myelin-basic-protein and 6-carboxyfluorescein

There is an allosteric relationship between the kinase and RNase domains of the ER stress sensor IRE1α. This relationship has been exploited to develop ATP-competitive inhibitors that are able to divergently modulate the RNase activity of IRE1α through its kinase domain. Here, we describe a series of biochemical methods for profiling the dual enzymatic activities of IRE1α. These methods can be used to ascertain how ATP-competitive inhibitors affect the kinase activity of IRE1α and for determining whether these ligands allosterically activate or inactivate RNase activity.

Topics: Adenosine Triphosphate; Allosteric Regulation; Animals; Baculoviridae; Catalytic Domain; Endoribonucleases; Enzyme Assays; Fluoresceins; Fluorescent Dyes; Gene Expression; Humans; Inhibitory Concentration 50; Ligands; Myelin Basic Protein; Phosphorus Radioisotopes; Phosphorylation; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Recombinant Proteins; Sf9 Cells; Spodoptera

Among several cholesterol oxidation products, 25-hydroxycholesterol is particularly potent in enhancing basic protein-induced carboxyfluorescence leakage from liposomes. Both myelin basic protein and poly(L-arginine) are effective at submicromolar concentrations in stimulating this vesicle rupture. Leakage is greatly augmented by the presence of only 1% of the 25-hydroxycholesterol. The possible relevance of these findings to myelin pathology is discussed.

Topics: Cholesterol; Fluoresceins; Humans; Hydroxycholesterols; Liposomes; Myelin Basic Protein; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Spectrometry, Fluorescence

The capacity of myelin basic protein or of poly-L-lysine to promote leakage of carboxyfluorescein from vesicles or the aggregation of vesicles was studied. The vesicles were composed of phosphatidylcholine as the sole or major lipid component. Addition of 10% sphingomyelin, 10% phosphatidylglycerol, 10% egg or bovine brain phosphatidylethanolamine, or 30% dodecanal had relatively little effect on the extent of carboxyfluorescein release in the presence of either myelin basic protein or poly-L-lysine. In contrast with these results, the extent of vesicle aggregation was very sensitive to lipid composition. Addition of 10% phosphatidylglycerol induced more aggregation than the other phospholipids tested. Admixing 10% of a partially degraded sample of bovine brain phosphatidylethanolamine also led to a large amount of aggregation induced by the myelin basic protein. This latter aggregation appeared more specific for the basic protein, as it occurred to a much smaller extent with poly-L-lysine. In general, the effects of the myelin basic protein on either carboxyfluorescein release or vesicle aggregation were similar to, although somewhat greater than, that of poly-L-lysine. The aggregation of vesicles containing degradation products of phosphatidylethanolamine can be ascribed largely to the presence of aliphatic aldehydes. The effect of aliphatic aldehydes was specific in that the aliphatic alcohol, hexadecanol, or the short-chain aldehydes, acetaldehyde or butyraldehyde, did not promote myelin basic protein-induced vesicle aggregation. In addition, poly-L-lysine was less effective than the basic protein in aggregating vesicles containing aliphatic aldehydes. (ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aldehydes; Dodecanol; Fatty Alcohols; Fluoresceins; Liposomes; Myelin Basic Protein; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Polylysine; Structure-Activity Relationship