Elrilruphoresls 1991, 12, 381 -382
Oliver Farnbacher Sebastian Weidinger Institut fur Anthropologie und Humangenetik der Universitat Munchen
AGIF of soluble glutamate-oxaloacetate transaminase
381
Description of a new variant in the glutamateoxaloacetate transaminase system by thin-layer agarose gel isoelectric focusing Thin-layer isoelectric focusing in agarose within the pH range of 4.0-6.5 has shown a high resolution of the soluble cytoplasmic glutamate-oxaloacetate trans aminase (GOT,) banding pattern.The complex pattern of the common GOT, phe, notype consists of eight bands with different intensities. A genetic variant of GOT,, which has been designated GOT, Mexico, could tentatively be identifiec'
Genetic polymorphism of human cytoplasmic glutamateoxaloacetate transaminase (GOT,-EC 2.6.1.1, aspartate aminotransferase) was first demonstrated in 1971 in red cells using starch gel electrophoresis [l]. One common phenotype (GOT, 1) and two rare ones (GOT, 2-1 and GOT, 3-1), which are controlled by three autosomal codominant alleles, were observed in a Japanese population. In 1982, Wurzinger and Mohrenweiser [2] described seven different electrophoretic variants in families from various racial and ethnic groups. In European populations, GOT, seems to be a monomorphic enzyme. According to a recent study [3] , using agarose gel isoelectric focusing (AGIF), the existence of some rare GOT, variants is also suspected in Europeans. The locus of the cytoplasmic enzyme has been mapped to chromosome 10q25.3 by somatic cell hybridization studies [4].Another form of the enzyme (GOT,) is associated with the mitochondria. In this paper we provide evidence of a further GOT, variant that is not detectable by conventional electrophoresis. Blood samples were obtained from healthy individuals in the course of paternity testing. Erythrocytes were washed twice in a physiological saline solution and frozen at -20 "C until use. After thawing, hemolysates were prepared by ultrasonification. One hundred pL of hemolysate were diluted with an equal volume of 0.04 M dithiothreitol (DTT) dissolved in distilled water. The DTT-treated samples were incubated for 1 h at room temperature. Thin-layer agarose gels (dimension 250 X 115 X 0.5 mm) were prepared on GelBond film (LKB, 1850-101) in a cassette with a U-shaped spacer. The gel solution consisted of 0.18 g agarose-IEF (Pharmacia, No. 17-0468-Ol), 4.5 mL glycerol (87%) and 13 mL distilled water. After melting, the mixture was degassed and cooled to 75 "C,followed by addition of 0.2 mL Pharmalytes pH 4.0-6.5 and 0.4 mL Pharmalytes pH 5-6. Before use the gel was precooled in the refrigerator for at least 2 h. As electrolytes, 0.05 M H,SO, and 1.0 M NaOH were used at the anode and cathode, respectively. For GOT, analysis 6 pL of samples were applied with an application strip near the cathode. Separation was carried out in an LKB-Multiphor I1 chamber within 150 min at
a temperature of 10°C. Maximum settings were 2000 V, 10 mA, and 15 W. The high concentration of glycerol (ca. 30% w/v) in the gel guaranteed its stability while focusing at 2000 V and sharpened the band patterns [5]. For the detection of GOT, phenotypes, a Whatman No. 1 filter paper was soaked in 5 mL 0.2 M sodium phosphate buffer, pH 7.5 containing 45 mg L-aspartate, 6 mg a-ketoglutarate, 6 mg NADH, 10 mg 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazoliumbromide (MTT), 0.5 mg pyridoxal 5-phosphate, and 6 pL malate dehydrogenase (MDH) (Boehringer, No. 127256).The filter paper was applied to the gel surface. The gel was incubated for 15 min at 37°C and then stained with a phenazine methosulfate (PMS) solution (0.1 Yo w/v). Using this technique the GOT, bands appeared clear on a blue background. Figure 1 presents the band patterns of GOT, isozymes obtained by AGIFin the pH range of 4.0-6.5.The common GOT, 1 phenotype was separated into eight bands, some with lower and some with higher intensities. In the course of paternity testing an unusual phenotype was observed in a mother and her child. This strongly suggests that the observed variation is a genetically controlled characteristic, designated GOT, Mexico according to the origin of the mother. The father had type GOT, 1. In comparison to GOT, 1,the variant phenotype shows two less sharp bands with a smaller corridor in the cathodal region and also a slight shift of these bands towards the anode.Presumably the variant Mexico has a similar isoelectric point as GOT, 1. In starch gel electrophoresis, type GOT, 1-Mexico was not distinguishable from the common GOT, 1. Both phenotypes show only a single band although a triple-banding pattern is normally obtained in GOT, heterozygotes.The varia-
Correspondence: Priv.-Doz. Dr. Sebastian Weidinger, Institut fur Anthropologie und Humangenetik der Universitat Munchen, Richard-WagnerStr. lO/l, DW-8000 Miinchen 2 , Germany Abbreviations: AGIF, agarose gel isoelectric focusing; DTT, dithiothreitol; GOT,, cytoplasmic glutamate-oxaloacetate transaminase; IEF, isoelectric focusing; MDH, malate dehydrogenase; MTT, 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; PMS, phenazine methosulfate
0VCH VerlagsgesellschaftmbH, D-6940 Weinheim, 1991
Figure 1. Erythrocyte GOT, phenotypes as obtained bq AGIF, p H range 4.0-6.5. Anode is at the top.From left to right: lanes (1),(2) and (3) GOT, 1, (4) GOT, 1-Mexico (mother), ( 5 ) GOT, 1-Mexico (child), and (6) and ( 7 ) GOT, 1. 0173-0835/91/0505-0381 $3.50+.25/0
382
Electrophoresis 1991, 12, 382-383
K.-K. Lee and A E. Ellis
tion that was observed for GOT, Mexico by AGIF was reproducible. Experiments carried out with smaller pH gradients have not given a higher resolution of GOT,. Using AGIFa further rare GOT, variant was revealed in the heterozygous state. To our knowledge a similar mutant has not yet been described. The method is easy and fast to carry out and reliable for the determination of GOT, phenotypes. It may become useful for population studies as well as for forensic and clinical investigations. Received January 22, 1991
Kuo-Kau Lee Anthony E. Ellis
References [l] Chen,S.H.andGiblett,E.R.,Amer.J.Hum.Genet.1971,23,419-424. [2] Wurzinger, K. H. and Mohrenweiser, H. W., A n n . Hum. Genet. 1982, 46, 191-201. [3] Weidinger, S., Farnbacher, O., Schwarzfischer, F. and Cleve, H., in: Radola, B. J. (Ed.), Electrophoresis Forum '89, Technical University Munich 1989, pp. 508-513. [4] Creagon, R., Tischfield, J., Mc Morris, F. A., Chen, S., Hirschi, M., Chen,T. R., Riciuti, F. and Ruddie, F. H., Cytogenef. Cell Genet. 1973, 12, 187. [5] Schlicht, C.,Fauth,M. and Radola, B. J., in: Radola,B. J. (Ed.), Electrophoresis Forum '89, Technical University Munich 1989. pp. 316320.
A novel method for specific visualisation of serum albumin in polyacrylamide gels by iodine staining
DAFS Marine Laboratory, Aberdeen Bovine serum, bovine serum albumins (delipidated or globulin free or Fraction V), rabbit serum, rabbit serum albumin, Atlantic salmon serum, purified Atlantic salmon serum albumin, human plasma a,-macroglobulin, hernocyanin, trypsin inhibitor, bovine transferrin and bovine lactoferrin were examined by a novel method for specific visualisation of albumins. In native polyacrylamide gel electrophoresis the albumins were visualised by iodine staining as a transparent spot against a brown background whilst the other proteins could not be visualised. It is suggested that the brown background was due to penetration of the gel by iodine while the chemical binding of iodine by albumin produced a decolourisation reaction. This novel method provides a fast and simple approach to identifying serum albumin in polyacrylamide gels. Serum albumin is a major serum protein in mammals and is well studied [ 11. However, the presence and characteristics of fish serum albumin have been controversial for a long time. Recently, two groups [2,3] have independently purified and identified a 68 kDa salmonid serum albumin in rainbow trout and Atlantic salmon, respectively, confirming the presence of salmonid serum albumin. Iodide binding protein(s) of teleost fish, which shared some characteristics of vertebrate serum albumin, have been previously reported [4, 51, but a fast and simple method of electrophoretic identification is still not established. In the present communication, we describe a novel method to identify vertebrate serum albumin in polyacrylamide gel electrophoresis by iodine staining.
ferrin, rabbit serum albumin (Fraction V), human plasma a,-macroglobulin, hemocyanin (from keyhole limpets), and trypsin inhibitor (from soybean) were purchased from Sigma (St. Louis, MO). Rabbit serum was collected from clotted blood of a New Zealand white rabbit weighing 2 kg. Fish serum was collected from four immature male sea water Atlantic salmon (Sulrno sularl.) weighing 200-300 g. The fish were anaesthetised with benzocaine and blood was withdrawn by hypodermic syringe from the caudal vessels. Atlantic salmon serum albumin was purified as previously described [3].The purified fraction, consisting of a single 68 kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, contained 0.40 mg/mL protein, 0.33 mg/ mL total lipids and 0.02 mg/mL total cholesterols.
Bovine serum was supplied by the Scottish Antibody Production Unit (Law Hospital, Carluke, Lanarkshire, Scotland). Bovine serum albumin (Fraction V), bovine serum albumin (essentially fatty acid free), bovine serum albumin (essentially globulin free), bovine transferrin, bovine lacto-
Phastgels (12.5 Yo polyacrylamide) were used in the PhastSystem (Pharmacia, Uppsala). The procedures were conducted according to the manufacturer's recommendations. After electrophoresis, gels were stained with Coomassie Brilliant Blue as recommended by the PhastSystem protocols or stained with saturated iodine solution (in physiological phosphate buffered saline, PBS, pH 7.2, Dulbecco's Ca2+and Mg2+free, Gibco). The staining by saturated iodine solution was performed as follows: the Phastgel (after
Correspondence: A. E. Ellis, DAFS Marine Laboratory, Victoria Road, Aberdeen, AB9 SDB, Scotland
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
0173-0835/91/0505-0382 $3,50+.25/0
Oliver Farnbacher Sebastian Weidinger Institut fur Anthropologie und Humangenetik der Universitat Munchen
AGIF of soluble glutamate-oxaloacetate transaminase
381
Description of a new variant in the glutamateoxaloacetate transaminase system by thin-layer agarose gel isoelectric focusing Thin-layer isoelectric focusing in agarose within the pH range of 4.0-6.5 has shown a high resolution of the soluble cytoplasmic glutamate-oxaloacetate trans aminase (GOT,) banding pattern.The complex pattern of the common GOT, phe, notype consists of eight bands with different intensities. A genetic variant of GOT,, which has been designated GOT, Mexico, could tentatively be identifiec'
Genetic polymorphism of human cytoplasmic glutamateoxaloacetate transaminase (GOT,-EC 2.6.1.1, aspartate aminotransferase) was first demonstrated in 1971 in red cells using starch gel electrophoresis [l]. One common phenotype (GOT, 1) and two rare ones (GOT, 2-1 and GOT, 3-1), which are controlled by three autosomal codominant alleles, were observed in a Japanese population. In 1982, Wurzinger and Mohrenweiser [2] described seven different electrophoretic variants in families from various racial and ethnic groups. In European populations, GOT, seems to be a monomorphic enzyme. According to a recent study [3] , using agarose gel isoelectric focusing (AGIF), the existence of some rare GOT, variants is also suspected in Europeans. The locus of the cytoplasmic enzyme has been mapped to chromosome 10q25.3 by somatic cell hybridization studies [4].Another form of the enzyme (GOT,) is associated with the mitochondria. In this paper we provide evidence of a further GOT, variant that is not detectable by conventional electrophoresis. Blood samples were obtained from healthy individuals in the course of paternity testing. Erythrocytes were washed twice in a physiological saline solution and frozen at -20 "C until use. After thawing, hemolysates were prepared by ultrasonification. One hundred pL of hemolysate were diluted with an equal volume of 0.04 M dithiothreitol (DTT) dissolved in distilled water. The DTT-treated samples were incubated for 1 h at room temperature. Thin-layer agarose gels (dimension 250 X 115 X 0.5 mm) were prepared on GelBond film (LKB, 1850-101) in a cassette with a U-shaped spacer. The gel solution consisted of 0.18 g agarose-IEF (Pharmacia, No. 17-0468-Ol), 4.5 mL glycerol (87%) and 13 mL distilled water. After melting, the mixture was degassed and cooled to 75 "C,followed by addition of 0.2 mL Pharmalytes pH 4.0-6.5 and 0.4 mL Pharmalytes pH 5-6. Before use the gel was precooled in the refrigerator for at least 2 h. As electrolytes, 0.05 M H,SO, and 1.0 M NaOH were used at the anode and cathode, respectively. For GOT, analysis 6 pL of samples were applied with an application strip near the cathode. Separation was carried out in an LKB-Multiphor I1 chamber within 150 min at
a temperature of 10°C. Maximum settings were 2000 V, 10 mA, and 15 W. The high concentration of glycerol (ca. 30% w/v) in the gel guaranteed its stability while focusing at 2000 V and sharpened the band patterns [5]. For the detection of GOT, phenotypes, a Whatman No. 1 filter paper was soaked in 5 mL 0.2 M sodium phosphate buffer, pH 7.5 containing 45 mg L-aspartate, 6 mg a-ketoglutarate, 6 mg NADH, 10 mg 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazoliumbromide (MTT), 0.5 mg pyridoxal 5-phosphate, and 6 pL malate dehydrogenase (MDH) (Boehringer, No. 127256).The filter paper was applied to the gel surface. The gel was incubated for 15 min at 37°C and then stained with a phenazine methosulfate (PMS) solution (0.1 Yo w/v). Using this technique the GOT, bands appeared clear on a blue background. Figure 1 presents the band patterns of GOT, isozymes obtained by AGIFin the pH range of 4.0-6.5.The common GOT, 1 phenotype was separated into eight bands, some with lower and some with higher intensities. In the course of paternity testing an unusual phenotype was observed in a mother and her child. This strongly suggests that the observed variation is a genetically controlled characteristic, designated GOT, Mexico according to the origin of the mother. The father had type GOT, 1. In comparison to GOT, 1,the variant phenotype shows two less sharp bands with a smaller corridor in the cathodal region and also a slight shift of these bands towards the anode.Presumably the variant Mexico has a similar isoelectric point as GOT, 1. In starch gel electrophoresis, type GOT, 1-Mexico was not distinguishable from the common GOT, 1. Both phenotypes show only a single band although a triple-banding pattern is normally obtained in GOT, heterozygotes.The varia-
Correspondence: Priv.-Doz. Dr. Sebastian Weidinger, Institut fur Anthropologie und Humangenetik der Universitat Munchen, Richard-WagnerStr. lO/l, DW-8000 Miinchen 2 , Germany Abbreviations: AGIF, agarose gel isoelectric focusing; DTT, dithiothreitol; GOT,, cytoplasmic glutamate-oxaloacetate transaminase; IEF, isoelectric focusing; MDH, malate dehydrogenase; MTT, 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; PMS, phenazine methosulfate
0VCH VerlagsgesellschaftmbH, D-6940 Weinheim, 1991
Figure 1. Erythrocyte GOT, phenotypes as obtained bq AGIF, p H range 4.0-6.5. Anode is at the top.From left to right: lanes (1),(2) and (3) GOT, 1, (4) GOT, 1-Mexico (mother), ( 5 ) GOT, 1-Mexico (child), and (6) and ( 7 ) GOT, 1. 0173-0835/91/0505-0381 $3.50+.25/0
382
Electrophoresis 1991, 12, 382-383
K.-K. Lee and A E. Ellis
tion that was observed for GOT, Mexico by AGIF was reproducible. Experiments carried out with smaller pH gradients have not given a higher resolution of GOT,. Using AGIFa further rare GOT, variant was revealed in the heterozygous state. To our knowledge a similar mutant has not yet been described. The method is easy and fast to carry out and reliable for the determination of GOT, phenotypes. It may become useful for population studies as well as for forensic and clinical investigations. Received January 22, 1991
Kuo-Kau Lee Anthony E. Ellis
References [l] Chen,S.H.andGiblett,E.R.,Amer.J.Hum.Genet.1971,23,419-424. [2] Wurzinger, K. H. and Mohrenweiser, H. W., A n n . Hum. Genet. 1982, 46, 191-201. [3] Weidinger, S., Farnbacher, O., Schwarzfischer, F. and Cleve, H., in: Radola, B. J. (Ed.), Electrophoresis Forum '89, Technical University Munich 1989, pp. 508-513. [4] Creagon, R., Tischfield, J., Mc Morris, F. A., Chen, S., Hirschi, M., Chen,T. R., Riciuti, F. and Ruddie, F. H., Cytogenef. Cell Genet. 1973, 12, 187. [5] Schlicht, C.,Fauth,M. and Radola, B. J., in: Radola,B. J. (Ed.), Electrophoresis Forum '89, Technical University Munich 1989. pp. 316320.
A novel method for specific visualisation of serum albumin in polyacrylamide gels by iodine staining
DAFS Marine Laboratory, Aberdeen Bovine serum, bovine serum albumins (delipidated or globulin free or Fraction V), rabbit serum, rabbit serum albumin, Atlantic salmon serum, purified Atlantic salmon serum albumin, human plasma a,-macroglobulin, hernocyanin, trypsin inhibitor, bovine transferrin and bovine lactoferrin were examined by a novel method for specific visualisation of albumins. In native polyacrylamide gel electrophoresis the albumins were visualised by iodine staining as a transparent spot against a brown background whilst the other proteins could not be visualised. It is suggested that the brown background was due to penetration of the gel by iodine while the chemical binding of iodine by albumin produced a decolourisation reaction. This novel method provides a fast and simple approach to identifying serum albumin in polyacrylamide gels. Serum albumin is a major serum protein in mammals and is well studied [ 11. However, the presence and characteristics of fish serum albumin have been controversial for a long time. Recently, two groups [2,3] have independently purified and identified a 68 kDa salmonid serum albumin in rainbow trout and Atlantic salmon, respectively, confirming the presence of salmonid serum albumin. Iodide binding protein(s) of teleost fish, which shared some characteristics of vertebrate serum albumin, have been previously reported [4, 51, but a fast and simple method of electrophoretic identification is still not established. In the present communication, we describe a novel method to identify vertebrate serum albumin in polyacrylamide gel electrophoresis by iodine staining.
ferrin, rabbit serum albumin (Fraction V), human plasma a,-macroglobulin, hemocyanin (from keyhole limpets), and trypsin inhibitor (from soybean) were purchased from Sigma (St. Louis, MO). Rabbit serum was collected from clotted blood of a New Zealand white rabbit weighing 2 kg. Fish serum was collected from four immature male sea water Atlantic salmon (Sulrno sularl.) weighing 200-300 g. The fish were anaesthetised with benzocaine and blood was withdrawn by hypodermic syringe from the caudal vessels. Atlantic salmon serum albumin was purified as previously described [3].The purified fraction, consisting of a single 68 kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, contained 0.40 mg/mL protein, 0.33 mg/ mL total lipids and 0.02 mg/mL total cholesterols.
Bovine serum was supplied by the Scottish Antibody Production Unit (Law Hospital, Carluke, Lanarkshire, Scotland). Bovine serum albumin (Fraction V), bovine serum albumin (essentially fatty acid free), bovine serum albumin (essentially globulin free), bovine transferrin, bovine lacto-
Phastgels (12.5 Yo polyacrylamide) were used in the PhastSystem (Pharmacia, Uppsala). The procedures were conducted according to the manufacturer's recommendations. After electrophoresis, gels were stained with Coomassie Brilliant Blue as recommended by the PhastSystem protocols or stained with saturated iodine solution (in physiological phosphate buffered saline, PBS, pH 7.2, Dulbecco's Ca2+and Mg2+free, Gibco). The staining by saturated iodine solution was performed as follows: the Phastgel (after
Correspondence: A. E. Ellis, DAFS Marine Laboratory, Victoria Road, Aberdeen, AB9 SDB, Scotland
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
0173-0835/91/0505-0382 $3,50+.25/0
