Search

The N-terminus of S. aureus staphylocoagulase (SC) triggers activation of host prothrombin (ProT), and the SC·ProT* complex cleaves host fibrinogen (Fbg) to form fibrin (Fbn) deposits, a hallmark of SC-positive endocarditis. The C-terminal domain of the prototypical Newman D2 Tager 104 SC contains 1 pseudo-repeat (PR) and 7 repeats (R1→R7) that bind Fbg/Fbn Fragment D (Frag D). This work defines affinities and stoichiometries of Frag D binding to single- and multi-repeat C-terminal constructs, using fluorescence equilibrium binding, NMR titration, Ala scanning, and native PAGE. Constructs containing PR and each single repeat bound Frag D with KD ~50 - 130 nM and a 1:1 stoichiometry, indicating a conserved binding site shared between PR and each repeat. NMR titration of PR-R7 with Frag D revealed that residues 22-49, bridging PR and R7, constituted the minimal peptide (MP) required for binding, corroborated by Ala scanning, and binding of labeled MP to Frag D. MP alignment with the PR-repeat and inter-repeat junctions identified conserved residues critical for binding. Labeled PR-(R1→R7) bound Frag D with KD ~7 - 32 nM and stoichiometry of 1:5; and PR-R1R2R3, PR-R1R6R7, PR-R3R4R7, and PR-R3R6R7 competed with PR-(R1→R7) for Frag D binding, with a 1:3 stoichiometry and KD ~7 - 42 nM. These findings are consistent with binding at the PR-R junctions with modest inter-repeat sequence variability. Circular dichroism of PR-R7 and PR-(R1→R7) suggested a largely disordered structure and conformational flexibility, allowing binding of multiple fibrin(ogen) molecules. This property facilitates pathogen localization on host fibrin networks.

Giant squid (Dosidicus gigas) proteins have appropriate functional properties, albeit of smaller magnitude in comparison to other marine species. Therefore, this research characterizes the ultrasound-induced (20 kHz; 20 and 40% amplitude; 0, 1, 3 and 5 min) changes to the physicochemical and foaming properties of mantle proteins. The changes in pH, electrophoretic profile, viscosity, surface hydrophobicity, particle size and zeta potential, as well as foaming capacity and stability were evaluated. A slight decrease (p ≥ 0.05) in the pH occurred as the ultrasound time increased. While no changes in SDS-PAGE (reducing and non-reducing) were detected, native PAGE revealed new bands. Ultrasound increased the viscosity and surface hydrophobicity, and decreased the particle size and net surface charge. Moreover, foaming capacity was improved and foaming stability was maintained at 100% for 1 h. Therefore, the application of ultrasound represents an alternative to improve the foaming properties.

The production of both exo- and endoinulinase by Aspergillus ficuum JNSP5-06 was investigated. Optimum fermentation conditions were found to be: inulin, 2%; yeast extract, 2%; (NH4)H2PO4, 0.5%; NaCl, 0.5%; MgSO4·7H2O, 0.05%; ZnSO4·7H2O, 0.01%; initial pH 6.5. A new and convenient method was developed to separate the inulinases by native-polyacrylamide gel electrophoresis (PAGE). Eight protein bands were obtained. Three bands were identified as exoinulinase and two bands were endoinulinase using TLC and HPLC.

Electrophoretic examination of dissociated haemocyanin subunits from a number of amphipod, decapod and isopod crustaceans supports the hypothesis that subunit composition is species-specific, despite marked within-species variation in many species. General patterns of heterogeneity on native PAGE gels were also evident between groupings within the Amphipoda. Gammarid amphipods could be split into two groups; one characterised by a high degree of heterogeneity and the other by a low degree of heterogeneity. The talitrid amphipods generally displayed a low degree of heterogeneity similar to, although still distinct from, the second gammarid category. Haemocyanin from the Hyalidae, a family allied to the talitrids was highly heterogeneous, similar to the first gammarid group and unlike the talitrids. Isopod haemocyanin banding patterns were more similar to one another than to any of the amphipod or decapod species examined. In general, the molecular weights of the amphipod Hcs tended to be greater than those of the isopods, with the decapods being lowest of all. It is suggested that Hc subunit heterogeneity may be a useful tool for investigating speciation and speciation events, and for reliably separating very closely-related species (e.g. Gammarus spp.), purely on the basis of their Hc subunit compositions.

Many proteins, including antibacterial peptides in the hemolymph, are induced by bacterial infections. We found two bacterially inducible carboxylesterases (CEs) in the hemolymph of the silkworm, Bombyx mori. CEs Est-1 and 2 were induced by lipopolysaccharide injection after 6 hours as well as E. coli infection. We found that bacterially inducible CEs clearly differed from noninducible CEs, including juvenile hormone esterases, in pI values, migration on analytical native PAGE, and inhibitor sensitivity. We are now studying the features and functions of these CEs.

Glutamic acid–salicylaldehyde Schiff-base metal complexes are bound into bovine serum albumin (BSA), which afforded BSA binding Schiff-base metal complexes (BSA-SalGluM, M Cu, Co, Ni, Zn). The BSA binding metal complexes were characterized by UV–vis spectra and Native PAGE. It showed that the protein structures of BSA kept after coordinating amino acid Schiff-bases metal complexes. The effect of the antioxidant activity was investigated. The results indicate that the antioxidant capacity of BSA increased more than 10 times after binding Schiff-base metal complexes.

Three types of cysteine synthase (CSase, EC 4.2.99.8) isozymes were purified from spinach leaves. Each isozyme was isolated to homogeneity by preparative PAGE. These isozymes were revealed to have different primary structures by amino-acid and proteinase digestion analyses, respectively. The enzymes designated as CSase 1, CSase 2 and CSase 3 with reference to the mobility on native PAGE were characterized with respect to physicochemical and enzymatic properties, and it was found that those enzymes had similar properties. It was also found that CSase 1 could be attributed to chloroplasts.

Polyacrylamide gel electrophoresis was performed on ultrapasteurized liquid whole egg (LWE), which was either homogenized or unhomogenized and heated at 64, 68, or 72 C each for 30, 60, or 95 s. Native PAGE was used to determine the effect of ultrapasteurization in combination with homogenization on LWE protein. The α-livetin band was absent at 72 C for 60 and 95 s processing times but remained present at the 30-s treatment time at 72 C and for all processing times at 64 and 68 C. Protein bands of the globulin/β-livetin and ovotransferrin/γ-livetin fractions appear to have faded in all heat-treated samples in comparison with the unheated samples, reflecting their heat instability. Homogenization had no effect on the presence of protein bands identified by PAGE.

We have developed a modified blue native polyacrylamide gel electrophoresis (PAGE) protocol that can overcome aggregation of lipases seen in native PAGE. We have shown that two lipases, Pseudomonas aeruginosa lipase and Candida rugosa lipase, which aggregate in the native gel, can be resolved using our protocol. Activity staining was done to test for the functionality of the two lipases.