Substrate Report for: Fluorescein-dibutyrate

Combinatorial libraries of the lid domain of Rhizopus oryzae lipase (ROL; Phe88Xaa, Ala91Xaa, Ile92Xaa) were displayed on the yeast cell surface using yeast cell-surface engineering. Among the 40,000 transformants in which ROL mutants were displayed on the yeast cell surface, ten clones showed clear halos on soybean oil-containing plates. Among these, some clones exhibited high activities toward fatty acid esters of fluorescein and contained non-polar amino acid residues in the mutated positions. Computer modeling of the mutants revealed that hydrophobic interactions between the substrates and amino acid residues in the open form of the lid might be critical for ROL activity. Based on these results, Thr93 and Asp94 were further combinatorially mutated. Among 6,000 transformants, the Thr93Thr, Asp94Ser and Thr93Ser, Asp94Ser transformants exhibited a significant shift in substrate specificity toward a short-chain substrate. Computer modeling of these mutants suggested that a unique oxyanion hole, which is composed of Thr85 Ogamma and Ser94 Ogamma, was formed and thus the substrate specificity was changed. Therefore, coupling combinatorial mutagenesis with the cell surface display of ROL could lead to the production of a unique ROL mutant.

The aim of the investigation was to study the changes in lipid content and lipoprotein lipase (LPL) activity of skeletal muscle of lactating and weaned rats, in order to gain insight on the role of skeletal muscle in the metabolism of triacylglycerols not used by the mammary gland after weaning. Sprague-Dawley rats fed ad libitum were killed 0, 8, 12, and 24 h after litter separation on the 14th day of lactation. Electron microscopy, as well as determinations of the total fat and phospholipid content were performed on muscles of the right hind limb. Lipoprotein lipase activity was determined in extracts of acetone powder of skeletal muscle, mammary gland, and adipose tissue using dibutyryl fluorescine as substrate. Fat droplets were identified in the muscle interfiber spaces of weaning rats. Muscle total fat and phospholipids were higher in weaned than in lactating rats. After litter separation, lipoprotein lipase activity increased significantly in muscle and in adipose tissue (fourfold), while activity in the mammary gland decreased. The increased muscle lipoprotein lipase activity in the weaned rats seems to be associated with the higher content of fat and with the presence of fat droplets.

The development patterns of hydrolytic enzymes during germination of barley, wheat, rye and sorghum were examined by a fluorescence staining technique. The method was based upon conversion of non-fluorescent fluorescein dibutyrate to highly fluorescent fluorescein, a reaction known to be preferentially catalyzed by lipase. The formation of hydrolases was compared with cell wall breakdown in germinating barley. Fluorescence microscopy of longitudional sections of germinated cereal seeds revealed that a considerable amount of hydrolytic enzymes reacting with fluorescein dibutyrate was produced as germination proceeded. The initial formation site of the enzymes was found to be in the scutellum. Later in germination the hydrolases gradually diffused into the entire endosperm. The diffusion pattern of hydrolases in barley was found to coincide with cell wall breakdown. It is concluded that the fluorescein dibutyrate method has a potential as an indicator of the transport of de novo synthetized enzymes in cereal seeds. Practical applications of the fluorescein dibutyrate method are discussed.

Diced electrophoresis gel (DEG) assay is a methodology to identify enzymes with a specified activity in complex cell or tissue lysates by means of two-dimensional separation using isoelectric focusing and native PAGE, followed by dicing of the gel into small pieces that are assayed separately, and digestion and peptide fingerprinting to identify the protein(s) of interest in positive wells. The existing hand-made system has some disadvantages, and here we describe the development and validation of an improved cutter-plate system that enables simple, reliable and reproducible DEG assay in a 384-well plate-based format with signal readout using fluorometric or LC-MS-based reaction monitoring. To illustrate the usefulness of this system, we describe its application to profile esterase activities in ovarian adenocarcinoma SKOV3 cell lysate and mouse liver lysate that activate a fluorogenic substrate, fluorescein dibutyrate (FDBu), as well as esterase activities in mouse liver lysate that activate S-bromobenzylglutathione dicyclopentyl ester (BBGDC), a prodrug of anti-tumor agent S-bromobenzylglutathione. The activity spot patterns detected for FDBu and BBGDC were completely different, indicating that different metabolic systems are involved in hydrolysis of these substrates. The major detected spot in each case was identified. The developed system provides a highly reproducible general assay platform that should be useful for characterizing novel protein functions in complex bio-samples, as well as enzymomics studies.

Tissue-restricted bioreactions can be utilized to design chemical-biological tools and prodrugs. We have developed a fluorescent-substrate-library-based enzyme discovery approach to screen tissue extracts for enzymatic activities of interest. Assay-positive candidate proteins were identified by diced electrophoresis gel assay followed by peptide mass fingerprinting. We discovered that pyruvyl anilide is specifically hydrolyzed by carboxylesterase 2 (CES2), which is predominantly localized in the liver and kidney. We show that the pyruvyl targeting group/CES2 enzyme pair can be used to deliver the 7-amino-4-methylcoumarin fluorophore specifically to the liver and kidney in vivo. Our screening approach should be useful to find other masking group/enzyme pairs suitable for development of fluorescent substrates and prodrugs.

Combinatorial libraries of the lid domain of Rhizopus oryzae lipase (ROL; Phe88Xaa, Ala91Xaa, Ile92Xaa) were displayed on the yeast cell surface using yeast cell-surface engineering. Among the 40,000 transformants in which ROL mutants were displayed on the yeast cell surface, ten clones showed clear halos on soybean oil-containing plates. Among these, some clones exhibited high activities toward fatty acid esters of fluorescein and contained non-polar amino acid residues in the mutated positions. Computer modeling of the mutants revealed that hydrophobic interactions between the substrates and amino acid residues in the open form of the lid might be critical for ROL activity. Based on these results, Thr93 and Asp94 were further combinatorially mutated. Among 6,000 transformants, the Thr93Thr, Asp94Ser and Thr93Ser, Asp94Ser transformants exhibited a significant shift in substrate specificity toward a short-chain substrate. Computer modeling of these mutants suggested that a unique oxyanion hole, which is composed of Thr85 Ogamma and Ser94 Ogamma, was formed and thus the substrate specificity was changed. Therefore, coupling combinatorial mutagenesis with the cell surface display of ROL could lead to the production of a unique ROL mutant.

The aim of the investigation was to study the changes in lipid content and lipoprotein lipase (LPL) activity of skeletal muscle of lactating and weaned rats, in order to gain insight on the role of skeletal muscle in the metabolism of triacylglycerols not used by the mammary gland after weaning. Sprague-Dawley rats fed ad libitum were killed 0, 8, 12, and 24 h after litter separation on the 14th day of lactation. Electron microscopy, as well as determinations of the total fat and phospholipid content were performed on muscles of the right hind limb. Lipoprotein lipase activity was determined in extracts of acetone powder of skeletal muscle, mammary gland, and adipose tissue using dibutyryl fluorescine as substrate. Fat droplets were identified in the muscle interfiber spaces of weaning rats. Muscle total fat and phospholipids were higher in weaned than in lactating rats. After litter separation, lipoprotein lipase activity increased significantly in muscle and in adipose tissue (fourfold), while activity in the mammary gland decreased. The increased muscle lipoprotein lipase activity in the weaned rats seems to be associated with the higher content of fat and with the presence of fat droplets.

rapid and sensitive analysis for determining pregerminated grains in barley is described. The method is based upon a visualization of enzyme activity in longitudinally divided half seeds using the lipase sensitive fluorochrome, fluorescein dibutyrate. Parallel morphological studies of seed halves from the same pregerminated grain by the fluorescein dibutyrate method and by the established EBC-method (starch agar/iodine) revealed good coincidence between the two methods. The fluorescein dibutyrate test is faster, more sensitive and more reproducible as compared to the EBC-method. Pregermination and germination analyses of barley seed lots with pregermination percentages of 0% to 46%, showed a significant inverse relation between viability and pregermination percentage after 12 months storage in the laboratory. It is concluded that pregermination percentages as low as 2-3% could cause a significant reduction in viable barley seeds after storage.

The development patterns of hydrolytic enzymes during germination of barley, wheat, rye and sorghum were examined by a fluorescence staining technique. The method was based upon conversion of non-fluorescent fluorescein dibutyrate to highly fluorescent fluorescein, a reaction known to be preferentially catalyzed by lipase. The formation of hydrolases was compared with cell wall breakdown in germinating barley. Fluorescence microscopy of longitudional sections of germinated cereal seeds revealed that a considerable amount of hydrolytic enzymes reacting with fluorescein dibutyrate was produced as germination proceeded. The initial formation site of the enzymes was found to be in the scutellum. Later in germination the hydrolases gradually diffused into the entire endosperm. The diffusion pattern of hydrolases in barley was found to coincide with cell wall breakdown. It is concluded that the fluorescein dibutyrate method has a potential as an indicator of the transport of de novo synthetized enzymes in cereal seeds. Practical applications of the fluorescein dibutyrate method are discussed.