Electrophoresis of Acid Phosphohydrolase lsozymes on Cellogel M. E. HODES, M. CRISP, AND E. GELB Department
Received August 9, 1976: accepted February 14, 1977. Methods are presented for the electrophoresis and detection of various acid phosphohydrolases on Cellogel. Isozymes of acid DNase, RNase, 3’phosphodiesterase, and nonspecific phosphodiesterase are readily detectable with these techniques. The enzymes studied were extracted from vertebrate spleens and leukocytes.
Isozymes of acid DNase and RNase have been detected following column chromatography (l-4), isoelectric focusing (2,5), and polyacrylamide-gel electrophoresis (6-8). No systematic search for isozymes of acid DNase, RNase, 3’-phosphodiesterase (3’-PDE), or nonspecific phosphodiesterase (NSPDE) has been published. This may be because the techniques used for preparation and detection of the enzymes were cumbersome and generally required large amounts of tissue. We present here methods for the separation and detection of a number of acid phosphohydrolases derived from spleen and leukocytes. The medium for separation is gelled cellulose acetate (Cellogel). The methods require 0.5-2.0 ml of blood or under 200 mg of spleen per determination. MATERIALS
Preparation of cell extracts. Blood was collected in heparinized, evacuated tubes ( Vacutainer) and the white cells were separated with the aid of Plasmagel (Laboratoire Roger Bellon, Neuilly, France) (9). Erythrocytes were lysed in ammonium chloride. The pellet containing leukocytes was resuspended in 1 ml of F-12 medium and the cells were counted, resedimented at low speed, resuspended in 100 ~1 of 0.1% Triton X-100 in distilled water, and sonicated for 15 set four times with the microtip of a Branson Sonifier, setting 6. The debris was removed by low-speed centrifugation and the supernatant fluid was used for the enzyme assays and electrophoretic separations. Spleen was extracted with an equal volume of distilled water in a blender for 1 min five times. Nonidet P-40 was added to a final concentration of 0.1% and blending continued for an additional minute. The supernatant 239 Copyright All rights
0 1977 by Academic Press. Inc. of reproduction in any form reserved.
HODES, CRISP, AND GELB
fluid obtained after centrifugation at 37,000g for 1 hr was used for the enzyme assays and electrophoretic separations. Partially purified DNase II and phosphodiesterase, obtained after CM-cellulose and isoelectric focusing procedures (5), were used for some of the experiments, but most were performed on crude extracts of leukocytes or spleen. Cellogel electrophoresis. This is performed on strips of Cellogel RS (Kalex Scientific Co., Manhasset, New York) presoaked in buffer for approximately 30 min as outlined in the pamphlet supplied with the strips. The buffer is one of the following: Buffer A, 0.04 M barbital, pH 8.6; Buffer B, the aluminum 1actate:urea buffer of Azen (lo), without urea in the electrophoresis chambers; Buffer C, 0.37 M glycine:acetic acid, pH 4. Samples of 1- 5 ~1 are applied to the blotted strip and placed in a Shandon electrophoresis chamber, and a potential difference is maintained for 1.52 hr with a Spinco Duostat power supply. Protein stain. Buffalo black NBR (Allied Chemical; 0.5 g/d1 in a mixture of 45 ml of methanol, 10 ml of acetic acid, and 45 ml of water) is applied for 10 min, and gels are destained in a mixture of 475 ml of methanol, 50 ml of acetic acid, and 475 ml of water. DNuse zymogram. The method is derived from that of Homey and Webster (11). The substrate consists of (a) methyl green:DNA at a final concentration of 0.3 mg of DNA/ml in 0.15 M sodium acetate, pH 5, containing 0.01 M EDTA and 0.01% sodium azide mixed with (b) an equal volume of 3% agarose in the same buffer and maintained at 55°C. The mixture is used to coat glass slides which are stored in the cold. Cellogel strips are laid on the agarose slides and incubated overnight at 37°C. The presence of DNase activity is indicated by clear areas in the green gel. RNuse zymogram. The substrate is prepared according to Uriel and Courcon (12). It contains RNA (2.5 g of Calbiochem C grade or 0.5 g of A grade) in a mixture of 50 ml of 0.2 M sodium acetate and 50 ml of 0.03 M acetic acid, pH 5.5. The Cellogel strip is subjected to electrophoresis as described above and covered with Whatman No. 1 filter paper saturated with substrate solution and incubated for 120 min at 37°C. The strip and filter paper are wrapped in Saran Wrap and aluminum foil during the incubation. The strip is fixed in a cold mixture of 25% ethanol and 5% acetic acid for 30 min and stained in a mixture of 2% aqueous pyronin B (10 ml), 0.4 M sodium acetate (45 ml), and 0.4 M acetic acid (45 ml) for 15 min. The strip is drained and destained for up to 24 hr in a mixture of equal parts of 0.4 M sodium acetate and acetic acid. It is further destained in methanol:water:acetic acid, 5:5: 1, for 5 min and returned to the acetate: acetic acid destaining solution. The procedure results in a uniform cranberry stain except at the site of RNase action. 3’-Phosphodiesteruse zymogrum. The enzyme substrate is prepared from a mixture of Solutions A and B. Solution A consists of 0.276 g of
OF ACID PHOSPHOHYDROLASES
thymidine 3’-nitrophenyl, ammonium salt (Calbiochem) in 1 ml of water. Solution B is a mixture of 16.7 ml of 0.5 M ammonium acetate buffer, pH 5.7, 50 ,ul of 1% Tween 80 (Atlas Powder Co., Wilmington, Delaware), 8.33 ml of 0.004 M sodium ethylenediaminetetraacetic acid, and 14.67 ml of water. For preparation of the substrate, 250 ~1 of Solution A is added to Solution B. After electrophoresis as described for DNase, the Cellogel strip is covered with Whatman No. 1 filter paper saturated with substrate, wrapped as described for RNase and incubated at 37°C for 90- 120 min. The strip is drained and covered with 0.1 M NaOH. A yellow band appears immediately at the site of PDE action. The yellow color can be preserved for a few hours if the strip is again enclosed in Saran Wrap. Nonspecijic phosphodiesterase zymogram. The method is derived from that of Hodes et al. (13). The enzyme substrate is prepared from a mixture of Solutions C and D. Solution C consists of 0.47 g of disodium [email protected]
)phosphate in 125 ml of water. Solution D is a mixture of 0.5 M sodium acetate buffer, pH 5.7 (30 ml), 0.3% Tween 80 (9 ml), and distilled water (15 ml). The enzyme substrate is a mixture of 40 ml of Solution C and
FIG. 1. Cellogel electrophoresis of human leukocyte and spleen DNase II. Leukocyte extract (5 ~1) was applied to columns 1 and 2 and spleen extract (2 ~1) to columns 3 and 4 at the origin (0 cm). The gels were run for 2 hr at 350 V in Buffer A. Column 4 was separated after electrophoresis and stained for protein and the remainder of the strip was laid on a methyl green:DNA agar plate overnight and removed prior to photography
HODES, CRISP, AND GELB
50 ml of Solution D. After electrophoresis as described for DNase, the Cellogel strip is overlaid with a piece of Whatman No. 1 filter paper saturated with substrate, wrapped as described for RNase, and incubated at 37°C for 2 hr. The strip is drained, saturated with cold 0.1 M NaOH or 2 M ammonium hydroxide. A yellow band appears immediately at the site of nonspecific PDE action. This color can be preserved as described for 3’-PDE. Acid phosphatase. This zymogram has been described by Hopkinson and Harris ( 14). Enzyme assays. These have been described (3). A unit of DNase or RNase activity is defined by an increase in absorption at 260 nm of l/ml/cm light path of the acid-soluble material released after digestion of the appropriate nucleic acid at 37°C for 1 min. A unit of phosphodiesterase
FIG.~. Cellogel elecrophoresis of human leukocyte and spleen acid RNase. Leukocyte extract (5 ~1) was applied to columns 2 and 3 and spleen extract (4 ~1 of a 1:5 dilution) to column 1. The origin is at 3.0 cm. Electrophoresis was in Buffer A at 350 V for 2 hr. Staining procedures are described in the methods section.
OF ACID PHOSPHOHYDROLASES
corresponds to the release of 1 pmol ofp-nitrophenol per minute at 37°C. Sensitivity ofassays. The methods can be used for quantitation and for evaluation of the limits of detection by serial dilution of the enzyme solution prior to electrophoresis as described by Klebe (15) for starch-gel electrophoresis. The limits of detection for DNase (16.5hr digestion), RNase (14-hr digestion), and PDE (2-hr digestion) were 0.004, 0.005, and 6 x 10e7 units. These limits can be extended by use of longer digestion times but a systematic appraisal of this was not undertaken. Photography. This was performed on Polacolor film (Polaroid Corp., Cambridge, Massachusetts) with appropriate filtration. The Polacolor prints were rephotographed for preparation of the illustrations. RESULTS
The separations evident after electrophoresis and development of DNase, RNase, 3’-PDE, and NSPDE are shown in Figs. 1, 2, 3, and 4
FIG. 3. Cellogel electrophoresis of human and bovine spleen 3’-phosphodiesterase. Partially purified bovine spleen extract and extracts of two different human spleens were applied (3 ~1) to columns 1, 2, and 3, respectively. Electrophoresis was in Buffer C at 200 V for 45 min. Development is described in the methods section. The origin is at 0 cm.
HODES, CRISP, AND GELB
FIG. 4. Cellogel electrophoresis of caprine leukocyte and bovine spleen nonspecific phosphodiesterase. Goat leukocyte extract (30 ~1) and partially purified bovine spleen NSPDE (5 ~1) was applied to columns 1 and 2, respectively. The origin is at 0 cm. Electrophoresis was in Buffer B at SO V for 55 mitt, and staining procedures are described in the methods section.
respectively. Leukocytes and spleen extracts from a number of species were the starting materials. A composite diagram showing the localization of some acid phosphohydrolases from human spleen and leukocytes is presented in Fig. 5. The zymogram of Fig. 1 illustrates clearly the separation of three isozymes (column 1) with acid DNase activity, only one of which corresponds to the splenic nuclease (column 3). The figure also reveals a difference in the zymograms of the two leukocyte donors of columns 1 and 2. The genetics of these differences is under investigation. Zymograms have been performed on cells derived from pig, mouse, goat, and cow. Differences among species are easily discernible using the technique described. Three different RNase isozymes are separable from human white blood cells (Fig. 2, columns 2 and 3), and four are separable from human spleen (column 1). Zymograms have been prepared for a number of species, and different isozyme patterns are clearly differentiable. The zymograms for NSPDE and 3’-PDE are dependent on the development of the yellow color thatp-nitrophenol assumes in alkali. The results obtained after cathodic runs for 3’-PDE and NSPDE are shown in Figs. 3
OF ACID PHOSPHOHYDROLASES
3’ PM RNase DNase 3’PDE
FIG. 5. Composite diagram showing localization of several acid phosphohydrolases after separation from human spleen or leukocytes in barbital buffer, pH 8.6, at 350 V for 2 hr. The lower 3’-PDE area was also the site of NSPDE action in this system.
and 4, respectively. The 3’-PDE has also been run anodically (not shown) as differentiation of the enzyme from some species requires use of the barbital buffer. Acid phosphatase has been studied extensively by others and was run for comparative purposes only. It was found in human spleen but not in leukocytes. DISCUSSION
The purpose of this communication is to illustrate the possibilities for separation of several acid phosphohydrolases obtained from different species and to show that isozymes of each enzyme are readily and quickly demonstrable. Preparation of the Cellogel, which consists merely of soaking in buffer, consumes much less time than does preparation of polyacrylamide or starch gels. The techniques, including extraction of the tissues and sample application, are simple and rapid, thus minimizing the production of artifacts of preparation. Relatively small amounts of blood or tissue are required for a single determination. The maximum requirement is 2 ml of blood for the determination of PDE. The quantity of tissue can be reduced by increasing the incubation time proportionately. This saving is achieved at the expense of definition, as the spots tend to widen owing to diffusion of the enzyme. The occurrence of isozymes of the acid RNases and DNases was documented previously by agarose, starch-gel, and polyacrylamide-gel but not by cellulose acetate electrophoresis. Ressler et al. (8) described a method for separating RNases by electrophoresis in agarose, and Pajdak
HODES, CRISP. AND GELB
and Lisiewicz (7) demonstrated four bands of RNase activity after electrophoresis of leukemic eosinophils in polyacrylamide gel. Zijllner and coworkers have separated the DNases of human parotid saliva (6) and sweat and urine (16) by microdisc electrophoresis, and Breynaert er al. (17) found two peaks of DNase II after electrophoresis of guinea pig liver on cellulose acetate. Zollner ef al. (6,lS) demonstrated two groups of acid DNase in human lymphocytes and found an increase in lymphocytic leukemia (19). This increase was mainly due to the faster-migrating activities (anodic run). Cellogel has apparently not been used for this type of study. There is less work on the phosphodiesterases. Evans ef al. (20) used apnitrophenyl ester of dTMP to detect alkaline phosphodiesterase in gels. Lerch (21) detected plant phosphodiesterase I in gels after digestion of the a-naphthyl ester of uridine 5’-phosphate but did not comment on sensitivity. Callahan et al. (22) separated acid phosphodiesterase from human tissues by DEAE-cellulose chromatography. There do not seem to be any studies of polymorphisms of either acid 3’-PDE or NSPDE. It is difficult to compare the sensitivity of the various methods proposed for electrophoresis of the phosphohydrolases. This is because the definition of activity, digestion time, and other conditions vary. Wilson (23) shortened and made more sensitive the method described by Wolf (24) for detection of RNase after electrophoresis in polyacrylamide gels. Wilson was able to detect less than 1 unit (defined by the release of 0.015 Fmol of acid-soluble nucleotide per minute at 37°C). Our unit corresponds to the release of approximately 0.1 pmol of acid-soluble nucleotide per minute at 37°C. We are able to detect 0.005 of our unit in 4 hr, or 0.033 Wilson unit, suggesting that the method described in this paper is about 30 times more sensitive. However, Wilson’s incubation time was only 12 min. The range of both methods can be increased by increasing the time of incubation with substrate, often with some loss of definition of the band. The method of Wilson was not applicable to crude plant extracts (23). The detection of DNases in polyacrylamide gels was described by Boyd and Mitchell (25). This was modified by Boyd and Boyd (26) and Boyd and Logan (27). The method, employing DNA incorporated in the gel, was not suitable for bovine spleen DNase II (25) although DNases active at low pH (ca. 4) were detectable in Drosophila. Grdina et al. (28) increased the sensitivity of the method of Boyd and Mitchell (25) 50-fold by substitution of ethidium bromide and viewing in fluorescent light for the methyl green or pyronin stains viewed in visible light by the latter authors. The method was not applied to DNase II. Zollner et al. (29) achieved sensitivity similar to that of Grdina et al. (28) by use of [3H]DNA gels and by measurement of tritium release following digestion. Zollner et al. (19) could detect DNase II activity in 1.6 x lo5 lymphocytes, whereas the present method can be used to determine the zymogram pattern in about lo4 leukocytes. Differences in sensitivity may be due to the longer digestion times used in
OF ACID PHOSPHOHYDROLASES
this paper (16.5 vs 4 hr). In addition the methods of Zijllner et al. (29) require slicing of the gels. Although not reported here, it is obvious that the techniques described can be used to separate the cognate enzymes with alkaline pH optima. Similarly, it is possible to add inhibitors to the substrate and differentiate certain of the isozymes. The techniques of Reddi (30) have been applied to the RNases separated from leukocytes by the techniques reported here and differences in the responses of the isozymes demonstrated (unpublished.) In sum, the methods as described have these advantages: They require less enzyme and less manipulation than most of the procedures described in the literature, can be used on crude cell extracts, and readily distinguish isozymes of the acid phosphohydrolases. AKCNOWLEDGMENTS This is publication No. 7624 from the Department of Medical Genetics and was supported in part by the Indiana University Human Genetics Center Grant PHS GM 2 1054. M. Z. Hodes and R. C. Karn helped with the early stages of the work.
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Sicard, P. J., and Barthelemy-Clavey, V. (1972) Enzymofogia 43, 227. Hodes, M. E., Yip, L. C., and Santos, F. R. (1967) Enzymologia 32, 16. Delaney, R. (1963) Biochemistry 2, 438. Hodes, M. E., Song, M. K.. Kam, R. C., Prah, G. L., and Hodes. M. Z. (1976) Comp.
6. Zollner, E. J., Klepsch, D. M.. Zahn, R. K., and Knepper, R. (1975) Enzyme 19, 60. 7. Pajdak, W., and Lisiewicz, J. (1973) Stand. .I. Haematol. 11, 325. 8. Ressler, N., Olivero, E., Thompson, G. R., and Joseph, R. R. (1966) Nature (London) 210, 695.
9. Amos, B. (1973) in Manual of Tissue Typing Techniques (Ray, J. G., Jr., Hare, D. B., and Kayhoe, D. E., eds.), DHEW Publication No. (NIH) 74.545. pp. 17-20, Department of Health, Education and Welfare, Bethesda, Md. 10. Azen, E. A. (1972) Science 176, 673. Il. Horney, D. L., and Webster. D. A. (1971) Biochim. Biophys. Acta 247, 54. 12. Uriel, J., and Courcon, J. (1961) C. R. Acud. Sci. 253, 1876. 13. Hodes, M. Z., Karn, R. C., and Hodes, M. E. (1975) Proc. Indiana Acad. Sci. 84, 194. 14. Hopkinson, D. A., and Harris, H. (1969) in Biochemical Methods in Red Cell Genetics (Yunis, J. J., ed.), pp. 337-354, Academic Press, New York. 15. Klebe, R. J. (1975) Biochem. Genet. 13, 805. 16. Beck, J. C.. Zollner, J., and Zahn, R. K. (1974) Arch. Dermatol. Forsch. 250, 65. 17. Breynaert, M. D., Derumez, P., and Biserte, G. (1969) Bull. Sot. Chim. Biol. (Paris) 50, 158. 18. Zollner. E. J., Storger, H.. Breter, H.-J., and Zahn. R. K. (1975) 2. Naturforsch. 36, 781. 19. Zbllner, E. J.. Beck, J.-D., Lemmel, E. M.. and Zahn, R. K. (1975)Concer Lctt. 1, 119. 20. Evans, W. H., Hood, D. 0.. and Gurd, J. W. (1973) Biochem. J. 135, 819.
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HODES, CRISP, AND GELB Lerch, B. (1968) Experientiu 24, 889. Callahan, J. W., Lassila, E. L., and Philippart, M. (1974) B&hem. Med. 11, 250. Wilson, C. W. (1969) Anal. Biochem. 31, 506. Wolf, G. (1968) Experientia 24, 890. Boyd, J. B., and Mitchell, H. K. (1965) Anal. Biochem. 13, 28. Boyd, J. B., and Boyd, H. (1970) Biochem. Genet. 4,447. Boyd, J. B., and Logan, W. R. (1972) Cell Differentiation 1, 107. Grdina, D. J., Lohman, P. H. M., and Hewitt, R. R. (1973) Anal. Biochem. 51, 255 Ziillner, E. J., Heicke, B., and Zahn, R. K. (1974) Anal. Biochem. 58, 71. Reddi, K. K. (1976) Biochem. Eiophys. Res. Commun. 68, 1119.
An Improved Counting Vial for Measurement of LowEnergy Gamma Rays in Liquid Scintillation Counters F. VERSLUIJS Pharmacological
University of Amsterdam, The Netherlands
Received August 20, 1976; accepted February 8, 1977 An improved counting vial for the measurement of low-energy gamma rays in liquid scintillation counters has been developed. In this vial the sample is separated from the scintillation cocktail. Within wide limits no correction is necessary for the height of the sample or for decreased efficiency in colored samples. A correction is needed only for the counting efficiency, which is a constant for each vial within wide limits of size and composition of the sample. A number of commercially available gamma-counting vials are compared with the improved counting vial. The new vial is particularly useful in the measurement of 125Iused in radioimmunoassays.
Liquid scintillation counters, which are developed for the measurement of beta particles, can also be used for counting gamma rays. However, in this latter case the efficiency is as a rule very low because of the construction of the counter and the low stopping power of the scintillation cocktail. Furthermore, the corrections are usually more difficult than for the counting of beta particles. The stopping power of the scintillation cocktail for gamma rays can be increased by the addition of organometallic compounds which raise the electron density. Preferentially, the sample and the scintillation cocktail should not be mixed, so that both can be used again (1,2), and the difficult preparation of the sample often necessary for liquid scintillation measurement can be omitted. Since 1972, a number of gamma-counting vials based on Ashcroft’s design (1) have been commercially available. However, using these vials the counting efficiency decreases as the sample height increases (2,3). Corrections for variation in sample height are inaccurate. Furthermore, in colored samples a very large decrease in counting rate may occur. This decrease in rate is caused by absorption of scintillation photons which must pass the sample in order to reach the photomultiplier tube(s). The 249 Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.