 labeled erythrocyte ghosts is displayed in Fig. 4.

FIG. 3. Time course of the incorporation of NEM into band III of human erythrocyte membranes. The reactions were carried out at 4°C for the times indicated and stopped as described in Methods, and the ghosts were solubilized and subjected to electrophoresis.

FIG. 4. A typical esr spectrum of normal human erythrocyte ghosts with which MAL-6 has been incubated overnight as described in Methods. The amplitudes of the M, = +l weakly and strongly immobilized lines are indicated W and S, respectively, and the measurement of A, is shown.

230

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ET AL.

This spectrum is a superposition of at least the S sites are labeled at the same rate. A two spectra, reflecting at least two differmore detailed discussion of the W/S ratio ent classes of sulfhydryl group sites in the is presented below in the discussion of RBC membrane. One type of site is found erythrocyte membrane protein sulfhydryl in an environment in which the spin label binding in myotonic muscular dystrophy. is essentially completely immobilized, reComputer-similated spectra further flected by the powder-like spectrum. The characterized the W and S sulfhydryl amplitude of the low-field line is indicated group sites. The powder spectrum compoby S (for strongly immobilized). The splitnent of Fig. 4 is simulated using a Lorenting of the outer hyperfme extrema of the tzian line shape of width 3.2 G (deterpowder portion of the spectrum is 32.4 t mined experimentally) and the principle 0.8 G, clearly indicative of a highly immo- T- and g-tensor values of doxyl propane. bilized label; this splitting may be com- This spectrum is then converted to an isopared to the T,, principle values of 32.9 tropic spectrum of the same integrated inand 31.8 G for doxyl propane (25) and di-ttensity and lineshape. By comparing the butyl nitroxide (26), respectively, and to 31 products of the peak-to-peak amplitude and the square of the peak-to-peak width G for tempone (27) as guests in appropriate host single crystals. of the resulting line with that of the experThe second environment indicated by imental W signal, also of Lorentzian the esr spectrum in Fig. 4 is one in which shape, the relative proportion of the two MAL-6 is only weakly immobilized; the kinds of SH sites can be estimated. A low-field peak-to-peak amplitude of this value of 0.31 for the fraction of sites in a weakly immobilized spectrum is indicated weakly immobilized environment is obby W (for weakly immobilized). Since the tained. Alternative experiments to deterlow- and high-field lines of the W environmine this fraction using 0.2 N sodium hyment are resolved, one-half the splitting droxide to convert all labeled S sites to Wbetween these lines is a measure ofA,, the like sites (12) yields a value of 0.41. The nitrogen isotropic hyperfme coupling con- discrepancy between this value and that stant. One obtains a value of 16.6 G forA, obtained from the computer simulated in the MAL-6 ghost spectrum, compared to spectrum may result from the non-loren16.7 and 13.8 G for MAL-6 in 5P8 and tzian lineshape which results from a dodecane, respectively. This result indiNaOH treatment of spin labeled ghosts, cates that the weakly immobilized envinecessitating the use of less accurate nuronment is a highly polar one and is theremerical approximations in the above calfore not likely to be in the lipid matrix of culations. the membrane but rather exposed to the Radical decay studies were performed to polar aqueous medium. help define the location of the labeled SH The ratio of the spectral amplitudes of groups. In order to ascertain the effect of MAL-6 attached to weakly immobilized ascorbate on the membrane, right-side-out sulfhydryl groups (W) to that of MAL-6 resealed ghosts were incubated with attached to strongly immobilized SH [14Clascorbate for 1 h, washed in buffer, groups (S) is a measure of the relative and then both the supernatant and the population of each class of SH sites. This packed resealed ghosts were separately statement assumes a two-state model and counted. After three washes, no radioachas been shown to be valid in systems such tivity was observed in either the supernaas ours for W/S less than 20 (12). In our tant or the membrane. In order to demonexperiments, W/S has a value of 5.5 ? 0.6 strate that ascorbate is not freely permeafor permeable erythrocyte ghosts. When ble through resealed ghosts, membranes W/S is calculated from spectra recorded were resealed in the presence of [14Clfrom ghosts that were incubated at 4°C ascorbate. The resealed ghosts were with MAL-6 for 30 min instead of the usual washed once and dialyzed against PBS for 18-h incubation, a value of approximately 24 h. No radioactivity was detected in the 7.0 is obtained, suggesting that not all of dialysate while the resealed membranes

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contained a constant, significant amount of [‘4Clascorbate during the entire period of dialysis. These results suggest that ascorbate does not penetrate the membrane from either side nor does it bind to the membrane. MAL-6 at a concentration of approximately lo-;’ M in 5P8 is reduced by excess ascorbate as described in the Methods section with a half-time of approximately 2 min. Using labeled open ghosts, the reduction half-time of the W peak is approximately 7-8 min and at long times this weakly immobilized component essentially disappears from the spectrum. The S signal has a decay half-time nearly twice t.hat of the W peak and after approximately 50% reduction no additional decay occurs at longer times. No detectable esr signals are observed from samples of lipids extracted from MAL-6 labeled erythrocyte ghosts. Total membrane SH group content of normal and MyD erythrocyte ghosts was estimated by the DTNB method of Sedlak and Lindsay (23). Within the resolution of this procedure no difference in SH content between control and MyD ghosts could be demonstrated. Due to the overlap of the central lines of the superimposed spectra and the small amplitudes of the high-field lines, we concentrated our analyses on the resolved low-field lines. The linewidths of the strongly immobilized signals are equal in control and MyD spectra (3.2 G, for the half-width at half-maximal amplitude of the S signal), as are also the linewidths of the weakly immobilized signals in the two cases (2.2 G for the peak-to-peak width). This result indicates that weakly immobilized labeled membrane protein SH groups have essentially the same mobility in control and MyD erythrocytes. A similar statement holds for the strongly immobilized labeled SH groups. AN and the splitting of the outer hyperfine extrema of the powder portion of the spectrum were also found to be the same in MyD and control ghosts. The ratio of the spectral amplitude of MAL-6 attached to weakly immobilized sulfhydryl groups (W) to that of MAL-6

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attached to strongly immobilized groups (S) is proportional to the relative population of each kind of site (12). W/S is significantly greater in MyD erythrocyte membranes compared to those of controls (Table I). The significance of the difference between the two means is calculated by a two-way analysis of variance method (28) to minimize the effect of possible variations between separate experiments that often occur in biological samples. The null hypothesis employed was that the difference between control and MyD means is zero (the value to be expected if the membrane proteins were identical in every way) and the alternative hypothesis was that this difference of W/S values was not equal to zero. A pooled estimate of the error was employed when the F ratio for interaction-to-error was less than unity. As is seen in the tables of results, W/S is highly significantly larger in MyD erythrocyte membranes (P = 0.001). The individual S and W amplitudes corrected for spectrometer gain and membrane protein content (as indicated in the Methods section) were also compared (Table II) by the two-way analysis of variance. MyD erythrocyte membrane proteins take up less MAL-6 in S sites than do those of controls (P = 0.025). This incorporation of MAL-6 into W sites is probably not different than controls (Table II); the P value indicates a possible difference of borderline significance. As opposed to the case of 18-h equilibration with MAL-6, esr spectra of ghosts incubated for 30 min with this spin label demonstrate no significant difTABLE COMPARISON 6 ATTACHED

I

OF esr SPECTRAL AMPLITUDES OF MALTO WEAKLY IMMOBILIZED SH GROUPS

(W) TO THAT OF MAL-6 IMMOBILIZED SH GROUPS MYOTONIC DISTROPHIC

ATTACHED TO STRONGLY (S) IN NORMAL (N) AND (MyD) ERYTHROCYTE

MEMBRANES mJIS)\,,,,

_____

5.53 0.63 17

x

SD n

-

6.07 0.98 15 P = 0.001”

“P value variance.

calculated

by

a two-way

analysis

of

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BU’ITERFIELD TABLE

II

COMPARISONS OF NORMAL (N) AND MyD SPECTRAL AMPLITUDES, CORRECTED FOR TOTAL PROTEIN CONTENT, OF MAL-6 ATTACHED TO STRONGLY (S) AND WEAKLY (W) IMMOBILIZED SULFHYDRYL GROUPS OF MEMBRANE PROTEINS IN ERYTHROCYTEF

S/mg P x SD

u The data weight-normalized b Calculated

P

N

MYD

N

MYD

1.00 0.18

0.78 0.16 7

5.28 0.98 9 0.05
4.44 0.59 7

9 ;b

Wlmg

0.025

< 0.10

are given relative to the mean of the S-signal for normals. from a two-way analysis of variance.

ference in W/S in MyD and control membranes. DISCUSSION

The function of membrane proteins is readily influenced by agents which interact with their SH groups. Conversely, the state of SH groups of membrane proteins is a sensitive reflection of protein conformation and the general and specific influences of local lipid and protein milieu upon such conformation. The present experiments demonstrate that MAL-6 can interact with membrane protein SH groups without grossly perturbing membrane structure. Furthermore, the absence of MAL-6 esr signals in lipids extracted from labeled RBC ghosts would indicate that its binding is indeed with protein constituents. The particular proteins to which MAL-6 is attached cannot be determined with certainty. As an indirect approach to MAL-6 binding to membrane protein sulfhydryl groups, we have employed [3H]NEM. The maximal incorporation of NEM and MAL6 appears with approximately the same time course. However, it is not possible to assert that both NEM and MAL-6 bind to identical sites and it is not possible at this time to relate the MAL-6 binding directly to spectrin, to protein III, or to the higher molecular weight proteins present in smaller amounts, as we can do with NEM. The esr spectrum of MAL-6 labeled erythrocyte membranes demonstrates at least two classes of sulfhydryl binding sites characterized by their rotational mo-

ET AL

bility and accessibility to both the spin label and ascorbate. The persistence of strongly immobilized signals in esr spectra of open ghosts incubated with membrane impermeable ascorbate suggests that the strongly immobilized sites are found both at the membrane surface and also sequestered deep within the lipid milieu. Weakly immobilized SH groups, which constitute approximately one-third of the total number of spin label binding sites, appear to be, however, located only near the membrane surface in a highly polar environment. Spectrin, which is located on the cytoplasmic surface of the erythrocyte membrane and which incorporates nearly onethird of the bound NEM (and presumably, MAL-6), may contain many of the W sites. It is of interest that while Hill plots of NEM inhibition of several RBC membrane bound enzymes demonstrated linear slopes, indicating only one type of binding (29), at least two different classes of SH groups are detected with MAL-6. The spin labeling method provides a description of the environment and location of labeled sulfhydryl groups of membrane proteins not obtainable from binding studies alone. The esr amplitude ratio of W to S sites combined with quantitative analysis of NEM incorporation in an erythrocyte membrane is a sensitive measurement of sulfhydryl group binding and environmental characteristics. These studies have been applied to erythrocyte membranes obtained from patients with the genetically derived disorder, myotonic muscular dystrophy, and have demonstrated alterations in protein conformation and/or membrane organization in ghost membranes in this disease. The current results cannot be attributed to a decreased amount of protein present in erythrocyte membranes since the total protein content and SDSpolyacrylamide gel electrophoretic patterns are not altered in MyD ghosts (6). In addition, the total SH group content in MyD erythrocyte membranes appears not to be different from that of controls within the sensitivity of the DTNB method, which is several orders of magnitude lower than that of the spin labeling technique. The increased value of W/S in MyD

ESR

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erythrocyte ghosts observed in the present spin labeling study results from a decreased incorporation of MAL-6 into strongly immobilized membrane protein sulfhydryl group sites. Changes in the W/ S ratio have been previously used by others to monitor erythrocyte membrane organizational alterations caused by various procedures and agents (10-15). The increased value of W/S in the esr spectra of membrane proteins in MyD erythrocytes demonstrated here therefore suggests membrane organizational or protein conformational changes to be present in this disease. The strongly immobilized sulfhydryl group sites appear to be located near the membrane surface and embedded deep within the lipid bilayer. The equality of W/S for normal and MyD ghosts labeled for only 30 min suggests that the MyD S sites that take up less MAL-6 spin label in the overnight labeling experiments are to be found buried in the lipid environment. Our previous lipid spin label studies demonstrated myotonic erythrocyte membranes to be more fluid than those of controls near the membrane surface (2, 3). It is not clear whether this increased surface membrane fluidity is the cause or the effect of the protein spin label changes demonstrated here. For instance, it is possible that changes in charge on the surface of the membrane or an alteration in lipid packing may make otherwise reactive, buried SH groups inaccessible to MAL-6. Likewise, changes in membrane protein conformation which result in a decreased binding by MAL-6 to S sites deep within the bilayer may be manifested in lipid changes near the membrane surface. Interrelationships of membrane lipids and proteins are probably involved. Protein conformational changes or alteration of protein-lipid interactions may help explain the difference in protein phosphorylation, ouabain-sensitive sodium efflux, and the response to fixation, all of which have been observed in MyD erythrocytes (4-7). ACKNOWLEDGMENTS the

We wish to thank Duke Department

Professor Donald of Mathematics

Burdick of for helpful

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discussions on statistical analysis, Michael Herbstreith and Marcia Butterfield for technical assistance, and Tracy Thieret far help with the computersimulated spectra. REFERENCES 1. LIPICKY, R. J., AND BRYANT, S. H. (1973) in New Developments in Electromyography and Clinical Neurophysiology (Desmedt, J. E., ed.), Vol. 1, pp. 451-463, Karger, Basel. 2. BUTTERFIELD, D. A., CHESNUT, D. B., ROSES, A. D., AND APPEL, S. H. (1974) Proc. Nat. Acad. Ski. USA 71, 909-913. 3. BUTTERFIELD, D. A., ROSES, A. D., COPPER, M. L., APPEL, S. H., AND CHESNUT, D. B. (1974) Biochemistry 13, 5078-5082. 4. HULL, K., ROSES, A. D., AND APPEL, S. H. (1975) J. Physiol., in press. 5. MILLER, S. E., ROSES, A. D., AND APPEL, S. H. (1975) Arch Neural., in press. 6. ROSES, A. D., AND APPEL, S. H. (1973)Proc. Nut. Acad. Sci. USA 70, 1855-1859. 7. ROSES, A. D., AND APPEL, S. H. 119751 J. Memb. Bid. 20,51-58. 8. BUTTERFIELD, D. A., ROSES, A. D., APPEL, S. H., AND CHESTNUT, D. B. (1976) Nature, in press. 9. JOCELYN, P. (1972) Biochemistry of the SH Group, Academic Press, New York. 10. CHAPMAN, D., BARRATT, M. D., AND KAMAT, V. B. (1969)Biochim. Biophys. Acta 173,154-157. 11. HOLMES, D. E., AND PIETTE, L. H. (1970) J. Pharm. Exp. Ther. 173, 78-84. 12. SCHNEIDER, H., AND SMITH, I. C. P. (1970) Biochim. Biophys. Acta 219, 73-80. 13. KIRKPATRICK, F. H., AND SANDBERG, H. E. (1973) Biochim. Biophys. Acta 298, 209-218. 14. KIRKPATRICK, F. H., AND SANDBERG, H. E. (1973) Arch. Biochem. Biophys. 156, 653-657. 15. SANDBERG, H. E., BRYANT, R. G., AND PIETTE, L. H. (1969) Arch. Biochem. Bioph.ys. 133, 144152. 16. CARTER, J. R., JR. (1973) Biochemistv 12, 171~ 176. 17. STECK, T. L., AND Yu, J. (1973) J. Supramol. Struct. 1, 220-232. 18. LENARD, J. (1970) Biochemistry 9, 5037-5040. 19. CARRAWAY, K. L., AND SHIN, B. C. (1972) J. Biol. Chern. 247, 2102-2108. 20. FAIRBANKS, G., STECK, T. L., AND WALLACH, D. F. H. (19711 Biochemistry 10, 2606-2617. 21. STECK, T. L., AND KANT, J. A. (1974) in Methods in Enzymology, Vol. 31, pp. 172-180, !.cademic Press, New York. 22. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 23. SEDLAK, J., AND LINDSAY, R. H. 11968) Anal. Biochem. 25, 192-205.

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CONNELL, H. M. (1965) J. Chem. Phys. 43, 2909-2910. 28. BROWNLEE, K. A. (1960) Statistical Theory and Methodology in Science and Engineering, Wiley, New York. 29. GODIN, D. V., AND SCHRIER, S. L. (1972)J. Membrane Bid. 7, 285-312.