Dipeptidyl Aminopeptidase Activity Research Articles

Dipeptidylamino-tripeptidylcarboxypeptidase NEMP3 and DPP3 (DPP III) are the same protein

Earlier it was shown that a group of extracellular low-specific metallopeptidases is present in the mammalian brain Kropotova and Mosevitsky (2016) [1]. These enzymes are weakly connected to the axonal ends of neurons. They were named Neuron bound Extracellular MetalloPeptidases (NEMP). The enzyme named NEMP3 turned out to be a unique exopeptidase that exhibits two activities: it removes the dipeptide from the N-end of the peptide, and it can also remove the tripeptide from the C-end of the peptide. Therefore, NEMP3 possesses the activities of dipeptidylaminopeptidase and of tripeptidylcarboxypeptidase. Mass spectrometry has revealed a homology of NEMP3 with DPP3 (DPP III, EC3.4.14.4), known as cytosolic dipeptidylaminopeptidase. We isolated DPP3 from rat and bovine liver and brain by the procedures used for this purpose by other authors. The effect of DPP3 on test peptides is the same as that of NEMP3. In particular, all DPP3 samples delete the tripeptide (AKF) from the C-end of the test peptide blocked at the N-end. The data obtained show that NEMP3 and DPP3 are the same protein (enzyme). Thus, DPP3 has two exopeptidase activities: the previously known activity of dipeptidylaminopeptidase and the activity of tripeptidylcarboxypeptidase discovered in this study. Another discovery is the extracellular activity of DPP 3 in the mammalian brain near synapses, which controls neuropeptides. DPP3 is involved in various processes, but in many cases its role remains to be clarified. The results obtained in this study will be useful for solving these questions.

Identification of Novel Natural Substrates of Fibroblast Activation Protein-alpha by Differential Degradomics and Proteomics.

Fibroblast activation protein-alpha (FAP) is a cell-surface transmembrane-anchored dimeric protease. This unique, constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities and can hydrolyze the post-proline bond. FAP expression is very low in adult organs but is upregulated by activated fibroblasts in sites of tissue remodeling, including fibrosis, atherosclerosis, arthritis and tumors. To identify the endogenous substrates of FAP, we immortalized primary mouse embryonic fibroblasts (MEFs) from FAP gene knockout embryos and then stably transduced them to express either enzymatically active or inactive FAP. The MEF secretomes were then analyzed using degradomic and proteomic techniques. Terminal amine isotopic labeling of substrates (TAILS)-based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CXCL-5, CSF-1, and C1qT6, that were confirmed in vitro In addition, differential metabolic labeling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation, metabolism and wound healing associated proteins. Plasma from FAP-deficient mice exhibited slower than wild-type clotting times. This study provides a significant expansion of the substrate repertoire of FAP and provides insight into the physiological and potential pathological roles of this enigmatic protease.

Plasmodium falciparum dipeptidyl aminopeptidase 3 activity is important for efficient erythrocyte invasion by the malaria parasite.

Parasite egress from infected erythrocytes and invasion of new red blood cells are essential processes for the exponential asexual replication of the malaria parasite. These two tightly coordinated events take place in less than a minute and are in part regulated and mediated by proteases. Dipeptidyl aminopeptidases (DPAPs) are papain-fold cysteine proteases that cleave dipeptides from the N-terminus of protein substrates. DPAP3 was previously suggested to play an essential role in parasite egress. However, little is known about its enzymatic activity, intracellular localization, or biological function. In this study, we recombinantly expressed DPAP3 and demonstrate that it has indeed dipeptidyl aminopeptidase activity, but contrary to previously studied DPAPs, removal of its internal prodomain is not required for activation. By combining super resolution microscopy, time-lapse fluorescence microscopy, and immunoelectron microscopy, we show that Plasmodium falciparum DPAP3 localizes to apical organelles that are closely associated with the neck of the rhoptries, and from which DPAP3 is secreted immediately before parasite egress. Using a conditional knockout approach coupled to complementation studies with wild type or mutant DPAP3, we show that DPAP3 activity is important for parasite proliferation and critical for efficient red blood cell invasion. We also demonstrate that DPAP3 does not play a role in parasite egress, and that the block in egress phenotype previously reported for DPAP3 inhibitors is due to off target or toxicity effects. Finally, using a flow cytometry assay to differentiate intracellular parasites from extracellular parasites attached to the erythrocyte surface, we show that DPAP3 is involved in the initial attachment of parasites to the red blood cell surface. Overall, this study establishes the presence of a DPAP3-dependent invasion pathway in malaria parasites.

Effect of supplemental yeast cell walls on growth performance, gut mucosal glutathione pathway, proteolytic enzymes and transporters in growing broiler chickens.

This study aimed to investigate the effect of supplemental yeast cell walls (YCW) on growth performance, gut mucosal glutathione pathway, proteolytic enzymes, and transporters in broiler chickens from 1 to 21 d of age. One-day-old broiler chickens (n = 480) were randomly allocated to 4 treatments with 6 replicates of 20 chicks each for diets containing YCW at 0, 0.5, 1.0, and 1.5 g/kg. The results showed that YCW supplementation increased (P < 0.05) ADFI and ADG by 15.3% and 16.0%, respectively, and the 2 higher doses of YCW had greater effects (P < 0.05) than the lower dose. For the glutathione pathway in the duodenal mucosa, the levels of reduced glutathione and glutathione reductase in the YCW treatments were increased (P < 0.05) by 15.6% and 17.4%, respectively, but glutathione S-transferases was not affected. Similarly, the YCW increased (P < 0.05) the mucosal activities of trypsin, dipeptidyl, and leucine aminopeptidase by 12.4%, 5.5%, and 17.3%, respectively, and the mRNA profiles of neutral, cationic, and oligopeptide transporters by 62.5%, 69.1%, and 11.5%, respectively. The YCW also increased (P < 0.05) the concentrations of Thr, Met, Ile, Leu, Lys, Arg, and Tyr in the blood by 8.8% to 39.2%. Additionally, the ADFI, ADG, reduced glutathione, trypsin, dipeptidyl aminopeptidase, leucine aminopeptidase, and cationic amino acid transporter increased linearly and quadratically (P < 0.05) with YCW supplementation. The results suggest that YCW supplementation can beneficially modulate intestinal glutathione pathway, proteolytic enzyme activity, and nutrient transport in growing animals.

Evaluation of a Commercial Sandwich Enzyme-Linked Immunosorbent Assay for the Quantification of Beta-Casomorphin 7 in Yogurt Using Solid-Phase Extraction Coupled to Liquid Chromatography-Tandem Mass Spectrometry as the "Gold Standard" Method.

This study investigated beta-casomorphin 7 (BCM7) in yogurt by means of LC-tandem MS (MS/MS) and enzyme-linked immunosorbent assay (ELISA) and use LC-MS/MS as the "gold standard" method to evaluate the applicability of a commercial ELISA. The level of BCM7 in milk obtained from ELISA analysis was much lower than that obtained by LC-MS/MS analysis and trended to increase during fermentation and storage of yogurt. Meanwhile, the results obtained from LC-MS/MS showed that BCM7 degraded during stages of yogurt processing, and its degradation may have been caused by X-prolyl dipeptidyl aminopeptidase activity. As a result, the commercial sandwich ELISA kit was not suitable for the quantification of BCM7 in fermented dairy milk.

Technological characterization of Lactobacillus in semihard artisanal goat cheeses from different Mediterranean areas for potential use as nonstarter lactic acid bacteria

The potential of 25 Lactobacillus isolates from 8 semihard artisanal goat cheeses manufactured in 4 different Mediterranean areas was examined for use as nonstarter lactic acid bacteria. The isolates were identified using 16S rDNA sequence analysis. Sixteen strains belonged to Lactobacillus paracasei and 9 to Lactobacillus rhamnosus. The isolates were first screened for salt tolerance, exopolysaccharide and diacetyl production, proteolytic and lipolytic activity, and acidification and autolyzing capacities. Most of the lactobacilli displayed strong salt tolerance [20 strains, including 13 of Lb. paracasei and 7 of Lb. rhamnosus, could grow at 6% (wt/vol) salt], low acidification activity (16 strains, including 9 of Lb. paracasei and 7 of Lb. rhamnosus, presented change in pH ≤0.4 U after 6 h of growth), and high autolytic activity (14 strains, including 9 of Lb. paracasei and 5 of Lb. rhamnosus, showed autolysis values ranging between 25 and 65%). Eleven Lb. paracasei and 6 Lb. rhamnosus produced exopolysaccharide, whereas 8 Lb. paracasei and 4 Lb. rhamnosus produced diacetyl. Moreover, 9 Lb. paracasei and 6 Lb. rhamnosus showed proteolytic activity; none of the isolates showed lipolytic activity. Based on the above characteristics, 8 strains were further evaluated for peptidase activity, including aminopeptidase, dipeptidyl aminopeptidase, and dipeptidase activities. The results indicated that all strains showed peptidase activity toward selected substrates. The substrate specificity and extent of peptidase activities were strain-dependent. Four strains (A-3, B-4, D-3, and D-8) presented the best characteristics and represented the most promising nonstarter lactic acid bacteria candidates for use in industrial manufacturing of goat cheese.

The Ginkgo biloba Extract Reverses the Renal Effects of Titanium Dioxide Nanoparticles in Adult Male Rats.

The Ginkgo biloba extract (GbE) is a commercial product used as a nutraceutic herbal remedy in Europe and US. It contains 27% of the polyphenols isorhamnetin, kaempferol, and quercetin, as antioxidants. We used male adult Wistar rats (200–300 g), divided into four groups: control group (treated with 5.0 mg/kg of sodium chloride, intravenous), titanium dioxide nanoparticles (TiO2-NPs) group (5.0 mg/kg, intravenous), GbE group (10 mg/kg, intraperitoneal), and GbE + TiO2-NPs group (treated 24 h before with 10 mg/kg of GbE, intraperitoneal), followed, 24 h later, by 5.0 mg/kg of TiO2-NPs intravenously. The statistical analysis was performed using Student's t-test for grouped data with ANOVA posttest. The GbE protected renal cells against the effects of TiO2-NPs because it reversed the increased activity of γ-glutamyltranspeptidase and the enzymatic activity of dipeptidylaminopeptidase IV at all times tested (0–5, 5–24, 24–48, and 48–72 h). Also it reversed the glucosuria, hypernatriuria, and urine osmolarity at three times tested (5–24, 24–48, and 48–72). Thus, we conclude that GbE has a beneficial activity in the cytoplasmic membranes of brush border cells on the renal tubules, against the adverse effects that can be produced by some xenobiotics in this case the TiO2-NPs, in experimental rats.

Characterization of protease activities in a crude extract of germinated cacao

Dynamic light scattering (DLS) was applied to the analysis of the hydrodynamic diameter distribution of proteins in extract of germinated cacao. The interactions among divalent cations and their relation with the activity of Xaa-prolyl dipeptidyl aminopeptidase 2 (Xaa-Pro-DAP2) enzyme were evaluated. The peptidases were isolated by 80% saturation of ammonium sulfate from acetone extract of germinated cacao seeds. The results showed a higher activity of Xaa-Pro-DAP2, besides aminopeptidase (APE) and carboxypeptidase (CP) enzymes. Using DLS analysis the size distribution was found to be multi-modal; however with Cu2+ and Cd2+ at 1 mM, the size distribution was found to be monomodal with a hydrodynamic diameter of 158.5 and 119 nm, respectively. The distribution remained constant from 60 to 80°C, suggesting that thermal stability is related to increase in Xaa-Pro-DAP2 activity an lower protein aggregation. APE activity was slightly activated by Co2+ at 1 mM, whereas no significant effect was observed on CP activity.

Acid adaptation to improve viability and X‐prolyl dipeptidyl aminopeptidase activity of the probiotic bacterium Lactobacillus fermentum HA6 exposed to simulated gastrointestinal tract conditions

SummaryIn this work, the viability of the probiotic bacterium Lactobacillus fermentum HA6 isolated from naturally fermented vegetables in Vietnam was improved by growing the bacterium into a mild acid condition (pH 4.0). Viability and probiotic functionality [X‐prolyl dipeptidyl aminopeptidase (PepX) activity] of the acid‐adapted bacterium exposed to simulated gastrointestinal conditions were investigated. After 180 min in the simulated gastric juice (0.3 g/L pepsin, pH 2.0), the viability of acid‐adapted L. fermentum HA6 (11.5%) was higher than that of control L. fermentum HA6 (2.2%). Specific PepX activity of acid‐adapted cells (24.5 U/mg) was higher than that of control cells (17.8 U/mg). After 180‐min exposure to the simulated small intestinal medium (0.3 g/L bile salts, 0.1 g/L pancreatin, pH 8.0), the viability of acid‐adapted L. fermentum HA6 (13.5%) was twofold as high as that of control L fermentum HA6 (8.0%). Our results suggested that acid adaptation has a key role in acquiring cross‐protection mechanism, which in this study resulted in higher survival of L. fermentum HA6 after simulated gastrointestinal stresses. The strategy of acid adaptation could be valuable for the production of robust probiotics.

E‐64c‐Hydrazide: A Lead Structure for the Development of Irreversible Cathepsin C Inhibitors

Cathepsin C is a papain-like cysteine protease with dipeptidyl aminopeptidase activity that is thought to activate various granule-associated serine proteases. Its exopeptidase activity is structurally explained by the so-called exclusion domain, which blocks the active-site cleft beyond the S2 site and, with its Asp 1 residue, provides an anchoring point for the N terminus of peptide and protein substrates. Here, the hydrazide of (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane (E-64c) (k2/Ki =140±5 M(-1) s(-1)) is demonstrated to be a lead structure for the development of irreversible cathepsin C inhibitors. The distal amino group of the hydrazide moiety addresses the acidic Asp 1 residue at the entrance of the S2 pocket by hydrogen bonding while also occupying the flat hydrophobic S1'-S2' area with its leucine-isoamylamide moiety. Furthermore, structure-activity relationship studies revealed that functionalization of this distal amino group with alkyl residues can be used to occupy the conserved hydrophobic S2 pocket. In particular, the n-butyl derivative was identified as the most potent inhibitor of the series (k2/Ki =56 000±1700 M(-1) s(-1)).

A Model to Assess Lactic Acid Bacteria Aminopeptidase Activities in Parmigiano Reggiano Cheese During Ripening

The aim of this work was to investigate in which phases of ripening of Parmigiano Reggiano cheese lactic acid bacteria aminopeptidases present in cheese extract could be involved in release of free amino acids and to better understand the behavior of these enzymes in physical-chemical conditions that are far from their optimum. In particular, we evaluated 6 different substrates to reproduce broad-specificity aminopeptidase N, broad-specificity aminopeptidase C, glutamyl aminopeptidase A, peptidase with high specificity for leucine and alanine, proline iminopeptidase, and X-prolyl dipeptidyl aminopeptidase activities releasing different N-terminal amino acids. The effects of pH, NaCl concentration, and temperature on the enzyme activities of amino acid β-naphthylamide (βNA)-substrates were determined by modulating the variables in 19 different runs of an experimental design, which allowed the building of mathematical models able to assess the effect on aminopeptidases activities over a range of values, obtained with bibliographic data, covering different environmental conditions in different zones of the cheese wheel at different aging times. The aminopeptidases tested in this work were present in cell-free Parmigiano Reggiano cheese extract after a 17-mo ripening and were active when tested in model system. The modeling approach shows that to highlight the individual and interactive effects of chemical-physical variables on enzyme activities, it is helpful to determine the true potential of an amino-peptidase in cheese. Our results evidenced that the 6 different lactic acid bacteria peptidases participate in cheese proteolysis and are induced or inhibited by the cheese production parameters that, in turn, depend on the cheese dimension. Generally, temperature and pH exerted the more relevant effects on the enzymatic activities, and in many cases, a relevant interactive effect of these variables was observed. Increasing salt concentration slowed down broad-specificity amino-peptidase C, glutamyl aminopeptidase A, proline iminopeptidase, and peptidase with high specificity for leucine and alanine. Interestingly, this variable did not affect broad-specificity aminopeptidase N and positively affected X-prolyl dipeptidyl aminopeptidase. The models elaborated varying pH, temperatures, and salt concentration and were a useful, low cost, and fast tool to understand the role of the main peptidases in the different phases of cheese ripening in relation to the major environmental factors influencing enzyme activity.

Transepithelial Transport and Stability in Blood Serum of Angiotensin-I-Converting Enzyme Inhibitory Dipeptides

The dipeptides Ala-Trp, Val-Phe, and Val-Tyr inhibit the angiotensin-I-converting enzyme. They are encrypted within the primary sequences of different food proteins, e.g. milk proteins. The angiotensin-I-converting enzyme inhibitory potency of these synthetic dipeptides was quantified using a spectrophotometric assay. The dipeptides showed no adverse effects on differentiated Caco-2 cells (model for human intestinal epithelium), as confirmed by transepithelial electrical resistance, microscopy and the activity of the brush-border enzyme dipeptidyl aminopeptidase IV. Furthermore, the transport of these bioactive dipeptides through intact Caco-2 monolayers and their stability to incubation in human blood serum has been demonstrated for the first time. Low molecular mass peptides represent the minimal structures required for angiotensin-I-converting enzyme inhibition which have a high potential bioavailability. Therefore, they may act as target peptides in enriched hydrolysates for the preparation of an angiotensin-I-converting enzyme inhibitory peptide and for the use in special formulations as functional foods/foods of specified health use.

Angiotensin I-converting enzyme inhibitory activity in milk fermented by wild-type and peptidase-deletion derivatives of Lactobacillus helveticus CNRZ32

Development of molecular methods has enabled detailed studies of the proteolytic system of Lactobacillus helveticus, which has a central role in the release of bioactive peptides during the fermentation of milk. The impact of general aminopeptidase (PepN) and X-prolyl dipeptidyl aminopeptidase (PepX) activity of L. helveticus on the level of angiotensin I-converting enzyme (ACE) inhibitory activity in fermented milk was elucidated by taking advantage of peptidase-negative derivatives of L. helveticus CNRZ32. According to the results, increased level of ACE-inhibitory activity was attained in milk fermented by the peptidase-deficient mutants. The ACE-inhibitory activity determined with the PepN-deficient strain decreased towards the end of fermentation, suggesting that PepN gives rise to a provisional accumulation of ACE-inhibitory peptides before their hydrolysis to shorter peptides or free amino acids. The ACE-inhibitory activity determined with the PepX-deficient strain increased throughout the fermentation, indicating specific blocking of the further hydrolysis of ACE-inhibitory peptides.

Enzymatic activities of lactic acid bacteria isolated from Cornetto di Matera sourdoughs

Forty-one strains of lactic acid bacteria (LAB) isolated from Cornetto di Matera sourdoughs were screened for their enzymatic activities, to elucidate their possible roles during the fermentation process. Urease, peptidase, phytase, phosphatase and β-glucosidase activities were measured spectrophotometrically using synthetic substrates. Proteolytic activities were examined in model doughs, using neutral and acidified sterile doughs as controls. All strains had low urease, glutamyl aminopeptidase and iminopeptidase activities, whereas differences within species were observed for the other enzymatic activities. Leuconostoc mesenteroides and Lactobacillus curvatus strains generally showed high aminopeptidase, X-prolyl dipeptidyl aminopeptidase, β-glucosidase and phytase activities, while the enzymatic activities of Lactobacillus plantarum, Lactobacillus pentosus and Weissella cibaria varied between strains. In order to classify the strains on the basis of similar enzymatic profiles, a hierarchical cluster analysis was carried out. Several strains of L. plantarum, L. curvatus and Leuc. mesenteroides showed an interesting combination of proteolytic, peptidase, β-glucosidase and phytase activities, suggesting their possible usefulness as a mixed starter culture in bread-making processes.

Analysis and expression of STE13ca gene encoding a putative X-prolyl dipeptidyl aminopeptidase from Candida albicans

Candida albicans STE13ca gene was identified by its homology to the Saccharomyces cerevisiae STE13 gene that encodes for the dipeptidyl aminopeptidase A (DAP A) involved in the maturation of α-factor mating pheromone. Our study revealed that C. albicans ATCC 10231 depicts dipeptidyl aminopeptidase activity. We also analyzed the expression of the STE13ca gene homologue from this pathogenic yeast. This gene of 2793 pb is homozygotic and encodes for a predicted protein of 930 amino acids with a molecular weight of 107,035 Da. The predicted protein displays significant sequence similarity to S. cerevisiae Ste13p. This C. albicans gene is located in chromosome R. STE13ca gene increases its levels of expression in conditions of nutritional stress (proline as nitrogen source) and during formation of the germinal tube, suggesting a basic biological function for the STE13ca in this yeast.

Genetic characterization of a polymorphic dipeptidyl aminopeptidase of Apis mellifera

Dipeptidyl aminopeptidase (DAP) activity towards L-leucylglycine-β-naphthylamide (Leu-Gly NA) was characterized in pupae and adult extracts of Apis mellifera. Enzyme activity was more conspicuous in pupae than in adult extracts and it seemed to be concentrated in digestive tract tissues. Two genetically determined electrophoretic variants were observed in honeybee samples from the USA and Chile; in Brazilian Africanized bees, two additional variants were observed. Segregational analyses showed no close linkage between Dap and Est-1, Lap D, Pgm-1, cd, and Ac loci of Apis mellifera. dipeptydil aminopeptidase / genetic variation / developmental distribution / tissue distribution / Apis mellifera

Dipeptidyl peptidase 9 has two forms, a broad tissue distribution, cytoplasmic localization and DPIV-like peptidase activity

Dipeptidyl peptidase (DP) IV has a distinct substrate specificity in hydrolyzing a post-proline bond. Here we present novel data on the sizes and tissue distribution of human and rat gene products and the peptidase activity of the DPIV-related gene DP9. A short cDNA of 2589 bp and a long cDNA of 3006 bp of DP9 were cloned. A ubiquitous predominant DP9 mRNA transcript at 4.4 kb represented the short form, whereas a less abundant 5.0-kb transcript present predominantly in muscle represented the long form. Both forms of DP9 have no transmembrane domain and two potential N-linked glycosylation sites. DP9 exhibited post-proline dipeptidyl aminopeptidase activity and was a cytoplasmic, 110-kDa monomer. Thus, the six DPIV gene family members have diverse characteristics: only DP9 and DP8 have exclusively cytoplasmic localization and only DP9, DP8, fibroblast activation protein (FAP) and DPIV have peptidase activity.

Synthetic peptides as substrate for assaying the proteolytic activity of Lactobacillus helveticus.

Four Lactobacillus helveticus strains were studied for proteolytic capacity and general aminopeptidase (AP) and X-Pro dipeptidyl aminopeptidase (DAP) activity. The rate of hydrolysis and the activity against synthetic substrates with N-terminal residues of Arg, Lys, Leu, Glu or Pro, varied markedly among the strains. The X-Pro DAP activity was consistently high. The crude cell-wall and cytoplasm extracts from strain Lb. helveticus ISLC59 were analysed thoroughly for their proteolysis ability by using four synthetic peptide substrates, including alpha(s)1-CN(f1-23). Peptides formed during in vitro hydrolysis of the synthetic substrates by cell wall and cytoplasm preparations were identified by LC-ESI/MS. In doing so, it was possible to infer a prevalent endopeptidase activity splitting Lys7-His8 and Gln13-Glu14 bonds in the cytoplasm, and to deduce a secondary activity, which hydrolysed Glu14-Val15, Leu16-Asn17, Glu18-Asn19 and Lys3-His4 bonds lacking in the cell-wall. The presence of exopeptidases, as mainly AP, DAP, and carboxypeptidase (CPase) was deduced from the formation of several N- and C-terminally truncated peptides sets. The AP activity was higher in the cell-wall layer, where CPase activity was absent. The in vitro assays with cell extracts of the Lb. helveticus ISLC59 strain revealed extensive exopeptidase and endopeptidase activities. In several cases, the hydrolytic system of Lb. helveticus that splits in vitro alpha(s)1-CN(f1-23) peptide bonds was similar to that of Lactococcus lactis. The effects were also compared with those occurring in vivo in hard cheese such as Grana Padano.

Protease, peptidase and esterase activities by lactobacilli and yeast isolates from Feta cheese brine.

The study of peptidase, esterase and caseinolytic activity of Lactobacillus paracasei subsp. paracasei, Debaryomyces hansenii and Sacchromyces cerevisiae isolates from Feta cheese brine. Cell-free extracts from four strains of Lact. paracasei subsp. paracasei, four strains of D. hansenii and three strains of S. cerevisiae, isolated from Feta cheese brine were tested for their proteolytic and esterase enzyme activities. Lactobacillus paracasei subsp. paracasei strains had intracellular aminopeptidase, dipeptidyl aminopeptidase, dipeptidase, endopeptidase and carboxypeptidase activities. Esterases were detected in three of four strains of lactobacilli and their activities were smaller with higher molecular weight fatty acids. The strains of yeasts did not exhibit endopeptidase as well as dipeptidase activities except on Pro-Leu. Their intracellular proteolytic activity was higher than that of lactobacilli. Esterases from yeasts preferentially degraded short chain fatty acids. Lactobacilli degraded preferentially beta-casein. Caseinolytic activity of yeasts was higher than that of lactobacilli. The results suggest that Lact. paracasei subsp. paracasei and yeasts may contribute to the development of flavour in Feta cheese. Selected strains could be used as adjunct starters to make high quality Feta cheese.

The acid phosphatase positive organelles of the Giardia lamblia trophozoite contain a membrane bound cathepsin C activity

We found a dipeptidyl aminopeptidase activity in the parasitic protozoan Giardia lamblia with properties similar to the lysosomal cathepsin C of rat-liver lysosomes. Subcellular fractionation of this parasite indicated that the cathepsin C activity is located in organelles not distinguishable from the ones containing acid phosphatase, a known marker enzyme of Giardia lysosome-like peripheral vesicles. Contrary to the rat lysosomal enzyme, Giardia cathepsin C behaved like a membrane protein. Moreover, the enzyme was not solubilized by Triton X-100 or Triton X-100/SDS at 0 °C but could be substantially solubilized by octylglucoside, Triton X-100 at 37 °C or by a pretreatment with the cholesterol complexing agent β-cyclodextrin before the Triton/SDS treatment carried out at 0 °C. These observations suggest that binding/anchorage of this enzyme to membranes occurs in cholesterol-rich microdomains.