The iron-binding protein lactoferrin is a multifunctional protein that has antibacterial, antifungal, antiviral, antitumour, anti-inflammatory, and immunoregulatory properties. All of these ...additional properties appear to be related to its highly basic N-terminal region. This part of the protein can be released in the stomach by pepsin cleavage at acid pH. The 25-residue antimicrobial peptide that is released is called lactoferricin. In this work, we review our knowledge about the structure of the peptide and attempt to relate this to its many functions. Microcalorimetry and fluorescence spectroscopy data regarding the interaction of the peptide with model membranes show that binding to net negatively charged bacterial and cancer cell membranes is preferred over neutral eukaryotic membranes. Binding of the peptide destabilizes the regular membrane bilayer structure. Residues that are of particular importance for the activity of lactoferricin are tryptophan and arginine. These two amino acids are also prevalent in "penetratins", which are regions of proteins or synthetic peptides that can spontaneously cross membranes and in short hexapeptide antimicrobial peptides derived through combinatorial chemistry. While the antimicrobial, antifungal, antitumour, and antiviral properties of lactoferricin can be related to the Trp/Arg-rich portion of the peptide, we suggest that the anti-inflammatory and immunomodulating properties are more related to a positively charged region of the molecule, which, like the alpha- and beta-defensins, may act as a chemokine. Few small peptides are involved in as wide a range of host defense functions as bovine and human lactoferricin.Key words: lactoferricin, penetratin, tryptophan, arginine, membrane perturbation, fluorescence spectroscopy, NMR spectroscopy, microcalorimetry.
Tpm1.1 (α) and Tpm2.2 (β) tropomyosins (39 amino acid substitutions) were isolated from adult rabbit skeletal muscle without chemical modification of cysteine, with negligible phosphorylation as ...assessed by two-dimensional polyacrylamide gel electrophoresis, and characterized biochemically. Reconstituted skeletal thin filaments composed of Tpm2.2 produce ∼30% less Ca(II)-induced activation of the steady-state actomyosin-S1MgATPase rate than Tpm1.1 does. This is observed at a high S1/actin ratio (6 μM myosin-S1A1, 3 μM thin filaments, pCa 4) and as a function of pCa (0.3 μM myosin-S1A1, 25 μM thin filaments). The two pCa versus MgATPase relationships are similar in terms of their steepness and midpoint. Isotype has a bearing on self-polymerization and interaction with troponin. Solutions (pH 7, ionic strength of ∼30 mM) of Tpm2.2 are more viscous than solutions of Tpm1.1, an observation explained by substitutions at the carboxy-terminal end of the molecule, including His276Asn and Met281Ile. Conversely, the enhancement of viscosity of Tpm1.1 by skeletal troponin is greater than that for Tpm2.2. Further, Tpm1.1 binds more strongly than Tpm2.2 to skeletal troponin-Sepharose, as evidenced by a later elution position in the salt gradient. Mixtures of tropomyosin and the amino-terminal CNBr fragment of troponin-T, CB1 (residues 1–151), were chromatographed on a size exclusion column in the presence of different concentrations of KCl. In 0.1 M salt, CB1 co-elutes with either isoform but is largely dissociated at 0.22 M. At intermediate salt concentrations, different degrees of complexation are observed, more extensive for Tpm1.1 than for Tpm2.2. Thus, the first reported variants of tropomyosin are distinct in their interactive and functional properties. The biochemical properties of Tpm2.2 are of particular relevance to the immature skeletal muscle thin filament.
Starch accumulation and degradation in chloroplasts is accomplished by a suite of over 30 enzymes. Recent work has emphasized the importance of multi-protein complexes amongst the metabolic enzymes, ...and the action of associated non-enzymatic regulatory proteins. Arabidopsis At5g39790 encodes a protein of unknown function whose sequence was previously demonstrated to contain a putative carbohydrate-binding domain.
We here show that At5g39790 is chloroplast-localized, and binds starch, with a preference for amylose. The protein persists in starch binding under conditions of pH, redox and Mg(+2) concentrations characteristic of both the day and night chloroplast cycles. Bioinformatic analysis demonstrates a diurnal pattern of gene expression, with an accumulation of transcript during the light cycle and decline during the dark cycle. A corresponding diurnal pattern of change in protein levels in leaves is also observed. Sequence analysis shows that At5g39790 has a strongly-predicted coiled-coil domain. Similar analysis of the set of starch metabolic enzymes shows that several have strong to moderate coiled-coil potential. Gene expression analysis shows strongly correlated patterns of co-expression between At5g39790 and several starch metabolic enzymes.
We propose that At5g39790 is a regulatory scaffold protein, persistently binding the starch granule, where it is positioned to interact by its coiled-coil domain with several potential starch metabolic enzyme binding-partners.
The main degradation products (furfural, hydroxymethylfurfural, acetate) derived from acid hydrolysis of hemicellulosic materials inhibit growth on and fermentation of xylose by Pachysolen ...tannophilus and Pichia stipitis, with the latter yeast being the more sensitive. The inhibitory effect was more severe when the inhibitors were present together in the medium. Agarose immobilization partially protected the yeasts from the deleterious effects of these compounds. Intracellular de-energization and acidification may be the mechanism by which these compounds exert their toxic effect on the yeast cells.PUBLICATION ABSTRACT
In
Daucus carota, N-acetylglutamate-5-phosphotransferase (NAGK; E.C. 2.7.2.8) specific activity was shown to correlate with the progression of somatic embryogenesis and was highest in the latter ...stages, where growth was most rapid. The enzyme was subsequently purified greater than 1200-fold using heat treatment, ammonium sulfate fractionation, gel filtration, anion exchange and dye ligand chromatography. Carrot NAGK was shown to have a subunit molecular weight of 31 kDa and form a hexamer. The Kms for NAG and ATP are 5.24 and 2.11 mM, respectively. Arginine (Arg) is a K-type allosteric inhibitor of the enzyme, and Hill coefficients in the order of 5 in the presence of Arg suggest that the enzyme is highly cooperative.
D. carota NAGK does not bind to
Arabidopsis thaliana PII affinity columns, nor does the
A. thaliana PII increase NAGK specific activity, indicating its cellular location is probably different.
Previous work on the metabolism of
Lactobacillus reuteri ATCC 55730 anticipated a variability in the use of organic electron acceptors as a means to relieve metabolic redox problems. Therefore, ...investigations focusing on this unique metabolism of
L. reuteri may reveal a basis for new probiotic properties. For instance,
L. reuteri may use reactive aldehydes and ketones as electron acceptors to balance their redox metabolism, which opens the possibility to exploit this bacterium for in vivo bioreduction of deleterious compounds in the gastrointestinal tract (GIT). Herein we demonstrate that
L. reuteri ATCC 55730 cultures on glucose are able to use furfural (1
g/L), and hydroxymethylfurfural (HMF) (0.5
g/L), as electron acceptors. The former enhances the growth rate by about 25% and biomass yield by 15%, whereas the latter is inhibitory. Furfural is stoichiometrically reduced to furfuryl alcohol by the culture. The conversion of furfural had no effect on the flux distribution between the simultaneously operating phosphoketolase and Embden–Meyerhof pathways, but initiated a flux to acetate production. In addition to furfural and HMF, cellular extracts showed potential to reoxidize NADH and/or NADPH with acrolein, crotonaldehyde, and diacetyl, indicating that conversion reactions take place intracellularly, however, utilization mechanisms for the latter compounds may not be present in this strain. The strain did not reduce other GIT-related reactive compounds, including acrylamide, glyoxal, and furan.
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