A hexameric form of the Neurospora crassa plasma membrane H+-ATPase |
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| Affiliation: | 1. Danish Research Institute of Translational Neuroscience (DANDRITE), Nordic EMBL Partnership for Molecular Medicine, Aarhus University, Aarhus, Denmark;3. Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark;4. EMBL, Notkestrasse 85, 22607 Hamburg, Germany;5. Aarhus Institute of Advanced Studies, Aarhus University, Denmark;1. Institute of Organic Chemistry, University of Münster, Corrensstrasse 40, D-48149 Münster, Germany;2. Institute of Medical Biochemistry, Center for Molecular Biology of Inflammation, University of Münster, Von-Esmarch-Strasse 56, D-48149 Münster, Germany;3. Institute of Biochemistry, University of Münster, Wilhelm-Klemm-Strasse 2, D-48149 Münster, Germany;4. Institute of Physics, University of Münster, Wilhelm-Klemm-Strasse 10, D-48149 Münster, Germany;1. Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas, USA;2. Department of Chemical & Petroleum Engineering, The University of Kansas, Lawrence, Kansas, USA;3. Department of Biochemistry, The University of Kansas, Lawrence, Kansas, USA;4. Kansas Vaccine Institute, The University of Kansas, Lawrence, Kansas, USA;5. Vaccine Research Center, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA;6. Department of Biochemistry & Molecular Biophysics, Columbia University, New York, New York, USA;7. Zuckerman Institute, Columbia University, New York, New York, USA;1. Department of Biointerface Chemistry, Faculty of Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan;2. National Institute of Standards and Technology Center for Neutron Research, Gaithersburg, MD 20899-6102, USA;3. Department of Materials Science and Engineering, University of Maryland, College Park, MD 20742-2115, USA;4. Department of Physics and Astronomy, University of Delaware, Newark, DE 19716, USA;5. Neutron Science and Technology Center, Comprehensive Research Organization for Science and Society (CROSS), 162-1 Shirakata, Tokai, Naka, Ibaraki 319-1106, Japan;6. Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan;1. Institut für Pharmazeutische Technologie, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438, Frankfurt, Germany;2. Hahn-Schickard Gesellschaft für angewandte Forschung e. V., Georges-Köhler-Allee 103, D-79110, Freiburg, Germany;3. Institute of Medical Genetics and Applied Genomics, University of Tuebingen, 72076, Tübingen, Germany;3. Department of Cell and Developmental Biology, the University of Michigan, Ann Arbor, Michigan 48109;5. the Department of Biology, the University of North Carolina, Chapel Hill, North Carolina 27599;4. the Department of Biology, the Johns Hopkins University, Baltimore, Maryland 21218;6. the Curriculum in Genetics and Molecular Biology, the University of North Carolina, Chapel Hill, North Carolina 27599 |
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| Abstract: | As isolated by our recently developed large-scale procedure, the Neurospora plasma membrane H+-ATPase exists as a homogeneous, oligomeric complex of 105,000-Da monomers with a molecular mass equivalent to a spherical protein of about 1 million Da, as judged by its behavior during chromatography on calibrated columns of Sepharose CL-6B and CL-4B. Treatment of this complex with the nonionic detergent, Tween 20, followed by Sepharose column chromatography in the presence of this detergent produces particles with an apparent molecular mass reduced by 100–300 kDa, and, importantly, when the isolated complex is treated with Tween 20 and then subjected to Sepharose chromatography in the absence of detergent, fully viable, largely detergent-free, homogeneous particles with a molecular mass equivalent to a spherical protein of 670,000 Da are formed. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, treatment of the particles isolated in the presence of Tween 20 with glutaraldehyde progressively yields dimers, trimers, tetramers, pentamers, and hexamers of the 105,000-Da monomer, with the expected precursor-product relationships, but no species larger than a hexamer is formed. These results thus strongly indicate that these particles are hexamers of 105,000-Da monomers. Glutaraldehyde crosslinking experiments with the ca. 1 million- and 670,000-Da particles indicate that they too are hexamers, suggesting that the differences in the apparent sizes of the three types of particles are most likely due to bound detergents. Possible implications of these findings are discussed. |
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