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Electrophoretic separation of enzymes of individual flour beetles, Tribolium castaneum, on polyacrylamide gel
ANALYTICAL
Electrophoretic Beetles,
Separation Tribolium
FRANCIS The
62, 321-326 (1974)
BIOCHEMISTRY
Genetics
of Enzymes
castaneum,
C. H. YEH Laboratory,
Received
on Polyacrylamide ELIYAHU
AND
10, 1972;
accepted
Flour Gel’
SCHEINBERC
Department of Biology, Calgary, Alberta, Canada May
of Individual
June
Vuivelsity
of Calgary.
10, 1974
A method of preparing a single-beetle homogenate for separating enzymes of individual flour beetles, Tribolium castaneum, by electroplioresis on polyacrylamide gel is described. This method can be adapted to the study of enzyme activity in organisms similar to T. castaneum in size and protein concentration (450 pg dry wt), or for the analysis of very small quantities of tissue and for whole organs from a slightly larger organism.
The introduction of polyacrylamide gel (1 and 2) as supporting medium in zone electrophoresis has provided a powerful tool for identifying some biochemical loci and for studying genetic variations in natural and laboratory animal populations. It is important to determine the electrophoretic variants within single organisms in an analysis of a gene pool variability. However, difficulties arise when dealing with small organisms such as the beetle, Tribolium. These difficulties are due to a small sample size that does not allow for a reliable sample preparation and to the relatively low sensitivity of the available analytic systems. Several modifications and advances of the method as applied to microelectrophoresis have subsequently been reported (3-7). In this paper a description of a rapid, sensitive, and reliable methcd of studying the activities of several enzymes in individuals of Triboliunz castaneum by electrophoresis on polyacrylamide gel is described. MATERIALS
Experimental
AND
METHODS
Populations
Individual beetles were obtained from a foundation population Tribolium custaneum derived in 1954 from the systematic crossing
of of
‘Contribution No. 5 from the Genetics Laboratory, University of Calgary. Supported in part by a National Research Council of Canada Grant, A5451 and a University of Calgary Grant in Aid of Research awarded to the second author. Copyright All rights
@ 1974 by Academic Press, of reproduction in any form
321 Inc. reserved.
322
YEH
AND
SCHEINBERG
TABLE 1 List of Ingredients of the Stacking Part (large pore 2.5% T, 7.4’% C) and the Separation Part (small pore, 3.77-2Oyo T, 2.6% C) of Gel Together with the Procedures for Their Preparation. Solutions A, B, and C are Stocks which can be Stored for Several Weeks Under Refrigeration in Broth Bottles. Solution D Should be Made Before Each Use. Solution E can be used Within 1 wk After Preparation if Kept Under Refrigeration in Brown Bottles Temp System A (stacks pH 8.3 runs at pH Small pore (3.75-2Oa/, T) Ingredient
Amt
= 15.5
at 9.3)
Large
pore
f
0.5”C System B (stacks at pH 8.3 runs at pH 6.6)
Small pore (3.75-20y0 T)
used
Amt
(4 Acrylamide Bisacrylamide Dist Hz0 to make
See (13)
10 g
0.8 g 50 ml
See (13)
(B)” 18.15
Ammonium Dist Hz0
persulfate to make
g -
24 ml
12.8 ml -
0.72 ml 300 ml
0.1 ml 100 ml
2w
-
-
4.8
48 ml 0.4 ml 200 ml
48 ml 0.4 ml 100 ml
-
25 ml
-
80 mg 25 ml
0.35 g 25 ml
(Working
to make
Proportion See (13)
_ a Davis
(9).
g -
0
-
fD) 0.14 g 25 ml (El
(E) Sucrose Dist Hz0
-
114 g -
CD) 0.25 g 25 ml
0.8 g 50 ml (B)
2.23
g
KY to make
pore
(4 10 g
Tris Glycine 1 M HSPO, ~NHCI ~NKOH Temed Dist Hz0 to make
Riboflavin Dist H20
Large used
24 g 60 ml solution) 2A 4B D 8E c
(Working
See (13)
24 g 60 ml solution) 2A B D 4E
EKZYMES
OF Tribolium
323
eight random-mat.ing laboratory stocks of different geographical origins. This synthetic stock has been propagated each generation with a minimum of 250 randomly chosen parents and maintained in a control chamber at 32.8 +- 1°C and 72 + 2% relative humidity, in half-pint milk bottles containing standard medium (8). Enzyme Preparation A Pyrex brand glass plate (Fisher No. 31, 748B) with 9 cavities, 22 mm by 7 mm, and compatible sized glass rods were used as the homogenizers. Individual beetles were ground in 0.1 ml of sample buffer containing 0.4 PM coenzyme in 20% sucrose solution (Table 1). The slurry was then collected into a microhematocrit tube and centrifuged for 10 min at 3000 rpm. The resulting supernatant (4.5 mg protein/ml) was used immediately or stored under refrigeration for up to G hr prior to electrophoresis. Each supernatant could be analyzed for three enzymes. Polyacrylamide
Gel Electrophoresis (PAGE)
The list of ingredients of the stacking part (large pore, 2.5% T, 7.4% C) and the separating part (small pore, 3.7520% T, 2.6% C) of the gel, together with their preparation procedures are listed in Table 1. PAGE was carried out with Biichler Polyanalyst (no. 3-1750, Biichler Instruments Inc.) following a modification of the procedures given by Davis (9). In order to minimize thermal contraction or expansion of the gel during electrophoresis, polymerization and electrophoresis were carried out at the same temperature (15°C 1 05°C) following the procedures given by Rodbard and Chrambach (11). The concentrations of polymerization catalysts (Table 1) were set so that polymerization was complet,ed in about 15 min. Electrophoresis was conducted at constant current of 1 ma/tube until the tracking dye had just migrated into the separating gel and continued at 2.5-3.5 mA/tube for 65-85 min depending on the enzyme (Table 2). Samples were introduced into the gel tubes directly from the microhematocrit tubes and carefully overlaid with upper buffer. The buffers used are listed in Table 2. Migration was toward the cathode for Isocitrate Dehydrogenase and anode for all other enzyme species (Table 2). Gels were stained overnight in the dark for enzymatic activities in culture tubes (10 X 75 mm) according to the methods of Brewer (19) except that 0.1 mg/ml staining solution of phenazine methosulfate was substituted to decrease background stain in gels. Gels were stored in dilute saline solution, with the exception of Lactate Dehydrogenase gels which were stored in 7% acetic acid. The relative mobility (R,) of all enzyme species were computed (II)
324
YEH
AND
SCHEINBERG
TABLE 2 Electrode Buffers for Polyacrylamide Gel Electrophoresis at 15.5 f 0.5%. A Quantity of 0.4 PM Coenzyme is Added to Upper Electrode Buffer During Each Run to Stabilize the Enzyme. Each Buffer Preparation can be used for 36 Samples When Stored Under Refrigeration
Enzyme
Upper buffer
Lower buffer
Systern
Alcohol dehydrogenase Glucose-8phosphate dehydrogenase I. 1 Lactate dehydrogenase 6-Phosphogluconate dehydroIsocitrate dehydrogenase
M
(PH
Tris-glyeine
.12
9.2)
0.182 M Glycine/ 0.356 M Lutidine (PH 8.3)
(PH
M
Tris-HCl 8.7)
0.182 M Glycine/ 0,356 M Lutidine (PH 8.3)
Current (mA/ tube)
Running time (mm)
3.5
65
3.0
85
2.5
80
3.0
85
3.0
80
k
B
and the values of retardation coefficient (&) and extrapolated Rf values when T = 0 (YO) for all enzyme species and their standard deviations were also computed (11). RESULTS
The KR and Y0 values and their standard deviations are given in Table 3 for Alcohol Dehydrogenase (ADH), Lactate Dehydrogenase (LDH) , Glucose 6-Phosphate Dehydrogenase (G6PD), 6-Phosphogluconate Dehydrogenase (6-PG DH) , and Isocitrate Dehydrogenase (IDH). ADH exhibited three unique bands designated as ADHl, ADHS, and ADH3 in order of decreasing mobility (Table 3). The KR and Y0 values of the three ADH species are significantly different at the 95% confidence level. ADH2 has a K, value (.062) intermediate between ADHl (0.053) and ADH3 (0.068). Similarly, the Y, value for ADH2 (0.76) is intermediate between ADHl (0.81) and ADH3 (0.64). This strongly suggests that ADH2 observed from pupal homogenate was a hybrid band composed of a mixture of ADHl and ADH3.
ENZYMES
Retardation Enzyme
Coefficients
(Ks)
OF
Tribolium.
TABLE 3 and Extrapolated
325
RI Values
when
2’ = 0 (Yo)
RR
‘KR
Yll
ADHl ADH2 ADH3
0.053 0.062 0.068
0.004 0.006 0.005
0.81 0.76 0.64
0.06 0.03 0.04
LDHI LDH2 LDH3 LDH4 LDH5
0.070 0.088 0.096 0.191 a
0.004 0.006 0.014 0.008 a
1.15 1.00 1.21 0.66 II
0.03 0.03 0.40 0.02 a
GGPDl G6PD2
0.070 0.091
0.005 0.007
0.89 0.77
0.13 0.10
6-PGDHl 6-PGDH2
0.071 0.078
0.004 0.005
0.73 0.58
0.03 0.01
IDHl IDH2 IDH3
0.064 0.071 0.078
0.005 0.010 0.004
0.67 0.60 0.51
0.02 0.01 0.01
a The KR and Yo values could not be computed could not be accurately measured.
since bands
were
WY0
faint
and
R, values
LDH exhibited four distinct bands designated as LDHl, LDHP, LDH3, and LDH4 in order of decreasing mobility (Table 3). The KR and Y0 values of the four LDH species are significantly different at the 95% confidence level. The slowest migrating band, LDH5, was observed. Since LDH5 was faint, its Rf value could not be accurately measured and computations for its KR and Y, values are omitted. GGPD exhibited two distinct bands designated as G6PDl and G6PD2 in order of decreasing mobility. The computed KR and Y,, values for these two GGPD speciesare significantly different at the 95% confidence level. GPGDH exhibited two distinct bands designated as GPGDHI and 6PGDH2 in order of decreasing mobility. The comput,ed Y, values for these two GPGDH species are significantly different while the KR values are indistinguishable at the 95% confidence level. IDH exhibited three distinct bands designated as IDHl, IDHB, and IDH3 in order of decreasing mobility. The K, and Y, values of these three IDH species are significantly different at the 95% confidence level. IDH2 has a KR value (0.071) intermediate between IDHl (0.064) and IDH3 (0.078). Similarly, the Y, value for IDH2 (0.60) is intermediate between IDHl (0.67) and IDH3 (0.51). This suggests that, IDH2 was a hybrid band composed of a mixture of IDHl and IDH3.
326
YEH
AND
BCHEINBERG
DISCUSSION
The computed KR and Y, values of the enzyme species allow the discrimination between fractionation based solely on size (intersecting Ferguson plots, constant YO), fractionation based solely on charge (parallel Ferguson plots, constant KR), and fractionation based on both (both YO’s and KE’s vary) (12). The estimated KE and Y, values of the enzyme species indicate that the 6-PGDH are charge isomers (same KR) while all other enzyme species listed in Table 3 differ both in charge and size. The results described here from various TriboZium isoenzymes may be very useful in separating and characterizing proteins extracted from small amounts of biological material. ACKNOWLEDGMENTS The authors are indebted to Dr. J. Due&en for providing the facilities protein weight determination and to the referees for their constructive criticisms.
for
REFERENCES 1. RAYMOND, S. AND WEINTRAUB, L. S. (1959) Science 30, 711. 2. ORNSTEIN, L. (1964) Ann. N. Y. Acad. Sci. 121, 321349. 3. FELGENHAUER, K. (1967) Biochim. Biophys. Acta 133, 165167. 4. GROSSBACH, U. AND WEINSTEIN, B. (1968) Anal. Biochem. 22, 311320. 5. NETJHOFF, V. (1968) Arzneim Forsch. 18, 35-39. 6. NEUHOFF, V. AND LEZIUS, A. (1968) 2. Naturforsch. B 23, 812-819. 7. GAINER, H. (1973) AnaZ. Biochem. 51, 646-650. 8. SCHEINBERG, E., BELL, A. E., AND ANDERSON, V. L. (1967) Genetics 55, 69-90. 9. DAVIS, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404427. 10. BREWER, G. J. (1970) An Introduction to Isoeyme Techniques, pp. 72, 13,
84, 97, 117. Academic Press, New York and London. 11. R~DBARD, D. AND CHRAMBACH, A. (1971) Anal. Biochem. 40, 95-134. 12. HENDRICK, J. L. AND SMITH, A. J. (1968) Arch. Biochem. Biophys. 126, 155-164. 13. WEBER, K., PRINGLE, J. R., AND OSBORN, M. (1972) Methods in Enzymotogy, Vol. 26, Part C, pp. 4, 5. Academic Press, New Ycrk and London.
Electrophoretic Beetles,
Separation Tribolium
FRANCIS The
62, 321-326 (1974)
BIOCHEMISTRY
Genetics
of Enzymes
castaneum,
C. H. YEH Laboratory,
Received
on Polyacrylamide ELIYAHU
AND
10, 1972;
accepted
Flour Gel’
SCHEINBERC
Department of Biology, Calgary, Alberta, Canada May
of Individual
June
Vuivelsity
of Calgary.
10, 1974
A method of preparing a single-beetle homogenate for separating enzymes of individual flour beetles, Tribolium castaneum, by electroplioresis on polyacrylamide gel is described. This method can be adapted to the study of enzyme activity in organisms similar to T. castaneum in size and protein concentration (450 pg dry wt), or for the analysis of very small quantities of tissue and for whole organs from a slightly larger organism.
The introduction of polyacrylamide gel (1 and 2) as supporting medium in zone electrophoresis has provided a powerful tool for identifying some biochemical loci and for studying genetic variations in natural and laboratory animal populations. It is important to determine the electrophoretic variants within single organisms in an analysis of a gene pool variability. However, difficulties arise when dealing with small organisms such as the beetle, Tribolium. These difficulties are due to a small sample size that does not allow for a reliable sample preparation and to the relatively low sensitivity of the available analytic systems. Several modifications and advances of the method as applied to microelectrophoresis have subsequently been reported (3-7). In this paper a description of a rapid, sensitive, and reliable methcd of studying the activities of several enzymes in individuals of Triboliunz castaneum by electrophoresis on polyacrylamide gel is described. MATERIALS
Experimental
AND
METHODS
Populations
Individual beetles were obtained from a foundation population Tribolium custaneum derived in 1954 from the systematic crossing
of of
‘Contribution No. 5 from the Genetics Laboratory, University of Calgary. Supported in part by a National Research Council of Canada Grant, A5451 and a University of Calgary Grant in Aid of Research awarded to the second author. Copyright All rights
@ 1974 by Academic Press, of reproduction in any form
321 Inc. reserved.
322
YEH
AND
SCHEINBERG
TABLE 1 List of Ingredients of the Stacking Part (large pore 2.5% T, 7.4’% C) and the Separation Part (small pore, 3.77-2Oyo T, 2.6% C) of Gel Together with the Procedures for Their Preparation. Solutions A, B, and C are Stocks which can be Stored for Several Weeks Under Refrigeration in Broth Bottles. Solution D Should be Made Before Each Use. Solution E can be used Within 1 wk After Preparation if Kept Under Refrigeration in Brown Bottles Temp System A (stacks pH 8.3 runs at pH Small pore (3.75-2Oa/, T) Ingredient
Amt
= 15.5
at 9.3)
Large
pore
f
0.5”C System B (stacks at pH 8.3 runs at pH 6.6)
Small pore (3.75-20y0 T)
used
Amt
(4 Acrylamide Bisacrylamide Dist Hz0 to make
See (13)
10 g
0.8 g 50 ml
See (13)
(B)” 18.15
Ammonium Dist Hz0
persulfate to make
g -
24 ml
12.8 ml -
0.72 ml 300 ml
0.1 ml 100 ml
2w
-
-
4.8
48 ml 0.4 ml 200 ml
48 ml 0.4 ml 100 ml
-
25 ml
-
80 mg 25 ml
0.35 g 25 ml
(Working
to make
Proportion See (13)
_ a Davis
(9).
g -
0
-
fD) 0.14 g 25 ml (El
(E) Sucrose Dist Hz0
-
114 g -
CD) 0.25 g 25 ml
0.8 g 50 ml (B)
2.23
g
KY to make
pore
(4 10 g
Tris Glycine 1 M HSPO, ~NHCI ~NKOH Temed Dist Hz0 to make
Riboflavin Dist H20
Large used
24 g 60 ml solution) 2A 4B D 8E c
(Working
See (13)
24 g 60 ml solution) 2A B D 4E
EKZYMES
OF Tribolium
323
eight random-mat.ing laboratory stocks of different geographical origins. This synthetic stock has been propagated each generation with a minimum of 250 randomly chosen parents and maintained in a control chamber at 32.8 +- 1°C and 72 + 2% relative humidity, in half-pint milk bottles containing standard medium (8). Enzyme Preparation A Pyrex brand glass plate (Fisher No. 31, 748B) with 9 cavities, 22 mm by 7 mm, and compatible sized glass rods were used as the homogenizers. Individual beetles were ground in 0.1 ml of sample buffer containing 0.4 PM coenzyme in 20% sucrose solution (Table 1). The slurry was then collected into a microhematocrit tube and centrifuged for 10 min at 3000 rpm. The resulting supernatant (4.5 mg protein/ml) was used immediately or stored under refrigeration for up to G hr prior to electrophoresis. Each supernatant could be analyzed for three enzymes. Polyacrylamide
Gel Electrophoresis (PAGE)
The list of ingredients of the stacking part (large pore, 2.5% T, 7.4% C) and the separating part (small pore, 3.7520% T, 2.6% C) of the gel, together with their preparation procedures are listed in Table 1. PAGE was carried out with Biichler Polyanalyst (no. 3-1750, Biichler Instruments Inc.) following a modification of the procedures given by Davis (9). In order to minimize thermal contraction or expansion of the gel during electrophoresis, polymerization and electrophoresis were carried out at the same temperature (15°C 1 05°C) following the procedures given by Rodbard and Chrambach (11). The concentrations of polymerization catalysts (Table 1) were set so that polymerization was complet,ed in about 15 min. Electrophoresis was conducted at constant current of 1 ma/tube until the tracking dye had just migrated into the separating gel and continued at 2.5-3.5 mA/tube for 65-85 min depending on the enzyme (Table 2). Samples were introduced into the gel tubes directly from the microhematocrit tubes and carefully overlaid with upper buffer. The buffers used are listed in Table 2. Migration was toward the cathode for Isocitrate Dehydrogenase and anode for all other enzyme species (Table 2). Gels were stained overnight in the dark for enzymatic activities in culture tubes (10 X 75 mm) according to the methods of Brewer (19) except that 0.1 mg/ml staining solution of phenazine methosulfate was substituted to decrease background stain in gels. Gels were stored in dilute saline solution, with the exception of Lactate Dehydrogenase gels which were stored in 7% acetic acid. The relative mobility (R,) of all enzyme species were computed (II)
324
YEH
AND
SCHEINBERG
TABLE 2 Electrode Buffers for Polyacrylamide Gel Electrophoresis at 15.5 f 0.5%. A Quantity of 0.4 PM Coenzyme is Added to Upper Electrode Buffer During Each Run to Stabilize the Enzyme. Each Buffer Preparation can be used for 36 Samples When Stored Under Refrigeration
Enzyme
Upper buffer
Lower buffer
Systern
Alcohol dehydrogenase Glucose-8phosphate dehydrogenase I. 1 Lactate dehydrogenase 6-Phosphogluconate dehydroIsocitrate dehydrogenase
M
(PH
Tris-glyeine
.12
9.2)
0.182 M Glycine/ 0.356 M Lutidine (PH 8.3)
(PH
M
Tris-HCl 8.7)
0.182 M Glycine/ 0,356 M Lutidine (PH 8.3)
Current (mA/ tube)
Running time (mm)
3.5
65
3.0
85
2.5
80
3.0
85
3.0
80
k
B
and the values of retardation coefficient (&) and extrapolated Rf values when T = 0 (YO) for all enzyme species and their standard deviations were also computed (11). RESULTS
The KR and Y0 values and their standard deviations are given in Table 3 for Alcohol Dehydrogenase (ADH), Lactate Dehydrogenase (LDH) , Glucose 6-Phosphate Dehydrogenase (G6PD), 6-Phosphogluconate Dehydrogenase (6-PG DH) , and Isocitrate Dehydrogenase (IDH). ADH exhibited three unique bands designated as ADHl, ADHS, and ADH3 in order of decreasing mobility (Table 3). The KR and Y0 values of the three ADH species are significantly different at the 95% confidence level. ADH2 has a K, value (.062) intermediate between ADHl (0.053) and ADH3 (0.068). Similarly, the Y, value for ADH2 (0.76) is intermediate between ADHl (0.81) and ADH3 (0.64). This strongly suggests that ADH2 observed from pupal homogenate was a hybrid band composed of a mixture of ADHl and ADH3.
ENZYMES
Retardation Enzyme
Coefficients
(Ks)
OF
Tribolium.
TABLE 3 and Extrapolated
325
RI Values
when
2’ = 0 (Yo)
RR
‘KR
Yll
ADHl ADH2 ADH3
0.053 0.062 0.068
0.004 0.006 0.005
0.81 0.76 0.64
0.06 0.03 0.04
LDHI LDH2 LDH3 LDH4 LDH5
0.070 0.088 0.096 0.191 a
0.004 0.006 0.014 0.008 a
1.15 1.00 1.21 0.66 II
0.03 0.03 0.40 0.02 a
GGPDl G6PD2
0.070 0.091
0.005 0.007
0.89 0.77
0.13 0.10
6-PGDHl 6-PGDH2
0.071 0.078
0.004 0.005
0.73 0.58
0.03 0.01
IDHl IDH2 IDH3
0.064 0.071 0.078
0.005 0.010 0.004
0.67 0.60 0.51
0.02 0.01 0.01
a The KR and Yo values could not be computed could not be accurately measured.
since bands
were
WY0
faint
and
R, values
LDH exhibited four distinct bands designated as LDHl, LDHP, LDH3, and LDH4 in order of decreasing mobility (Table 3). The KR and Y0 values of the four LDH species are significantly different at the 95% confidence level. The slowest migrating band, LDH5, was observed. Since LDH5 was faint, its Rf value could not be accurately measured and computations for its KR and Y, values are omitted. GGPD exhibited two distinct bands designated as G6PDl and G6PD2 in order of decreasing mobility. The computed KR and Y,, values for these two GGPD speciesare significantly different at the 95% confidence level. GPGDH exhibited two distinct bands designated as GPGDHI and 6PGDH2 in order of decreasing mobility. The comput,ed Y, values for these two GPGDH species are significantly different while the KR values are indistinguishable at the 95% confidence level. IDH exhibited three distinct bands designated as IDHl, IDHB, and IDH3 in order of decreasing mobility. The K, and Y, values of these three IDH species are significantly different at the 95% confidence level. IDH2 has a KR value (0.071) intermediate between IDHl (0.064) and IDH3 (0.078). Similarly, the Y, value for IDH2 (0.60) is intermediate between IDHl (0.67) and IDH3 (0.51). This suggests that, IDH2 was a hybrid band composed of a mixture of IDHl and IDH3.
326
YEH
AND
BCHEINBERG
DISCUSSION
The computed KR and Y, values of the enzyme species allow the discrimination between fractionation based solely on size (intersecting Ferguson plots, constant YO), fractionation based solely on charge (parallel Ferguson plots, constant KR), and fractionation based on both (both YO’s and KE’s vary) (12). The estimated KE and Y, values of the enzyme species indicate that the 6-PGDH are charge isomers (same KR) while all other enzyme species listed in Table 3 differ both in charge and size. The results described here from various TriboZium isoenzymes may be very useful in separating and characterizing proteins extracted from small amounts of biological material. ACKNOWLEDGMENTS The authors are indebted to Dr. J. Due&en for providing the facilities protein weight determination and to the referees for their constructive criticisms.
for
REFERENCES 1. RAYMOND, S. AND WEINTRAUB, L. S. (1959) Science 30, 711. 2. ORNSTEIN, L. (1964) Ann. N. Y. Acad. Sci. 121, 321349. 3. FELGENHAUER, K. (1967) Biochim. Biophys. Acta 133, 165167. 4. GROSSBACH, U. AND WEINSTEIN, B. (1968) Anal. Biochem. 22, 311320. 5. NETJHOFF, V. (1968) Arzneim Forsch. 18, 35-39. 6. NEUHOFF, V. AND LEZIUS, A. (1968) 2. Naturforsch. B 23, 812-819. 7. GAINER, H. (1973) AnaZ. Biochem. 51, 646-650. 8. SCHEINBERG, E., BELL, A. E., AND ANDERSON, V. L. (1967) Genetics 55, 69-90. 9. DAVIS, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404427. 10. BREWER, G. J. (1970) An Introduction to Isoeyme Techniques, pp. 72, 13,
84, 97, 117. Academic Press, New York and London. 11. R~DBARD, D. AND CHRAMBACH, A. (1971) Anal. Biochem. 40, 95-134. 12. HENDRICK, J. L. AND SMITH, A. J. (1968) Arch. Biochem. Biophys. 126, 155-164. 13. WEBER, K., PRINGLE, J. R., AND OSBORN, M. (1972) Methods in Enzymotogy, Vol. 26, Part C, pp. 4, 5. Academic Press, New Ycrk and London.