Metabolism of aspartame by human and pig intestinal microvillar peptidases.
The artificial sweetener aspartame (N-L-alpha-aspartyl-L-phenyl-alanine-1-methyl ester; Nutrasweet), its decomposition product alpha Asp-Phe and the related peptide alpha Asp-PheNH2 were rapidly hydrolysed by microvillar membranes prepared from human duodenum, jejunum and ileum, and from pig duodenum and kidney. The metabolism of aspartame by the human and pig intestinal microvillar membrane preparations was inhibited significantly (> 78%) by amastatin or 1,10-phenanthroline, and partially (> 38%) by actinonin or bestatin, and was activated 2.9-4.5-fold by CaCl2. The inhibition by amastatin and 1,10-phenanthroline, and the activation by CaCl2 are characteristic of the cell-surface ectoenzyme aminopeptidase A (EC 3.4.11.7) and a purified preparation of this enzyme hydrolysed aspartame with a Km of 0.25 mM and a Vmax of 126 mumol/min per mg. A purified preparation of aminopeptidase W (EC 3.4.11.16) also hydrolysed aspartame but with a Km of 4.96 mM and a Vmax of 110 mumol/min per mg. However, rentiapril, an inhibitor of aminopeptidase W, caused only slight inhibition (maximally 19%) of the hydrolysis of aspartame by the microvillar membrane preparations. Similar patterns of inhibition and kinetic parameters were observed for alpha Asp-Phe and alpha Asp-PheNH2. Two other decomposition products of aspartame, beta Asp-PheMe and cyclo-Asp-Phe, were essentially resistant to hydrolysis by both the human and pig intestinal microvillar membrane preparations and the purified preparations of aminopeptidases A and W. Although the relatively selective inhibitor of aminopeptidase N (EC 3.4.11.2), actinonin, partially inhibited the metabolism of aspartame, alpha Asp-Phe and alpha Asp-PheNH2 by the human and pig intestinal microvillar membrane preparations, these peptides were not hydrolysed by a purified preparation of aminopeptidase N. Membrane dipeptidase (EC 3.4.13.19) only hydrolysed the unblocked dipeptide, alpha Asp-Phe, but the selective inhibitor of this enzyme, cilastatin, did not block the metabolism of alpha Asp-Phe by the microvillar membrane preparations.
Hooper
N M
NM
Department of Biochemistry and Molecular Biology, University of Leeds, UK.
Hesp
R J
RJ
Tieku
S
S
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001218:Aspartame / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D043384:Glutamyl Aminopeptidase; D006801:Humans; D006868:Hydrolysis; D007422:Intestines / Q000201:enzymology*; D007668:Kidney / Q000201:enzymology; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008871:Microvilli / Q000201:enzymology; D010455:Peptides; D010618:Phenanthrolines / Q000494:pharmacology; D013552:Swine
D000900:Anti-Bacterial Agents; D010455:Peptides; D010618:Phenanthrolines; C016977:amastatin; D000626:Aminopeptidases; C047029:aminopeptidase W; D043384:Glutamyl Aminopeptidase; D007930:Leucine; C012211:ubenimex; D002122:Calcium Chloride; C025205:1,10-phenanthroline; D001218:Aspartame