Cloning of the Fatty Acid Synthetase β Subunit from Fission Yeast, Coexpression with the α Subunit, and Purification of the Intact Multifunctional Enzyme Complex

1998 ◽  
Vol 13 (3) ◽  
pp. 403-413 ◽  
Author(s):  
Hajime Niwa ◽  
Eisaku Katayama ◽  
Mitsuhiro Yanagida ◽  
Kosuke Morikawa
Keyword(s):  
Fatty Acid ◽  
Fission Yeast ◽  
Fatty Acid Synthetase ◽  
Enzyme Complex ◽  
Β Subunit ◽  
Α Subunit ◽  
Multifunctional Enzyme

scholarly journals Regulation of enzyme turnover during tissue differention. Studies on the effects of hormones on the turnover of fatty acid synthetase in rabbit mammary gland in organ culture

1975 ◽  
Vol 148 (2) ◽  
pp. 309-320 ◽  
Author(s):  
B K Speake ◽  
R Dils ◽  
R J Mayer
Keyword(s):  
Mammary Gland ◽  
Fatty Acid ◽  
Organ Culture ◽  
Half Life ◽  
Culture Medium ◽  
Fatty Acid Synthetase ◽  
Enzyme Complex ◽  
Rapid Fall ◽  
Enzyme Turnover ◽  
Rabbit Milk

1. Explants of mammary gland from mid-pregnant rabbits were cultured with insulin, prolactin and cortisol. 2. Antibodies raised to fatty acid synthetase were used to measure the amount as well as the rate of synthesis and the rate of degradation of the enzyme in the explants over defined periods in organ culture. These measurements were also made after the hormones had been removed from the culture medium. The changes which occur in the activity of fatty acid synthetase are due to changes in the amount of the enzyme present. They are not due to activation or inactivation of the enzyme. 3. The rate of lipogenesis (measured from [1-14C]acetate) in the explants during culture varies independently of the amount of fatty acid synthetase both in the presence and after removal of the hormones. Hence the amount of fatty acid synthetase does not limit lipogenesis. The proportion of medium-chain fatty acids C8:0 and C10:0 (which are characteristic of rabbit milk) synthesized by the explants in the presence of hormones increases at about the same rate as the amount of fatty acid synthetase present. However, when hormones are removed from the medium the proportion of these acids synthesized declines as rapidly as the rate of lipogenesis and not as the amount of fatty acid synthetase presen. 4. The rates of synthesis of fatty acid synthetase and of the total particulate-free supernatant protein in the explants were compared by measuring the incorporation of L-[U-14C]leucine into the protein of the explants. These rates increase by 5-fold and 3.6-fold respectively when explants are cultured with hormones, and they then reach approximately constant rates. When the hormones are removed there is a rapid fall in the rate of synthesis of fatty acid synthetase and of the total particulate-free supernatant protein to values which are similar to those obtained with freshly prepared explanted tissue. 5. In unstimulated explants fatty acid synthetase appears to be degraded with a half-life of 15-21h. During the hormonally stimulated differentiation of the tissue the rate of degradation of the enzyme is considerably decreased or is switched off completely. After the amount of fatty acid synthetase has increased to a maximum the enzyme complex is again degraded with a half-life of 23-29h. The removal of hormones after the explants have been hormonally stimulated for different times results in an increase in the rate of degradation of fatty acid synthetase. However, this increase only occurs if degradation was previously proceeding at a considerably decreased rate. The degradation of the total particulate-free supernatant protein continues throughout the period of differentiation of the explant tissue in culture. It appears to be somewhat decreased during the period of rapid maturation of the tissue during culture.

scholarly journals Aberrant mitosis in fission yeast mutants defective in fatty acid synthetase and acetyl CoA carboxylase.

1996 ◽  
Vol 134 (4) ◽  
pp. 949-961 ◽  
Author(s):  
S Saitoh ◽  
K Takahashi ◽  
K Nabeshima ◽  
Y Yamashita ◽  
Y Nakaseko ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fission Yeast ◽  
Fatty Acid Synthesis ◽  
Fatty Acid Synthetase ◽  
Acid Synthesis ◽  
Temperature Sensitive ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Nuclear Structures ◽  
Yeast Mutants

Two fission yeast temperature-sensitive mutants, cut6 and lsd1, show a defect in nuclear division. The daughter nuclei differ dramatically in size (the phenotype designated lsd, large and small daughter). Fluorescence in situ hybridization (FISH) revealed that sister chromatids were separated in the lsd cells, but appeared highly compact in one of the two daughter nuclei. EM showed asymmetric nuclear elongation followed by unequal separation of nonchromosomal nuclear structures in these mutant nuclei. The small nuclei lacked electron-dense nuclear materials and contained highly compacted chromatin. The cut6+ and lsd1+ genes are essential for viability and encode, respectively, acetyl CoA carboxylase and fatty acid synthetase, the key enzymes for fatty acid synthesis. Gene disruption of lsd1+ led to the lsd phenotype. Palmitate in medium fully suppressed the phenotypes of lsd1. Cerulenin, an inhibitor for fatty acid synthesis, produced the lsd phenotype in wild type. The drug caused cell inviability during mitosis but not during the G2-arrest induced by the cdc25 mutation. A reduced level of fatty acid thus led to impaired separation of non-chromosomal nuclear components. We propose that fatty acid is directly or indirectly required for separating the mother nucleus into two equal daughters.

scholarly journals Reconstitution, morphology and crystallization of a fatty acid β-oxidation multienzyme complex from Pseudomonas fragi

1997 ◽  
Vol 328 (3) ◽  
pp. 815-820 ◽  
Author(s):  
Momoyo ISHIKAWA ◽  
Yuriko MIKAMI ◽  
Jiro USUKURA ◽  
Hiroshi IWASAKI ◽  
Hideo SHINAGAWA ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Gene Product ◽  
Multienzyme Complex ◽  
Β Subunit ◽  
Pseudomonas Fragi ◽  
Α Subunit ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Α And Β Subunits ◽  
Product Forms ◽  
Enoyl Coa Hydratase

The fatty acid β-oxidation multienzyme complex from Pseudomonas fragi, HDT, exhibits predominantly the three enzymic activities of 2-enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and 3-oxoacyl-CoA thiolase (EC 2.3.1.16). The HDT complex is encoded by the faoAB operon, consisting of the faoA and faoB genes that encode two individual constituents, the α-subunit and the β-subunit. We have constructed Escherichia coli overexpression systems for the faoAB gene product (coexpression of the α- and β-subunits), the α-subunit alone and the β-subunit alone, and have purified the three respective products. Gel-filtration analysis revealed that the faoAB gene product forms a heterotetrameric structure, α2β2, identical with the native HDT oligomeric state from P. fragi, whereas the α-subunit and β-subunit individually form dimers. Electron microscopy demonstrated that each protein morphologically adopts the above oligomeric structures. The HDT complex, reconstituted in vitro from the isolated α- and β-subunits, exhibits the three original enzymic activities and yields the same crystal as those from the native enzyme. CD measurements indicated that the α- and β-dimers hardly alter their global conformations upon the formation of the HDT complex. Interestingly, the β-dimer alone does not exhibit 3-oxoacyl-CoA thiolase activity, whereas the α-dimer alone exhibits both the 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities. These results suggest that the contact between the α- and β-subunits is essential for the thiolase activity. We have identified several structurally important proteolytic sites within each subunit, which are protected in the intact heterotetrameric molecule. These findings allow the possible location of the interface between the two subunits, which should be crucial for the exhibition of thiolase activity.

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