The effects of substrates, products and other ligands on the susceptibility of inositol monophosphatase to proteolysis by endoprotease lys-C

AbstractInositol monophosphatase is cleaved by endoprotease lys-C at a single site (Lys36—Ser37). The rate of proteolysis is greatly reduced in the presence of substrate (D,L-Ins(I)P) and Mg2+, and less so in the presence of Pi and Mg2+, consistent with protection of the susceptible bond in the E—P or E·Pi states of the enzyme. Potentiation by Li+ of the protection afforded by a substrate analogue, 1S-phosphoryloxy-2R,4S-dihydroxycyclohexane, and Mg2+ supports the idea that Li+ binds to the E—P state

Bovine inositol monophosphatase: proteolysis and structural studies

AbstractBovine brain inositol monophosphatase is inactivated when trypsin catalyses the cleavage of a single peptide bond between Lys-36 and Ser-37. This proteolysis is closely followed by cleavage at two other sites in the protein between Lys-78 and Ser-79 and between Lys-156 and Ser-157 suggesting that all of these sites are exposed in the native conformation of the protein. All of these residues are predicted to lie at the ends of α helices. The most susceptible bond (Lys-36-Ser-37) is predicted to lie in a highly flexible region of the protein. Circular dichroism studies suggest that approximately 40% of the secondary structure of this protein is helical which is similar to that predicted by the algorithm of Gamier et al. [(1978) J. Mol. Biol. 120, 97-120]

Bovine inositol monophosphatase The identification of a histidine residue reactive to diethylpyrocarbonate

AbstractThe inositol monophosphatase from bovine brain is inactivated by the histidine-specific reagent diethylpyrocarbonate. Using 4 mM reagent at pH 6.5, the reaction results in the modification of 3 equivalents of histidine per polypeptide chain. The loss of activity occurs at the same rate as the slowest reacting of these residues. Site directed mutagenesis studies have been used to generate a mutated enzyme species bearing a His-217→Gln replacement and have shown that it is the modification of histidine 217 which results in the inactivation of the enzyme

Functional mitochondria are required for amyloid beta-mediated neurotoxicity

The role of mitochondria in amyloid bpeptide (Ab)-induced cytotoxicity is unclear. We therefore exposed NT2 cells, a clonal human teratocarcinoma cell line capable of differentiation into terminal neurons, to Ab 25-35 or to Ab 1-42 to evaluate cell viability and altered mitochondrial function. A 24-h incubation of native NT2 cells (r+ cells) with Ab 25-35 or with Ab 1-42 produced a dose-dependent decline in MTT reduction. Ab 1-42 was shown to be more toxic compared with Ab 25-35. Ab 25-35 toxicity was prevented or diminished by a 22-h preincubation with antioxidants (vitamin E, melatonin, and idebenone), as well as by simultaneous incubation with GSH or the nicotinic receptor agonist nicotine. Ab 25-35 exposure was also associated with (1) inhibition of mitochondrial respiratory chain complexes (I, NADH-ubiquinone oxidoreductase; II/III, succinate-cytochrome c oxidoreductase; and IV, cytochrome c oxidase), (2) ATP depletion, and (3) reduction of the mitochondrial membrane potential. In contrast, NT2 cells rendered incapable of oxidative phosphorylation via depletion of their mitochondrial DNA (r0 cells) were unaffected by exposure to Ab 25-35 or Ab 1-42. These data indicate that Ab can disrupt mitochondrial function and that such disruption causes oxidative stress. It is further suggested that a functional mitochondrial respiratory chain is required for Ab toxicit

Isolation of the rotenone-sensitive NADH-ubiquinone reductase (complex I) from red beet mitochondria

Complex 1 of the respirator) chain (EC 1.6.531, measured as NADH-duroquinone and NADH-ubiquinone, reductase activities, was isolated from purified red beetroot (Beta vulgaris L.I mitochondria. The mitochondria were disrupted by freeze-thawing and inner membrane vesicles were pelleted. After solubilization of the vesicles with Triton X-100, the enzyme complex was purified 11-fold (compared to the activity in the inner membrane vesicles) by size-exclusion chromatography on a Sephacryl S-400 HR column and then by ion-exchange chromatography on a DEAE-Sepharose CL-6B column. Triton X-100 was present throughout the purification procedure. Tire purified complex showed approximately 30 bands on SDS-PAGE and about 15 polypeptides including those at 80. 54, 53. 51. 27. 25 and 22 kDa cross-reacted with polyclonal antibodies raised against complex I from Neurospora crassa. This is similar lo the pattern obtained with complex I from Neurospera crassa.Analysis by nativc-SDS 2-dimensional PAGE revealed the existence of several molecular mass forms of the purified complex.After reconstitution of the purified complex into phosphatidylcholine vesicles, the NADH-ubiquinone reductase activity had a Km (NADH) of about I μM and was inhibited by both rotenone and dicyclohexylcarbodiimide