1. The routes of elimination of Ng-methylarginine, Ng, Ng-dimethylarginine and Ng, Ng-dimethylarginine were investigated in the rabbit. 2. Analyses showed low plasma concentrations of these amino acids (around 1 nmol/ml) and ratios similar to those found in tissue proteins. The concentrations of these amino acids in extracts of brain, kidney, liver and spleen were similar except that liver had a lower concentration of Ng-methylarginine and Ng, Ng-dimethylarginine. Cerebrospinal fluid contained traces of each amino acid.
Reaction of N-benzyloxycarbonyl derivatives of aliphatic amino acids, and threonine, aspartic, and glutamic acids whose side-chains were protected with the t-butyl group, gave the corresponding Nmethylamino acid derivatives in good yields. The methionine derivative could be obtained by using only one mol of methyl iodide. Derivatives of threonine, and aspartic and glutamic acids whose side-chains were not protected could not be methylated. Analysis of the crude products of methylation in three cases showed that they contained 0-1% of racemized material.La rCaction des dCrivCs N-benzyloxycarbonyle d'acides aminis aliphatiques, de la thrConine, et des acides aspartique et glutamique dont les chaines IatCrales furent bloqukes avec le groupe I-butyle, a conduit aux dCrivCs N-mCthylCs correspondants des acides aminks avec d'excellents rendements. Le derivC de la mCthionine a pu &tre obtenu en utilisant seulement une mole d'iodure de mCthyle. Les dCrivCs de la thrtonine et des acides aspartique et glutamique dont les chaines IatCrales ne sont pas protCgCes, n'ont pu ittre methyles. L'analyse des produits bruts de la mkthylation a montrC que dans trois cas, ceux-ci contenaient de 0 a 1% de produits racemisis.[Traduit par le journal]Can.
The racemization of an N-methylamino-acid residue during peptide-bond formationand mixed-anhydride activation has been investigated using Ala-MeLeu-Gly and Ala-MeLeu as model peptides. The results were compared with those for Ala-Leu-Gly, Ala-Leu, and Ala-Pro. The extents of racemization were determined by analysis of the diastereomeric products of the reactions after deprotection, using an amino-acid analyzer. Extensive racemization was detected after the hydrolysis of the mixed anhydrides of Bz-MeLeu, Z-Ala-MeLeu, and Z-Ala-Leu, but not of Boc-Ala-Pro and Z-MeIle. Significant racemization (2.8-39%) was observed when Z-Ala-MeLeu was coupled with Gly-OBzl by various methods in the presence of salts such a s triethylamine hydrochloride orp-toluenesulfonate. Only coupling through the N-hydroxysuccinimide (HONSu) ester gave stereochemically pure product. In the absence of salt, less racemization was observed, but only couplings using N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline and N,N1-dicyclohexylcarbodiimide-HONSu gave essentially pure products. Polar solvents promoted racemization, but excess base did not. Chemical evidence that the racemization intermediate is an oxazolium-5-oxide has been obtained by trapping the intermediate a s an addition product (a pyrrole) in 85% yield.The yields obtained by various coupling methods have been determined for several model peptides. Couplings at the carboxyl group of an N-methylamino acid gave high yields only in the absence of salt, except for coupling by the HONSu ester method. Couplings to an N-methylamino group gave high yields, except for coupling by thep-nitrophenyl ester method. Couplings to Ala-MeLeu-OBu' gave higher yields than couplings to Ala-MeLeu-OBzl, presumably due to piperazine-dione formation by the latter. Une racemisation importante a CtC dCtectte apres hydrolyse des anhydrides mixtes de Bz-MeLeu, Z-Ala-MeLeu et Z-Ala-Leu mais pas pour Boc-Ala-Pro e t Z-MeIle. Une racemisation notables (2.b39%) a etC observCe lors du couplage du Z-Ala-MeLeu sur le Gly-OBzl par les diverses mithodes en prisence de sels tels que le chlorohydrate ou para toluene sulfonate de triethylamine. Seul le couplage par I'ester d e La N-hydroxy succinimide (HONSu) conduit 2 un produit d e puretC sttreochimique. En l'absence de sel, la racemisation est plus faible mais seuls les couplages par le NCthoxycarbonyl Cthoxy-2 dihydro-1,2 quinoleine et le complexe N,N'-dicyclohexylcarbodiimide-HONSu donnent des produits essentiellement purs. Les solvants polaires favorisent la racCmisation mais pas un exces d e base. L'intermCdiaire de racemisation oxyde-5 d'oxazolium a it6 rnis en evidence par piegeage sous forme de produits d'addition (un pyrrole) avec un rendement de 85%.Les rendements obtenus dans les divers couplages ont ete dCterminCs sur differents peptides modeles.
We examined the degradation of Alzheimer's beta-amyloid protein (1-40) by soluble and synaptic membrane fractions from post mortem human and fresh rat brain using HPLC. Most of the activity at neutral pH was in the soluble fraction. The activity was thiol and metal dependent, with a similar inhibition profile to insulin-degrading enzyme. Immunoprecipitation of insulin-degrading enzyme from the human soluble fraction using a monoclonal antibody removed over 85% of the beta-amyloid protein degrading activity. Thus insulin-degrading enzyme is the main soluble beta-amyloid degrading enzyme at neutral pH in human brain. The highest beta-amyloid protein degrading activity in the soluble fractions occurred between pH 4-5, and this activity was inhibited by pepstatin, implicating an aspartyl protease. Synaptic membranes had much lower beta-amyloid protein degrading activity than the soluble fraction. EDTA (2mM) caused over 85% inhibition of the degrading activity but inhibitors of endopeptidases -24.11, -24.15, -24.16, angiotensin converting enzyme, aminopeptidases, and carboxypeptidases had little or no effect.
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