Aminopeptidase chromatography

Dipeptidyl aminopeptidase (from rat brain) [9031-94-1] [EC 3.4.11.10]. Purified about 2000-fold by column chromatography on CM-cellulose, hydroxylapatite and Gly-Pro AH-Sepharose. [Imai et al. J Biochem (Tokyo)93 431 1983.]... 

H. F. M. Hermes, and J. Kamphuis, Determination of biocatalyst consumption in an aminopeptidase process using automated sample preparation and high-performance liquid chromatography,... 

Dipeptidyl peptidase IV (E.C. 3.4.14.5) cleaves off N-terminal dipeptides from peptides when a proline or alanine is at the penultimate position. The enzyme was purified from human seminal plasma and prostasomes by a two-step scheme of ion-exchange chromatography on DEAE-Sepharose, followed by affinity chromatography on adenosine deaminase (E.C. 3.5.4.4)-Sepharose [7]. This scheme resulted in a pure, native protein with an overall yield ranging from 35% to 55%. The preparation obtained was free of contaminating aminopeptidase activity. 

Although the Edman method is by far the most common method of sequencing peptides from the /V-terminus, some enzymic methods are used occasionally. Several aminopeptidases are available commercially, which differ in their specificities. One aminopeptidase from porcine kidney preferentially releases amino acids such as leucine with hydrophobic side-chains. This enzyme does not release /V-terminal Arg or Lys or any amino acid that is followed by Pro. Another enzyme, aminopeptidase M, which is obtained from the microsomal fraction of porcine kidney cells, is less specific and perhaps more useful. It is advisable to examine aliquots of the hydrolysate at intervals by chromatography to determine the order in which amino acids are being released. 

Furthermore, enzymatic hydrolysis of model isopeptides N -oligo(L-methionyl)-l-lysine from Bio-beads13031 by pepsin, chymotrypsin, cathepsin C (dipeptidyl peptidase IV) and intestinal aminopeptidase N was investigated using high-performance liquid chromatography to identify and quantify the hydrolysis products 3041. 

In an effort to generate methionine aminopeptidase inhibitors, a chromatography-free route for Suzuki coupling reactions has been developed, taking advantage of automation and microwave heating (Scheme 4.17). The approach involves initial immobilization of carboxylic-acid-functionalized heteroaryl halides on a polymer-supported 2-fert-Butylimino-2-diethylamino-l,3-dimethylperhydro-l,3,2-... 

Acetoxymercuri)aniline (36) Affinity chromatography of dipeptidyl aminopeptidase I 333 ... 

Cole [6]. The N2 fragment is known to contain the cAMP-dependent phosphorylation site, serine residue 38 [7]. We confirmed this with cAMP dependency phosphoiylated and subsequently N-bromosuccinimide-treated histone HI. The [ P]ADP-ribosylated N2 fragment was further treated with three peptidases in the order of cathepsin D, aminopeptidase M, and carboxypeptidase B, and the product was analyzed by high performance liquid chromatography. The radioactive product was identified as ADP-ribose-arginine adduct [8]. 

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