Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A method to provide near-constant sustained release of high molecular weight, water-soluble proteins from polyanhydride microspheres is described. The polyanhydrides used were poly(fatty acid dimer) (PFAD), poly(sebacic acid) (PSA), and their copolymers [P(FAD-SA)]. P(FAD-SA) microspheres containing proteins of different molecular sizes--lysozyme, trypsin, heparinase, ovalbumin, albumin, and immunoglobulin--were prepared by a solvent evaporation method using a double emulsion. The microspheres containing proteins were spherical, with diameters of 50-125 microns, and encapsulated more than 80% of the protein, irrespective of the protein used. Enzymatic activity studies showed that encapsulation of enzymes inside polyanhydride microspheres can protect them from activity loss. When not placed inside polyanhydride microspheres, trypsin lost 80% of its activity in solution at 37 degrees C at pH 7.4 in 12 hr, whereas inside the polyanhydride microspheres the activity loss was less than 10% under these conditions. About 47% of the enzymatic activity of heparinase encapsulated in the microspheres was lost at 37 degrees C in 24 hr, while in solution it lost over 90% of its activity. The protein-loaded microspheres displayed near-zero-order erosion kinetics over 5 days as judged by the release of sebacic acid (SA) from the microspheres. The microspheres degraded to form SA and FAD monomers. All proteins were released at a near-constant rate without any large initial burst, irrespective of polymer molecular weight and protein loading. The period of protein release was longer than that of SA and continued protein release was observed even after the microsphere matrix had completely degraded. Differential scanning calorimetric studies demonstrated an interaction between protein and the FAD monomers produced with microsphere degradation. It is likely that the protein interaction with FAD monomers permits formation of water-insoluble protein aggregates which slowly dissolve and diffuse out of the matrix, leading to delayed protein release. For trypsin-loaded microspheres, trypsin lost 40% of its activity during microsphere preparation. Activity studies demonstrated that the sonication process was primarily responsible for activity loss. A reduction in the period of ultrasound exposure decreased the loss of protein activity to around 20%.
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PMID:Controlled delivery systems for proteins using polyanhydride microspheres. 848 30

Both metalloprotein and flavin-linked sulfhydryl oxidases catalyze the oxidation of thiols to disulfides with the reduction of oxygen to hydrogen peroxide. Despite earlier suggestions for a role in protein disulfide bond formation, these enzymes have received comparatively little general attention. Chicken egg white sulfhydryl oxidase utilizes an internal redox-active cystine bridge and a FAD moiety in the oxidation of a range of small molecular weight thiols such as glutathione, cysteine, and dithiothreitol. The oxidase is shown here to exhibit a high catalytic activity toward a range of reduced peptides and proteins including insulin A and B chains, lysozyme, ovalbumin, riboflavin-binding protein, and RNase. Catalytic efficiencies are up to 100-fold higher than for reduced glutathione, with typical K(m) values of about 110-330 microM/protein thiol, compared with 20 mM for glutathione. RNase activity is not significantly recovered when the cysteine residues are rapidly oxidized by sulfhydryl oxidase, but activity is efficiently restored when protein disulfide isomerase is also present. Sulfhydryl oxidase can also oxidize reduced protein disulfide isomerase directly. These data show that sulfhydryl oxidase and protein disulfide isomerase can cooperate in vitro in the generation and rearrangement of native disulfide pairings. A possible role for the oxidase in the protein secretory pathway in vivo is discussed.
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PMID:Sulfhydryl oxidase from egg white. A facile catalyst for disulfide bond formation in proteins and peptides. 1042 77

Thermal inactivation of glucose oxidase (GOD; beta-d-glucose: oxygen oxidoreductase), from Aspergillus niger, followed first order kinetics both in the absence and presence of additives. Additives such as lysozyme, NaCl, and K2SO4 increased the half-life of the enzyme by 3.5-, 33.4-, and 23.7-fold respectively, from its initial value at 60 degrees C. The activation energy increased from 60.3 kcal mol-1 to 72.9, 76.1, and 88.3 kcal mol-1, whereas the entropy of activation increased from 104 to 141, 147, and 184 cal x mol-1 x deg-1 in the presence of 7.1 x 10-5 m lysozyme, 1 m NaCl, and 0.2 m K2SO4, respectively. The thermal unfolding of GOD in the temperature range of 25-90 degrees C was studied using circular dichroism measurements at 222, 274, and 375 nm. Size exclusion chromatography was employed to follow the state of association of enzyme and dissociation of FAD from GOD. The midpoint for thermal inactivation of residual activity and the dissociation of FAD was 59 degrees C, whereas the corresponding midpoint for loss of secondary and tertiary structure was 62 degrees C. Dissociation of FAD from the holoenzyme was responsible for the thermal inactivation of GOD. The irreversible nature of inactivation was caused by a change in the state of association of apoenzyme. The dissociation of FAD resulted in the loss of secondary and tertiary structure, leading to the unfolding and nonspecific aggregation of the enzyme molecule because of hydrophobic interactions of side chains. This confirmed the critical role of FAD in structure and activity. Cysteine oxidation did not contribute to the nonspecific aggregation. The stabilization of enzyme by NaCl and lysozyme was primarily the result of charge neutralization. K2SO4 enhanced the thermal stability by primarily strengthening the hydrophobic interactions and made the holoenzyme a more compact dimeric structure.
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PMID:Thermal inactivation of glucose oxidase. Mechanism and stabilization using additives. 1271 78

Mia40 participates in oxidative protein folding within the mitochondrial intermembrane space (IMS) by mediating the transfer of reducing equivalents from client proteins to FAD-linked oxidoreductases of the Erv1 family (lfALR in mammals). Here we investigate the specificity of the human Mia40/lfALR system towards non-cognate unfolded protein substrates to assess whether the efficient introduction of disulfides requires a particular amino acid sequence context or the presence of an IMS targeting signal. Reduced pancreatic ribonuclease A (rRNase), avian lysozyme, and riboflavin binding protein are all competent substrates of the Mia40/lfALR system, although they lack those sequence features previously thought to direct disulfide bond formation in cognate IMS substrates. The oxidation of rRNase by Mia40 does not limit overall turnover of unfolded substrate by the Mia40/lfALR system. Mia40 is an ineffective protein disulfide isomerase when its ability to restore enzymatic activity from scrambled RNase is compared to that of protein disulfide isomerase. Mia40's ability to bind amphipathic peptides is evident by avid binding to the isolated B-chain during the insulin reductase assay. In aggregate these data suggest that the Mia40/lfALR system has a broad sequence specificity and that potential substrates may be protected from adventitious oxidation by kinetic sequestration within the mitochondrial IMS.
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PMID:Mia40 is a facile oxidant of unfolded reduced proteins but shows minimal isomerase activity. 2601 36