DOI: 10.1046/j.1471-4159.1999.0731422.x. Purification of Glutathione Reductase from Bovine Brain, Generation of an Antiserum, and Immunocytochemical Localization of the Enzyme in Neural Cells

Purification of Glutathione Reductase from Bovine Brain, Generation of an Antiserum, and Immunocytochemical Localization of the Enzyme in Neural Cells

10.1046/j.1471-4159.1999.0731422.x

Crossref journal-article

Wiley

Journal of Neurochemistry (311)

Abstract

Abstract : Glutathione reductase (GR) is an essential enzyme for the glutathione‐mediated detoxification of peroxides because it catalyzes the reduction of glutathione disulfide. GR was purified from bovine brain 5,000‐fold with a specific activity of 145 U/mg of protein. The homogeneity of the enzyme was proven by sodium dodecyl sulfate‐polycrylamide gel electrophoresis and silver staining of the gel. The purified GR from bovine brain is a dimer of two subunits that have an apparent molecular mass of 55 kDa. The purified GR was used to generate a rabbit antiserum with the intention to localize GR in brain cells. The antiserum was useful for the detection of GR by double‐labeling immunocytochemical staining in astroglia‐rich and neuron‐rich primary cultures from rat brain. In homogenates of these cultures, no significant difference in the specific activities of GR was determined. However, not all cell types present in these cultures showed identical staining intensity for GR. In astrogliarich primary cultures, strong GR immunoreactivity was found in cells positive for the cellular markers galactocerebroside and C3b (antibody Ox42), indicating that oligodendroglial and microglial cells, respectively, contain GR. In contrast, only weak immunoreactivity for GR was found in cells positive for glial fibrillary acidic protein. In neuron‐rich primary cultures, GAP43‐positive cells stained with the antiserum against GR. These data demonstrate that, in cultures of neural cells, neurons, oligodendroglial cells, and microglial cells express high levels of GR.

Bibliography

Gutterer, J. M., Dringen, R., Hirrlinger, J., & Hamprecht, B. (1999). Purification of Glutathione Reductase from Bovine Brain, Generation of an Antiserum, and Immunocytochemical Localization of the Enzyme in Neural Cells. Journal of Neurochemistry, 73(4), 1422â1430. Portico.

Authors 4

Jan Mirko Gutterer (first)
Ralf Dringen (additional)
Johannes Hirrlinger (additional)
Bernd Hamprecht (additional)

References 52 Referenced 62

10.1016/0014-5793(89)80687-8
10.1159/000468877 / Enzyme / Kinetic properties of sheep brain glutathione reductase. by Acan N.L. (1991)
10.1006/bmme.1995.1005
10.1016/S0165-0173(97)00045-3
{'key': 'e_1_2_8_6_2', 'first-page': '265', 'article-title': 'Function and regulation of the pentose phosphate pathway in brain.', 'volume': '29', 'author': 'Braquer N.Z.', 'year': '1988', 'journal-title': 'Curr. Top. Cell. Regul.'} / Curr. Top. Cell. Regul. / Function and regulation of the pentose phosphate pathway in brain. by Braquer N.Z. (1988)
10.1159/000112127
10.1046/j.1471-4159.1996.66062329.x
10.1016/0005-2744(78)90348-0
10.1016/0024-3205(95)00055-B
10.1016/0003-2697(76)90527-3
10.1016/0003-2697(81)90398-5
10.1016/S0076-6879(85)13062-4
10.1016/0014-5793(87)80305-8
CooperA.J.L.(1997)Glutathione in the brain : disorders of glutathione metabolism inThe Molecular and Genetic Basis of Neurological Disease(Rosenberg R. N. Prusiner S. B. DiMauro S. Barchi R. L. and Kunk L. M. eds) pp.1195–1230.Butterworth‐Heinemann Boston.
10.1007/BF00968004
10.1083/jcb.111.4.1559
10.1016/S0006-8993(97)00233-3
10.1002/(SICI)1098-1136(199806)23:2<139::AID-GLIA5>3.3.CO;2-T
10.1046/j.1471-4159.1999.0722523.x
{'key': 'e_1_2_8_21_2', 'first-page': 'S139A', 'article-title': 'Purification of glutathione reductase from bovine brain.', 'volume': '378', 'author': 'Gutterer J.M.', 'year': '1997', 'journal-title': 'Biol. Chem.'} / Biol. Chem. / Purification of glutathione reductase from bovine brain. by Gutterer J.M. (1997)
{'key': 'e_1_2_8_22_2', 'first-page': 'S27', 'article-title': 'Generation of an antiserum against brain glutathione reductase : use for Western blots and immunocytochemistry.', 'volume': '379', 'author': 'Gutterer J.M.', 'year': '1998', 'journal-title': 'Biol. Chem.'} / Biol. Chem. / Generation of an antiserum against brain glutathione reductase : use for Western blots and immunocytochemistry. by Gutterer J.M. (1998)
10.1111/j.1471-4159.1992.tb10990.x
HamprechtB.&DringenR.(1995)Energy metabolism inNeuroglia(Kettenmann H. and Ransom B. R. eds) pp.473–487.Oxford University Press New York.
10.1016/0076-6879(85)09097-8
10.1042/bj0261406
10.1111/j.1471-4159.1962.tb07491.x
10.1016/0003-9861(68)90108-2
10.1016/0006-8993(95)00209-9
10.1016/0024-3205(93)90429-7
10.1016/S0149-7634(96)00005-X
10.1002/(SICI)1098-1136(199804)22:4<371::AID-GLIA6>3.0.CO;2-6
10.1016/0022-2836(87)90191-4
10.1002/(SICI)1096-9861(19960916)373:2<157::AID-CNE1>3.0.CO;2-2
10.1111/j.1471-4159.1993.tb03309.x
10.1038/227680a0
10.1002/(SICI)1098-1136(199810)24:2<252::AID-GLIA10>3.0.CO;2-Z
10.1016/0006-8993(86)91006-1
10.1111/j.1432-1033.1979.tb13210.x
Lopez‐BareaJ. BarcenaJ.A. BocanegraJ.A. FlorindoJ. Garcia‐AlfonsoC. Lopez‐RuizA. Martinez‐GalisteoE. PeinadoJ.(1990)Structure mechanism functions and regulatory properties of glutathione reductase inGlutathione : Metabolism and Physiological Functions(Vina J. ed) pp.105–116.CRC Press Boca Raton Florida.
10.1111/j.1471-4159.1973.tb12143.x
10.1016/S0021-9258(19)52451-6
10.1046/j.1471-4159.1994.62010045.x
10.1146/annurev.bi.52.070183.003431
10.1073/pnas.79.8.2709
10.1111/j.1471-4159.1990.tb01194.x
SchirmerR.H. Krauth‐SiegelR.L. SchulzG.E.(1989)Glutathione reductase inGlutathione : Chemical Biochemical and Medical Aspects—Part A(Dolphin D. Poulson R. and Avramovic O. eds) pp.553–595.Wiley New York.
10.1006/bbrc.1997.7153
10.1111/j.1432-1033.1990.tb15431.x
VogelR. WiesingerH. HamprechtB.(1997)Postnatal development of malic enzyme isoforms in rat brain inNeurochemistry(Teelken A. W. and Korf J. eds) pp.765–769.Plenum Press New York. (10.1007/978-1-4615-5405-9_127)
10.1046/j.1471-4159.1998.71020844.x
10.1016/S1874-6047(08)60241-3
10.1111/j.1432-1033.1976.tb10654.x

Dates

Type	When
Created	22 years, 5 months ago (March 12, 2003, 1:41 a.m.)
Deposited	1 year, 10 months ago (Oct. 28, 2023, 12:10 p.m.)
Indexed	2 months, 1 week ago (June 27, 2025, 6:51 a.m.)
Issued	25 years, 11 months ago (Oct. 1, 1999)
Published	25 years, 11 months ago (Oct. 1, 1999)
Published Online	23 years, 7 months ago (Jan. 17, 2002)
Published Print	25 years, 11 months ago (Oct. 1, 1999)

Funders 0

None

BibTeX

@article{Gutterer_1999, title={Purification of Glutathione Reductase from Bovine Brain, Generation of an Antiserum, and Immunocytochemical Localization of the Enzyme in Neural Cells}, volume={73}, ISSN={1471-4159}, url={http://dx.doi.org/10.1046/j.1471-4159.1999.0731422.x}, DOI={10.1046/j.1471-4159.1999.0731422.x}, number={4}, journal={Journal of Neurochemistry}, publisher={Wiley}, author={Gutterer, Jan Mirko and Dringen, Ralf and Hirrlinger, Johannes and Hamprecht, Bernd}, year={1999}, month=oct, pages={1422–1430} }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        6,
        27
      ]
    ],
    "date-time": "2025-06-27T10:51:44Z",
    "timestamp": 1751021504539
  },
  "reference-count": 52,
  "publisher": "Wiley",
  "issue": "4",
  "license": [
    {
      "start": {
        "date-parts": [
          [
            2002,
            1,
            17
          ]
        ],
        "date-time": "2002-01-17T00:00:00Z",
        "timestamp": 1011225600000
      },
      "content-version": "vor",
      "delay-in-days": 839,
      "URL": "http://onlinelibrary.wiley.com/termsAndConditions#vor"
    }
  ],
  "content-domain": {
    "domain": [],
    "crossmark-restriction": false
  },
  "published-print": {
    "date-parts": [
      [
        1999,
        10
      ]
    ]
  },
  "abstract": "<jats:p> <jats:bold>Abstract</jats:bold> : Glutathione reductase (GR) is an essential enzyme for the glutathione\u2010mediated detoxification of peroxides because it catalyzes the reduction of glutathione disulfide. GR was purified from bovine brain 5,000\u2010fold with a specific activity of 145 U/mg of protein. The homogeneity of the enzyme was proven by sodium dodecyl sulfate\u2010polycrylamide gel electrophoresis and silver staining of the gel. The purified GR from bovine brain is a dimer of two subunits that have an apparent molecular mass of 55 kDa. The purified GR was used to generate a rabbit antiserum with the intention to localize GR in brain cells. The antiserum was useful for the detection of GR by double\u2010labeling immunocytochemical staining in astroglia\u2010rich and neuron\u2010rich primary cultures from rat brain. In homogenates of these cultures, no significant difference in the specific activities of GR was determined. However, not all cell types present in these cultures showed identical staining intensity for GR. In astrogliarich primary cultures, strong GR immunoreactivity was found in cells positive for the cellular markers galactocerebroside and C3b (antibody Ox42), indicating that oligodendroglial and microglial cells, respectively, contain GR. In contrast, only weak immunoreactivity for GR was found in cells positive for glial fibrillary acidic protein. In neuron\u2010rich primary cultures, GAP43\u2010positive cells stained with the antiserum against GR. These data demonstrate that, in cultures of neural cells, neurons, oligodendroglial cells, and microglial cells express high levels of GR.</jats:p>",
  "DOI": "10.1046/j.1471-4159.1999.0731422.x",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2003,
        3,
        12
      ]
    ],
    "date-time": "2003-03-12T06:41:40Z",
    "timestamp": 1047451300000
  },
  "page": "1422-1430",
  "source": "Crossref",
  "is-referenced-by-count": 62,
  "title": "Purification of Glutathione Reductase from Bovine Brain, Generation of an Antiserum, and Immunocytochemical Localization of the Enzyme in Neural Cells",
  "prefix": "10.1111",
  "volume": "73",
  "author": [
    {
      "given": "Jan Mirko",
      "family": "Gutterer",
      "sequence": "first",
      "affiliation": []
    },
    {
      "given": "Ralf",
      "family": "Dringen",
      "sequence": "additional",
      "affiliation": []
    },
    {
      "given": "Johannes",
      "family": "Hirrlinger",
      "sequence": "additional",
      "affiliation": []
    },
    {
      "given": "Bernd",
      "family": "Hamprecht",
      "sequence": "additional",
      "affiliation": []
    }
  ],
  "member": "311",
  "published-online": {
    "date-parts": [
      [
        2002,
        1,
        17
      ]
    ]
  },
  "reference": [
    {
      "key": "e_1_2_8_2_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0014-5793(89)80687-8"
    },
    {
      "key": "e_1_2_8_3_2",
      "doi-asserted-by": "crossref",
      "first-page": "121",
      "DOI": "10.1159/000468877",
      "article-title": "Kinetic properties of sheep brain glutathione reductase.",
      "volume": "45",
      "author": "Acan N.L.",
      "year": "1991",
      "journal-title": "Enzyme"
    },
    {
      "key": "e_1_2_8_4_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1006/bmme.1995.1005"
    },
    {
      "key": "e_1_2_8_5_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0165-0173(97)00045-3"
    },
    {
      "key": "e_1_2_8_6_2",
      "first-page": "265",
      "article-title": "Function and regulation of the pentose phosphate pathway in brain.",
      "volume": "29",
      "author": "Braquer N.Z.",
      "year": "1988",
      "journal-title": "Curr. Top. Cell. Regul."
    },
    {
      "key": "e_1_2_8_7_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1159/000112127"
    },
    {
      "key": "e_1_2_8_8_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1046/j.1471-4159.1996.66062329.x"
    },
    {
      "key": "e_1_2_8_9_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0005-2744(78)90348-0"
    },
    {
      "key": "e_1_2_8_10_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0024-3205(95)00055-B"
    },
    {
      "key": "e_1_2_8_11_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0003-2697(76)90527-3"
    },
    {
      "key": "e_1_2_8_12_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0003-2697(81)90398-5"
    },
    {
      "key": "e_1_2_8_13_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0076-6879(85)13062-4"
    },
    {
      "key": "e_1_2_8_14_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0014-5793(87)80305-8"
    },
    {
      "key": "e_1_2_8_15_2",
      "unstructured": "CooperA.J.L.(1997)Glutathione in the brain : disorders of glutathione metabolism inThe Molecular and Genetic Basis of Neurological Disease(Rosenberg R. N. Prusiner S. B. DiMauro S. Barchi R. L. and Kunk L. M. eds) pp.1195\u20131230.Butterworth\u2010Heinemann Boston."
    },
    {
      "key": "e_1_2_8_16_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1007/BF00968004"
    },
    {
      "key": "e_1_2_8_17_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1083/jcb.111.4.1559"
    },
    {
      "key": "e_1_2_8_18_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0006-8993(97)00233-3"
    },
    {
      "key": "e_1_2_8_19_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/(SICI)1098-1136(199806)23:2<139::AID-GLIA5>3.3.CO;2-T"
    },
    {
      "key": "e_1_2_8_20_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1046/j.1471-4159.1999.0722523.x"
    },
    {
      "key": "e_1_2_8_21_2",
      "first-page": "S139A",
      "article-title": "Purification of glutathione reductase from bovine brain.",
      "volume": "378",
      "author": "Gutterer J.M.",
      "year": "1997",
      "journal-title": "Biol. Chem."
    },
    {
      "key": "e_1_2_8_22_2",
      "first-page": "S27",
      "article-title": "Generation of an antiserum against brain glutathione reductase : use for Western blots and immunocytochemistry.",
      "volume": "379",
      "author": "Gutterer J.M.",
      "year": "1998",
      "journal-title": "Biol. Chem."
    },
    {
      "key": "e_1_2_8_23_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1471-4159.1992.tb10990.x"
    },
    {
      "key": "e_1_2_8_24_2",
      "unstructured": "HamprechtB.&DringenR.(1995)Energy metabolism inNeuroglia(Kettenmann H. and Ransom B. R. eds) pp.473\u2013487.Oxford University Press New York."
    },
    {
      "key": "e_1_2_8_25_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0076-6879(85)09097-8"
    },
    {
      "key": "e_1_2_8_26_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1042/bj0261406"
    },
    {
      "key": "e_1_2_8_27_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1471-4159.1962.tb07491.x"
    },
    {
      "key": "e_1_2_8_28_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0003-9861(68)90108-2"
    },
    {
      "key": "e_1_2_8_29_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0006-8993(95)00209-9"
    },
    {
      "key": "e_1_2_8_30_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0024-3205(93)90429-7"
    },
    {
      "key": "e_1_2_8_31_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0149-7634(96)00005-X"
    },
    {
      "key": "e_1_2_8_32_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/(SICI)1098-1136(199804)22:4<371::AID-GLIA6>3.0.CO;2-6"
    },
    {
      "key": "e_1_2_8_33_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0022-2836(87)90191-4"
    },
    {
      "key": "e_1_2_8_34_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/(SICI)1096-9861(19960916)373:2<157::AID-CNE1>3.0.CO;2-2"
    },
    {
      "key": "e_1_2_8_35_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1471-4159.1993.tb03309.x"
    },
    {
      "key": "e_1_2_8_36_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/227680a0"
    },
    {
      "key": "e_1_2_8_37_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/(SICI)1098-1136(199810)24:2<252::AID-GLIA10>3.0.CO;2-Z"
    },
    {
      "key": "e_1_2_8_38_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0006-8993(86)91006-1"
    },
    {
      "key": "e_1_2_8_39_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1432-1033.1979.tb13210.x"
    },
    {
      "key": "e_1_2_8_40_2",
      "unstructured": "Lopez\u2010BareaJ. BarcenaJ.A. BocanegraJ.A. FlorindoJ. Garcia\u2010AlfonsoC. Lopez\u2010RuizA. Martinez\u2010GalisteoE. PeinadoJ.(1990)Structure mechanism functions and regulatory properties of glutathione reductase inGlutathione : Metabolism and Physiological Functions(Vina J. ed) pp.105\u2013116.CRC Press Boca Raton Florida."
    },
    {
      "key": "e_1_2_8_41_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1471-4159.1973.tb12143.x"
    },
    {
      "key": "e_1_2_8_42_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0021-9258(19)52451-6"
    },
    {
      "key": "e_1_2_8_43_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1046/j.1471-4159.1994.62010045.x"
    },
    {
      "key": "e_1_2_8_44_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1146/annurev.bi.52.070183.003431"
    },
    {
      "key": "e_1_2_8_45_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.79.8.2709"
    },
    {
      "key": "e_1_2_8_46_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1471-4159.1990.tb01194.x"
    },
    {
      "key": "e_1_2_8_47_2",
      "unstructured": "SchirmerR.H. Krauth\u2010SiegelR.L. SchulzG.E.(1989)Glutathione reductase inGlutathione : Chemical Biochemical and Medical Aspects\u2014Part A(Dolphin D. Poulson R. and Avramovic O. eds) pp.553\u2013595.Wiley New York."
    },
    {
      "key": "e_1_2_8_48_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1006/bbrc.1997.7153"
    },
    {
      "key": "e_1_2_8_49_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1432-1033.1990.tb15431.x"
    },
    {
      "key": "e_1_2_8_50_2",
      "doi-asserted-by": "crossref",
      "unstructured": "VogelR. WiesingerH. HamprechtB.(1997)Postnatal development of malic enzyme isoforms in rat brain inNeurochemistry(Teelken A. W. and Korf J. eds) pp.765\u2013769.Plenum Press New York.",
      "DOI": "10.1007/978-1-4615-5405-9_127"
    },
    {
      "key": "e_1_2_8_51_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1046/j.1471-4159.1998.71020844.x"
    },
    {
      "key": "e_1_2_8_52_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S1874-6047(08)60241-3"
    },
    {
      "key": "e_1_2_8_53_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1432-1033.1976.tb10654.x"
    }
  ],
  "container-title": "Journal of Neurochemistry",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1046%2Fj.1471-4159.1999.0731422.x",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "text-mining"
    },
    {
      "URL": "https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1471-4159.1999.0731422.x",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2023,
        10,
        28
      ]
    ],
    "date-time": "2023-10-28T16:10:23Z",
    "timestamp": 1698509423000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "https://onlinelibrary.wiley.com/doi/10.1046/j.1471-4159.1999.0731422.x"
    }
  },
  "subtitle": [],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        1999,
        10
      ]
    ]
  },
  "references-count": 52,
  "journal-issue": {
    "issue": "4",
    "published-print": {
      "date-parts": [
        [
          1999,
          10
        ]
      ]
    }
  },
  "alternative-id": [
    "10.1046/j.1471-4159.1999.0731422.x"
  ],
  "URL": "http://dx.doi.org/10.1046/j.1471-4159.1999.0731422.x",
  "relation": {},
  "ISSN": [
    "0022-3042",
    "1471-4159"
  ],
  "subject": [],
  "container-title-short": "Journal of Neurochemistry",
  "published": {
    "date-parts": [
      [
        1999,
        10
      ]
    ]
  }
}