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The quantitation of actin in human lymphocytes by isoelectric focusing
BIOCHEMICAL
MEDICINE
27, 200-206
( 1982)
The Quantitation of Actin in Human Lymphocytes by Isoelectric Focusing F. LIEBES,’ DENISE NEVRLA, AND ROBERT
RICHARD STARK, LEONARD
SILBER’ Department
of Medicine.
New
York Uniraersity School of Medicine, New York, NCM~ York 10016
550 First
Avenue.
Received July 7, 1981
Actin is a major intracellular protein which is functionally important in a wide range of cellular activities. Changes in actin content have been related to the state of cellular proliferation or differentiation in a number of culture systems (l-3). Recently, alterations in the P:r ratio of nonmuscle actin during embryogenesis have been shown using isoelectric focusing (4,5). In this report, a relatively simple yet sensitive technique is described for quantifying the content and the l3:~ ratio of actin in human-lymphocyte extracts. In the present method, lymphocyte proteins are resolved in isoelectric focusing gels; the gels are scanned following staining with Coomassie brilliant blue R250, and the actin content is determined densitometrically. With this method, actin was measured in lymphocyte preparations from 24 normal donors.
MATERIALS AND METHODS Sample preparation. Peripheral blood lymphocytes were obtained as previously described (6). Informed consent was obtained according to the provisions of the Helsinki Conference. Cell pellets containing 0.1-I .O x IO* cells were stored at -70°C until used. At this temperature, the actin content was stable for up to 6 months. The cell pellets were thawed and sonicated in a lysis solution containing 9.5 M urea, 5% l3-mercaptoethanol, and 0.5% NP40 at a concentration of 1.0 x lo” cells/ml solution. Cells were disrupted using a Heat Systems sonifier equipped with a microprobe at 4°C with a IO-set burst at 60-W power. Separate aliquots I Scholar of the Leukemia Society of America. * Address reprint requests to: Robert Silber, M.D., Department of Medicine, New York University School of Medicine, 550 First Avenue, New York. N.Y. 10016. 200 OOOG2944/82/020200-07$02.00/O Copyright All rights
Q 1982 by Academic Press. Inc. of reproduction in any form reserved.
LYMPHOCYTEACTINCONTENT
201
of cells were used for protein determinations. Actin to be used as a standard was purified to 95% homogeneity from rabbit skeletal muscle (RSM) according to the method of Spudich and Watt (7). The actin in a concentration of 1 to 2 mg/ml was stored as aliquots in 3 mM Tris, 0.1 mM CaCl,, 0.5 mM ATP, 0.75 mM P-mercaptoethanol, pH 7.5, at -70°C and was stable for up to 2 months. Actin was purified from lymphocytes by a modification of the method of Gordon et al. (8) as applied to lymphocyte actin by Liebes et al. (9). Electrophoresis. Isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis were performed according to the method of O’Farrell (10) with the following modifications. A Hoeffer tube electrophoresis unit with a capacity of 18 tubes was used. The gels were poured into 0.3 x 13 cm tubes. Ampholines pH 5-7 and 4-6 (LKB or Bio-Rad) were added in equal amounts to a final concentration of 2%. The final concentration of acrylamide and bis-acrylamide in the gels was 7.5 and 0.14%, respectively. The lower concentration of bis-acrylamide allowed swelling of the gels to a 0.5cm diameter prior to scanning. Sodium dodecyl sulfate (SDS) electrophoresis in the second dimension was carried out according to the method of Neville and Glossman (11) using a 3% stacking gel and a 10% running gel. Gel staining and destaining. Isoelectric focusing gels were fixed and stained by a minor modification of Vesterberg’s method (12) in which gels were stained with Coomassie brilliant blue R250 at room temperature for 6 hr and destained in a solution containing acetic acid : ethanol : H,O; 5 : 35 : 60; v/v/v. Destaining generally required 48-70 hr with rotary shaking. Prior to scanning, the gels were allowed to expand in 10% acetic acid for 2 hr. SDS slab gels were stained overnight in 0.25% Coomassie blue and destained as described above. Quantitation. Segments of gel containing actin standard or the p and y components of actin from cell extracts were excised and aligned in a glass cuvette with a 4-mm diameter. The gels were scanned at 576 nm with a Beckman Acta II spectrophotometer. The areas under the peaks corresponding to actin were measured using a Houston Omniscribe twochannel recorder equipped with an electronic integrator. To adjust for variations in the staining and destaining of the gels, a standard curve ranging from 0.5 to 5.0 pg protein was generated for each experiment using rabbit skeletal muscle actin. For all experiments, only p and y peak values falling within this range of linearity were used for quantitation. The total protein concentration in cells and in actin standards was determined according to the method of Lowry et al. (13). RESULTS
The use of a mixture of pH 4-6 and 5-7 Ampholines generated a reproducible pH gradient over a range of 4.5 to 6.5 (5). Actin, purified
202
STARK ET AL.
FIG. 1. Isoelectric focusing gels of purified actin and lymphocyte sonicates stained with Coomassie blue. (1) 1.5 p,g Rabbit-skeletal-muscle actin; (2,3) 15 pg purified lymphocyte actin; (4-11) lymphocyte sonicates from different preparations of lymphocytes. Each gel contains protein from 2.5 X 10’ cells (-100 kg total protein).
from rabbit skeletal muscle and from lymphocytes, focused in the middle of this gradient (Fig, 1). As was found with other vertebrate nonmuscle cells, actin purified from blood lymphocytes was resolved into only p and y species on isoelectric focusing. Over the 4.5-6.5 pH gradient, the p and y actins in whole cell sonicates were consistently resolved from other polypeptides (Figs. 1 and 2). To determine if other polypeptides were present in sufficient concentration in the region containing the actin bands after isoelectric focusing, an overloaded gel was subjected to SDSelectrophoresis in a second dimension (Fig. 3). Almost all of the detectable protein at a pZ from 5.4 to 5.5 migrated as actin with a molecular weight of 43,000. Quantitation of the vertical segment of the gel containing the actin band demonstrated that 95% of the material appeared to be actin on the basis of this molecular weight. Thus, a valid quantitation of the total cell-actin content could be obtained by measuring the p and y bands. The relation of integrated area units obtamed using rabbit skeletal muscle (RSM) actin versus the protein applied per gel was linear from 0.5 to 5.0 kg (Fig. 4). Values below 0.25 ).~g were difficult to quantify
LYMPHOCYTE
I
I
I
203
ACTIN CONTENT
I,
1
I
012345678 LENGTH
!
I
I
9
10
km)
FIG. 2. Densitometric scans of isoelectric focusing gels. Upper scan, whole cell sonicate from 1.5 x IO6 cells; lower scan, 3 ug purified lymphocyte actin. The region scanned is representative of pH values of 5.0 to 6.0. Gels were scanned at a rate of 1.5 cm/min, using a 0.05 -mm slit with the absorbance at 576 nm determined on the 0.5 A range.
with accuracy with the scanning system employed. Above 5 pg the linearity was lost although values as high as 7.5 p,g could be determined. Above 7.5 kg, nonlinear staining of the gels as a result of overloading occurred. The linearity of the assay was evaluated by applying varying volumes of a cell sonicate ranging from 5 to 25 ~1 to the isoelectric focusing gels. Figure 4 shows the linear relationship between the integration values obtained with the actin bands and the volume of extract applied to the gel. The actin content of human peripheral blood lymphocytes was determined for 24 samples from normal subjects. Each cell preparation was analyzed in duplicate or triplicate. The standard error of the mean of
FIG. 3. Two-dimensional electrophoresis of polypeptides from 7.5 x 106 lymphocytes (350 ug protein). Isoelectric focusing was performed in the horizontal direction and SDSelectrophoresis was performed in the vertical direction using a 10% polyacrylamide slab gel as described under Materials and Methods. The gel was stained with Coomassie blue. The molecular weights of the standards are as indicated. The arrow denotes actin.
204
STARK ET AL
pg RSM I 5
ACTIN
I I I I 10 15 20 25 /II CELL EXTRACT
FIG. 4. Demonstration of linearity of the isoelectric focusing assay. A comparison is made between the areas corresponding to rabbit-skeletal-muscle standard or the f3 and y actins from a whole cell sonicate. The area units are obtained as described under Materials and Methods. (0) Rabbit-skeletal-muscle actin, 0.5 to 5.0 ug; (A) p actin; (m) y actin in lymphocyte sonicate (10’ cells/p1 sonicate).
these determinations was usually less than 15% of the mean value. The actin content was 2.1 t 0.4 mg/lO’ cells (mean r SD). The percentage of actin of the total cellular-protein content (obtained on cell pellets not applied to isoelectric focusing gels) was 6.3 ? 1.6%. The P/r ratio was 2: 1. DISCUSSION A number of methods used to measure actin have been described. Most commonly, densitometric scanning of stained SDS-electrophoresis gel is employed. This approach assumes equivalent staining of all polypeptides and the absence of other proteins comigrating with actin. Utilizing SDS-electrophoresis with gels containing 10% acrylamide, we obtained values for the actin content of lymphocytes that ranged from 15 to 20% of the total protein (unpublished data). These values probably represented significant overestimates because of contaminating polypeptides with a molecular weight similar to actin. Using a IO-20% acrylamide gradient, better resolution was obtained and a mean actin content of 8.3% was found (9). Difficulties were still encountered in the somewhat subjective assignment of limits to the actin peak. There is also a radioimmunoassay (14) and an assay based upon inhibition of DNase I activity (15) to measure actin. These are performed under conditions which may not completely solubihze actin and as such may underestimate the actin content. A method described by Anderson is based upon the measurement of unique radiolabeled peptides (16). Although highly specific for actin, this technique involves many steps and would not be well suited for the measurement of a large number of specimens. The method does demonstrate, however, that when quanti-
LYMPHOCYTEACTIN
CONTENT
205
tated by SDS electrophoresis, only about one-half of the protein in the band comigrating with actin is truly actin. The staining of purified actin using the dye Fast Green in isoelectric focusing gels has been described in a recent study (17). While linearity of dye uptake with purified actin was shown, ply quantitation was not reported. We did not encounter the problems of Coomassie-blue staining of Ampholines described by these authors. Since Coomassie-blue staining has a greater sensitivity, this dye was used in the present method. The advantages of the method described above are as follows. (1) The cells are sonicated in a lysis solution designed to maximize solubilization and minimize proteolytic degradation of polypeptides. (2) No extraction or precipitation which might lead to a loss of protein is required. (3) Isoelectric focusing over a narrow pH gradient clearly resolves the p and y forms of actin from other polypeptides. (4) Purified rabbit-skeletalmuscle actin is easily obtainable in pure form; since its molecular weight and charge are nearly identical to those of nonmuscle actins, it is an ideal standard. (5) The sensitivity of Coomassie-blue staining allows quantitation of as little as 0.5 kg actin; as such actin can be reliably measured in I x lo6 lymphocytes. SUMMARY A simple and sensitive technique for quantitating the actin content of lymphocytes is described. The method uses isoelectric focusing over a pH gradient of 4.5 to 6.5 to resolve actin from other polypeptides followed by densitometric scanning of Coomassie-blue stained gels. The dye uptake of purified rabbit-skeletal-muscle actin standards was linear from 0.5 to 5.0 kg of applied protein. Cell protein was solubilized in a lysis solution containing urea, P-mercaptoethanol, and NP40. Linear results were obtained when homogenates prepared from 5 to 25 x IO5 cells were applied. The actin content of human peripheral blood lymphocytes determined with this assay was 2.1 + 0.3 mg/109 cells and represented 6.3 ? 1.6% of the total cellular protein. The ratio of P:r actin in human lymphocytes was 2 : 1. ACKNOWLEDGMENTS This work was supported by a grant from the National Institutes of Health, CA11655.
REFERENCES I. Hoffman-Lieberman, B., and Sachs. L.. Ce// 14, 825 (1978). 2. Rubin, R. W., Warren, R. H., Lukeman, D. S., and Clements, E., J. Cell B;o/. 78, 28 (1978). 3. Bet-tram, J. S., Libby, P. R.. and LeStourgeon. W. M., Cancer RPS. 37, 4104 (1977). 4. Saborio, J. L., Segura, M., Flares, M.. Garcia. R., and Palmer, E., J. Biof. them. 254, 11119 (1979).
206
STARK
ET Al
5. Flanagan. M. D.. and Lin. S.. J. h’errtwhem. 32. 1037 (iY7Y). 6. Petty. H. R.. Ware. B. R.. Liebes. L. F.. Pelle. E.. and Silber. R.. Blocrcl 57, 750 (1981). 7. Spudich, J. A., and Watt, S.. J. Biol. Chem. 246, 4866 (1971). 8. Gordon, D. J.. Eisenberg. E.. and Korn. E. D.. 1. Bid. C‘hem. 251, 4778 (1978). 9. Liebes. L., Burger. J.. Elsbdch. B.. Conklyn. M., Stark. R.. and Silber. R.. B/o& 54, 88a ( 1979). IO. O’Farrell. P. H.. J. Biol. Cherr7. 250. 4007 (1975). I I. Neville, D. M.. and Glossman. H.. .I. Biol. Chrm. 246, 6335 f 1971). I?. Vesterberg. 0.. Biochim. Biophy.~. Acrrt 243, 345 (I971 ). 13. Lowry. 0. H., Rosebrough. N. J.. Farr. A. I... and Randall, R. J.. J. Bid. Cl7ew7. 193, 265 (1951). 14. Lessard, J. L.. Carlton, D., Rein. D. C.. and Akeson. R., And. Biochrm. 94. I40 (1979). 15. Blikstad, I.. Markey. F.. Carlsson, L., Persson. T.. and Lindberg. U.. Cell 15, 935 (1978). 16. Anderson, P. J., Binchem. J. 179, 425 (1979). 17. Allen, R. E.. Masak, K. C.. and McAllister. P. K.. Anal. Biochetn. 104. 494 (1980).
MEDICINE
27, 200-206
( 1982)
The Quantitation of Actin in Human Lymphocytes by Isoelectric Focusing F. LIEBES,’ DENISE NEVRLA, AND ROBERT
RICHARD STARK, LEONARD
SILBER’ Department
of Medicine.
New
York Uniraersity School of Medicine, New York, NCM~ York 10016
550 First
Avenue.
Received July 7, 1981
Actin is a major intracellular protein which is functionally important in a wide range of cellular activities. Changes in actin content have been related to the state of cellular proliferation or differentiation in a number of culture systems (l-3). Recently, alterations in the P:r ratio of nonmuscle actin during embryogenesis have been shown using isoelectric focusing (4,5). In this report, a relatively simple yet sensitive technique is described for quantifying the content and the l3:~ ratio of actin in human-lymphocyte extracts. In the present method, lymphocyte proteins are resolved in isoelectric focusing gels; the gels are scanned following staining with Coomassie brilliant blue R250, and the actin content is determined densitometrically. With this method, actin was measured in lymphocyte preparations from 24 normal donors.
MATERIALS AND METHODS Sample preparation. Peripheral blood lymphocytes were obtained as previously described (6). Informed consent was obtained according to the provisions of the Helsinki Conference. Cell pellets containing 0.1-I .O x IO* cells were stored at -70°C until used. At this temperature, the actin content was stable for up to 6 months. The cell pellets were thawed and sonicated in a lysis solution containing 9.5 M urea, 5% l3-mercaptoethanol, and 0.5% NP40 at a concentration of 1.0 x lo” cells/ml solution. Cells were disrupted using a Heat Systems sonifier equipped with a microprobe at 4°C with a IO-set burst at 60-W power. Separate aliquots I Scholar of the Leukemia Society of America. * Address reprint requests to: Robert Silber, M.D., Department of Medicine, New York University School of Medicine, 550 First Avenue, New York. N.Y. 10016. 200 OOOG2944/82/020200-07$02.00/O Copyright All rights
Q 1982 by Academic Press. Inc. of reproduction in any form reserved.
LYMPHOCYTEACTINCONTENT
201
of cells were used for protein determinations. Actin to be used as a standard was purified to 95% homogeneity from rabbit skeletal muscle (RSM) according to the method of Spudich and Watt (7). The actin in a concentration of 1 to 2 mg/ml was stored as aliquots in 3 mM Tris, 0.1 mM CaCl,, 0.5 mM ATP, 0.75 mM P-mercaptoethanol, pH 7.5, at -70°C and was stable for up to 2 months. Actin was purified from lymphocytes by a modification of the method of Gordon et al. (8) as applied to lymphocyte actin by Liebes et al. (9). Electrophoresis. Isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis were performed according to the method of O’Farrell (10) with the following modifications. A Hoeffer tube electrophoresis unit with a capacity of 18 tubes was used. The gels were poured into 0.3 x 13 cm tubes. Ampholines pH 5-7 and 4-6 (LKB or Bio-Rad) were added in equal amounts to a final concentration of 2%. The final concentration of acrylamide and bis-acrylamide in the gels was 7.5 and 0.14%, respectively. The lower concentration of bis-acrylamide allowed swelling of the gels to a 0.5cm diameter prior to scanning. Sodium dodecyl sulfate (SDS) electrophoresis in the second dimension was carried out according to the method of Neville and Glossman (11) using a 3% stacking gel and a 10% running gel. Gel staining and destaining. Isoelectric focusing gels were fixed and stained by a minor modification of Vesterberg’s method (12) in which gels were stained with Coomassie brilliant blue R250 at room temperature for 6 hr and destained in a solution containing acetic acid : ethanol : H,O; 5 : 35 : 60; v/v/v. Destaining generally required 48-70 hr with rotary shaking. Prior to scanning, the gels were allowed to expand in 10% acetic acid for 2 hr. SDS slab gels were stained overnight in 0.25% Coomassie blue and destained as described above. Quantitation. Segments of gel containing actin standard or the p and y components of actin from cell extracts were excised and aligned in a glass cuvette with a 4-mm diameter. The gels were scanned at 576 nm with a Beckman Acta II spectrophotometer. The areas under the peaks corresponding to actin were measured using a Houston Omniscribe twochannel recorder equipped with an electronic integrator. To adjust for variations in the staining and destaining of the gels, a standard curve ranging from 0.5 to 5.0 pg protein was generated for each experiment using rabbit skeletal muscle actin. For all experiments, only p and y peak values falling within this range of linearity were used for quantitation. The total protein concentration in cells and in actin standards was determined according to the method of Lowry et al. (13). RESULTS
The use of a mixture of pH 4-6 and 5-7 Ampholines generated a reproducible pH gradient over a range of 4.5 to 6.5 (5). Actin, purified
202
STARK ET AL.
FIG. 1. Isoelectric focusing gels of purified actin and lymphocyte sonicates stained with Coomassie blue. (1) 1.5 p,g Rabbit-skeletal-muscle actin; (2,3) 15 pg purified lymphocyte actin; (4-11) lymphocyte sonicates from different preparations of lymphocytes. Each gel contains protein from 2.5 X 10’ cells (-100 kg total protein).
from rabbit skeletal muscle and from lymphocytes, focused in the middle of this gradient (Fig, 1). As was found with other vertebrate nonmuscle cells, actin purified from blood lymphocytes was resolved into only p and y species on isoelectric focusing. Over the 4.5-6.5 pH gradient, the p and y actins in whole cell sonicates were consistently resolved from other polypeptides (Figs. 1 and 2). To determine if other polypeptides were present in sufficient concentration in the region containing the actin bands after isoelectric focusing, an overloaded gel was subjected to SDSelectrophoresis in a second dimension (Fig. 3). Almost all of the detectable protein at a pZ from 5.4 to 5.5 migrated as actin with a molecular weight of 43,000. Quantitation of the vertical segment of the gel containing the actin band demonstrated that 95% of the material appeared to be actin on the basis of this molecular weight. Thus, a valid quantitation of the total cell-actin content could be obtained by measuring the p and y bands. The relation of integrated area units obtamed using rabbit skeletal muscle (RSM) actin versus the protein applied per gel was linear from 0.5 to 5.0 kg (Fig. 4). Values below 0.25 ).~g were difficult to quantify
LYMPHOCYTE
I
I
I
203
ACTIN CONTENT
I,
1
I
012345678 LENGTH
!
I
I
9
10
km)
FIG. 2. Densitometric scans of isoelectric focusing gels. Upper scan, whole cell sonicate from 1.5 x IO6 cells; lower scan, 3 ug purified lymphocyte actin. The region scanned is representative of pH values of 5.0 to 6.0. Gels were scanned at a rate of 1.5 cm/min, using a 0.05 -mm slit with the absorbance at 576 nm determined on the 0.5 A range.
with accuracy with the scanning system employed. Above 5 pg the linearity was lost although values as high as 7.5 p,g could be determined. Above 7.5 kg, nonlinear staining of the gels as a result of overloading occurred. The linearity of the assay was evaluated by applying varying volumes of a cell sonicate ranging from 5 to 25 ~1 to the isoelectric focusing gels. Figure 4 shows the linear relationship between the integration values obtained with the actin bands and the volume of extract applied to the gel. The actin content of human peripheral blood lymphocytes was determined for 24 samples from normal subjects. Each cell preparation was analyzed in duplicate or triplicate. The standard error of the mean of
FIG. 3. Two-dimensional electrophoresis of polypeptides from 7.5 x 106 lymphocytes (350 ug protein). Isoelectric focusing was performed in the horizontal direction and SDSelectrophoresis was performed in the vertical direction using a 10% polyacrylamide slab gel as described under Materials and Methods. The gel was stained with Coomassie blue. The molecular weights of the standards are as indicated. The arrow denotes actin.
204
STARK ET AL
pg RSM I 5
ACTIN
I I I I 10 15 20 25 /II CELL EXTRACT
FIG. 4. Demonstration of linearity of the isoelectric focusing assay. A comparison is made between the areas corresponding to rabbit-skeletal-muscle standard or the f3 and y actins from a whole cell sonicate. The area units are obtained as described under Materials and Methods. (0) Rabbit-skeletal-muscle actin, 0.5 to 5.0 ug; (A) p actin; (m) y actin in lymphocyte sonicate (10’ cells/p1 sonicate).
these determinations was usually less than 15% of the mean value. The actin content was 2.1 t 0.4 mg/lO’ cells (mean r SD). The percentage of actin of the total cellular-protein content (obtained on cell pellets not applied to isoelectric focusing gels) was 6.3 ? 1.6%. The P/r ratio was 2: 1. DISCUSSION A number of methods used to measure actin have been described. Most commonly, densitometric scanning of stained SDS-electrophoresis gel is employed. This approach assumes equivalent staining of all polypeptides and the absence of other proteins comigrating with actin. Utilizing SDS-electrophoresis with gels containing 10% acrylamide, we obtained values for the actin content of lymphocytes that ranged from 15 to 20% of the total protein (unpublished data). These values probably represented significant overestimates because of contaminating polypeptides with a molecular weight similar to actin. Using a IO-20% acrylamide gradient, better resolution was obtained and a mean actin content of 8.3% was found (9). Difficulties were still encountered in the somewhat subjective assignment of limits to the actin peak. There is also a radioimmunoassay (14) and an assay based upon inhibition of DNase I activity (15) to measure actin. These are performed under conditions which may not completely solubihze actin and as such may underestimate the actin content. A method described by Anderson is based upon the measurement of unique radiolabeled peptides (16). Although highly specific for actin, this technique involves many steps and would not be well suited for the measurement of a large number of specimens. The method does demonstrate, however, that when quanti-
LYMPHOCYTEACTIN
CONTENT
205
tated by SDS electrophoresis, only about one-half of the protein in the band comigrating with actin is truly actin. The staining of purified actin using the dye Fast Green in isoelectric focusing gels has been described in a recent study (17). While linearity of dye uptake with purified actin was shown, ply quantitation was not reported. We did not encounter the problems of Coomassie-blue staining of Ampholines described by these authors. Since Coomassie-blue staining has a greater sensitivity, this dye was used in the present method. The advantages of the method described above are as follows. (1) The cells are sonicated in a lysis solution designed to maximize solubilization and minimize proteolytic degradation of polypeptides. (2) No extraction or precipitation which might lead to a loss of protein is required. (3) Isoelectric focusing over a narrow pH gradient clearly resolves the p and y forms of actin from other polypeptides. (4) Purified rabbit-skeletalmuscle actin is easily obtainable in pure form; since its molecular weight and charge are nearly identical to those of nonmuscle actins, it is an ideal standard. (5) The sensitivity of Coomassie-blue staining allows quantitation of as little as 0.5 kg actin; as such actin can be reliably measured in I x lo6 lymphocytes. SUMMARY A simple and sensitive technique for quantitating the actin content of lymphocytes is described. The method uses isoelectric focusing over a pH gradient of 4.5 to 6.5 to resolve actin from other polypeptides followed by densitometric scanning of Coomassie-blue stained gels. The dye uptake of purified rabbit-skeletal-muscle actin standards was linear from 0.5 to 5.0 kg of applied protein. Cell protein was solubilized in a lysis solution containing urea, P-mercaptoethanol, and NP40. Linear results were obtained when homogenates prepared from 5 to 25 x IO5 cells were applied. The actin content of human peripheral blood lymphocytes determined with this assay was 2.1 + 0.3 mg/109 cells and represented 6.3 ? 1.6% of the total cellular protein. The ratio of P:r actin in human lymphocytes was 2 : 1. ACKNOWLEDGMENTS This work was supported by a grant from the National Institutes of Health, CA11655.
REFERENCES I. Hoffman-Lieberman, B., and Sachs. L.. Ce// 14, 825 (1978). 2. Rubin, R. W., Warren, R. H., Lukeman, D. S., and Clements, E., J. Cell B;o/. 78, 28 (1978). 3. Bet-tram, J. S., Libby, P. R.. and LeStourgeon. W. M., Cancer RPS. 37, 4104 (1977). 4. Saborio, J. L., Segura, M., Flares, M.. Garcia. R., and Palmer, E., J. Biof. them. 254, 11119 (1979).
206
STARK
ET Al
5. Flanagan. M. D.. and Lin. S.. J. h’errtwhem. 32. 1037 (iY7Y). 6. Petty. H. R.. Ware. B. R.. Liebes. L. F.. Pelle. E.. and Silber. R.. Blocrcl 57, 750 (1981). 7. Spudich, J. A., and Watt, S.. J. Biol. Chem. 246, 4866 (1971). 8. Gordon, D. J.. Eisenberg. E.. and Korn. E. D.. 1. Bid. C‘hem. 251, 4778 (1978). 9. Liebes. L., Burger. J.. Elsbdch. B.. Conklyn. M., Stark. R.. and Silber. R.. B/o& 54, 88a ( 1979). IO. O’Farrell. P. H.. J. Biol. Cherr7. 250. 4007 (1975). I I. Neville, D. M.. and Glossman. H.. .I. Biol. Chrm. 246, 6335 f 1971). I?. Vesterberg. 0.. Biochim. Biophy.~. Acrrt 243, 345 (I971 ). 13. Lowry. 0. H., Rosebrough. N. J.. Farr. A. I... and Randall, R. J.. J. Bid. Cl7ew7. 193, 265 (1951). 14. Lessard, J. L.. Carlton, D., Rein. D. C.. and Akeson. R., And. Biochrm. 94. I40 (1979). 15. Blikstad, I.. Markey. F.. Carlsson, L., Persson. T.. and Lindberg. U.. Cell 15, 935 (1978). 16. Anderson, P. J., Binchem. J. 179, 425 (1979). 17. Allen, R. E.. Masak, K. C.. and McAllister. P. K.. Anal. Biochetn. 104. 494 (1980).