EIeclrophoresis 1990, I I . 509-5 10
Katsunori Akiyama Departmentof Legal Medicine,Tokyo Womens’s Medical College, Tokyo
isoelectric focusing of 6-PGD
509
Analysis of 6-phosphogluconatedehydrogenase isozymes by polyacrylamide gel isoelectric focusing Three common phenotypes of 6-phosphogluconate dehydrogenase (6-PGD) were identified by enzyme visualization following isoelectric focusing in photochemically polymerized polyacrylamide gels whereas in chemically polymerized gels no activity could be detected. Gene frequencies calculated from 429 individuals were 0.9 15 and 0.085 for 6-PGD*A and 6-PGD*C, respectively, similar to the gene frequencies of Japanese samples previously reported.
6-Phosphogluconate dehydrogenase (EC 1.1.1.44; 6-PGD) is an important metabolic enzyme in the hexose monophosphate shunt pathway catalyzing the conversion of 6-phosphogluconate to ribulose-5-phosphate. The genetic polymorphism of 6-PGD in human red blood cell was first described by Fildes and Parr [ ll. They presented evidence that the two common alleleproducts, 6-PGD*A and 6-PGD*C (at that time called 6-PGD*B) could be identified by starch gel electrophoresis and enzyme activity staining procedures. Electrophoretic variants of the rare 6-PGD alleles have also been detected in various human populations of different ethnic origin, including the 6-PGD*Wantoat, 6-PGD*Kadar, 6-PGD*Bombay [21, 6-PGD*Elcho 131, 6-PGD*Singapore [41, 6-PGD*Thai [5], 6-PGD*Oshakati [6], 6-PGD*Richmond [ 7 , 81, 6-PGD*Hackney [ 7 ] , 6-PGD*Freiburg [9], and the 5-PGD*Lowell [ 101 alleles. In Japan, the distribution of 6-PGD phenotypes using starch gel electrophoresis has been described by several authors I1 1-231. Harada [241 has described the 6-PGD phenotypes and a rare 6-PGD*Singapore variant after using isoelectric focusing (IEF) in acetate membranes with Ampholine carrier ampholytes and sucrose. In this report, the analysis of 6-PGD phenotypes in Japanese blood samples by polyacrylamide gel I E F in Ampholine carrier ampholytes is described. Hemolysates (429 blood samples) were prepared by freezing and thawing of washed and packed fresh red blood cells. The
Correspondence:Dr. Katsunori Akiyama, Department of Legal Medicine, Tokyo Women’s Medical College, Tokyo 162, Japan Abbreviations: IEF, isoelectric focusing; 6-PGD, 6-phosphogluconate dehydrogenase
samples for electrophoresis were a 1:10dilution ofhemolysate in 0.05 M dithiothreitol (DTT), and incubated for 10 min at room temperature. Polyacrylamide gels (1 10 x 240 x 0.2 mm) were prepared according to Finney [251. This gel consisted of 3.45 mL acrylamide (29.1 %), 5.25 mL N,N’-methylenebisacrylamide (0.9 %), 0.2 mL riboflavin (20 mg/%), 0.68 g 3-(N-morpholino)propanesulfonicacid (MOPS), 2.5 g sucrose, 1.5 mL carrier ampholytes (Ampholine, LKB) pH 5-7 and p H 7-9 in a 1:2 ratio, and was diluted to 20 mL with water. IEF was carried out at 1200 V, 50 mA and 25 W for 4 h with cooling at 4 “C. The 6-PGD enzyme activity staining procedures were carried out according to Harris and Hopkinson [261. Figure 1 shows the three common 6-PGD phenotypes, 6P G D A, 6-PGD AC and 6-PGD C, separated by I E F in aphotochemically polymerized polyacrylamide gel. Chemically polymerized I E F gels are not recommended. Figure 2 is a schematic representation of the I E F patterns obtained from the common 6-PGD phenotypes in a p H 5.8-6.2 gradient. For each 6-PGD phenotype characterized by starch gel electrophoresis, it was observed that the 6-PGD A type consisted of one high activity component (a), the 6-PGD AC type showed a dimeric pattern consisting of two high activity components, (a) and (c2), and one low activity component (cl), and the 6P G D C type consisted of two high activity components, (cl) and (c2), and one low activity component (a). Using the low voltage I E F electrophoresis method reported by Yuasa et al. [271, component (a) of the 6-PGD A type could be separated into two bands, but then the ( c l ) and (c2) bands ofthe 6-PGD AC and 6-PGD C types could not be detected. It was found that the 6-PGD band patterns described in the present study were similar to the patterns reported by Harada 1241 after using IEF on cellulose acetate membranes.
7igure 1. Three common 6-PGD phenoypes separated by IEF in a photochemially potymerized polyacrylamide gel. A) Enzyme visualization, (B) Schematic epresentation. 0 173-0835/90/0606-0509 $02.5010
5 10
Electrophoresis 1990,l I , 5 10-5 13
A. G. Lenz er nl.
Gene frequencies calculated from 429 unrelated J apanese red blood samples were 0.915 and 0.085 for 6-PGD*A and 6PGD*C, respectively. In our samples, there were n o variant types deviating from the three commoin phenotypes. These results did not differ from J apanese samples reported by other authors in Japan. The 6-PGD isozyme system found by polyacrylamide gel IEF could be applied in the field of forensic and legal medicine. Received February 14, 1990
References 111 Fildes, R. A. and Parr, C. W., Nature 1963,200,890-891.
[21 Blake, N. M., Saha, N., McDermid, E. M. and Kirk, R. L., Hum. Genet. 1974,21,347-354. 131 Blake, N. M. and Kirk, R. L., Nature 1969,221,278-279. 141 Blake, N. M., McDermid, E. M., Kirk, R. L ,Ong, Y. W. and Simons. M. J., Sing. Med. J. 1973, 14, 2-8. 151 Tuchinda, S., Rucknagel, D. L., Nakorn, S. N. and Prawase, W., Biochem. Genet. 1968,2,253-264. 161 Jenkins, T. and Nurse, G. T., Ann. Hum. Genet. 1974,38, 19-29. 171 Parr, C. W., Nature 1966,210,487-489. I81 Davidson, R. G., Ann. Hum. Genet. 1967,30,355-361. [91 Tariverdian, G., Ropers, H., Op't Hof, J. and Ritter, H., Hum. Genet. 1970,10,355-357. [ 101 Nelson, M. S., Hum. Genet. 1982,62, 333--336.
Anke-Gabriele Lenz Barbara Meyer Hans Weber Konrad Maier Projekt Inhalation, Arbeitsgruppe Biochemie, Gesellschaft fur Strahlenund UmweltforschungMunchen
1 1 11 Omoto.K.andHarada,S.,Jpn.J.HumanGenet. 1970.14,298-305.
1121 Omoto, K., Ishizaki, K., Harada, S., Akaishi, S., Kudo, T. and Takahashi, K.,J.Anthrop. Sac. Nippon 1973,81,159-173. [ 131 Omoto, K., Ishimoto, G., Harada, S. and Misawa,S.,J.Anthrop. Sac. Nippon 1975,83,253-260. 1141 Ishimoto, G., Jpn. J. Hum. Genet. 1970,15,26-34. ll51 Ishimoto, G. and Kuwata, M., J. Anthrop. Sac. Nippon 1973, 81, 153- 158. [ 161 Ishimoto,G. and Kuwata,M.,Jpn.J.LegalMed.1973,27,134-I4 1. 1171 Ishimoto,G. andKuwata,M.,Jpn.J. LegalMed. 1973,27,346-350. [ 181 Harada, S., Akaishi, S., Kudo, T. and Omoto, K., J. Anthrop. SOC. Nippon 1971,79,356-366. [I91 Harada, S. and Misawa, S.,Jpn. J. Hum. Genet. 1976,21, 23-27. 1201 Toyomasu. T., Katayama, K., Miyazaki, T. and Matsumoto, H., J. Anthrop. SOC.Nippon 1971.85, 31 1-323. 1211 Toyomasu, T. and Katayama, K., J. Anthrop. Soc. Nippon 1979,87, 71-76. 1221 Oya, M., Ito, H., Kido, A,, Suzuki, O., Katsumata, Y. and Yada, S., Forensic. Sci. 1978, 11, 135-138. 1231 Ikemoto, S.,Yoshida,H., Goto,K.,Taminato.C.,Matsumoto,T. and Tomita, K., Jichi. Med. School J . 1982,5, 145-147. 1241 Harada, S., Clin. Chim. Acta 1975,63, 275-283. [251 Finney, S. T., Renshaw, N. A. and Werrett, D. .I., Forensic. Sci. Ins. 1985,27,237-245. 1261 Harris, H. and Hopkinson, D. A,, Handbook of Enzyme Electrophoresis in Human Genetics, North-Holland, Amsterdam 1976. 1271 Yuasa, I., Tamaki, N., Inoue, T. and Okada, K., Forensic. Sci. Int. 1985,28,63-67.
Two-dimensionalelectrophoresis of dog bronchoalveolar lavage fluid proteins Proteins of dog bronchoalveolar lavage fluid, obtained by washing the epithelial lining layer of lungs with phosphate-buffered saline, were separated by two-dimensional electrophoresis. Due to the low protein and high salt content of the bronchoalveolar lavage fluid, samples had to be concentrated and desalted. Following electrophoresis the protein spots were visualized by silver staining. Comparing the two-dimensional protein patterns of bronchoalveolar lavage fluid with that from serum, several lungspecific proteins were detected. The most prominent protein, most probably a surfactant-associated protein, showed isoforms with isoelectric points in the range of pH 4.2-4.8, and a molecular mass of 32 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction with dithiothreitol.
Two-dimensional electrophoresis (2-DES) of the proteins of different body fluids was described as, a possible specific diagnostic test for different diseases [ 1-61. Only littleis known about the 2-DE protein pattern from human or animal bronCorrespondence: Dr. Anke-Gabriele Lenz, Gesellschaft fur Strahlen- und Umweltforschung, Projekt Inhalation, Arbeitsgruppe Biochemie, Ingolstadter Landstr. 1, D-8042 Neuherberg, Federal Republic of Germany Abbreviations: BAL, bronchoalveolar lavage; DTT, dithiothreitol; IEF, isoelectric focusing; IPG, immobilized pH gradient; M,, molecular weight; NP-40, Nonidet P-40; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; SP-A, surfactant protein A ; 2-DE, two-dimensional electrophoresis 0VCH Verlagsgeseilschaft rnbH D-6940 Weinheim, 1990
choalveolar lavage (BAL) fluids. The soluble proteins washed out from the epithelial lining layer of the lungs may, however, provide helpful information in determining the presence of lung injury t71. Such evaluations require the knowledge of the composition of BAL fluid from normal individuals. Comparing the 2-DE pattern of BAL fluid proteins with that of serum proteins the detection of lung-specific proteins may be possible. Miiller and von Wichert [81 used gradient gel electrophoresis, isoelectric focusing (IEF) and immunological studies on BAL fluid and serum of humans and rats to classify proteins present only in BAL fluid and proteins identical to serum proteins. However, IEF and gradient gel electrophoresis were not combined in a 2-DE approach. Bell et al. [9]sepa0173-0835/90/0606-05 10 %OZ.SO/O
Katsunori Akiyama Departmentof Legal Medicine,Tokyo Womens’s Medical College, Tokyo
isoelectric focusing of 6-PGD
509
Analysis of 6-phosphogluconatedehydrogenase isozymes by polyacrylamide gel isoelectric focusing Three common phenotypes of 6-phosphogluconate dehydrogenase (6-PGD) were identified by enzyme visualization following isoelectric focusing in photochemically polymerized polyacrylamide gels whereas in chemically polymerized gels no activity could be detected. Gene frequencies calculated from 429 individuals were 0.9 15 and 0.085 for 6-PGD*A and 6-PGD*C, respectively, similar to the gene frequencies of Japanese samples previously reported.
6-Phosphogluconate dehydrogenase (EC 1.1.1.44; 6-PGD) is an important metabolic enzyme in the hexose monophosphate shunt pathway catalyzing the conversion of 6-phosphogluconate to ribulose-5-phosphate. The genetic polymorphism of 6-PGD in human red blood cell was first described by Fildes and Parr [ ll. They presented evidence that the two common alleleproducts, 6-PGD*A and 6-PGD*C (at that time called 6-PGD*B) could be identified by starch gel electrophoresis and enzyme activity staining procedures. Electrophoretic variants of the rare 6-PGD alleles have also been detected in various human populations of different ethnic origin, including the 6-PGD*Wantoat, 6-PGD*Kadar, 6-PGD*Bombay [21, 6-PGD*Elcho 131, 6-PGD*Singapore [41, 6-PGD*Thai [5], 6-PGD*Oshakati [6], 6-PGD*Richmond [ 7 , 81, 6-PGD*Hackney [ 7 ] , 6-PGD*Freiburg [9], and the 5-PGD*Lowell [ 101 alleles. In Japan, the distribution of 6-PGD phenotypes using starch gel electrophoresis has been described by several authors I1 1-231. Harada [241 has described the 6-PGD phenotypes and a rare 6-PGD*Singapore variant after using isoelectric focusing (IEF) in acetate membranes with Ampholine carrier ampholytes and sucrose. In this report, the analysis of 6-PGD phenotypes in Japanese blood samples by polyacrylamide gel I E F in Ampholine carrier ampholytes is described. Hemolysates (429 blood samples) were prepared by freezing and thawing of washed and packed fresh red blood cells. The
Correspondence:Dr. Katsunori Akiyama, Department of Legal Medicine, Tokyo Women’s Medical College, Tokyo 162, Japan Abbreviations: IEF, isoelectric focusing; 6-PGD, 6-phosphogluconate dehydrogenase
samples for electrophoresis were a 1:10dilution ofhemolysate in 0.05 M dithiothreitol (DTT), and incubated for 10 min at room temperature. Polyacrylamide gels (1 10 x 240 x 0.2 mm) were prepared according to Finney [251. This gel consisted of 3.45 mL acrylamide (29.1 %), 5.25 mL N,N’-methylenebisacrylamide (0.9 %), 0.2 mL riboflavin (20 mg/%), 0.68 g 3-(N-morpholino)propanesulfonicacid (MOPS), 2.5 g sucrose, 1.5 mL carrier ampholytes (Ampholine, LKB) pH 5-7 and p H 7-9 in a 1:2 ratio, and was diluted to 20 mL with water. IEF was carried out at 1200 V, 50 mA and 25 W for 4 h with cooling at 4 “C. The 6-PGD enzyme activity staining procedures were carried out according to Harris and Hopkinson [261. Figure 1 shows the three common 6-PGD phenotypes, 6P G D A, 6-PGD AC and 6-PGD C, separated by I E F in aphotochemically polymerized polyacrylamide gel. Chemically polymerized I E F gels are not recommended. Figure 2 is a schematic representation of the I E F patterns obtained from the common 6-PGD phenotypes in a p H 5.8-6.2 gradient. For each 6-PGD phenotype characterized by starch gel electrophoresis, it was observed that the 6-PGD A type consisted of one high activity component (a), the 6-PGD AC type showed a dimeric pattern consisting of two high activity components, (a) and (c2), and one low activity component (cl), and the 6P G D C type consisted of two high activity components, (cl) and (c2), and one low activity component (a). Using the low voltage I E F electrophoresis method reported by Yuasa et al. [271, component (a) of the 6-PGD A type could be separated into two bands, but then the ( c l ) and (c2) bands ofthe 6-PGD AC and 6-PGD C types could not be detected. It was found that the 6-PGD band patterns described in the present study were similar to the patterns reported by Harada 1241 after using IEF on cellulose acetate membranes.
7igure 1. Three common 6-PGD phenoypes separated by IEF in a photochemially potymerized polyacrylamide gel. A) Enzyme visualization, (B) Schematic epresentation. 0 173-0835/90/0606-0509 $02.5010
5 10
Electrophoresis 1990,l I , 5 10-5 13
A. G. Lenz er nl.
Gene frequencies calculated from 429 unrelated J apanese red blood samples were 0.915 and 0.085 for 6-PGD*A and 6PGD*C, respectively. In our samples, there were n o variant types deviating from the three commoin phenotypes. These results did not differ from J apanese samples reported by other authors in Japan. The 6-PGD isozyme system found by polyacrylamide gel IEF could be applied in the field of forensic and legal medicine. Received February 14, 1990
References 111 Fildes, R. A. and Parr, C. W., Nature 1963,200,890-891.
[21 Blake, N. M., Saha, N., McDermid, E. M. and Kirk, R. L., Hum. Genet. 1974,21,347-354. 131 Blake, N. M. and Kirk, R. L., Nature 1969,221,278-279. 141 Blake, N. M., McDermid, E. M., Kirk, R. L ,Ong, Y. W. and Simons. M. J., Sing. Med. J. 1973, 14, 2-8. 151 Tuchinda, S., Rucknagel, D. L., Nakorn, S. N. and Prawase, W., Biochem. Genet. 1968,2,253-264. 161 Jenkins, T. and Nurse, G. T., Ann. Hum. Genet. 1974,38, 19-29. 171 Parr, C. W., Nature 1966,210,487-489. I81 Davidson, R. G., Ann. Hum. Genet. 1967,30,355-361. [91 Tariverdian, G., Ropers, H., Op't Hof, J. and Ritter, H., Hum. Genet. 1970,10,355-357. [ 101 Nelson, M. S., Hum. Genet. 1982,62, 333--336.
Anke-Gabriele Lenz Barbara Meyer Hans Weber Konrad Maier Projekt Inhalation, Arbeitsgruppe Biochemie, Gesellschaft fur Strahlenund UmweltforschungMunchen
1 1 11 Omoto.K.andHarada,S.,Jpn.J.HumanGenet. 1970.14,298-305.
1121 Omoto, K., Ishizaki, K., Harada, S., Akaishi, S., Kudo, T. and Takahashi, K.,J.Anthrop. Sac. Nippon 1973,81,159-173. [ 131 Omoto, K., Ishimoto, G., Harada, S. and Misawa,S.,J.Anthrop. Sac. Nippon 1975,83,253-260. 1141 Ishimoto, G., Jpn. J. Hum. Genet. 1970,15,26-34. ll51 Ishimoto, G. and Kuwata, M., J. Anthrop. Sac. Nippon 1973, 81, 153- 158. [ 161 Ishimoto,G. and Kuwata,M.,Jpn.J.LegalMed.1973,27,134-I4 1. 1171 Ishimoto,G. andKuwata,M.,Jpn.J. LegalMed. 1973,27,346-350. [ 181 Harada, S., Akaishi, S., Kudo, T. and Omoto, K., J. Anthrop. SOC. Nippon 1971,79,356-366. [I91 Harada, S. and Misawa, S.,Jpn. J. Hum. Genet. 1976,21, 23-27. 1201 Toyomasu. T., Katayama, K., Miyazaki, T. and Matsumoto, H., J. Anthrop. SOC.Nippon 1971.85, 31 1-323. 1211 Toyomasu, T. and Katayama, K., J. Anthrop. Soc. Nippon 1979,87, 71-76. 1221 Oya, M., Ito, H., Kido, A,, Suzuki, O., Katsumata, Y. and Yada, S., Forensic. Sci. 1978, 11, 135-138. 1231 Ikemoto, S.,Yoshida,H., Goto,K.,Taminato.C.,Matsumoto,T. and Tomita, K., Jichi. Med. School J . 1982,5, 145-147. 1241 Harada, S., Clin. Chim. Acta 1975,63, 275-283. [251 Finney, S. T., Renshaw, N. A. and Werrett, D. .I., Forensic. Sci. Ins. 1985,27,237-245. 1261 Harris, H. and Hopkinson, D. A,, Handbook of Enzyme Electrophoresis in Human Genetics, North-Holland, Amsterdam 1976. 1271 Yuasa, I., Tamaki, N., Inoue, T. and Okada, K., Forensic. Sci. Int. 1985,28,63-67.
Two-dimensionalelectrophoresis of dog bronchoalveolar lavage fluid proteins Proteins of dog bronchoalveolar lavage fluid, obtained by washing the epithelial lining layer of lungs with phosphate-buffered saline, were separated by two-dimensional electrophoresis. Due to the low protein and high salt content of the bronchoalveolar lavage fluid, samples had to be concentrated and desalted. Following electrophoresis the protein spots were visualized by silver staining. Comparing the two-dimensional protein patterns of bronchoalveolar lavage fluid with that from serum, several lungspecific proteins were detected. The most prominent protein, most probably a surfactant-associated protein, showed isoforms with isoelectric points in the range of pH 4.2-4.8, and a molecular mass of 32 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction with dithiothreitol.
Two-dimensional electrophoresis (2-DES) of the proteins of different body fluids was described as, a possible specific diagnostic test for different diseases [ 1-61. Only littleis known about the 2-DE protein pattern from human or animal bronCorrespondence: Dr. Anke-Gabriele Lenz, Gesellschaft fur Strahlen- und Umweltforschung, Projekt Inhalation, Arbeitsgruppe Biochemie, Ingolstadter Landstr. 1, D-8042 Neuherberg, Federal Republic of Germany Abbreviations: BAL, bronchoalveolar lavage; DTT, dithiothreitol; IEF, isoelectric focusing; IPG, immobilized pH gradient; M,, molecular weight; NP-40, Nonidet P-40; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; SP-A, surfactant protein A ; 2-DE, two-dimensional electrophoresis 0VCH Verlagsgeseilschaft rnbH D-6940 Weinheim, 1990
choalveolar lavage (BAL) fluids. The soluble proteins washed out from the epithelial lining layer of the lungs may, however, provide helpful information in determining the presence of lung injury t71. Such evaluations require the knowledge of the composition of BAL fluid from normal individuals. Comparing the 2-DE pattern of BAL fluid proteins with that of serum proteins the detection of lung-specific proteins may be possible. Miiller and von Wichert [81 used gradient gel electrophoresis, isoelectric focusing (IEF) and immunological studies on BAL fluid and serum of humans and rats to classify proteins present only in BAL fluid and proteins identical to serum proteins. However, IEF and gradient gel electrophoresis were not combined in a 2-DE approach. Bell et al. [9]sepa0173-0835/90/0606-05 10 %OZ.SO/O
