300

Biochimica et Biophysica Acta, 432 (1976) 300--311

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98594 INVESTIGATION OF HUMAN LYMPHOCYTE PLASMA MEMBRANE ASSOCIATED NUCLEIC ACID

PETER W. MELERA and A.P. CRONIN-SHERIDAN Sloan-Kettering Institute for Cancer Research, Walker Laboratory, 145 Boston Post Road, Rye, N.Y. 10580 (U.S.A.)

(Received December 15th, 1975)

Summary Plasma membranes were prepared from the h u m a n l y m p h o c y t e cell line WIL23A by h y p o t o n i c swelling, Dounce homogenization, differential and equilibrium centrifugation. The resulting vesiculated membrane fragments were f o u n d to have densities of 1.10 and 1.17 g/ml, and were defined by lactoperoxidase mediated whole cell iodination, L-[3H] fucose incorporation, 5'-nucleotidase activity (EC 3.1.3.5) and electron micrographic visualization. Recovery of plasma membrane from whole cell homogenates was estimated to be approximately 30--35% as judged by the recovery of '2SI-labeled cell surface protein. When plasma membranes were prepared from cells which had been incubated for 18 h in the presence of 0.5 pCi/ml [3H] thymidine such that greater than 109 acid insoluble counts could be demonstrated in the whole cell homogenates, no [3H] thymidine label and presumably, therefore, no DNA, could be shown to be coincident with either the 1.10 or 1.17 density. Similar experiments with [3H] uridine suggested t h a t 90% of the plasma membranes did n o t contain RNA, while 10% remained questionable.

Introduction Since several reports on the association of DNA with the plasma membrane of h u m a n l y m p h o c y t e s had been published [1--4] and had suggested the possible involvement of deoxyribose nucleic acid in regulatory mechanisms mediated by the cell surface (i.e., mitogen or antigen response, cell-cell recognition, etc.), we began a study designed to measure the effects of physiological stress and environmental change on this nucleic acid fraction. In a similar sense, we also sought to characterize any RNA which might be found associated with the plasma membrane.

301 Materials and Methods

Cell maintenance WIL23A cells were received from Dr. R.A. Lerner of the Dept. of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, Calif. These cells were derived from a clone of WI-L2, a line of diploid cells established from the spleen of a patient having hereditary spherocytic anemia [5]. The cells were maintained in logarithmic growth (18--22 h doubling time) as suspension cultures in screw-capped Erlenmeyer flasks, at concentrations of l 0 s to 106 per ml of Minimal Essential Media (Eagle) with Earle's salts, supplemented with 10% nonessential amino acids solution (100 X), 10% sodium pyruvate solution (100 X) and 10% fetal calf serum (Grand Island Biological Company). In addition, the cultures contained 100 units per ml of penicillin and 100 gg per ml pf streptomycin. Trypan blue exclusion assay indicated that the cultures were routinely greater than 90% viable.

Preparation of plasma membranes Logarithmically growing cells were collected by centrifugation and washed twice with phosphate buffered saline (Dulbecco's phosphate buffered saline) at room temperature. All subsequent steps were carried out at 4°C. Approximately 2 • 109 cells were suspended in 30 ml of 0.01 M Tris/HC1 (pH 7.4) + 0.005 M MgC12 and allowed to swell for 5 min. The cell suspension was then adjusted to 0.015 M NaC1 b y addition of 3 ml of cold 0.15 M NaC1, and the cells disrupted by 21 strokes of the loose-fitting B pestle of a 50 ml glass Dounce homogenizer (Vitro, Inc.). Microscopic examination of the cells at various points indicated 21 strokes to be optimal and to produce the most n u m b e r of broken cells while minimizing obvious nuclear damage. It should be noted here that our concern over nuclear damage and subsequent contamination of our preparations with nuclear DNA p r o m p t e d us to sacrifice quantitative cell lysis in favor o f nuclear integrity. Under the conditions described here, recovery of nuclei and, therefore, elimination of nuclear DNA as a contaminant was excellent (greater than 96%); cell lysis was approximately 65%. After disruption, the cell suspension was centrifuged at 800 X g for 5 min, and the resulting supernatant centrifuged at 8000 X g for 10 min. This postmitochondrial supernatant was layered over a 5 ml 60% sucrose cushion in a SW 25.1 tube and centrifued for 1 h at 25 000 rev./min. The resulting clearly visible " m e m b r a n e cushion" (i.e., microsomal cushion) was collected and its sucrose concentration adjusted to 25% before layering over a 25--50% linear sucrose gradient in a SW 40 tube. Gradients were run to equilibrium in 18 h at 39 000 rev./min. All sucrose solutions were made in 0.01 M Tris/HC1 (pH 7.4) + 0.005 M MgC12 + 0.015 M NaC1. Ultrapure nuclease-free sucrose (SchwartzMann) was used in all experiments. Density gradients were collected from the top and optically scanned at 254 nm or 280 nm. Approximately 20 fractions of 0.65 ml each were collected and kept on ice. Linearity of density gradients was verified b y refractive index determinations of every other fraction. Protein was determined by the m e t h o d of L o w r y et al. [6]. If samples were to be assayed for radioactivity, aliquots

302 were either counted directly in 10 ml Scintisol (Isolab, Inc., Akron, Ohio) or the entire fraction precipitated by addition of an equal volume of 10% trichloroacetic acid. After at least 30 min in ice, precipitates were collected on glass fiber filters (Glass Fiber, Type B, Millipore, Inc.), washed twice with ethanol, dried and counted in toluenefluor. If samples were to be assayed for enzyme markers or examined in the electron microscope, fractions of similar density from several gradients were pooled, diluted and centrifuged for 18 h in the SW 50.1 rotor at 45 000 rev./min. The resulting pellets were then dispersed in the proper enzyme assay buffers or fixed for electron microscope evaluation.

Lactoperoxidase 12sI labeling The m e t h o d used here was essentially that described by Marchalonis et al. [7]. 2 • 109 cells were washed three times with 50 ml sodium phosphate-buffered saline. 2 • 107 Cells were removed and washed twice more in an a t t e m p t to remove as much media protein as possible. The iodination reaction mixture contained l 0 T cells in 0.25 ml of sodium phosphate-buffered saline, 20 pl Na12SI (1 mCi) carrier-free, New England Nuclear), 10 pl H202 (0.03% solution), and 25 pl lactoperoxidase {0.05 mg/ml solution, Calbiochem). Incubation was continued for 5 min at 30°C and was stopped by addition of 10 ml cold sodium phosphate-buffered saline. Microscopic examination of iodinated cells revealed little cell lysis and maintenance of cell viability was confirmed by trypan blue exclusion. After two washes in cold sodium phosphate-buffered saline, both batches of iodinated cells were mixed with the remaining unlabeled cells and membranes prepared as usual. Fucose labeling was performed as follows: 109 cells in 10 ml of media were incubated for 2 h at 37°C with 2 pCi/ml L-[3H] fucose (3.73 Ci/mmol, New England Nuclear). Cells were harvested by centrifugation, washed twice with 50 ml phosphate buffered saline and membranes prepared as usual. Results

Conditions for cell disruption and membrane preparation The protocol for membrane preparation used here represents a compromise between m a n y recognized factors which affect membrane isolation. Recent articles [8,9,10] have pointed o u t the considerable problems involved with plasma membrane isolation and t h a t in the final analysis, the methods of cell disruption, buffer composition, membrane markers used, etc., are best determined separately for each cell or tissue type studied. Our use of lymphocytes, cells with a notably large nuclear-to-cytoplasmic ratio, and whose plasma membrane lies in relatively close proximity to the nucleus and nuclear membrane, suggested the use of hypotonic disruption buffers. Inclusion of 5 mM MgCl2 in the buffer to stabilize nuclei during cell swelling and disruption was suggested by Dr. R.H.F. Peterson and proved to be a crucial part of our procedure. The data of Fig. 1 show the effect of magnesium on sucrose equilibrium density profiles as indicated by A2s4 scanning and [3H] thymidine incorporation. Table I summarizes the magnesium effect on nuclear recovery and on the distribution of incorporated [all] thymidine during membrane preparation. The

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results show that when Mg 2+ was added to the preparations, nuclear recovery substantially increased, and the a m o u n t of [3H] thymidine associated with the membrane cushion substantially decreased (Table I). When prepared in the presence of Mg2+, the membrane cushion contained no detectable [3H]thymidine at sucrose densities less than 1.20 (Fig. 1), whereas membrane cushions prepared in the absence of Mg 2÷ did. Experiments not presented here showed that free DNA pelleted under our gradient conditions.

Location o f plasma membrane fractions in sucrose density gradients The data of Fig. 2 were obtained from a single gradient in which 20 pl aliquots of each fraction were counted directly in Scintisol, and the remainder of each fraction trichloroacetic acid precipitated and washed with lipid solvents, as described in the Figure Legend. Although recovery of 12sI radioactivity initially p u t on the gradient was greater than 90%, as calculated from the 20 #1 samples, the a m o u n t of trichloroacetic acid precipitable, lipid solvent stable

304 TABLE

I

EFFECT

O F M g 2+ O N N U C L E A R

RECOVERY

A N D [3Ft] T H Y M I D I N E

DISTRIBUTION

C e i l s w e r e g r o w n i n t h e p r e s e n c e o f 0 . 5 # C i / m l [ 3 H ] t h y m i d i n e f o r 1 8 - - 2 0 h, c o l l e c t e d , w a s h e d , a n d d i v i d e d into two equal fractions. Viabihty of cells was routinely checked at this point to measure the extent of "tritium death," if any, during the labeling period. Trypan blue exclusion indicated that under our condit i o n s t h e v i a b i l i t y o f l a b e l e d c u l t u r e s w a s c o m p a r a b l e t o t h a t o f c o n t r o l s , i.e., g r e a t e r t h a n 9 0 % . M e m b r a n e s w e r e p r e p a r e d f r o m o n e f r a c t i o n w i t h M g 2+ as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . T h e o t h e r f r a c t i o n w a s t r e a t e d i d e n t i c a l l y e x c e p t M g 2+ w a s n o t a d d e d . I n o r d e r t o m i n i m i z e q u e n c h i n g p r o b l e m s , r a d i o activity measurements were taken from oxidized samples (Oxymat, Intertechnique Co.) which had first been trichloroacetic acid precipitated onto glass fiber filters, appropriately washed and air-dried.

Total incorporation Nuclear pellet Mitochondrial pellet Membrane cushion supernatant Membrane cushion Total cpm recovered: S t a r t i n g cell n u m b e r Nuclei and whole cells recovered in 800 X g pellet

( - - ) M g 2+

( + ) M g 2+

1.31 640 525 0.62 0.7 1.17

1.27 1197 2 0.074 0.05 1.19

" 109 • 106 • 106 • 106 • 106 • 106

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F i g , 2. L a c t o p e r o x i d a s e mediated iodination of the lymphocyte cell s u r f a c e . C e l l s w e r e h a r v e s t e d b y centrifugation and washed with sodium phosphate-buffered saline. Two samples of 107 cells each were r e m o v e d a n d i o d i n a t e d as d e s c r i b e d i n t h e t e x t . I o d i n a t e d c e l l s w e r e m i x e d w i t h u n l a b e l e d c e l l s a n d m e m b r a n e s p r e p a r e d as u s u a l ( F i g . l b ) . S a m p l e s w e r e p r e c i p i t a t e d o n t o g l a s s f i b e r f i l t e r s w i t h c o l d t r i c h l o r o a c e t i c a c i d as u s u a l , b u t w e r e t h e n e x t e n s i v e l y w a s h e d t w i c e a t r o o m t e m p e r a t u r e w i t h 1 5 m l o f e a c h o f the following: absolute ethanol, then ether and finally acetone. The final acetone wash removed no detect a b l e 125I r a d i o a c t i v i t y f r o m t h e f i l t e r s . A 2 S 4 ( ); 125I c o u n t s b e f o r e l i p i d e x t r a c t i o n ( o . . . . . . o), 125I c o u n t s a f t e r l i p i d e x t r a c t i o n ( o . . . . . . o).

305 radioactivity recovered amounted to approximately 3.0% of the total radioactivity p u t on the gradient. Trichloroacetic acid precipitation and lipid extraction of the samples revealed the presence of 2 peaks of 125I incorporation at densities of 1.10 and 1.17. The 1.10 peak, also evident when sample aliquots were counted directly, accounted for 90% of the trichloroacetic acid precipitable lipid solvent stable 12sI, the 1.17 peak accounted for the remaining 10%. No significant 12sI label was found associated with densities greater than 1.17 in any of our experiments which included several gradient runs on cushion material obtained from three separately iodinated samples. Although a 97% loss of ~2sI label from the gradient fractions may appear excessive, several points must be considered: (1)vesiculation of membrane fragments during cell disruption probably traps free 12sI; (2) the plasma membrane contains a significant a m o u n t of lipid [11] which could conceivably become iodinated [ 12] ; (3) intracellular lipids and unsaturated fatty acids, not membrane-associated but present as contaminants in the particulate fractions, could also be subject to iodination [12], and (4) free ~2sI probably contaminates the membrane cushion to some extent. Trapping of 12sI in membrane vesicles most likely accounts for the large majority of label removed by trichloroacetic acid precipitation and lipid solvent extraction. In any event, the remaining lipid solvent stable radioactivity can be attributed to iodinated tyrosine residues of cell surface protein [7,13]. Table II shows the distribution of '2sI label in the pelleted fractions obtained during the membrane isolation after trichloroacetic acid precipitation and lipid extraction. Since enzymatic iodination of whole cells has been shown to be specific for cell surface proteins [7,13], and since we can reasonably assume that it proceeds in random fashion over the cell surface, the data of Table II suggested that our membrane cushion contained about 25% of the total plasma membranes of the cell. However, as noted in Table I, we routinely recovered whole cells in our nuclear pellets. Although variable, the number of whole cells on the average accounted for 30 to 40% of the starting cell number. Appropriate calculations suggested, therefore, that the 25% figure actually represented a recovery of 30--35% of the disrupted plasma membrane. It was later found that this figure was in good agreement with the recovery of [3H] fucose as reported below. T A B L E II C O M P A R I S O N O F 125I L A B E L I N P A R T I C U L A T E F R A C T I O N S A F T E R T R I C H L O R O A C E T I C A C I D PRECIPITATION AND LIPID EXTRACTION Cells were i o d i n a t e d , m e m b r a n e s i s o l a t e d , and fractions prepared for c o u n t i n g as described in Materials and M e t h o d s and Fig. 2. R a d i o a c t i v e samples w e r e c o u n t e d in a t o l u e n e base scintillation fluid (toluenePPO) using the 32p channel o f a B e c k m a n L S - 3 5 0 c o u n t e r . 125I (cprn)

Nuclear pellet Mitochondrial pellet Membrane c u s h i o n Relative percentage o f m e m b r a n e c u s h i o n radioactivity to t o t a l particulate radioactivity

254 400 162 350 1 4 0 797

25%

306

The results of the [3H] fucose labeling experiments are not presented in graphic form since they were essentially identical with the data obtained from the 12sI labeling experiments. In the graph of Fig. 2, which shows the trichloroacetic acid precipitable lipid extraction stable, ~2sI profile is very similar to the radioactivity profile obtained when cushion material from [3H] fucose-labeled cells was analyzed, although the resolution between the 1.10 and 1.17 peaks was not quite as good as that in Fig. 2. The distribution of counts between the peaks was similar to that found with the 125I label, and the a m o u n t of trichloroacetic acid precipitable [3HI fucose label recovered in the membrane cushion accounted for 35--40% of that found in the whole cell homogenates. 5'-Nucleotidase (EC 3.1.3.5) has been shown to be a useful marker enzyme for mammalian l y m p h o c y t e plasma membrane [14]. The data of Table III indicated that, for what we felt were technical reasons, we could not detect the enzyme in whole cell homogenates, we could find the activity in the membrane cushion and were able to record a 10-fold enhancement of the activity at the 1.10 density and a 3-fold enhancement at the 1.17 density. Appropriate calculations indicated t h a t the sum of the 1.10 and 1.17 activities accounted for approximately 75% of the total cushion activity. Although the final specific activity of our 1.10 fraction was comparable to other reported values for l y m p h o c y t e plasma membrane 5'-nucleotidase activity [14], we had to use 800 pg of membrane cushion protein to obtain acceptable levels of activity for our assay. Considering the dilution in going from cushion to whole cell homogenate and estimating 109 cells to yield approximately 150 mg of protein, it would have been necessary for us to have used a m i n i m u m of 20 mg of homogenate protein to detect the activity. Technically, this was not feasible under the conditions of these experiments. When taken together with our electron micrograph data, not shown here but graciously provided by Dr. Etienne deHarven, and which confirmed the exclusive presence of smooth vesiculated membrane at the 1.10 density and the presence of smooth vesiculated membranes and ribosome-like granules at the 1.17 density, our ~2sI, [3H] fucose and enzyme marker data strongly suggest that under our conditions, vesiculated plasma membrane fragments are found

TABLE nI DISTRIBUTION

OF 5P-NUCLEOTIDASE

ACTIVITY

A p p r o x i m a t e l y 1 . 5 • 1 0 9 cells w e r e h a r v e s t e d , w a s h e d and m e m b r a n e s prepared as d e s c r i b e d i n M a t e r i a l and M e t h o d s . 1 . 1 0 and 1 . 1 7 f r a c t i o n s w e r e p o o l e d s e p a r a t e l y and p e l l e t e d as p r e v i o u s l y d e s c r i b e d . Pellets w e r e t h e n s u s p e n d e d in the a p p r o p r i a t e b u f f e r s f o r p r o t e i n and e n z y m e a c t i v i t y a n a l y s i s . A c t i v i t y w a s a s s a y e d b y t h e m e t h o d o f B o d a n s k y and S c h w a r t z [ 3 1 ] .

Specific activity * mg Protein Total activity

W h o l e cell homogenate

Membrane cushion

1.10

1.17

density

density

Not detectable

2.3 4.03 9.27

20.3 0.239 4.85

7.5 0.297 2.23

192 mg --

Recovery of cushion activity * I n ~ m o l P i r e l e a s e d • h -1 • m g - 1 p r o t e i n .

75%

307 at equilibrium sucrose densities of 1.10 and 1.17, with the large majority (90%) located at 1.10. In addition and on the basis of the recovery of iodinated surface protein and [3H] fucose, our membrane preparations accounted for approximately 30--35% of the total plasma membrane of our cells, the remaining 65--70% being lost in nuclear and mitochondrial pellets. Association o f DNA and R N A with the lymphocyte plasma membrane DNA: Combination of the data from Fig. 1 and Fig. 2 clearly indicates that there is no coincidence between plasma membrane and DNA in our preparations. Although only one gradient containing DNA label is shown (Fig. 1), 18 h [3H] thymidine-labeled cells have been run through these procedures at least six times. We have never observed significant DNA label coincident with the plasma membrane densities of 1.10 or 1.17. Since we are dealing with approximately 30--35% of the total plasma membrane of our cells, it seems unreasonable to suggest that we have specifically isolated only that plasma membrane which does not contain DNA, although we cannot strictly rule it out. It would seem more reasonable to suggest, however, that WIL23A lymphocyte plasma membranes do not contain significant amounts of DNA. RNA: The data of Fig. 3A show the A2s4 and radioactivity profiles which

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308 resulted when plasma membranes were prepared from cells incubated for 18 h in the presence of [3H] uridine. RNA label was found t h r o u g h o u t the entire gradient with obvious coincidence found with the major A254 peaks. Considerable amounts of RNA were found towards the b o t t o m of the gradients and half of the [3H] uridine counts were found tightly pelleted. RNAs coincident with the 1.10 and 1.17 densities were extracted from the gradient fractions with phenol sodium dodecyl sulfate and bentonite (buffer B in Melera and Rusch [15] and subjected to electrophoretic analysis in both 2.6% and 7.5% polyacrylamide gels [15,16]. The RNA species found at the two densities were entirely different (data n o t shown). Only 4 S RNA was found in the 1.10 fractions while the presence of 28 S and 18 S RNA dominated the 1.17 fraction. Several repetitions of these experiments gave similar data for the 1.10 fraction while the 1.17 fraction seemed to be somewhat variable as to the relative amounts of 28 S and 18 S RNA present. It was also found that the distribution of 28 S and 18 S RNA within the 1.17 density region (1.16--1.19) was dependent upon the fraction studied and, in some instances, was sensitive to the sample size placed on the gradient. The results were consistent with the idea t h a t the 1.17 region of the gradient contained a trailing edge of the prominent 1.18--1.19 polyribosome peak. Depending upon the gradient load, the resolution of what were probably free ribosomal subunits mixed with other ribosomal subunits associated with rough endoplasmic reticulum or other membrane fragments varied. The use of poly(U)-Sepharose chromatography to determine the presence of poly(A)-containing RNA indicated that while no poly(A) RNA was present at a density of 1.16 or less, it was detectable in l o w ' a m o u n t s at the 1.17 density and in all density fractions greater than 1.17. The results of these experiments, although inconclusive, suggest that the presence of 28 S and 18 S RNA and poly(A)-containing RNA at the 1.17 density could very well be fortuitous due to the close promixity of the polyribosome containing 1.18--1.19 density peak. Conclusions of a similar nature have also been reached by others [17,181. When electrophoresed with authentic yeast or Physarum tRNA, the 1.10 coincident RNA was shown to be homogeneous in size and to have a mobility similar to tRNA (data not shown). Since the electrophoretic profiles of our gradient RNA revealed no evidence of degradation, nuclease breakdown of large molecules to form the 1.10 4 S RNA was ruled out. The tRNA-like mobility of the 4 S RNA plus its ability to accept amino acid, albeit poorly and under suboptimal conditions (Melera, unpublished observations), suggested that the 1.10 4 S RNA was probably l y m p h o c y t e tRNA. Association of nucleic acid with a membrane fragment through a covalent or other formal bond should render t h a t complex somewhat stable. Therefore, the 1.10 membrane-RNA complex should have moved to its density of 1.10 when run ascendingly. Fig. 3B shows the profiles which resulted when [3H] uridine-labeled cushion material was placed at the b o t t o m of a 25--50% gradient and asked to move up to its apparent 1.10 density. When L-[3H] fucose or 12SI-labeled cushion material was run ascendingly, radioactivity profiles and A2s0 profiles were similar to those obtained from the descending runs (Sheridan and Melera, unpublished observations). Clearly this was n o t the case with the ascending [3H] uridine and A2s4 profiles. Neither the A2s4 nor [3H] uridine counts moved to the 1.10 densi-

309 ty, indicating a lack of association between nucleic acid and the membrane known to be present at the 1.10 density. Taken together, these results suggest that the R N A coincident with the plasma membrane at density 1.10 is probably soluble t R N A of cytoplasmic origin and n o t membrane associated or specific. Its initial presence in the membrane cushion was probably caused b y a combination of factors, including trapping during cell disruption and membrane vesiculation, and b y direct contamination of the cushion during collection by the supernatant material directly above and in contact with it. Discussion Our approach to the study of plasma membrane nucleic acid was predicated on our ability to prepare partially purified human l y m p h o c y t e plasma membranes. The data presented here indicates that we have accomplished that goal and, in general, have obtained data similar to those reported by others for l y m p h o c y t e plasma membrane [19]. Appropriate calculations based on an average yield of 150 mg total protein per 109 cells and on the amount o f protein found at the 1.10 density suggested that the plasma membrane accounts for a b o u t 0.2% of the total l y m p h o c y t e cell protein. The distribution of plasma membrane into two density regions was similar to other reports for plasma membranes isolated from human platelets [ 2 0 ] , human lymphocytes [21], mouse and rat liver [18] and pig lymphocytes [17]. The significance of the dual densities, if any, is n o t understood. Our primary interest at the outset of this work was to attempt to purify and study plasma membrane associated DNA. However, we were n o t able to demonstrate the presence of DNA in the human l y m p h o c y t e plasma membrane. One might argue that we have chosen preparative conditions which select against finding DNA in plasma membranes since we open the cells and centrifuge them in the presence of 0.005 M MgC12. Mg2÷-requiring DNAase(s) might possibly be activated under these conditions and the membrane DNA degraded. Several considerations lead us to believe that this is not the case. (1) In the original report on the presence of DNA in plasma membranes of WIL: cells [1], membranes were prepared in the presence of Mg 2÷ y e t it was estimated that 0.5% of the cellular DNA was associated with the plasma membrane. (2) According to Table I (and assuming that all cellular DNA is labeled to approximately the same specific activity after exposure of the cells to [3H] thymidine for one doubling time), 0.5% of the total DNA would contain approximately 6.5 • 106 cpm. If associated with the plasma membrane, these counts should have been found in the membrane cushion. Correcting for 35% recovery of plasma membrane, only 2.5% of the counts expected were present in the cushion when Mg 2÷ was used in the membrane preparation. Even in the absence of Mg 2÷ only 30% of the counts expected were found in the cushion. (3) Most importantly, when Mg 2÷ was used in the preparations, the loss of counts from the membrane cushion was compensated for by an increase in counts in the nuclear pellet, not in the membrane cushion supernatant where one would expect to find free DNA or nucleotides; i.e., the products of DNA degradation by DNAase. We consider these arguments sufficient to suggest that Mg 2÷ activated DNAase

310

did n o t play a significant role in our results insofar as recovery of membrane associated DNA was concerned. Reference to the literature has revealed several papers which do [20,22--24] and several papers which do not [17,19,25,26] report the presence of DNA in plasma membranes. Although there does not seem to be a consistent difference in preparation techniques which could account for these variable results, we suggest that the differences are due to the degree of nuclear damage during cell disruption and that reported plasma membrane DNA is most likely "nuclear membrane-attached DNA", as described in detail by Franke et al. [27]. Clearly our DNA data contradict those of Lerner et al. [1] who, using the same cell line used here, reported DNA to be associated with 100% of the membrane fragments obtained after cellular disruption by NP-40. The differences in the results are difficult to reconcile, but our experience with detergent lysis (Melera, unpublished observations) and the data of others [28,29] suggest that the disruption, delipidation and micelle formation caused by nonionic detergents such as NP-40 and Triton X-100 may lead to reproducible but artifactual results when attempting to ascribe sites of origin in vivo to cellular fractions obtained in vitro. The problems may in fact be multiplied when the lipoprotein matrix of plasma and nuclear membranes are considered. A recent paper [30] has reported the histochemical localization of what appears to be DNA on the surface of tumorigenic cells, while not finding this nucleic acid on the surface of nontumorigenic cells. Nineteen different cell lines were tested and only those cells strongly tumorigenic gave positive results. Unless, and in the unlikely event that our " n o r m a l " WIL23A cells have become strongly tumorigenic in culture, our data are in agreement with the nontumorigenic aspect of these recent findings. Acknowledgments The authors acknowledge the cooperation of Dr. L.F. Cavalieri in whose laboratory this work was carried out. We also thank Ms. J. Murcott for excellent technical assistance and Dr. R.H.F. Peterson for m a n y helpful discussions. This work was supported in part by NCI grant CA-08748 and AEC contract AT(111)-3521). References 1 L e r n e r , R . A . , M e i n k e , W. a n d G o l d s t e i n , D . A . ( 1 9 7 1 ) P r o e . N a t l . A c a d . Sci. U.S. 6 8 , 1 2 1 2 - - 1 2 1 6 2 Hall, M . R . , M e i n k e , W., G o l d s t e i n , D . A . a n d L e m e r , R . A . ( 1 9 7 1 ) N a t . N e w Biol. 2 3 4 , 2 2 7 - - 2 2 9 3 M e i n k e , W., H a l l , M . R . , G o l d s t e i n , D . A . , K o h n e , D . E . a n d L e r n e r , R . A . ( 1 9 7 3 ) J. Mol. Biol. 7 8 , 43--56 4 M e i n k e , W. a n d G o l d s t e i n , D . A . ( 1 9 7 4 ) J. Mol. Biol. 8 6 , 7 5 7 - - 7 7 3 5 L e v y , J . A . , V i r o l a n e n , M. a n d D e f e n d i , V. ( 1 9 6 8 ) C a n c e r 2 2 , 5 1 6 - - 5 2 4 6 L o w r y , O . H . , R o s e b r o u g h , N . S . , F a r r , A . L . a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 6 9 7 M a r c h a l o n i s , J . J . , C o n e , R . E . a n d S a n t e r , V. ( 1 9 7 1 ) B i o c h e m . J. 1 2 4 , 9 2 1 - - 9 2 7 8 S o l y o m , A. a n d T r a m s , E . G . ( 1 9 7 2 ) E n z y m e 1 3 , 3 2 9 - - 3 7 2 9 W a l l a c h , D . F . H . a n d L i r a , P.S. ( 1 9 7 3 ) B i o e h i m . B i o p h y s . A e t a 3 0 0 , 2 1 1 - - 2 5 4 1 0 D e Pierre, J . W . a n d K a t n o v s k y , M . L . ( 1 9 7 3 ) J. Cell Biol. 5 6 , 2 7 5 - - 3 0 3 11 W a r l e y , A. a n d C o o k , G.M.W. ( 1 9 7 3 ) B i o e h i m . B i o p h y s . A c t a 3 2 3 , 5 5 - - 6 8 1 2 F i d g e , N . H . a n d P o u l i s , P. ( 1 9 7 4 ) C l i n i c a C h i m i e a A c t a 5 2 , 1 5 - - 2 6 1 3 H u b b a r d , A . L . a n d C o h n , Z . A . ( 1 9 7 2 ) J . Cel Biol. 5 5 , 3 9 0 - - 4 0 5

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Misra, D.H., Gill, III, T.J. and Estes, L.W. (1974) Biochim. Biophys. Acta 352,455---461 Melera, P.W. and Rusch, H.P. (1973) Exp. Cell Res. 82, 197--209 Melera, P.W. and Rusch, H.P. (1973) Biochemistry 12, 1307--1311 Allan, D. and C r u m p t o n , M.J. (1970) Biochem. J. 120, 133--143 Evans, W.H. (1970) Biochem. J. 1 1 6 , 8 3 3 - - 8 4 2 C r u m p t o n , M.S, and Snary, D. (1974) Preparation and Properties of L y m p h o c y t e Plasma Membrane, in C o n t e m p o r a r y Topics in Molecular I m m u n o l o g y , Aga, G.L. ed., Vol. 3, pp. 27--56, Plenum Press, New York Barber, A.J. and Jamieson, G.A. (1970) J. Biol. Chem. 245, 6 3 5 7 - - 6 3 6 5 Demus, H. (1973) Biochim. Biophys. Acta 291, 93--106 Ferber, E., Resch, K., Wallach, D.F.H. and Imm, W. (1973) Biochim. Biophys. Aeta 2 6 6 , 4 9 4 - - 5 0 4 Ladoulis, C.T., Misra, D.H., Estes, L.W. and Gill, T.J., III (1974) Biochim. Biophys. Acta 356, 27--35 Kornfeld, R. and Siemers, C. (1974) J. Biol. Chem. 249, 1295--1301 Marique, D. and Hillebrand, J. (1973) Cancer Res. 33, 2761--2767 Gospodarowicz, D. (1973) J. Biol. Chem. 248, 5050--5056 Franke, W.W., Deumling, B., Zentgraf, H., Falk, H. and Rae, P.M.M. (1973) Exp. Cell Res. 81, 365-392 Helenius, A. and Simons, K. (1975) Biochim. Biophys. Acta 415, 2 9 - - / 9 Atkinson, P.H. and Summers, D.F. (1971) J. Biol. Chem. 246, 5 1 6 2 - - 5 1 7 5 Aggarwal, S.K., Wagner, R.W., McAllister, P.K. and Rosenberg, B. (1975) Proe. Natl. Acad. Sci. U.S. 72,928--932 Bodansky, O. and Schwartz, M.K. (1963) J. Biol. Chem. 238, 3420--3426

Investigation of human lymphocyte plasma membrane associated nucleic acid.

Plasma membranes were prepared from the human lymphocyte cell line WIL23A by hypotonic swelling, Dounce homogenization, differential and equilibrium c...
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