Isolation of lactoperoxidase, lactoferrin, α-lactalbumin, β-lactoglobulin B and β-lactoglobulin A from bovine rennet whey using ion exchange chromatography

A mild and rapid method is described for isolating various milk proteins from bovine rennet whey. β-Lactoglobulin from bovine rennet whey was easily adsorbed on and desorbed from a weak anion exchanger, diethylaminoethyl-Toyopearl. However, α-lactalbumin could not be adsorbed onto the resin. α-Lactalbumin and β-lactoglobulin from rennet whey could also be adsorbed and separated using a strong anion exchanger, quaternary aminoethyl-Toyopearl. The rennet whey was passed through a strong cation exchanger, sulphopropyl-Toyopearl, to separate lactoperoxidase and lactoferrin. α-Lactalbumin and β-lactoglobulin were adsorbed onto quaternary aminoethyl-Toyopearl. α-Lactalbumin was eluted using a linear (0–0.15 M) concentration gradient of NaCl in 0.05 M Tris–HCl buffer (pH 8.5). Subsequently, β-lactoglobulin B and β-lactoglobulin A were eluted from the column with 0.05 M Tris–HCl (pH 6.8), using a linear (0.1–0.25 M) concentration gradient of NaCl. The yields were 1260 mg α-lactalbumin, 1290 mg β-lactoglobulin B and 2280 mg β-lactoglobulin A from 1 l rennet whey.     

Unusual evolution of 11β- and 17β-hydroxysteroid and retinol dehydrogenases

11β-hydroxysteroid dehydrogenases regulate glucocorticoid concentrations and 17β-hydroxysteroid dehydrogenases regulate estrogen and androgen concentrations in mammals. Phylogenetic analysis of the sequences from two 11β-hydroxysteroid dehydrogenases and four mammalian 17β-hydroxysteroid dehydrogenases indicates unusual evolution in these enzymes. Type 1 11β- and 17β-hydroxysteroid dehydrogenases are on the same branch; Type 2 enzymes cluster on another branch with β-hydroxybutyrate dehydrogenase, 11-cis retinol dehydrogenase and retinol dehydrogenase; Type 3 17β-hydroxysteroid dehydrogenase is on a third branch; while the pig dehydrogenase clusters with a yeast multifunctional enzyme on a fourth branch. Pig 17β-hydroxysteroid dehydrogenase appears to have evolved independently from the other three 17β-hydroxysteroid dehydrogenase dehydrogenases; in which case, the evolution of 17β-hydroxysteroid dehydrogenase activity is an example of functional convergence. The phylogeny also suggests that independent evolution of specificity toward C11 substituents on glucocorticoids and C17 substituents on androgens and estrogens has occurred in Types 1 and 2 11β- and 17β-hydroxysteroid dehydrogenases.     

Hydrolysis of β-lactoglobulin by thermolysin and pepsin under high hydrostatic pressure

Hydrolysis of β-lactoglobulin with thermolysin and pepsin at pressures ranging between 0.1 and 350 MPa showed a significant increase of cleavage rates. Pressure-induced changes of susceptibility to hydrolysis of β-lactoglobulin proteolytic sites were also observed. The pressure, raised to 200 MPa, accelerates the hydrolysis of β-lactoglobulin by thermolysin and changes obtained peptide profiles. Initially, higher pressure makes the N-terminal, and to a smaller extent, C-terminal peptide fragments of β-lactoglobulin molecule, more susceptible to removal by thermolysin. This indicates combined influence of pressure-induced thermolysin activation and partial unfolding of β-lactoglobulin by compression at neutral pHs. The rates of hydrolysis of β-lactoglobulin by pepsin (negligible at 0.1 MPa) are increased considerably with pressure up to 300 MPa. The Susceptibility of β-lactoglobulin proteolytic sites to peptic cleavage remains constant over all the studied pressure range. The lack of significant qualitative changes in the peptic peptide profiles produced at different pressures and at clearly pressure-dependent rates points to negative reaction volume changes as the major factor in peptic hydrolysis of β-lactoglobulin under high pressure. Thus the β-lactoglobulin molecule resists pressure-induced unfolding in acid pHs and yields to it in neutral pHs. © 1995 John Wiley & Sons, Inc.     

Stabilizing effects of 2-methylalanine residues on β-turns and α-helices

¹³C-, ¹H-nmr, CD, and x-ray crystallography revealed β-turns of type III for Boc-Gly-L-Ala-Aib-OMe, Boc-L-Ala-Aib-L-Ala-OMe; the 3₁₀-helix for Boc-Aib-L-Ala-Aib-L-Ala-Aib-OMe; and antiparallel arranged α-helices for Boc-L-Ala-Aib-Ala-Aib-Ala-Glu(OBzl)-Ala-Aib-Ala-Aib-Ala-OMe. An N-terminal rigid α-helical segment is found in the polypeptide antibiotics alamethicin, suzukacillin, and trichotoxin. The α-helix dipole is essential for their voltage-dependent pore formation in lipid bilayer membranes, which is explained by a flip-flop gating mechanism based on dipole–dipole interactions of parallel and antiparallel arranged α-helices within oligomeric structures.     

β‐Bulges: Extensive structural analyses of β‐sheets irregularities

β‐Sheets are quite frequent in protein structures and are stabilized by regular main‐chain hydrogen bond patterns. Irregularities in β‐sheets, named β‐bulges, are distorted regions between two consecutive hydrogen bonds. They disrupt the classical alternation of side chain direction and can alter the directionality of β‐strands. They are implicated in protein‐protein interactions and are introduced to avoid β‐strand aggregation. Five different types of β‐bulges are defined. Previous studies on β‐bulges were performed on a limited number of protein structures or one specific family. These studies evoked a potential conservation during evolution. In this work, we analyze the β‐bulge distribution and conservation in terms of local backbone conformations and amino acid composition. Our dataset consists of 66 times more β‐bulges than the last systematic study (Chan et al. Protein Science 1993, 2:1574–1590). Novel amino acid preferences are underlined and local structure conformations are highlighted by the use of a structural alphabet. We observed that β‐bulges are preferably localized at the N‐ and C‐termini of β‐strands, but contrary to the earlier studies, no significant conservation of β‐bulges was observed among structural homologues. Displacement of β‐bulges along the sequence was also investigated by Molecular Dynamics simulations.     

Secondary structure changes and peptic hydrolysis of β-lactoglobulin induced by diols

The addition of ethylene glycol, and 1,2- and 1,3-propanediol, decreases the bulk dielectric constant of the medium, and according to CD measurements, increases significantly the proportion of helical structure in β-lactoglobulin. The medium-induced folding changes followed by limited peptic hydrolysis show that the cleavage of β-lactoglobulin by pepsin is triggered by structural transformations induced by ethylene glycol only and not by 1,2- and 1,3-propanediol. Density measurements, at constant chemical potential and constant molality, demonstrate that all diols are present in the immediate domain of the protein. They are engaged in hydrophobic interactions with the amino acids of β-lactoglobulin core inducing the formation of additional α-helices. © 1996 John Wiley & Sons, Inc.     

Structural classification of αββ and ββα supersecondary structure units in proteins

We present a fully automatic structural classification of supersecondary structure units, consisting of two hydrogen-bonded β strands, preceded or followed by an α helix. The classification is performed on the spatial arrangement of the secondary structure elements, irrespective of the length and conformation of the intervening loops. The similarity of the arrangements is estimated by a structure alignment procedure that uses as similarity measure the root mean square deviation of superimposed backbone atoms. Applied to a set of 141 well-resolved nonhomologous protein structures, the classification yields 11 families of recurrent arrangements. In addition, fragments that are structurally intermediate between the families are found; they reveal the continuity of the classification. The analysis of the families shows that the α helix and β hairpin axes can adopt virtually all relative orientations, with, however, some preferable orientations; moreover, according to the orientation, preferences in the left/right handedness of the α–β connection are observed. These preferences can be explained by favorable side by side packing of the α helix and the β hairpin, local interactions in the region of the α–β connection or stabilizing environments in the parent protein. Furthermore, fold recognition procedures and structure prediction algorithms coupled to database-derived potentials suggest that the preferable nature of these arrangements does not imply their intrinsic stability. They usually accommodate a large number of sequences, of which only a subset is predicted to stabilize the motif. The motifs predicted as stable could correspond to nuclei formed at the very beginning of the folding process. Proteins 30:193–212, 1998. © 1998 Wiley-Liss, Inc.     

Thioflavin T fluorescence assay for β-lactoglobulin fibrils hindered by DAPH

The molecule 4,5-dianilinophthalimide was recently found to be an efficient compound in disaggregating amyloid fibrils involved in the Alzheimer’s disease. In this study we have investigated whether the compound 4,5-dianilinophthalimide was able to disaggregate fibrils derived from β-lactoglobulin. In addition to a Thioflavin T fluorescence assay, flow-induced birefringence was used as an independent technique to measure the total length concentration of the fibrils. An additional advantage of the latter technique is that not only the total length concentration, but also the length distribution of the fibrils can be measured. The results from flow-induced birefringence showed that the total amount of fibrils and also the length distribution of the fibrils was not influenced by the addition of 4,5-dianilinophthalimide, even though this was suggested by the results of the Thioflavin T assay. The results of flow-induced birefringence were confirmed by rheological measurements and transmission electron microscopy. Our findings show that the use of a Thioflavin T assay in order to probe the possible disaggregating effect of certain compounds can give misleading results.     

Role of the I-domain in collagen binding specificity and activation of the integrins α1β1 and α2β1

Adhesion to collagens by most cell types is mediated by the integrins α1β1 and α2β1. Both integrin α subunits belong to a group which is characterized by the presence of an I domain in the N-terminal half of the molecule, and this domain has been implicated in the ligand recognition. Since purified α1β1 and α2β1 differ in their binding to collagens I and IV and recognize different sites within the major cell binding domain of collagen IV, we investigated the potential role of the α1 and α2 I domains in specific collagen adhesion. We find that introducing the α2 I domain into α1 results in surface expression of a functional collagen receptor. The adhesion mediated by this chimeric receptor (α1-2-1β1) is similar to the adhesion profile conferred by α2β1, not α1β1. The presence of α2 or α1-2-1 results in preferential binding to collagen I, whereas α1 expressing cells bind better to collagen IV. In addition, α1 containing cells bind to low amounts of a tryptic fragment of collagen IV, whereas α2 or α1-2-1 bearing cells adhere only to high concentrations of this substrate. We also find that collagen adhesion of NIH-3T3 mediated by α2β1 or α1-2-1β1, but not by α1, requires the presence of Mn²⁺ ions. This ion requirement was not found in CHO cells, implicating the I domain in cell type-specific activation of integrins. J. Cell. Physiol. 176:634–641, 1998. © 1998 Wiley-Liss, Inc.     

Simultaneous determination of retinol, β-carotene and α-tocopherol in adipose tissue by high-performance liquid chromatography

A method is described for evaluation of fat-soluble vitamin in human adipose tissue with the aim to obtain, accurately and within the shortest analysis time, a time-integrated measure of exposure to vitamins from the diet. Fat tissue was deproteinized with ethanol and extracted with n-hexane. Normal-phase HPLC was performed in a Lichrosorb Si60 column with a gradient of n-hexane–2-propanol at 1 ml/min. Detection was accomplished using a diode-array system (for retinol and β-carotene) in series with a fluorescence detector (α-tocopherol). The method was validated and applied to human adipose tissue in a total of 140 subjects. The mean contents found were 0.43, 0.84, 240.3 μg/g for retinol, β-carotene and α-tocopherol, respectively. The method is sensitive enough for detecting the compounds in 1.6 mg of adipose tissue considering the lowest concentration found.     

Fe2+ binding on amyloid β‐peptide promotes aggregation

The metal ions Zn²⁺, Cu²⁺, and Fe²⁺ play a significant role in the aggregation mechanism of Aβ peptides. However, the nature of binding between metal and peptide has remained elusive; the detailed information on this from the experimental study is very difficult. Density functional theory (dft) (M06‐2X/6‐311++G (2df,2pd) +LANL2DZ) has employed to determine the force field resulting due to metal and histidine interaction. We performed 200 ns molecular dynamics (MD) simulation on Aβ_1‐42‐Zn²⁺, Aβ_1‐42‐Cu²⁺, and Aβ_1‐42‐Fe²⁺ systems in explicit water with different combination of coordinating residues including the three Histidine residues in the N‐terminal. The present investigation, the Aβ_1‐42‐Zn²⁺ system possess three turn conformations separated by coil structure. Zn²⁺ binding caused the loss of the helical structure of N‐terminal residues which transformed into the S‐shaped conformation. Zn²⁺ has reduced the coil and increases the turn content of the peptide compared with experimental study. On the other hand, the Cu²⁺ binds with peptide, β sheet formation is observed at the N‐terminal residues of the peptide. Fe²⁺ binding is to promote the formation of Glu22‐Lys28 salt‐bridge which stabilized the turn conformation in the Phe19‐Gly25 residues, subsequently β sheets were observed at His13‐Lys18 and Gly29‐Gly37 residues. The turn conformation facilitates the β sheets are arranged in parallel by enhancing the hydrophobic contact between Gly25 and Met35, Lys16 and Met35, Leu17 and Leu34, Val18 and Leu34 residues. The Fe²⁺ binding reduced the helix structure and increases the β sheet content in the peptide, which suggested, Fe²⁺ promotes the oligomerization by enhancing the peptide‐peptide interaction. Proteins 2016; 84:1257–1274. © 2016 Wiley Periodicals, Inc.     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

Calorimetric studies of p-nitrophenol binding to α- and β-cyclodextrin

The thermodynamics of the binding of p-nitrophenol to both α- and β-cyclodextrin has been investigated as a function of pH and solvent composition using the technique of flow microcalorimetry. A preferential binding of the anionic form of the ligand is found for both cyclodextrins. It is postulated that the additional affinity for p-nitrophenolate is due to a dispersion interaction between the cyclodextrin cavity and the delocalized charge of the ligand. Studies of the binding of p-nitrophenol analogs and of the binding of p-nitrophenol as a function of ionic strength and DMSO cosolvent composition are consistent with this theory.     

β Propellers: structural rigidity and functional diversity

Recently solved structures and proposed models have helped to reveal the structural characteristics of the β-propeller fold, as well as the features that contribute to its high rigidity and stability. Possible strategies for identifying β-propeller proteins in newly characterised sequences are helping to overcome the problems of predicting the β-propeller fold from amino acid sequences.     

Isolation and purification of serum and interfacial peptides of a trypsinolyzed β-lactoglobulin oil-in-water emulsion

Information on the conformation of proteins adsorbed to an oil–water interface is usually determined by following the time course of enzymatic hydrolysis of the protein in an oil-in-water emulsion. Unlike previous works reported in the literature, the research presented in this paper provides information on which peptides are actually in contact with the lipid bilayer (interfacial peptides) and those segments that project into the aqueous phase (serum peptides). In order to achieve this classification of peptides, we present a method to separate serum peptides from interfacial peptides by initial centrifugation steps followed by reversed-phase high-performance liquid chromatography. The effectiveness of the method was ascertained by performing proteolysis on β-lactoglobulin adsorbed to an oil-water interface in a soybean oil-water emulsion. It was found that more peptides are qualitatively and quantitatively found adsorbed to the oil–water interface as compared to peptides released into the serum.     

Selection and structural analysis of de novo proteins from an α3β3 genetic library

The construction of novel functional proteins has been a key area of protein engineering. However, there are few reports of functional proteins constructed from artificial scaffolds. Here, we have constructed a genetic library encoding α3β3 de novo proteins to generate novel scaffolds in smaller size using a binary combination of simplified hydrophobic and hydrophilic amino acid sets. To screen for folded de novo proteins, we used a GFP‐based screening system and successfully obtained the proteins from the colonies emitting the very bright fluorescence as a similar intensity of GFP. Proteins isolated from the very bright colonies (vTAJ) and bright colonies (wTAJ) were analyzed by circular dichroism (CD), 8‐anilino‐1‐naphthalenesulfonate (ANS) binding assay, and analytical size‐exclusion chromatography (SEC). CD studies revealed that vTAJ and wTAJ proteins had both α‐helix and β‐sheet structures with thermal stabilities. Moreover, the selected proteins demonstrated a variety of association states existing as monomer, dimer, and oligomer formation. The SEC and ANS binding assays revealed that vTAJ proteins tend to be a characteristic of the folded protein, but not in a molten‐globule state. A vTAJ protein, vTAJ13, which has a packed globular structure and exists as a monomer, was further analyzed by nuclear magnetic resonance. NOE connectivities between backbone signals of vTAJ13 suggested that the protein contains three α‐helices and three β‐strands as intended by its design. Thus, it would appear that artificially generated α3β3 de novo proteins isolated from very bright colonies using the GFP fusion system exhibit excellent properties similar to folded proteins and would be available as artificial scaffolds to generate functional proteins with catalytic and ligand binding properties.