No abstract

Chronic imbalance between production and degradation of the human amyloid-beta peptide (Abeta) is assumed to play an important role in pathogenesis of Alzheimer's disease (AD). Post-translational modifications of Abeta could influence its interactions with specifically cleaving proteases and, therefore, perturb the Abeta homeostasis. The angiotensin-converting enzyme (ACE) was previously shown to degrade non-modified Abeta in vitro and in cells. In the presented work, we investigated the effect of isomerization of Asp-7, a common non-enzymatic age-related modification found in AD-associated Abeta species, on hydrolysis of Abeta by ACE. Two synthetic peptides corresponding to the Abeta region 1-16 with either Asp or isoAsp residues in position 7 were examined as monomeric soluble substrates for the N- as well as for the C-domain of ACE. The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) coupled with the (18)O-labeled internal standard approach has allowed us to show that (i) the N-domain of ACE (N-ACE), but not the C-domain, selectively cleaves the Arg-5-His-6 bond in both peptides, and that (ii) N-ACE hydrolyzes the isoAsp-7 analogue more efficiently than the non-modified one. Our results demonstrate a new endopeptidase activity of N-ACE as well as high preference of the domain to recognize and hydrolyze the isomerized Abeta species that were earlier suggested to promote AD pathogenesis. The results suggest the need for further analysis of biological effects of isomerized Abeta and its interaction with ACE in AD pathogenesis.

No abstract

Duodenase, a serine protease from bovine duodenum mucosa, was located in endoplasmic reticulum, the Golgi secretory granules of epithelial cells and ducts of Brunner's glands by the A-gold iinmunocytochemical method. Duodenase exhibits trypsin-like and chyinotrypsin-like specificities with a preference for substrates having lysine at the P1 and proline at the P2 positions. The kinetic constants for the hydrolysis of 21 potential duodenase substrates are reported. The best substrates were found to be a-N-tosylglycylprolyllysine 4-nitroanilide (kJK,,, of 35 000 M-' s-'), a-N-succinylthreonylprolyllysine 4-nitroanilide (kc4,/K1,! of 18 000 M-' s-') and a-N-serylprolyllysine 4-nitroanilide (k,JK,, of 2600 M-' s-I), all of which contain the P I -P3 sequence of the enteropeptidase zymogen/activation site. On the basis of its catalytic properties and sites of localization, duodenase has been postulated to be an activator of the enteropeptidase precursor. A tetradecapeptide (LVTQEVSPKIVGGS) having the P9 -PS'sequence of the cleavage site of zymogen activation of bovine proenteropeptidase was synthesized, and kinetic parameters of its hydrolysis by duodenase were determined (K,,, of 87 pM; k,;,, of 1.4 s-I; k,,,/K,,, of 16000 M-' s-I). Crystals of duodenase frozen in a stream of liquid nitrogen diffracted synchrotron Xrays to 0.2-nm resolution.

Quantitation by mass spectrometry is increasingly used to monitor protein levels in biological samples. Most of the current methods are based on the relative comparison of protein quantities but are not suited for the determination of the absolute amount of a given protein. Here we describe a method for the absolute quantitation of proteins that is based on amino acid analysis by matrix-assisted laser desorption/ionization mass spectrometry. Proteins are completely hydrolyzed by acid hydrolysis and then mixed with standards of isotopically labeled amino acids. For the presented study, lysine, leucine, and two different types of labeled arginine were examined as standards. Quantitation of proteins is then achieved by measuring the ratios of labeled and unlabeled amino acids. The method has a sensitivity down to the low-femtomole range and can be applied to quantitate proteins separated by gel electrophoresis. Furthermore, we demonstrate that a mixture of two proteins can be quantitated using two labeled amino acids simultaneously.

The method of direct introduction 18O isotopes in peptides and proteins carboxylic groups through the exchange with H218O in the presence of TFA is shown. The isotope label is steady enough in awide range of pH. Because the labeled compounds retain their physical and chemical characteristics, they can be used as an internal standard in quantitative determination of authentic compounds in the analyzed sites by mass spectrometry methods. The technique may be applicable for quantitative analysis of peptides and proteins in biological environments, for quantitative study of the kinetics of metabolism and enzymatic activity. For polypeptides and proteins the quantitative analysis is combined with trypsinolysis. If necessary, the isotope label may be introduced simultaneously in all peptides and proteins control bioassays, making it suitable for use as a standard for the comparative analysis of experimental bioassays.

A series of relatively short (GCC)n triplet repeats (n = 3–30) located within regulatory regions of many mammalian genes may be considered as putative cis‐acting transcriptional elements (GCC‐elements). Fragile X‐mental retardation syndrome is caused by an expansion of (GCC)n triplet repeats within the 5′‐untranslated region of the human fragile X‐mental retardation 1 (FMR1) gene. The present study aimed to characterize a novel human (GCC)n‐binding protein and investigate its possible role in the regulation of the FMR1 gene. A novel human (GCC)n‐binding protein, p56, was isolated and identified as a Krüppel‐like transcription factor, ZF5, by MALDI‐TOF analysis. The capacity of ZF5 to specifically interact with (GCC)n triplet repeats was confirmed by the electrophoretic mobility shift assay with purified recombinant ZF5 protein. In cotransfection experiments, ZF5 overexpression repressed activity of the GCC‐element containing mouse ribosomal protein L32 gene promoter. Moreover, RNA interference assay results showed that endogenous ZF5 acts as a repressor of the human FMR1 gene. Thus, these data identify a new class of ZF5 targets, a subset of genes containing GCC‐elements in their regulatory regions, and raise the question of whether transcription factor ZF5 is implicated in the pathogenesis of fragile X syndrome.

The primary structure of three major cationic peptides from porcine neutrophils has been determined. The sequencing was made by the combined use of electrospray ionization mass spectrometry and Edman degradation. The determined sequences unambiguously show that these peptides can not be considered as defensins.