Peptidomic Investigation of the Interplay between Enzymatic Tenderization and the Digestibility of Beef Semimembranosus Proteins.

This work investigated the influence of enzymatic tenderization on digestibility changes of beef semimembranosus proteins using peptidomics methods. Hydrolysis by proteinase K and bromelain elevated the average bitterness index of identified peptides by generating high-Q values peptides (1714-1790 Cal/mol), including KDLFDPIIQ, LIDDHFLFDKPVSPL, and QLIDDHFLFDKPVSPLLL. Proteolysis during enzymatic tenderization acted as a "pre-digestion" step and significantly elevated the degree of hydrolysis of beef protein (by 4.5-17.3%) in subsequent simulated gastrointestinal digestion. Peptidomics analysis of digests revealed large variations in the peptide composition, which was positively correlated with the degree of proteolysis during enzymatic tenderization. Enzymatic tenderization with proteinase K- (for 0.5 h) or bromelain-treated samples largely increased the survival rate (by 65.5 or 82.8%) of peptides during simulated digestion, possibly because of the "secondary enzyme-substrate interaction" effect. This work could provide a new sight into the possible influence of enzymatic tenderization on meat nutrition.

Catalytic activity, structure and stability of proteinase K in the presence of biosynthesized CuO nanoparticles.

Here, CuO nanoparticles were synthesized using Sambucus nigra (elderberry) fruit extract. Further, the binding of proteinase K, as a model enzyme with green synthesized nanoparticles was investigated. The results demonstrated that the structural changes in enzyme were induced by the binding of nanoparticles. These changes were accompanied by the decrease in the Michaelis-Menten constant at 298 K. This means that the enzyme affinity for the substrate was increased. Thermodynamic parameters of protein stability and protein-ligand binding were estimated from the spectroscopic measurements at 298-333 K. Depending on the temperature, CuO nanoparticles showed a dual effect on the thermodynamic stability and binding affinity of enzyme. Nanoparticles increase the stability of the native state of enzyme at room temperature. On the other hand, nanoparticles stabilize the unfolded state of enzyme at 310-333 K. An overall favorable Gibbs energy change was observed for the binding process at 298-333 K. The enzyme-nanoparticle binding is enthalpically driven at room temperature. It was concluded that hydrogen bonding plays a key role in the interaction of enzyme with nanoparticles at 298-310 K. At higher temperatures, the protein-ligand binding is entropically driven. This means that hydrophobic association plays a major role in the proteinase K-CuO binding at 310-333 K.

Generation of Infectious Prions and Detection with the Prion-Infected Cell Assay.

Cell lines propagating prions are an efficient and useful means for studying the cellular and molecular mechanisms implicated in prion disease. Utilization of cell-based models has led to the finding that PrPC and PrPSc are released from cells in association with extracellular vesicles known as exosomes. Exosomes have been shown to act as vehicles for infectivity, transferring infectivity between cell lines and providing a mechanism for prion spread between tissues. Here, we describe the methods for generating a prion-propagating cell line with prion-infected brain homogenate, cell lysate, conditioned media, and exosomes and also detection of protease-resistant PrP with the prion-infected cell assay.

Antiquorum sensing and antibiofilm potential of Capparis spinosa.

BACKGROUND: Emergence of antibiotic resistance among bacterial pathogens often leads to the failure of existing antibiotics to treat bacterial infections; thus, there is a need to seek alternative treatment measures. The aim of this study was to evaluate the anti-quorum sensing (anti-QS) and antibiofilm potential of Capparis spinosa to prevent the onset of bacterial infections as an alternate to antibiotics. METHODS: The methanolic extract of the dried fruits of C. spinosa was assessed for its activity in inhibiting QS-depedent phenomenon such as violacein pigment production in Chromobacterium violaceum, biosurfactant production in Pseudomonas aeruginosa PAO1, swimming and swarming motility, exopolysaccharide production (EPS) and biofilm formation in Escherichia coli, Proteus mirabilis, Serratia marcescens and PAO1. RESULTS: Extract of C. spinosa showed a higher degree of anti-QS activity in a dose dependent manner without affecting the bacterial growth. At 2 mg/mL, this extract significantly (p ≤0.005) inhibited the biofilm formation to 79, 75, 73, 70% and EPS production to 58, 46, 66 and 67% in S. marcescens, PAO1, E. coli and P. mirabilis, respectively. It also exhibited inhibition in swimming and swarming motility of bacterial pathogens. The non-enzymatic nature of the anti-QS compound in C. spinosa was confirmed by proteinase K and heat treatment. CONCLUSIONS: Because the methanolic extract of C. spinosa demonstrated anti-QS and antibiofilm activity at 0.5-2 mg/mL, it could be further exploited for novel molecules to treat the emerging infections of antibiotic resistant bacterial pathogens.

Cloning and biochemical characterization of APIT, a new l-amino acid oxidase from Aplysia punctata.

The purple ink of the sea hare Aplysia punctata contains a 60 kDa protein with tumoricidal activity. This A. punctata ink toxin (APIT) kills tumor cells within 6--8h in an apoptosis independent manner by the production of high amounts of hydrogen peroxide which induce a necrotic form of oxidative stress. Here, we describe the biochemical features of APIT associated with its anti-tumor activity. APIT is a weakly glycosylated FAD-binding L-amino acid oxidase that catalyzes the oxidative deamination of L-lysine and L-arginine and thereby produces hydrogen peroxide (H(2)O(2)), ammonia (NH(4)(+)) and the corresponding alpha-keto acids. The tumoricidal effect is completely abrogated in the absence of the amino acids L-lysine and L-arginine. The enzyme is stable at temperatures from 0 to 50 degrees C. Similar to other FAD-binding enzymes, it is resistant against tryptic digest. Even digest with proteinase K fails to degrade the enzyme. Cloning of the APIT gene and subsequent sequencing revealed a FAD-binding domain followed by a so-called GG-motif, which is typical for L-amino acid oxidases. Strongest homology exists to escapin, aplysianin A precursor, the cyplasins L and S and achacin.

Specific structural requirements for the inhibitory effect of thapsigargin on the Ca2+ ATPase SERCA.

Mutational analysis of amino acid residues lining the thapsigargin (TG) binding cavity at the interface of the membrane surface and cytosolic headpiece was performed in the Ca(2+) ATPase (SERCA-1). Specific mutations such as F256V, I765A, and Y837A reduce not only the apparent affinity of the ATPase for TG but also the maximal inhibitory effect. The effect of mutations is dependent on the type and size of the substitute side chain, indicating that hydrophobic partitioning of TG and complementary molecular shapes are involved not only in binding but also in the inhibitory mechanism. A major factor determining the inhibitory effect of bound TG is its interference with conformational changes that are required for the progress of the ATPase cycle. Most prominent and specific is the TG interference with a wide displacement of the Phe-256 side chain that is associated with the E2 to E1.2Ca(2+) transition. The specificity of the TG inhibitory mechanism is emphasized by the finding that the F256V mutation does not interfere at all with the effect of 2,5-di-(t-butyl)-hydroquinone, which is another SERCA inhibitor bound by hydrophobic partitioning. The specificity of the inhibitory mechanism is also emphasized by the observation that within the concentration range producing total inhibition of wild-type SERCA-1, TG produces a 4-fold stimulation of the P-glycoprotein (multidrug transporter) ATPase.

The crystallographic structure of the subtilisin protease from Penicillium cyclopium.

The major extracellular protease from the fungus Pencillium cyclopium was crystallized in the presence of p-phenylmethanesulfonyl fluoride (PMSF) and investigated by X-ray diffraction analysis. It was subsequently cloned and the amino acid sequence deduced from its cDNA. Although the sequence is only 49% identical to that of proteinase K of Tritirachium album, the three-dimensional structures of the two proteases are virtually identical. The model for P. cyclopium protease was refined by simulated annealing to an R of 18% at 1.7 A resolution. The greatest variation from the proteinase K polypeptide is in loop 114-134 and is due to the absence of a disulfide bridge in the P. cyclopium protease that is present in proteinase K. A difference was also observed in the orientation of the histidine in the catalytic triad, though this could be due to the presence of PMSF at the active site. The coordination geometry of the strongly bound calcium in the P. cyclopium protease is octahedral and uses some different protein ligands than does proteinase K. In the protease from P. cyclopium there is no cysteine near the active site, nor is there a second calcium binding site as is found in proteinase K, suggesting that neither is important to catalytic activity.