Small-angle neutron-scattering and electron microscope studies of the chicken liver fatty acid synthase

A structural model for the chicken liver fatty acid synthase is proposed based on electron microscope and small-angle neutron-scattering studies of the enzyme. The model has the overall appearance of two side by side cylinders with dimensions of 160 X 146 X 73 A, with each subunit 160 A in length and 73 A in diameter. The model was constructed by dividing each cylinder into three domains having lengths of 32, 82, and 46 A, with the domain structures in the two subunits being related to each other by a dyad axis. The model is consistent with chemical cross-linking studies which indicated that the subunits are arranged in a head to tail fashion. The cross-linking studies further showed that the beta-ketoacyl synthase active site contains a cysteine and a pantetheine residue from adjacent subunits. It is proposed that the domains which catalyze the addition of C2 units from malonate to the growing fatty acid chain lie in the crevice between the two subunits and that the two independent sets of fatty acid-synthesizing centers lie on the major axis of the model on opposite ends of the molecular dyad.     

Yeast fatty acid synthase: structure to function relationship

The yeast fatty acid synthase is a multifunctional enzyme composed of two nonidentical subunits in an alpha 6 beta 6 complex that is active in synthesizing fatty acids. The seven catalytic activities required for fatty acid synthesis are divided between the alpha and beta subunits such that the alpha 6 beta 6 complex has six complements of each activity. It has been proposed that these are organized into six centers for fatty acid synthesis. There are different opinions regarding the operation of these centers in the alpha 6 beta 6 complex, on view being that they are functionally independent and the other proposes half-sites activity for the complex. We have attempted to distinguish between these proposals by the most direct method of active site titration, i.e., quantitation of fatty acyl product in the absence of turnover. This was accomplished by using p-nitrophenyl thioacetate and thiophenyl malonate (in place of the coenzyme A analogues) as substrates along with NADPH, thereby depriving the yeast synthase of coenzyme A required to release product as fatty acyl coenzyme A. The amount of fatty acyl product formed was quantitated by gas-liquid chromatography, as well as by direct estimation of radioactivity in the product when p-nitrophenyl thio [1-14C] acetate was used as a substrate. In both cases, a stoichiometry of close to six was found for mole of fatty acid synthesized per mole of alpha 6 beta 6 complex. This indicates that there are six functional centers for fatty acid synthesis in the multifunctional yeast alpha 6 beta 6 fatty acid synthase and that these centers operate independently.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of yeast fatty acid synthetase by modifying the beta-ketoacyl reductase active lysine residue with pyridoxal 5'-phosphate

Treatment of yeast fatty acid synthetase with pyridoxal 5'-phosphate inhibited the enzyme. Assays of the partial activities of the pyridoxal phosphate-treated synthetase showed that only the beta-ketoacyl reductase was significantly inhibited. NADPH prevented inactivation of the enzyme by pyridoxal phosphate, indicating that pyridoxal modifies a residue near or in the beta-ketoacyl reductase site. The pyridoxal-treated synthetase shows a fluorescence spectrum with a maximum of 426 nm after uv irradiation at 325 nm. Binding of the pyridoxal phosphate to the synthetase is reversible as shown by the disappearance of the fluorescence band after dialysis of pyridoxal-treated enzyme. Reduction with NaBH4 of the pyridoxal-treated enzyme eliminates this fluorescence maximum and causes the appearance of a new band at 393 nm. These observations suggest that pyridoxal phosphate interacts with the synthetase by forming a Schiff base with lysine residue at the beta-ketoacyl reductase site. Amino acid analyses of the HCl hydrolysates of the borohydride-reduced, pyridoxal-treated synthetase showed the presence of 6 mol of N6-pyridoxal derivative of lysine per mole of fatty acid synthetase, indicating the presence of six sites of beta-ketoacyl reductase in the native enzyme. Autoradiography of sodium dodecyl sulfate-polyacrylamide gels of the pyridoxal phosphate enzyme reduced with NaB3H4 indicates that the alpha subunit contains the beta-ketoacyl reductase domain. These findings are consistent with the proposed structure of the alpha 6 beta 6 complex required for palmitoyl-CoA synthesis.     

Characterization of recombinant thioesterase and acyl carrier protein domains of chicken fatty acid synthase expressed in Escherichia coli

Fatty acid synthase of animal tissue is a multifunctional enzyme comprised of two identical subunits, each containing seven partial activities and a site for the prosthetic group, 4'-phosphopantetheine (acyl carrier protein). We have recently isolated cDNA clones of chicken fatty acid synthase coding for the dehydratase, enoyl reductase, beta-ketoacyl reductase, acyl carrier protein, and thioesterase domains (Chirala, S.S., Kasturi, R., Pazirandeh, M., Stolow, D.T., Huang, W.Y., and Wakil, S.J. (1989) J. Biol. Chem. 264, 3750-3757). To gain insight into the structure and function of the various domains, the portion of the cDNA coding for the acyl carrier protein and thioesterase domains was expressed in Escherichia coli by using an expression vector that utilizes the phage lambda PL promoter. The recombinant protein was efficiently expressed and purified to near homogeneity using anion-exchange and hydroxyapatite chromatography. As expected from the coding capacity of the cDNA expressed, the protein has a molecular weight of 43,000 and reacts with antithioesterase antibodies. The recombinant thioesterase was found to be enzymatically active and has the same substrate specificity and kinetic properties as the native enzyme of the multifunctional synthase. Treatment of the recombinant protein with alpha-chymotrypsin results in the cleavage of the acyl carrier protein and thioesterase domain junction sequence at exactly the same site as with native fatty acid synthase. The amino acid composition of the purified recombinant protein revealed the presence of 0.6 mol of beta-alanine/mol of protein, indicating partial pantothenylation of the recombinant acyl carrier protein domain. These results indicate that the expressed protein has a conformation similar to the native enzyme and that its folding into functionally active domains is independent of the remaining domains of the multifunctional synthase subunit. These conclusions are consistent with the proposal that the multifunctional synthase gene has evolved from fusion of component genes.     

Assay for the terminal enzyme of the stearoyl coenzyme A desaturase system using chick embryo liver microsomes.

The NADH-dependent stearoyl CoA desaturase of hepatic microsomes (EC 1.14.99.5) is an enzyme system consisting of cytochrome b5 reductase (EC 1.6.2.2), cytochrome b5, and the terminal desaturase. We have developed a simple method for routine assay of the terminal enzyme based on complementation of the enzyme with chick embryo liver microsomes lacking desaturase activity. Desaturation of [1-14C]stearoyl CoA by the enzyme-microsome mixture is then assayed by thin-layer chromatography of the reaction products and determination of the amount of oleate formed. Microsomes from the livers of starved-refed rats were used as the source of the stearoyl CoA desaturase. The enzyme alone, solubilized and free from cytocrome b5 reductase and cytochrome b5, was unable to catalyze the desaturation of stearoyl CoA. However, after preincubation with chick embryo liver microsomes in the presence of 1% Triton X-100, the enzyme was active. The enzyme activity was linear with time and desaturase protein under the conditions described and depended on the concentrations of Triton X-100 present in the preincubation and the assay. The optimum concentrations of Triton X-100 were 1% for the preincubation and 0.1-0.15% in the assay. The desaturation activity was dependent on NADH and O2, and was inhibited 95% by 1 mM KCN. The use of chick embryo liver microsomes in this method eliminates the need to use purified cytochrome b5 reductase, cytochrome b5, and liposomes for routine assays and greatly reduces the complexities of timing and order of addition encountered in the existing assays.     

Detergent dissection of membrane proteins of Entamoeba histolytica and its effect on lymphokine release in in vitro.

Fifty-two amoebic liver abscess cases were assessed for the release of lymphokines (LMIF) using detergent dissected membrane proteins (DDMP) of axenic Entamoeba histolytica (NIH:200) obtained with sodium deoxycholate treatment. Lymphokines release by T lymphocytes in response to both DDMP and whole amoebic lysate (WAL) was tested by leukocyte migration inhibition test on blood samples from amoebic liver abscess cases. A significant increase was noted in the release of LMIF and 100% positivity was observed with DDMP compared to whole amoebic extract with a positivity of 73%. The difference between means of the above two with regards to release of LMIF was found to be highly significant (P less than 0.005). This shows the patients had high degree of leukocyte sensitization to surface antigens of E. histolytica compared to the whole amoebic lysate. These findings suggest that the antigens shed might have important role as a potent antigen in elicitation of CMI response in amoebic liver abscess cases.     

On the 4'-phosphopantetheine content of chicken and rat liver fatty acid synthetases.

The finding that animal synthetases are complexes consisting of two polypeptide chains (Stoops, J.K., Arslanian, M.J., Oh, Y.H., Vanaman, T.C., and Wakil, S.J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 1940-1944) led us to investigate their 4'-phosphopantetheine content. We have found that the chicken and rat synthetases contain 1.6 to 2.2 mol of 4'-phosphopantetheine per mol of the complex. The implications of this finding concerning the structure of the complex and the biosynthetic pathway of fatty acid synthesis are discussed.     

Optimization of methods for peripheral blood mononuclear cells isolation and expansion of human gamma delta T cells

Human Vg9/Vδ2 T cells (γδ T cells) are immune surveillance cells both in innate and adaptive immunity and are a possible target for anticancer therapies, which can induce immune responses in a variety of cancers. Small non-peptide antigens such as zoledronate can do activation and expansion of T cells in vitro. It is evident that for adoptive cancer therapies, large numbers of functional cells are needed into cancer patients. Hence, optimization of methods needs to be carried out for the efficient expansion of these T cells. Standardization of peripheral blood mononuclear cells (PBMCs) isolation was devised. Cytokines (interleukin 2 (IL-2) and interleukin 15 (IL-15)) and zoledronate were also standardized for different concentrations. It was found that an increased number of PBMCs were recovered when washing was done at 1100 revolution per minute (rpm). Significantly high expansion fold was (2524 ± 787 expansion fold) achieved when stimulation of PBMCs was done with 1 µM of zoledronate and both cytokines IL-2 and IL-15 supported the expansion and survival of cells at the concentrations of 100 IU/ml and 10 ng/ml respectively. 14-day cultures showed highly pure (91.6 ± 5.1%) and live (96.5 ± 2.5%) expanded γδ T cells. This study aimed to standardize an easy to manipulate technique for the expansion of γδ T cells, giving a higher yield.