In processes of barley plant development, various endo- and exopeptidases are involved. To determine the type and number of aminopeptidases that could participate in barley seed germination and ...tissue growth, their activities in extracts of non-germinated and germinated barley (Hordeum vulgare L. cv. Angora) grains and young tissues have been examined, and some of their properties determined. Aminopeptidases (AP) hydrolysing 2-naphthylamides of various amino acids were present in dry and germinated grains, roots, seedlings and leaves, showing preferences for amino acids phenylalanine (Phe), arginine (Arg), leucine (Leu) and methionine (Met), and lower activity towards alanine (Ala), proline (Pro), glycine (Gly) and histidine (His). Levels and ratios of AP activities changed during germination and tissue development, indicating that APs of different specificities are required at different stages of germination and in young tissues. Thus, the increase of all aminopeptidase activities during the first 24 hours of germination and subsequent decrease show significant involvement in seed primary metabolism restoration. The activities of Arg- and HisAP are equally important in green malt. Seedlings and leaves have pronounced substrate specificity for Phe, Leu, Ala and Pro, while roots have the lowest AP specific activities. From the activities and determined properties, the presence of at least six aminopeptidases optimally active at pH=7.4-8.2 could be discerned in dry and germinated grains, and young tissues of Angora barley. Two aminopeptidases are most probably of broad substrate specificity, three show narrow preference with dominating Leu, Phe, or Pro/His, while one is specific for Arg. Key words: aminopeptidase activities, barley, germination, gel filtration, isoelectric focusing (IEF)
Applicability of electrophoretic separations of albumins/globulins, followed by nonspecific protein staining and specific glycoprotein and aminopeptidase detection has been examined for barley ...variety discrimination. Albumins/globulins extracted from dry grains and green malt of six barley varieties were separated by polyacrylamide gel electrophoresis (10 % T, pH=8.9) of native and sodium dodecyl sulfate denatured proteins, and by isoelectric focusing in pH gradient of 3.5-9.5 and 4.0-6.5. Analysis of dry grain extracts gave better results than green malt extracts. Obtained data indicate that SDS-PAGE of albumins/globulins and IEF in pH gradient 3.5-9.5 followed by Coomassie Blue staining could be useful in Croatian barley variety discrimination. Angora barley could be clearly distinguished from the other varieties which were grouped as follows: Rodnik/Sladoran, Barun/Rex, and Martin. Glycoprotein patterns did not improve the recognition of individual varieties. Broad specificity, phenylalanine and leucine preferring, and arginine specific aminopeptidase were not found as applicable markers for discrimination of examined barley varieties.
Complex microbiological and biochemical changes take place during cheese ripening process, having a significant impact on the formation of organoleptic characteristics of the final product. ...Proteolytic enzymes degraded casein to larger and smaller peptides and free amino acids. The quantity and the ratios of particular amino acids and soluble peptides significantly influence the texture and organoleptic properties of cheese. The products of amino acid degradation in cheese are alcohols, aldehydes, esters, acids and sulphur compounds, which form specific aromas of various cheese types. More than 200 different volatile components have been identified in cheese. Cheese flavour is concentrated in the watersoluble fraction (peptides, amino acids, organic acids and amines), while aroma is mainly concentrated in the volatile fraction (organic acids, aldehydes, amines, esters). Many cheeses contain the same or similar components, but in different concentrations and ratios. Specific characteristics of cheese aroma are not based only on one specific compound, but on combination of different compounds produced in the maturing process. The technological process of cheese production can be accelerated and modified by influencing proteolytic processes, which are therefore the subject of intense research.
Applicability of polyacrylamide gel electrophoresis and isoelectric focusing of hordeins for discrimination of six two-rowed winter barley varieties (Angora, Sladoran, Rodnik, Rex, Martin and Barun) ...has been investigated. Hordeins extracted from dry grains and green malt of barley varieties were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in gradient gel of 8-18% T, and by isoelectric focusing in pH gradient of 3.5-9.5 and 5.5-8.5, respectively. In all separation experiments better resolution of proteins was achieved with dry grain extracts, than with malt extracts. Angora, Sladoran and Martin variety could be distinguished from other varieties by differences in hordein patterns obtained by gradient gel SDS-PAGE (8-18% T), and Angora, Sladoran, Martin and Rodnik by isoelectric focusing in pH gradient 5.5-8.5.
Aminopeptidases (APs) of the same substrate specificities have been detected in the mycelia and culture filtrate of Streptomyces rimosus. To compare extracellular and intracellular prolyl, leucyl and ...arginyl AP, dynamics of their biosynthesis, excretion and localization were analyzed during submerged cultivation of two S. rimosus strains, T55 and ZGL3, in several media. AP activity in mycelia reached maximum in the stationary phase, and decreased to different extent at a later stage. The accumulation of APs, except prolyl aminopeptidase (ProAP), in the culture filtrate followed the growth of bacteria and decreased later on, when peptide-richer medium was used. When S. rimosus was grown in glucose-richer medium, the accumulation of APs in the medium started at the late log phase and continued to the end of cultivation, due to cell lysis. The combined addition of calcium and ammonium salts to tryptone soy broth increased the AP activity in S. rimosus ZGL3 culture filtrates up to two times. The AP intracellular activity was significantly higher compared to its intercellular activity (2 to 24 times). Mycelium/medium AP activity ratio decreased with the age of the culture, its change being dependent on the S. rimosus strain, growth medium composition and AP specificity. Leucyl AP (LeuAP) was the most prone to be released from the mycelium, suggesting that part of the enzyme could be excreted by active transport. Determination of AP distribution within cell compartments has confirmed that the three APs are intracellular enzymes residing in cytosol, but also suggested their partial association with cytoplasmic membrane. Key words: prolyl, leucyl and arginyl aminopeptidases; localization; Streptomyces rimosus
Dipeptidyl peptidase (DPP III) was purified from rat and human erythrocytes using an identical procedure. Electrophoretic analyses revealed the same molecular size and pI for both enzymes. The ...molecular mass of the human enzyme, measured by matrix-assisted laser desorption/ionization MS, was 82500+/-60 Da. Its tryptic peptide mass profile was determined using the same technique, and the amino acid sequence of two internal peptides was obtained by tandem MS and Edman degradation. A search of databases revealed a high similarity between the human erythrocyte and rat liver DPP III: 21 matches out of 34 detected peptides were found, covering 40% of the total sequence. Matched peptides included the peptide harboring the characteristic HELLGH sequence motif, and a stretch of 19 identical amino acids, containing Glu, a putative ligand of active site zinc. Both enzymes preferred Arg-Arg-2-naphthylamide, and were activated by micromolar Co2+, differing in their pH optima and kcat/Km. Zn2+ ions, sulfhydryl reagents, and aminopeptidase inhibitors, especially probestin, inhibited the rat DPP III more potently. The two enzymes showed the highest affinity for angiotensin III (Ki < 1 microM) and a preference for ahydrophobic residue at the P1' site. However, significant differences in the binding constants for several peptides indicated non-identity in the active site topography of human and rat erythrocyte DPP III.
The aim of this study was to determine the content of free amino acids (
FAA
) and their ratio in ewe's milk cheese from the island
K
rk during its ripening.
FAA
content was determined by reversed ...phase
HPLC
(
RP
‐
HPLC
) of cheese aqueous/ethanol extracts after
FAA
were transformed into their 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate derivatives. Their concentration increased during ripening, reaching the value of 5% in cheese dry matter. The dominant
FAA
were glutamic acid>leucine>valine>aspartic acid>phenylalanine>serine>proline, and higher content of nonessential vs essential
FAA
was revealed.
K
rk cheese has, in relation to other cheeses, higher values for glutamic acid/leucine, glutamic acid/phenylalanine, glutamic acid/proline and smaller values for leucine/aspartic acid, valine/aspartic acid, phenylalanine/aspartic acid ratios, while other ratios are comparable to those of other hard ovine cheeses.
The aim of this study was to determine the content of free amino acids (FAA) and their ratio in ewe's milk cheese from the island Krk during its ripening. FAA content was determined by reversed phase ...HPLC (RP‐HPLC) of cheese aqueous/ethanol extracts after FAA were transformed into their 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate derivatives. Their concentration increased during ripening, reaching the value of 5% in cheese dry matter. The dominant FAA were glutamic acid>leucine>valine>aspartic acid>phenylalanine>serine>proline, and higher content of nonessential vs essential FAA was revealed. Krk cheese has, in relation to other cheeses, higher values for glutamic acid/leucine, glutamic acid/phenylalanine, glutamic acid/proline and smaller values for leucine/aspartic acid, valine/aspartic acid, phenylalanine/aspartic acid ratios, while other ratios are comparable to those of other hard ovine cheeses.
Lapstatin, a low-molecular-weight aminopeptidase inhibitor, was purified to homogeneity from Streptomyces rimosus culture filtrates. The purification procedure included extraction with methanol, ...followed by chromatography on Dowex 50WX4, AG50WX4, and HPLC RP C18 columns. By amino acid analysis, mass spectrometry, and NMR spectroscopy, the structure of lapstatin was shown to be 3-amino-2-hydroxy-4-methylpentanoylvaline. Lapstatin inhibited the extracellular leucine aminopeptidases from Streptomyces rimosus, Streptomyces griseus, and Aeromonas proteolytica with an IC50 in the range of 0.3-2.4 microM. IC50 values for other enzymes tested were at least tenfold higher. Leucine aminopeptidase from Streptomyces griseus was inhibited in a competitive manner, with an inhibition constant of 5 x 10(-7) M. Lapstatin is the first low-molecular-weight compound isolated from streptomycetes shown to inhibit an autogenous aminopeptidase.
U sirevima se tijekom zrenja odvijaju kompleksne mikrobiološke i biokemijske promjene koje imaju značajan utjecaj na formiranje organoleptičkih karakteristika gotovog proizvoda. Proteolitički
enzimi ...razgrađuju kazein do većih i manjih peptida te slobodnih aminokiselina. Količina i omjeri pojedinih aminokiselina i topljivih peptida znatno utječu na teksturu i organoleptička svojstva sira. Razgradnjom aminokiselina u siru nastaju alkoholi, aldehidi, esteri, kiseline i sumporni spojevi koji formiraju specifične arome raznih vrsta sireva. U siru je identificirano više od 200 različitih hlapljivih
komponenti. Okus sira je koncentriran u frakciji topljivoj u vodi (peptidi, aminokiseline, organske kiseline i amini), dok je aroma uglavnom koncentrirana u hlapljivoj frakciji (organske kiseline, aldehidi, amini, esteri). Mnogi sirevi sadrže iste ili slične komponente, ali u različitim koncentracijama i odnosima. Posebnosti arome sira ne zasnivaju se samo na jednom specifičnom spoju već na kombinaciji
različitih spojeva nastalih tijekom zrenja. Djelovanjem na proteolitičke procese može se ubrzati i modificirati tehnološki proces proizvodnje sira, te ih se stoga intenzivno proučava.
