A rapid and highly resolving method for protein subunit separation.
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Biomedical subjects
Publications and source records attributed to F Wieland.
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1. The multi-enzyme complex of fatty acid synthetase, Mr 2300,000, was dissociated by acylation with dimethyl maleic anhydride under conditions which lead to an acylation of about 30% of the epsilon amino groups of lysine. The complete dissociation into the subunits alpha and beta is demonstrated by analytical ultracentrifugation as well as disc gel electrophoresis. 2. This dissociation is reversible. Hydrolysis of the resulting protein dicarboxylic acid monoamides under mildly acidic conditions leads to the unmodified subunits, which can be reconstituted to form a complex displaying about 60% of the original activity. 3. The subunits were isolated by sucrose-density-gradient centrifugation and studied for the different partial enzyme activities involved in long-chain fatty acid synthesis: malonyl, palmitoyl and acetyl transferase, enoyl reductase and dehydratase were shown to be exclusive functions of the beta chains of the complex, confirming a pentafunctional role of this subunit.
Rabbit and goat antibodies against the isolated alpha and beta subunits of yeast fatty acid aynthetase were raised and characterized. The purified IgG fractions were studied as to their capability to precipitate their antigens and the holoenzyme and to inhibit the partial reactions involved in overall fatty acyl-CoA synthesis. The specificity of the antibodies was investigated by immunodiffusion and by immunotitration. Native enzyme was crosslinked with each of the antibodies, and dimeric and oligomeric groups of IgG-crosslinked fatty acid synthetase molecules were isolated by sucrose density gradient centrifugation. Electron microscopic investigation of the crosslinked material as well as other data led us to suggest a three-dimensional model of yeast fatty acid synthetase.
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