Analysis of Protein-Protein Interactions in the Arabidopsis Flavonoid Enzyme System

点击翻译按钮获取中文摘要

WPCM 2 B P Z Courier 10cpi #| x =V x 6 X @ 8 ; X @ Canon LBP-8III (Additional) CALB8IAD.PRS x @ 0J zX @ #| x 2 Z B % X F ` Canon LBP-8III (Additional) CALB8IAD.PRS x @ 0J zX @Courier 10cpi Courier Footnote 16cpi . ? x x x , x 6 X @ 8 ; X @ + H H H , S , H 6 X @ ; @ t" t } r } r 6e t t } r 6h t } r . s u t a s } r' I ut r PQV 6e L 2 K 9308279 delalleramoya The maintenance of proper intracellular levels of cholesterol is dependent on a dynamic equilibrium between cholesterol efflux and uptake of the lipid component after having bound to an extracellular acceptor in serum. To study this phenomenon, a method was developed to assay the combination of "released" cholesterol with serum acceptor molecules. The objective of the present research is to extend the current assay in order to obtain more information on other parameters that can influence cholesterol efflux. These factors include intracellular cholesterol content and the relative activities of serum components, other than those lipoproteins already known to regulate cholesterol homeostasis such as lecithin cholesterol ester hydrolase (LCAT) and cholesterol ester transfer protein (CETP). To begin to monitor the movement of cholesterol after entry into the extracellular acceptor pool, methods will be developed to assay intracellular acyl cholesterol ester transferase (ACAT). Changes in ACAT activity have been shown to correlate with cell cholesterol in numerous cell systems. With the ACAT enzyme assay in place, cholesterol efflux values will be correlated with changes in serum components such as high density lipoprotein (HDL) subclasses, other lipoproteins, and standard parameters of LCAT and CETP. Finally, the new assay method will be scaled down for screening small volume serum samples from genetically engineered mice, that have extra, or missing copies, of genes for serum proteins that play a crucial role in cholesterol metabolism. %%% A better method will be developed to measure changes in the circulating total concentration of cholesterol in serum. This is difficult to do because cholesterol exists in combination with a variety of different circulating serum components, and in this combined state, it is not detected by ordinary procedures now in use. Development of such an assay system and the identification of more of the different extracellular cholesterol absorbing components in serum will provide a better understanding of cholesterol metabolism in vertebrate animals. *** 0*0*0* 9306526 Bode Ascorbic acid is maintained in some mammalian tissue cells at concentrations almost 20 fold higher than in plasma. In the mammalian system, ascorbate exists in either of two states; ascorbic acid, the reduced form, which may autooxidize to produce dehydroascorbic acid (DHAA). DHAA is toxic, and when it is taken up by cells, it is stored intracellularly in the reduced form. The mechanism of DHAA reduction to ascorbic acid inside cells is not clear. DHAA may be reduced nonenzymatically by a variety of SH containing compounds, such as reduced glutathione, glutathione together with NADPH, or NADPH alone. In some cells, however, the rate of DHAA reduction to ascorbic acid is 2 5 times above the rate of the glutathione dependent chemical reduction reaction. This suggests that a specific DHAA ascorbic acid reduction system may be at work in some cells. In this laboratory, unique reducing activities have been detected in at least one type of tissue. Preliminary data indicate that DHAA reduction activiti es are attributable to at least two separate proteins; one protein appears in the cytosol and the other is membrane associated. In the present proposal we will attempt to isolate and characterize the action and synthetic regulation these presumptive DHAA reduction proteins and determine their role in normal cell function. %%% Ascorbic acid is as important compound in animal cells. However, on the way to the cells, ascorbic acid may react with oxygen and form a toxic derivative called dehydroascorbic acid (DHAA). To protect themselves, when the cells take up DHAA, it is converted to ascorbic acid. The question arises whether ascorbic acid is formed from DHAA by a chemical reaction inside cells, or whether there is a specific enzyme system the causes the reaction to occur. The latter case may be correct, because some cells can convert DHAA at a faster rate than can be explained by just an ordinary chemical reaction. Preliminary results in this laboratory suggest that two proteins do exist in certain cells, which may form a system to change DHAA to ascorbic acid. One protein exists in the cell cytoplasm and the other one seems to be bound to cell membrane material. We will try to purify these two proteins and determine how they operated to change DHAA into ascorbic acid. We will also try to determine what causes the cell to form these proteins and whether they are important for the cell to have. *** 0*0*0* 9306820 Borgeson We have discovered a novel delta 12 desaturase and have begun the biochemical characterization of this enzyme that places the second double bond in the essential fatty acid linoleic acid. Vertebrates require the 18:2(n 6) acid in their diet, where it becomes a structural component of membranes and is a precursor for eicosanoids. Until recently, all animals, including insects, were considered unable to synthesize linoleic acid. Only plants, protozoa and some fungi readily synthesize the fatty acid. Studies in our laboratory demonstrated, howev er, that a number of insect species do possess a delta 12 desaturase and are able to produce linoleic acid. The discovery of the desaturase in insects presents an opportunity to examine the evolution of a key enzyme in the lipid biosynthetic pathway. One of the long term goals of our research is to examine the delta 12 desaturase in different ? species, and to determine if the process of delta 12 desaturation is a primitive metabolic activity, subsequently lost by most animals, or whether possession of this enzyme reflects an example of convergent evolution. A second goal is to examine the regulation of desaturase expression at the transcriptional and translational level. Studies are described in the present proposal to clone the enzyme from two representative insect species. Once the gene coding for the enzyme in insects is isolated, future experiments designed to answer fundamental questions about the evolution and regulation of this key enzyme can begin. %%% Linoleic acid is a type of lipid, or fat, that is required in the walls of animal cells. The lipid molecule is also required in animals for the synthesis of eicosanoids, which operate in intracellular communication. Originally it was believed that linoleic acid was only synthesized by plants, protozoa and certain fungi, and that animals had to ingest the lipid molecule in their diet. However, a key enzyme in the synthesis of linoleic acid, the delta 12 desaturase, was recently found by this laboratory in insects. This discovery provides an opportunity to learn more about the evolutionary history of the linoleic acid synthetic pathway. Could it be an ancient pathway lost during the evoulation of the animal cell, or is it a property that has been acquired more recently by the insects. Experiments will be undertaken, first to clone and sequence the insect delta 12 desaturase gene(s), and th