Structure of fatty acid synthetase from the Harderian gland of guinea pig. Proteolytic dissection and electron microscopic studies.
Limited proteolysis and electron microscopic observation of fatty acid synthetase from the Harderian gland of guinea pig was performed to elucidate the higher-order structures of this multifunctional protein. Staphylococcus aureus V8 protease dissected the 250,000 Mr subunit of fatty acid synthetase into 120,000, 70,000, 35,000 and 30,000 Mr fragments, which were aligned in this order from the NH2 terminus. Some of the protease-resistant fragments produced with elastase, trypsin and lysyl endopeptidase were purified and fragment-specific antibodies (A40L, A33E and A25T) were prepared. A25T and A33F specifically bound the 35,000 and 30,000 Mr fragments, and A40L recognized the region between the 120,000 and 70,000 Mr fragments. Electron microscopic studies employing rotary shadowing, unidirectional shadowing and negative staining revealed that the overall dimension of the enzyme was 22 nm x 15 nm x 7 nm, and that two elongated subunits mainly composed of three subregions were in contact with each other at a few, three at most, points with two holes between them. The outer two attachment sites were often not in contact, indicating a certain flexibility of subunits at their ends. Immunocomplexes composed of fatty acid synthetase and fragment-specific antibodies were isolated and observed under the electron microscope. The attachment sites of A40L and A33E were located at the end of the minor and the major axes of the ellipsoidal contour of the molecule, respectively. Based on these results, the three-dimensional structure of animal fatty acid synthetase is discussed.
Kitamoto
T
T
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Nishigai
M
M
Sasaki
T
T
Ikai
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001665:Binding Sites; D064429:Fatty Acid Synthases / Q000378:metabolism*; D006168:Guinea Pigs; D006243:Harderian Gland / Q000378:metabolism*; D006868:Hydrolysis; D015151:Immunoblotting; D007765:Lacrimal Apparatus / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data
D000906:Antibodies; D064429:Fatty Acid Synthases