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Esterases of chicken serum located on an acrylamide gel
Comp. Biochem. Physiol., 1973, Vol. 45B, pp. 109 to 111. Pergamon Press. Printed in Great Britain
ESTERASES OF CHICKEN SERUM LOCATED ON AN ACRYLAMIDE GEL BRUCE GLICK Poultry Science Department, Mississippi State University, State College, Mississippi 39762 (Received 18 September 1972)
A b s t r a c t - - 1 . Esterase (ES) activity was observed in six regions of an acrylamide gel. 2. ES-1, ES-2, ES-4, ES-5 and ES-6 were associated with five serum proteins: Rho-2, and bands 2, 14, 16 and 17. 3. ES-4 was eliminated by eserine (10 -8 M).
INTRODUCTION AN PLECTROPrmROORAM has been developed in our laboratory b y employing disc electrophoresis (Glick, 1968). O f the twenty bands included in the electropherogram, nine bands have been identified with specific s e r u m proteins. F u r t h e r characterization of these bands as well as possible identification of the remaining bands has been a research goal of this laboratory. Since esterase activity of poultry plasma and s e r u m has been revealed b y starch gel electrophoresis (Kaminski, 1964; Kaminski & Jeanne-Rose, 1964; Kimura, 1969, 1970; Petrovsky et al., 1970), experiments were designed to determine the esterase activity of the protein bands in our disc electropherogram. T h e identification of esterase activity and the differentiation of aliesterases and acetylcholinesterases will be reported in this paper. MATERIALS AND M E T H O D S A New Hampshire strain of chickens developed by Professor L. J. Dreesen was used in these experiments. Birds were fed a basal ration devoid of antibiotics and other medication (Morgan & Glick, 1972). Serum was collected at 6 weeks of age. The serum was electrophoresed within 24 hr of collection. The disc electrophoresis (30 rain, 5 mA/gel) was performed in three separate polymerized layers of polyacrylamide gel: sample gel, stacking gel and separating gel. The stock solutions used in making the gels and the electrophoretic procedure have been described (Davis, 1964). Our procedure for identifying the bands have been previously outlined (Glick, 1968). The esterase procedure included presoaking the gels in Trls-HC1 buffer (pH 7"4) for 20 rain (22°C) and transferring the gel to a solution containing I ml of the "stock substrate", 24 ml of Tris-HC1 buffer and 25 mg fast blue B. The gels were incubated in the latter solution for 20 rain at 37°C. Additional bands did not develop following longer periods of incubation. The "stock substrate" contained 3 ml ethyl alcohol, 2 ml Tris-HC1 buffer and 46.5 nag B naphthylacetate. Eserine (10 -a M) was added during the presoaking period to 109
110
BRUCE GLICK
identify acetylcholinesterases. T h e results with eserine were duplicated with acetylthiocholine. A pink to orange color accompanied by some flocculation occurred immediately upon adding 1 ml of the "stock substrate" to the buffer-dye mixture. Following electrophoresis, duplicate gels were stained for protein and esterase activity or a single gel was sliced longitudinally and one-half stained for protein and the other for esterase activity. T h e esterase active bands were designated ES-1 (P2), ES-2 (No. 2), ES-3 (No. 12), ES-4 (No. 14), ES-5 (No. 16) and ES-6 (No. 17). T h e numbers in parentheses correspond to the protein stained bands. RESULTS AND DISCUSSION
Six areas exhibiting esterase activity were identified (Figs. 1 and 2). The two most rapidly migrating bands ES-1 and ES-2 were associated with P2 and band 2, respectively, and were always diffuse in their appearance. ES-3 migrated 9-10 m m from the origin and varied in its association with band 12 (Fig. 1). The most ORIGIN
__
__
__
ORIGIN
17
ES-
16
ES-$
6
ES-4
14
12
. . . . . . . . . .
ES-3
. . . . . . . . . . . . . . . . . . . .
ES-2
--, ,,-,,,-, --, --,
lo
2 1 Pz
i
I lii li
i iii i i
Pl
mluutuntnnlull
i
. . . . . . . . . .
ES-1
FIG. 1. A partial drawing of an electropherogram of serum protein (left) from a cyclophosphamide-treated bird compared with bands developed following staining for serum esterase (right). T h e numbers on the left are according to Glick (1968). F o r reference, bands 1 and 10 are albumin and major transferrin, respectively. T h e dash-dot notation indicates diffuse bands.
intensely staining band was ES-4 which migrated between 4 and 5 m m from the origin. This is in the region occupied by immunoglobulin G (IgG). However, since IgG masks bands 14 and 13, one cannot determine from the serum of a normal bird which protein exhibited esterase activity (Glick, 1968). An agammaglobulinemic bird has been produced in our laboratory by injecting 4 mg of cyclophosphamide (Cy) on the first 2 days after hatching (Glick, 1971). Using the serum from Cy-treated birds, ES-4 and band 14 were identically located in the gel (Fig. 2). Two thin lines, pink to orange, were observed at the points occupied by bands 17 and 16 (IgM) and designated ES-5 and ES-6, respectively.
ESTERASES OF CHICKEN SERUM LOCATED ON AN ACRYLAMIDE GEL BRUCE GLICK Poultry Science Department, Mississippi State University, State College, Mississippi 39762 (Received 18 September 1972)
A b s t r a c t - - 1 . Esterase (ES) activity was observed in six regions of an acrylamide gel. 2. ES-1, ES-2, ES-4, ES-5 and ES-6 were associated with five serum proteins: Rho-2, and bands 2, 14, 16 and 17. 3. ES-4 was eliminated by eserine (10 -8 M).
INTRODUCTION AN PLECTROPrmROORAM has been developed in our laboratory b y employing disc electrophoresis (Glick, 1968). O f the twenty bands included in the electropherogram, nine bands have been identified with specific s e r u m proteins. F u r t h e r characterization of these bands as well as possible identification of the remaining bands has been a research goal of this laboratory. Since esterase activity of poultry plasma and s e r u m has been revealed b y starch gel electrophoresis (Kaminski, 1964; Kaminski & Jeanne-Rose, 1964; Kimura, 1969, 1970; Petrovsky et al., 1970), experiments were designed to determine the esterase activity of the protein bands in our disc electropherogram. T h e identification of esterase activity and the differentiation of aliesterases and acetylcholinesterases will be reported in this paper. MATERIALS AND M E T H O D S A New Hampshire strain of chickens developed by Professor L. J. Dreesen was used in these experiments. Birds were fed a basal ration devoid of antibiotics and other medication (Morgan & Glick, 1972). Serum was collected at 6 weeks of age. The serum was electrophoresed within 24 hr of collection. The disc electrophoresis (30 rain, 5 mA/gel) was performed in three separate polymerized layers of polyacrylamide gel: sample gel, stacking gel and separating gel. The stock solutions used in making the gels and the electrophoretic procedure have been described (Davis, 1964). Our procedure for identifying the bands have been previously outlined (Glick, 1968). The esterase procedure included presoaking the gels in Trls-HC1 buffer (pH 7"4) for 20 rain (22°C) and transferring the gel to a solution containing I ml of the "stock substrate", 24 ml of Tris-HC1 buffer and 25 mg fast blue B. The gels were incubated in the latter solution for 20 rain at 37°C. Additional bands did not develop following longer periods of incubation. The "stock substrate" contained 3 ml ethyl alcohol, 2 ml Tris-HC1 buffer and 46.5 nag B naphthylacetate. Eserine (10 -a M) was added during the presoaking period to 109
110
BRUCE GLICK
identify acetylcholinesterases. T h e results with eserine were duplicated with acetylthiocholine. A pink to orange color accompanied by some flocculation occurred immediately upon adding 1 ml of the "stock substrate" to the buffer-dye mixture. Following electrophoresis, duplicate gels were stained for protein and esterase activity or a single gel was sliced longitudinally and one-half stained for protein and the other for esterase activity. T h e esterase active bands were designated ES-1 (P2), ES-2 (No. 2), ES-3 (No. 12), ES-4 (No. 14), ES-5 (No. 16) and ES-6 (No. 17). T h e numbers in parentheses correspond to the protein stained bands. RESULTS AND DISCUSSION
Six areas exhibiting esterase activity were identified (Figs. 1 and 2). The two most rapidly migrating bands ES-1 and ES-2 were associated with P2 and band 2, respectively, and were always diffuse in their appearance. ES-3 migrated 9-10 m m from the origin and varied in its association with band 12 (Fig. 1). The most ORIGIN
__
__
__
ORIGIN
17
ES-
16
ES-$
6
ES-4
14
12
. . . . . . . . . .
ES-3
. . . . . . . . . . . . . . . . . . . .
ES-2
--, ,,-,,,-, --, --,
lo
2 1 Pz
i
I lii li
i iii i i
Pl
mluutuntnnlull
i
. . . . . . . . . .
ES-1
FIG. 1. A partial drawing of an electropherogram of serum protein (left) from a cyclophosphamide-treated bird compared with bands developed following staining for serum esterase (right). T h e numbers on the left are according to Glick (1968). F o r reference, bands 1 and 10 are albumin and major transferrin, respectively. T h e dash-dot notation indicates diffuse bands.
intensely staining band was ES-4 which migrated between 4 and 5 m m from the origin. This is in the region occupied by immunoglobulin G (IgG). However, since IgG masks bands 14 and 13, one cannot determine from the serum of a normal bird which protein exhibited esterase activity (Glick, 1968). An agammaglobulinemic bird has been produced in our laboratory by injecting 4 mg of cyclophosphamide (Cy) on the first 2 days after hatching (Glick, 1971). Using the serum from Cy-treated birds, ES-4 and band 14 were identically located in the gel (Fig. 2). Two thin lines, pink to orange, were observed at the points occupied by bands 17 and 16 (IgM) and designated ES-5 and ES-6, respectively.