Detection of erythrocyte membrane protein alterations in hereditary spherocytosis through the use of thermal stress: A spin label study

Life Sciences, Vol. 29, pp. 2071-2079 Printed in the U.S.A.

Pergamon Press

DETECTION OF ERYTHROCYTE MEMBRANE PROTEIN ALTERATIONS IN HEREDITARY SPHEROCYTOSIS THROUGH THE USE OF THERMAL STRESS: A SPIN LABEL STUDY L. W.-M. Fung,

M. S. Ostrowskl, and S. Sarnalk T

W. A. Meena

*Department of Chemistry, Wayne State Unlversity, Detroit, MI 48202 +Department of Pediatrics, Wayne State Unlverslty School of Medlclne (Received in final form September 21, 1981) Summary The membrane protelns of normal and heredltary spherocytosis have been labelled wlth a malelmlde-analog nltroxlde spln label and studied by electron paramagnetlc resonance technlques. The spectral amplitude ratios from weakly and strongly ~mmobIllzed labels differed sllghtly at 20 ° and 40 ° . Increasing the temperature to 47 ° and incubatlng for long tlme periods markedly accentuated the difference. It is suggested that the apparent differences in heat sensltlvlty between normal and heredltary spherocytosls erythrocyte membrane protelns reflect a latent structural alteratlon(s) of hereditary spherocytosls erythrocyte membrane proteins. Such structural alterations may result ~n altered functlonal behavlor when the membrane is subjected to stress. Heredltary spherocytosls (HS) red cells exhlblt a varlety of abnormal propertles ranglng from morphologlcal and hemolytlc aberratlons to enzyme deflclencles and abnormal 1on-permeab111ty, but none seems to be a fundamental defect (i). It is generally belleved that the abnormalltles are assoclated wlth the erythrocyte membrane (2);and a search for a defect In membrane protelns, such as spectrln, seems to be promising (3,4). One of the major functions of spectrln-actln molecules in the membrane Is belleved to be the maintenance of the blconcave shape of the erythrocyte (5,6). However, electrophoresls patterns of the membrane protelns in polyacrylamlde gels do not show slgniflcant dlfferences between the normal and HS membranes, indlcating no major difference in molecular welght (slze) of the membrane protelns. In the search for other molecular abnormalltles in HS cells, dlfferent results have been found In the red cells from dlfferent patlents. For example, among the blood samples of 12 patlents, two samples showed severe restrlction in the release of spectrin molecules (4). Fatty acld composltlons In some HS erythrocytes were found to be normal (7) wNereas earller reports suggested that the phosphollplds in HS cells lack long chaln fatty acld conjugates (8). The llpid fluldlty has also been reported to be decreased in HS erythrocyte membranes, as compared to normal controls in a spln label study (9). However, another group, uslng a fluorescent probe, has concluded that the fluldlty of HS membranes is normal (i0). It ~s posslble that HS may be a heterogeneous dlsease wlth heterogeneous structural and/or functlonal alteratlons In the erythrocyte membranes. In add~tlon, these flndlngs suggest that, in some cases of HS, there exlst subtle, but observable, changes in the erythrocyte membranes. The membrane structure and membrane component organlzatlon, as described 0024-3205/81/202071-09502.00/0 Copyrlght (c) 1981 Pergamon Press Ltd.

2072

E P R Studies

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Proteins

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1981

by the fluid m o s a i c model, are dynamic in nature. Thus, the detection of dynamic propertles of m e m b r a n e components is a rather sensitive assay for determining the physical state of membranes, allowing us to m o n i t o r m o l e c u l a r similarlties and differences b e t w e e n normal and HS membranes. We have used spin label electron paramagnetic resonance (EPR) to study the motional behavior of spin labels attached to proteins ~n m e m b r a n e s of both normal and HS cells Materials

and Methods

The m e m b r a n e s of three HS patients were used. Two patients (M T. and D.T.) were full siblings; the thlrd (M.C.) was unrelated. In early chlldhood all three patients showed hemolytic anemia characterized by h e m o g l o b i n values ranging from 7 to 12 grams percent, r e t l c u l o c y t e counts of 8 to i@%, n u m e r a b l e spherocytes on perlpheral smear and mild elevations of Indlrect-acting billrubln. Osmotic fragility curves were left-shifted, indlcatlng increased fragillty, this was worsened upon incubation. They each had palpable splenomegaly. M.T. and D.T. were splenectomlzed at a p p r o x i m a t e l y slx years of age w h i c h resulted in the expected resolutlon of hemolysis with p o s t - s p l e n e c t o m y h e m o g l o b i n levels and r e t i c u l o c y t e counts returning to normal. The number of spherocytes on the peripheral smear increased slightly in each case after splenectomy. M C. was not splenectomized at the time of the study HS and control (gender and age matched) blood samples w e r e drawn w i t h i n ten minutes of each other. Fresh blood samples from Red Cross Blood Bank were also used as controls. All controls gave similar results. In order to characterize the HS cells used in our studies, we performed osmotic fragility and "incubated" osmotic fragility tests on our HS cells as well as the s p e c t r l n - r e l e a s e test on the HS m e m b r a n e ghosts. Osmotlc fragillty test" The methods of Godal (ll) were followed NaCI solutions at concentratlons in the range of 0.i to 0.9% were prepared by dissolving NaCI in distilled water. 5~ of packed cells or lOu~ of whole blood were added to 2m~ of NaCI solutlon already equilibrated at room temperature. The suspensions w e r e incubated for 60 mln. at room temperature prior to eentrlfugatlon. The optlcal a b s o r p t i o n at 577 nm was used to m o n i t o r the concentratlon of h e m o g l o b i n in the supernatant. Spectrin release: The m e t h o d s of R a l s t o n (4) were used. Fresh m e m b r a n e s were prepared wlth 5mM phosphate buffer and dlalyzed against 0 imM EDTA, pH 7.5, for 72 hours followed by u l t r a c e n t r i f u g a t l o n at 35,000g for I hour. Proteln concentratlons of the supernatant and pellet were determlned by the Lowry m e t h o d (13). M e m b r a n e ~reparatlon: 10m% of fresh whole blood samples were centrifuged and the blood serum and buffy coat w e r e removed to give about 5m~ of packed cells. The cells were w a s h e d wlth 3 volumes of Isotonlc buffer of 0.15M NaCI, 10mM Trls and 0.2mM EDTA at pH 7.4 (20o). After three washes, the cells w e r e lysed w i t h 20 v o l u m e s of 10mM Trls buffer at pH 7.4. The h e m o l y s a t e solutlon was centrlfuged for i0 mln. at 21,000g and the supernatant was removed. The membranes were w a s h e d three m o r e tlmes to glve w h l t e m e m b r a n e ghosts, and then spln labelled w l t h N - ( l - o x y l - 2 , 2 , 6 , 6 - t e t r a m e t h y l - 4 - p l p e r l d l n y l ) malelmlde (Mal-6). The spln label (from Syva, CA) was stored In acetonitrlle, w h l e h was evaporated b e f o r e the additlon of m e m b r a n e sample, 30-50 ~g M a l - 6 / m g proteln. After 1-hour incubatlon in the dark, the m e m b r a n e samples were washed w i t h 40 volumes of Trls buffer to remove excess spin labels. After the thlrd wash, EPR spectra were taken, and w a s h l n g was termlnated w h e n the EPR spectrum of a sample was the same as the one taken after the prevlous wash. Some of the

Vol.

29, No.

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1981

EPR Studles

of HS Membrane

Proteins

2073

spln labelled m e m b r a n e s were stored in small vials, whlch were submerged into liquld n l t r o g e n and then stored at -80 ° . For EPR experlments on these samples, the m e m b r a n e s were thawed and used immedlately. All procedures were carrled out at 4 ° in a cold room or on ice except as otherwlse stated. Normal control and HS samples w e r e prepared in parallel. EPR samples: 50UZ glass cap111arles were washed in 0.01% sodlum azlde solutlon, dried and used as EPR samples tubes. (Irreproducible data were occaslonally obtalned when sample tubes were not pre-washed In azlde solution). A V a r l a n EIO9E EPR spectrometer equlpped w l t h a varlable temperature controller was used for all EPR measurements. The temperature of the sample was m o n l t o r e d dlrectly by placlng a thermocouple inside the sample tube, just above the sample surface. A N1colet 535 tlme averager was interfaced wlth the EPR spectrometer to ~mprove s~gnal to nolse ratios. The EPR m e a s u r e m e n t s were always started at 20 ° , wlth samples belng stored on ice untll being placed in the EPR cavlty. After the 20 ° measurement, the temperature was raised to 40 ° , and then to 47 ° . After the measurement at 47 ° , the samples In the EPR tubes were transferred to a constant temperature bath held at 47 ± 0.i ° for 20 hours and EPR m e a s u r e m e n t s were taken e~ther at two-hour intervals or at the end of the lncubatlon perlod, all at 47 ° . Procedures for EPR m e a s u r e m e n t s were slmllar to those descrlbed previously (14). Results

and D 1 s c u s s l o n

Figure 1 shows a typlcal plot of the percent hemolysls of both normal and HS cells as a functlon of NaCI concentratlon in the suspension media, 1 hour and 5 days after blood belng drawn. The osmotic fragility of HS cells is left-shlfted in both fresh and incubated cells. Both sets of data are In good agreement w l t h the diagnostlc abnormallties of HS cells (12).

I00

i 80 Zt 20 &

& FIG.

~4

&

i

Osmotlc frag111ty curves of both normal and HS cells. N and HS denote the curves for fresh cells, N' and HS' denote the curves for Incubated cells. O denotes fresh (one hour after belng drawn) normal cells, A fresh HS cells, • incubated (5-day-old) normal cells, and ; incubated HS cells.

In spectrln release supernatant of m e m b r a n e s m e m b r a n e samples agalnst used in thls study seems

experiments, slmllar amounts of protelns in the of HS and normal cells are found after dialysls of the low lonlc strength buffer at 4 ° . The HS cells to be slmllar to most of the HS cells that R a l s t o n

2074

EPR Studies

of HS M e m b r a n e

used in hls study (i0 samples release of spectr~n m o l e c u l e s

Proteins

out of 12), namely, (4).

Vol.

no severe

29, No.

restrlctlon

20, 1981

in the

In the spln label study, we have used an N-ethyl m a l e l m l d e (hEM) analog, hal-6, to alkylate some of the sulfhydryl (SH) groups. Even though it has been reported that the blndlng of some SH agents, llke hEM, to erythrocyte m e m b r a n e s produces spherlng of normal erythrocytes (16), the spln label does not seem to perturb the m e m b r a n e s (15). The total amount of SH groups in membranes has been reported to be 131 nmoles per mg protelns (17). The hal-6 molecule, whlch is bulkler than the hEM molecule, blnds only about 25 nmoles of SH per mg protelns under our labelllng condltlon (15) About 80% of the hal-6 blnds to the protelns on the cytoplasmlc surface of the m e m b r a n e (15). The EPR slgnal intensltles (obtalned from double integratlon of the a b s o r p t l o n spectra) of labelled m e m b r a n e s of HS cells show that slmllar numbers of SH groups (25 nmoles of SH per mg protelns) are alkylated by hal-6 In the HS membranes. The results lndlcate that the m e m b r a n e protelns that are m a l n l y at the cytoplasmlc surface and are accesslble to hal-6, ~ncludlng spectrln-actln, have slmllar chemlcal envlronments and have no gross structural dlfferences in the normal and the HS cells Thls flndlng Is In good agreement w l t h the results obtalned In our spectrln release experlments and also wlth a recent EPR study (9), where no dlfferences are found in both cases T~e EPR spectra show two types of spln label slgnals, one component ~s a w e a k l y immobilized (W) component and the other a strongly immoblllzed (S) component, as shown in the Inset of Figure 2. The m a 3 o r l t y (about 90%) is the S component, whlch is broad and characterlstlc of very slow motlons (~ > 10-7 sec). The remalnlng mlnor portlon (W component) exhlblts c o m p a r a t l v e T y narrow llne wldths. The ratlo of the amplltudes of these two components, W/S, have been used to study the red cell m e m b r a n e s of M u s c u l a r D y s t r o p h y patlents (19). Since the W component glves rlse to a narrow llne and the S component to a broad llne, the W/S ratlo is very sensltlve to any transltlons between W and S components, desplte the fact that the W component Is only a m~nor port~on

N

I

18

14

< |

i

i/' i

18 T~mo (hr)

FIG.

2

The W/S values of both normal and HS frozen m e m b r a n e s at 47 ° as a function tlme of a typlcal experlmental run. * denotes normal and o HS membranes. Inset A portlon of the EPR spectrum of membranes in lOmM Trls at pH 7.4, 25 °

of

Vol.

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20, 1981

E P R Studies

of HS Membrane

Proteins

2075

of the total Integrated signal intensity. Therefore, we are able to use the W/S ratlo to follow some small changes in label mob111tles on the protelns. The immobillzatlon of a spln label by a proteln or the loosenlng of a label blndlng slte must Involve at least m~nor structural alterations of the labelled protelns. Prevlously we have found that the W/S ratio is very senslt~ve to experlmental conditions, such as the temperature of the EPR m e a s u r e m e n t s and the ionlc strength and pH of the samples, that affect the dynamlc and structural properties of the proteins in m e m b r a n e s (18). Table I gives the m e a n W/S ratios for fresh and frozen m e m b r a n e samples of both normal and HS cells at 20 ° , 40 ° , 47 ° , and 47 ° after Incubating for about 20 hours. The m e a n values of comblned W/S ratlos of fresh and frozen samples are also presented. The mean values appear to dlffer slightly between HS and normal membranes, with those of normal m e m b r a n e s generally exhlbiting the hlgher values. The Student's t-test for differences in the mean W/S ratlos between normal and HS m e m b r a n e s ylelds slgniflcance at the P 0.05 level for several entries in Table I, namely at 20 ° frozen, at 47 ° frozen and comblned, and at 47o/20 hours fresh, frozen and combined. A typical paired run of normal and HS samples is shown in more detail in Figure 2. Upon incubatlon at 47 °, the W/S ratios of both samples increase as a function of tlme and then eventually level off asymptotically toward limltlng values. Thls set of data shows that the W/S ratlos for normal membranes are slgnlflcantly hlgher than those for HS m e m b r a n e s at the end of the incubation perlod. The total EPR slgnal intensity remains constant before and after the 20-hour incubatlon. Figure 3a shows the W/S values at 47 ° as a functlon of tlme for all of the samples examlned here. Data points appear scattered In this flgure. Due to the sensltiVities of the W/S ratios to experlmental cond~tlons, it is not surprlslng to see a sllght overlap of the ranges of values exhiblted by the

24 / N

s

~m

p

m~ lO-

7

o

% o

W 1 2 "~ / $

,, 8

r

,

i

Ii

o

o°o o

!

•

O"

2

r n c

0

I •

I

IO Time

20

(hr) FIG.3a

a.

b.

I 30

•

e

~ 8

l-

le

1

I 30

Time (hr) FIG.3b

The W/S ratios of normal (*) and HS (o) membranes at 47 ° as a function of tlme for eight experimental runs. Each run consists of a normal and HS samples. Both fresh and frozen samples are included. The palred dlfferences of the W/S ratlos, W/S(N) - W/S(HS), as shown in Figure 3a, at 47 ° as a functlon of time.

2076

EPR Studies

of HS M e m b r a n e

Proteins

Vol.

29, No.

20, 1981

two membranes, but at any one t~me the average value for W/S of normal ms s u b s t a n t ~ a l l y h~gher than that for HS. The W/S ratlo standard d e v l a t ~ o n ms about 10-15% of the W/S v a l u e for all of the data in thls study. A p p r o x i m a t e l y half of th~s v a r i a t l o n appears to be due to m ~ n o r d i f f e r e n c e s ~n sample h a n d l ~ n g during d~fferent preparations. For several equivalent membrane samples prepared separately, but in parallel, the standard d e v i a t i o n ~s g e n e r a l l y about 5 to 8% of the W/S value. Th~s suggests that an e x a m i n a t i o n of the palred d ~ f f e r e n c e s b e t w e e n the W/S ratios for normal and HS erythrocyte m e m b r a n e s m a y e l ~ m ~ n a t e some of the v a r l a n c e due to sllght e x p e r i m e n t a l d~fferences. The m e a n pa~red d~fferences, W/S(N) - W/S(HS), at 20 ° , 40 ° , 47 ° and at 47 ° after 20-25 hours ~ n c u b a t ~ o n are g~ven in T a b l e II. From these data, ~t can be seen that the d l f f e r e n c e ~ncreases w l t h ~ncreaslng temperature, and ~s s ~ g n ~ f ~ c a n t l y enhanced by ~ n c u b a t ~ o n at hlgh t e m p e r a t u r e Student's t-test of the m e a n pa~red d l f f e r e n c e against the null h y p o t h e s i s y ~ e l d s s ~ g n l f ~ c a n c e at the P < 0.005 level for several of the entrles and appear, ~n general, to increase by about a factor of ten over those for the grouped sample d ~ f f e r e n c e s (Table I). The W/S ratlo palred d l f f e r e n c e s b e t w e e n normal and HS e r y t h r o c y t e m e m branes u p o n i n c u b a t l o n are shown in Figure 3b. The data of thls flgure suggest that the d l f f e r e n c e b e t w e e n normal and HS m e m b r a n e W/S ratlos increases llnearly w l t h tlme. A llnear least square r e g r e s s l o n ylelds the stralght llne shown in F i g u r e 3b. An F test for the s l g n l f l c a n c e of the r e g r e s s l o n ylelds P < 0.001. For thls tlme serles the palred d l f f e r e n c e w i l l remove W/S changes w l t h tlme w h l c h are common to both normal and HS membranes. Thus the increasIng d l f f e r e n c e b e t w e e n normal and HS m e m b r a n e s observed here suggests that thermal stress m a y be acting to u n m a s k latent s t r u c t u r a l or dynamlc alteratlons in HS e r y t h r o c y t e m e m b r a n e protelns, as compared to those of normal erythrocytes. The freezlng process m a y have also Introduced some stress ~n m e m b r a n e s to g ~ v e the small but o b s e r v a b l e d l f f e r e n c e s in W/S ratlos in frozen normal and HS samples (Tables I and II). However, the d i f f e r e n c e s are m u c h smaller than those caused by thermal stress. The n a t u r e of the thermal stress is not clear. It may be r e l a t e d to d e g r a d a t i o n of m e m b r a n e s , o r p r o t e ~ n releases, by e n d o g e n e o u s proteases, for example. It ~s clear, though, that the thermal stress produces d l f f e r e n t results ~n normal and HS m e m b r a n e s as ~ndlcated by the m o b ~ l ~ t y of the labelled protelns. The gradual increase in W/S ratlo as we increase t e m p e r a t u r e indlcates an increase in thermal energy to glve faster m o l e c u l a r m o t i o n and a g r a d u a l m e m brane s t r u c t u r a l t r a n s f o r m a t l o n to allow m o r e spln labels to be in the w e a k l y I m m o b 1 1 1 z e d m o t l o n a l range than at lower temperature. The s t r u c t u r a l transformatlon is perhaps related to the loss of hellcal content In m e m b r a n e protelns upon h e a t l n g (20) and/or d l s s o c l a t l o n of m e m b r a n e components from the m e m b r a n e m a t r l x upon heatlng. The EPR data suggest that protelns in HS m e m branes may be "locked" in c e r t a l n s t r u c t u r e s and exhlblt less u n f o l d l n g and/ or less m e m b r a n e component d l s s o c l a t l o n as a f u n c t l o n of tlme at 47 ° . Consequently, HS m e m b r a n e s b e c o m e m o r e rlgld than normal m e m b r a n e s under slmilar thermal stress (incubated at 47 ° for 20 hours). These d l f f e r e n c e s are dlfflcult to observe, as shown by the s p e c t r l n r e l e a s e experlment and slmllar W/S ratlos at 20 ° , but are a m p l i f l e d after the m e m b r a n e samples are stressed by heat and b e c o m e clearly d e t e c t a b l e by the EPR method. Such s t r u c t u r a l alteratlons are subtle, however, they may result in a l t e r e d f u n c t l o n a l b e h a v l o r w h e n the m e m b r a n e is subjected to stress. It has been shown that another klnd of p e r t u r b a t l o n , namely a g g r e g a t l o n of s p e c t r l n - a c t l n due to increased lonlc strength or divalent catlon concentratlon, also brlngs out the d l f f e r e n c e s b e t w e e n HS and n o r m a l m e m b r a n e s (21). It has been reported that the heat treatment r e s p o n s e of HS cells was llke that of normal cells (22). The lack of o b s e r v e d d l f f e r e n c e s in thls case m a y be r e l a t e d to the sensltlvlty of the d e t e c t l o n m e t h o d or may be due to h e t e r o g e n e l t y in HS cells Recently, it has

Vol.

29, No.

20,

1981

EPR Studles

been reported that spectrln shows heredltary elllptocytosls (23).

small differences

TABLE Comparison

of HS Membrane

of M e a n W/S Ratlos

in thermal

2077

stablllty

in

I of Normal

and HS Membranes

N

I

Proteins

HS

Temp.

W/S

± S.E.M.

20 °

3.06 ± 0.i0

n

W/S ± S.E.M.

n

P

3.13 # ± 0.i0

6

-

17

(2.70) 40 ° 47-0 §

7.14 ± 0.29

i0

7.38 i 0.31

5

-

12.80 ± 0.30

2

12.20 ± 0.40

2

-

2

0.028

0.029

(12.60) 47-20 §§

18.50 ± 0.42

2

14.75 ± 0.78 (14.20)

II

III

20 °

3.06 ± 0.i0

22

2.70 ± 0.09

i0

40 °

6.66 ± 0.24

15

6.28 ± 0.34

6

-

47-0

13.26 ± 0.48

14

11.52 ± 0.68

9

0.045

47-20

19.05 ± 1.07

4

14.60

± 1.14

4

0.030

20 °

3.06 i 0.07

39

2.86 ± 0.09

16

40 °

6.85 ± 0.19

25

6.78 ± 0.28

ii

47-0

13.20 ± 0.42

16

11.65 ± 0.56

ii

0.034

47-20

18.87 ± 0.69

6

6

0.002

14.65

+ 0.74

P is the slgnlflcance of dlfferences between the m e a n W/S ratios and HS. Values greater than 0.05 are not glven.

I:

t

Fresh Membranes; II: frozen)Membranes.

of HS patlents

M.T.

#%of HS patlent M.C., 547-0

and D.T.

(slbllngs),

not splenectomlzed.

is 47 ° at 0 hour.

§§47-20

Frozen Membranes;

is 47 ° at 20 hours.

III:

Comblned

-

of normal

(fresh

both splenectomlzed.

and

2078

EPR Studles

of HS M e m b r a n e Protelns

TABLE Comparison

I

II

III

See T a b l e

A(W/S)

29, No.

i S.E.M.

W/S(N)-W/S(HS)

n

2"

20 °

-0 07 ± 0.13

6

-

40 °

0.33 ± 0 28

3

-

47-0

0 60 + 0.i0

2

-

47-20

3.75 ± 0.25

2

0.046

20 °

0.25 + 0.06

i0

0.002

40 °

0.65 ± 0.20

6

0.021

47-0

1.90 ± 0.38

9

0.001

47-20

4.28 ± 0.91

3

0.042

20 °

0.13 ± 0.07

16

-

40 °

0.54 + 0.16

9

0.010

47-0

1.66 ± 0.35

ii

0.0007

47-20

4.13 ± 0.53

5

0.002

I footnotes

for symbol

20, 1981

II

of M e a n Paired D i f f e r e n c e s ,

Temp.

Vol.

illustrations.

In summary, we have studied the e r y t h r o c y t e m e m b r a n e proteins of HS cells e x t e n s l v e l y by a rather s e n s i t i v e EPR m e t h o d and have c o n c e n t r a t e d the studies on two patients from the same family to e l i m i n a t e some of the p o s s i b l e intrlnsic h e t e r o g e n e l t i e s that may exist in sample cells. Even though the HS cells clearly have a d i f f e r e n t m o r p h o l o g y and different p r o p e r t i e s of hemolysis, the EPR data show that the m e m b r a n e proteins, e s p e c i a l l y those on the cytop l a s m i c surface, of HS and normal are similar at 20o-40 ° range. However, after i n c u b a t i o n at 47 ° for 20 hours, the W/S ratlo is c o n s i d e r a b l y larger in n o r m a l than in HS m e m b r a n e s , s u g g e s t i n g a latent s t r u c t u r a l alteratlon(s) of HS e r y t h r o c y t e m e m b r a n e proteins. Acknowledgements We thank Mr David Cragg for p e r f o r m l n g the osmotic fragility tests Thls r e s e a r c h was s u p p o r t e d by C o t t r e l l R e s e a r c h Grant from R e s e a r c h Corp o r a t l o n and r e s e a r c h grants from M l c h l g a n Grant A s s o c l a t l o n and the N a t i o n a l Institutes of H e a l t h (HL22432)

Vol. 29, No. 20, 1981

EPR Studies of HS Membrane Proteins

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References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23

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