ELECTROPHORETIC SYSTEMS FOR PREPARATIVE FRACTIONATION OF PROTEIN PRECURSORS OF BIOACTIVE PEPTIDES FROM COW’S MILK

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.

Physiological mechanism epistatic interaction of resistance genes to acaricides of various chemical classes in the interline hybrids of two-spotted spider mite

Background. The presence in interline hybrids two-spotted spider mite Tetranychus urticae Koch two genes determining resistance to acaricides of various chemical classes significantly increases their sensitivity to the action of each these toxicants. Materials and methods. The resistant and susceptible to malathion, bifenthrin and abamectin inbred lines of spider mite by disruptive selection cycles were obtained. The toxicological tests were performed by diagnostic concentrations of acaricides. The protein marker gene of resistance to malathion was determined by poliacrylamide disc-electrophoresis. Results. The epistatic interaction of resistance genes to different acaricides is not manifestation at the stages of transcription and translation of genetic information. Conclusion. The epistatic effect of another gene on the resistance gene to the current acaricide is a different consequence of metabolism processes encoded by each gene at the stage of phenotypic regulation.

Variety-specificity of soluble proteins of potato tubers

Separation of soluble tuber proteins from six potato clones and twelve varieties cultivated in Poland has been accomplished by disc electrophoresis. It was found that electrophoretic pattern was unique for a given clone or variety. Data obtained confirm results of the other authors for the other varieties and indicate that electrophoretic analysis of potato tuber proteins can be a useful method for taxonomic studies. Such analysis however cannot be used for genetic research since no correlations were found between electrophoretic patterns and genetic origin of respective clones and varieties.

Acrylamide gel electrophoresis of proteins, acid phosphatases and RN-ases from three potato varieties

Studies on variety differences in the protein and acid phosphatase patterns as well as ribunuclease activity distribution were carried out by disc electrophoresis on saline extracts of three varieties of the potato <i>Solanum tuberosum</i> (L.). The protein bands varied in number, position and relative abundance. One main zone of the acid phosphatase activity was detected consisting of 2-3 electrophoretically different bands. Variety differences were concerned with the number and relative abundance of these bands. RNase activity was detected in 4 main zones, in some of them additional subbands were visible. Differences between the three examined varieties were reflected in the occurence of the particular activity zones or their subbands.

Metabolism of excised embryos of Lupinus luteus L. VI. An electrophoretic analysis of some dehydrogenases in cultured embryos as compared with the normal seedling axes

The electrophoretic patterns (disc electrophoresis) of the studied dehydrogenases: glucose-6-phosphate - (A), malate - (B), glutamate - (C), alcohol - (D) and lactate dehydrogenase (E), in the axial organs of isolated <i>Lupinus luteus</i> embryos and seedlings cultivated over 12 days are characterized by great similarities. With time, after the third day of cultivation the patterns begin to become less deyeloped. Analyses performed during the first 10 hours of imbibition of seed parts indicate that the maximal development of isozyme patterns occurs during the third hour after which the patterns become poorer. The most uniform type of pattern. and the lowest number of isozymes was shown by glutamate dehydrogenase, the richest pattern was shown by malate dehydrogenase. No band common for a 11 the 27 experimental elements was found.

Heterogeneity of the acid phosphatase and ribonuclease from protocorms of the orchids Cymbidium Sw. and changes occurring after treatment with streptomycin

The molecular forms of the acid phosphatase and RNase in protocorms of <i>Cymbidium</i> Sw. were studied by disc electrophoresis. The effect of streptomycin added to the culture medium on both enzymes was investigated. Significant changes in enzyme activity and electrophoretic patterns occured after addition of streptomycin at the beginning of culture growth. This indicates that the enzymes are affected by streptomycin in early stages of development of the protocorms.

Preliminary studies on ribonucleases from Poa pratensis seeds

Ribonuclease was extracted from <i>Poa pratensis</i> seeds with 0.1 M acetate buffer, pH 5.1, and then precipitated with alcohol. The enzyme was separated into 5 fractions (I-V) after chromatography on DEAE-cellulose at pH 5.1. The enzymes were stable at 50°C, at pH 7.1. The activity of ribonucleases I, II, III and V were optimal at pH 7.1-7.3, and that of ribonuclease IV at pH 8.1. Ali enzymes were inhibited by Ca²⁺ and EDTA. Mg²⁺ inhibited the activity of ribonucleases II, III, IV, and had no influence on that of ribonucleases I and V. Ribonucleases IV and V showed only one activity band in disc electrophoresis, whereas ribonucleases, I, II and III were found to be heterogenous.