The recognition of phosphatidylserine (PS) on the surface of any apoptotic cell is considered to be a key event for its clearance. We challenge this concept by showing that pretreatment of neutrophils with either host or bacterial protease affects their uptake by human monocyte-derived macrophages without having an effect on cell-surface PS presentation. Specifically, whereas preincubation of apoptotic neutrophils with cathepsin G or thrombin significantly inhibited their uptake, gingipains R or clostripain enhanced phagocytosis by macrophages. Moreover, bacterial proteinases sensitized healthy neutrophils for uptake by macrophages, whereas endogenous proteinases were unable to elicit this effect. This stimulation was apparently owing to the combined effect of proteolytic cleavage of an antiphagocytic signal (CD31) and the generation of a novel 'eat-me' signal on the neutrophil surface. These results argue that neutrophil recognition and phagocytosis by macrophages is mediated by a protein ligand whose proteolytic modification could affect the local inflammatory process.
The article considers the possibility of obtaining purified fractions-precursors of bioactive peptides from milk proteins by the method of preparative electrophoresis. To choose an electrophoretic system, a comparative study has been carried out of four methods of electrophoresis in polyacrylamide gel that are used to analyse milk proteins (disc-electrophoresis without disaggregating agents, and disc-electrophoresis in the presence of sodium dodecylsulfate in homogeneous and gradient gel, and electrophoresis in homogeneous gel with urea). Electrophoresis of the total milk protein has shown that none of these systems allows separating effectively all protein precursors of bioactive peptides. The next stage was obtaining two main groups of milk proteins – caseins and serum proteins for electrophoretic fractionation. With the help of analytical electrophoresis, it has been established that each of the obtained groups had a typical proteins composition. Then, the proteins groups obtained were fractionated by preparative electrophoresis using the four electrophoretic systems listed above. In this case, the casein proteins that differ in the primary structure (αS1-, αS2-, β-, and ϰ-caseins) can be effectively separated by preparative electrophoresis on the basis of a homogeneous gel system in the presence of urea. The composition of this electrophoretic system was simplified. Unlike the analytical variant of a homogeneous polyacrylamide gel system, the toxic 2-mercaptoethanol was excluded, and the urea concentration was reduced. For the fractionation of serum proteins, a disc-electrophoresis without disaggregating agents can be used as a basis. It allows obtaining the main precursors of bioactive peptides from milk serum proteins: β-lactoglobulin, α-lactalbumin, serum albumin, and immunoglobulins. The protein precursors obtained by preparative electrophoresis were used to develop the biotechnology of obtaining bioactive phosphopeptides and inhibitors of the angiotensin-converting enzyme.
Introduction. The primary objective of this review is to analyze and summarize the existing scientific information about the structure features, formation conditions and properties of proteinases and peptidases of lactic acid microorganisms, which are widely used in the production of dairy products. Material and methods. The proteolysis of milk proteins, occured by lactic acid microorganisms, is an investigation object of this review article. Scientific articles as well as theses and monographs of microbiology, biochemistry and dairy science have been analysed. Methodology of the investigation is based upon the use of the methods of analysis, comparison and synthesis. Results and discussion. Cleavage of proteins and amino acids with enzymes of lactic acid and propionic acid bacteria promotes the enrichment of dairy products with nitrogen-containing and nitrogenfree compounds, and as a result, the product obtains necessary consistency, taste and smell. In addition to providing organoleptic properties, the formation of a large number of peptides with different types of biological activity occurs also in the process of proteolysis of milk proteins in the production of dairy products. The proteolytic system of lactic acid bacteria consists of three parts: proteinases, which that provide the initial cleavage of casein to peptides with formation of a large number of oligopeptides; peptidases, which cleavage peptides to amino acids; transport system, which provides transfer of proteolysis products through the cytoplasmic membrane. Proteinases function outside microbial cells, produce them, and peptidases-in cells of lactic acid bacteria. By the specificity of the effect on the fractions of the casein complex of milk proteinases of lactic acid microorganisms are divided into 2 types-РІ and РІІІ. Proteinases РІ are able to cleavage β-caseins and don't cleavage α S1-and κ-caseins, but proteinases РІІІ hydrolyse all three fractions: S-, and caseins. None of the peptidases with carboxypeptidase activity were revealed among large number of lactic acid bacteria peptidases. PepN, PepС, РерА are referred to the aminopeptidases, found in lactic acid microorganisms. In addition to aminopeptidase, dipeptidases and tripeptidases were revealed in lactobacilli. Conclusion. It is recommended to use systematized characteristics of proteinases and peptidases of appropriate microorganisms with the purpose of providing quality organoleptic parameters of dairy products as well as the formation of biologically active peptides in the process of selecting the species composition of starter cultures.
Introduction. The aim of this work is studying of the proteolytic processes features in the production of natural phosphopeptides of the milk casein complex. Materials and methods. Casein substrate was isolated by isoelectric sedimentation from skimmed milk. Proteolysis was performed using pancreatin. The degree of proteolysis was determined spectrophotometrically by the absorption of low molecular weight products of proteolysis at λ = 280 nm. The yield of phosphopeptides was determined gravimetrically after precipitation with ethanol in the presence of calcium ions. Electrophoresis of phosphoproteins of the substrate and products of proteolysis was carried out in an alkaline system of homogeneous polyacrylamide gel in the presence of urea. Results and discussion. Natural phosphopeptides were obtained by proteolysis of the phosphoproteins of the casein complex with pancreatin (E:S = 1:100) under physiological conditions (37° C, pH 7.9). At different stages of proteolysis, the yield of phosphopeptides and peptides soluble in 10% trichloroacetic acid was determined. Degree of proteolysis has increased monotonically throughout the studied period. The yield of phosphopeptides reaches its maximum in the 90th minute of proteolysis and then decreases continuously. The yield of phosphopeptides is lower than it was used proteolytic preparations of microbial origin. The results obtained by gel filtration and electrophoresis in a polyacrylamide gel indicate that a decrease in the yield of phosphopheptides after the 90th minute of proteolysis may be caused by the phosphopeptide's molecular weight decrease. Most of phosphopeptides obtained in the 90th minute of proteolysis have a molecular weight up to 2000 Da, which is characteristic for the already known biologically active phosphopeptides. Conclusions. During the proteolysis of casein with pancreatin in physiological conditions, the total yield of proteolysis products increases monotonously. The yield of phosphopeptides has maximum. Gel filtration and electrophoresis data indicate that this is due to a decrease in the molecular mass.
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