Characterization of a novel microsomal fatty acid synthetase (FAS) compared to a cytosolic FAS in the housefly, Musca domestica.
A novel membrane-bound fatty acid synthetase (FAS) associated with the microsomal fraction from the housefly, Musca domestica, was solubilized and purified to homogeneity. The microsomal FAS was solubilized by 0.75 M KCl in phosphate buffer and was purified to homogeneity by the sequential use of ammonium sulfate precipitation followed by Sepharose CL-6B, DEAE Sephacel and Red Agarose (dye ligand affinity) chromatography. The specific activity of the microsomal FAS was increased 1,440-fold to 6,522 U/mg during purification. The cytosolic FAS from the housefly was also purified by similar methods and the specific activity increased 183-fold to 7,533 U/mg. The relative molecular mass of the microsomal and cytosolic FAS are 419 +/- 22 kDa and 405 +/- 18 kDa, respectively, for the dimers as determined by gel permeation chromatography. The microsomal and the cytosolic FAS yield different tryptic digestion maps and have slightly different amino acid compositions, which demonstrate structural differences between the two FASs. In addition, there are differences between the two FASs in their kinetic characteristics and their ability to incorporate methylmalonylCoA into the growing fatty acyl chain.
Gu
P
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Department of Biochemistry/MS 330, University of Nevada, Reno 89557-0014, USA.
Welch
W H
WH
Guo
L
L
Schegg
K M
KM
Blomquist
G J
GJ
D000214:Acyl Coenzyme A / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis*; D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D011232:Chemical Precipitation; D003600:Cytosol / Q000201:enzymology*; D064429:Fatty Acid Synthases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D006793:Houseflies / Q000201:enzymology*; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D011189:Potassium Chloride / Q000494:pharmacology; D012995:Solubility; D014357:Trypsin / Q000737:chemistry
D000214:Acyl Coenzyme A; D000596:Amino Acids; C015357:methylmalonyl-coenzyme A; D011189:Potassium Chloride; D064429:Fatty Acid Synthases; D014357:Trypsin; D000645:Ammonium Sulfate