Arginine metabolising enzymes as therapeutic tools for Alzheimer's disease: peptidyl arginine deiminase catalyses fibrillogenesis of beta-amyloid peptides.
The accumulation of arginine in the cerebrospinal fluid and brains of patients suffering from acute neurodegenerative diseases like Alzheimer's disease, point to defects in the metabolic pathways involving this amino acids. The deposits of neurofibrillary tangles and senile plaques perhaps as a consequence of fibrillogenesis of beta-amyloid peptides has also been shown to be a hallmark in the aetiology of certain neurodegenerative diseases. Peptidylarginine deiminase (PAD II) is an enzyme that uses arginine as a substrate and we now show that PAD II not only binds with the peptides Abeta(1-40), Abeta(22-35), Abeta(17-28), Abeta(25-35) and Abeta(32-35) but assists in the proteolytic degradation of these peptides with the concomitant formation of insoluble fibrils. PAD was purified in 12.5% yield and 137 fold with a specific activity of 59 micromol min(-1) mg(-1) from bovine brain by chromatography on diethylaminoethyl (DEAE)-Sephacel. Characterisation of the enzyme gave a pH and temperature optima of 7.5 degrees C and 68 degrees C, respectively, and the enzyme lost 50% activity within 38 min at this temperature. Michaelis-Menten kinetics established a V(max) and K(m) of 1.57 micromol min(-1) ml(-1) and 1.35 mM, respectively, with N-benzoyl arginine ethyl ester as substrate. Kinetic analysis was used to measure the affinity (K(i)) of the amyloid peptides to PAD with values between 1.4 and 4.6 microM. The formation of Abeta fibrils was rate limiting involving an initial lag time of about 24 h that was dependent on the concentration of the amyloid peptides. Turbidity measurements at 400 nm, Congo Red assay and Thioflavin-T staining fluorescence were used to establish the aggregation kinetics of PAD-induced fibril formation.
Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, P.O. Box 94, Grahamstown, 6140, South Africa.
Peter
P
Mohlake
Chris G
CG
Whiteley
D000544:Alzheimer Disease / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D002417:Cattle; D006801:Humans; D006867:Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016874:Neurofibrillary Tangles / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D000076342:Protein-Arginine Deiminases
D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; D001120:Arginine; D006867:Hydrolases; D000076342:Protein-Arginine Deiminases