[Proteolytic activity of IgG-antibodies of mice, immunized by calf thymus histones].

The main goal of the study was to determine the ability of histones to induce production of the proteolytically active IgG-antibodies in BALB/c mice. In order to perform this study 8 mice were immunized with the fraction of total calf thymus histones. IgGs were isolated from the serum of the immunized and not immunized animals by means of precipitation with 33% ammonium sulfate, followed by affinity chromatography on protein G-Sepharose column. Histones, myelin basic protein (MBP), lysozyme, BSA, ovalbumin, macroglobulin, casein and cytochrome c served as substrates for determining the proteolytic activity. It was found that IgGs from the blood serum of immunized mice are capable of hydrolyzing histone H1, core histone and MBP. On the contrary, the proteolytic activity of IgGs from the blood serum of not immunized mice was not detected. The absence of proteolytical enzymes in the fraction of IgGs was proven by HPLC chromatography. High levels of proteolytic activity toward histones have been also detected in affinity purified IgGs from blood serum of patients with rheumatoid arthritis, but not in healthy donors. These data indicate that eukaryotic histones may induce production of protabzymes in mammals. The possible origin of these protabzymes and their potential biological role in mammalians is discussed.

[The analysis of biochemical markers of MCF-7 cells apoptosis induced by recombinant analog of lactaptin].

Earlier we isolated and characterized human milk pro-apoptotic peptide - lactaptin and generated its engineered analog - RL2. It was shown that both lactaptin and RL2 are capable to induce apoptotic death of MCF-7 cells. In this report we have analyzed biochemical markers of RL2 induced MCF-7 apoptosis. The activation of initiator and effector caspases as well as mitochondrial membrane potential and cytoplasm membrane changes were analyzed using flow cytometry and Western-blot methods. We have found that RL2 induced apoptotic death of MCF-7 cells was accompanied by PS exposure on the plasma membrane surface. It also was shown that RL2 has induced dissipation of mitochondrial membrane potential and resulted in activation of initiator caspases 8, 9 and effector caspase 7.

[Biological features of immunoglobulins of class G in mice fed with cattle brains for a long time].

It was found that mice fed with brain of cattle or brain of cattle with a meat-bone flour (1:1), were capable of producing immunoglobulins which interacted with DNA, and protein and lipid fractions of mouse brain. These immunoproteins also induced death of cells of L1210 line of murine leukemia via apoptosis accompanied by changes in the animal level of proteins Bcl-2, p53 and procaspase 3. The potentials of using this experimental animal model for studying cytopathogenetic mechanisms of transmissible spongiform encephalopathies are discussed.

[Proteolytic activity of blood serum IgG in patients with systemic lupus erythematosis].

Proteolytic activity of blood serum IgGs of 10 patients with systemic lupus erythematosis (SLE) was studied in comparison with such activity in 10 clinically healthy donors. Antibodies were precipitated from blood serum by saturation with 50% (NH4)2SO4 and IgG was isolated by the affinity chromatography on protein G-sepharose column. Histone H1 and core histones from the calf thymus, bovine myelin basic protein (MBP), lysozyme of chicken egg and bovine serum albumin (BSA) were used as substrates for proteolytic action. It was found that 4 of 10 preparations of IgGs possess an ability to hydrolyze both histone H1 and MBP. These antibodies practically did not cleave lysozyme of the chicken egg and BSA. Gel-filtration of antibodies under acidic condition and following examination of proteolytic activity of chromatographic fractions showed that histone H1 and MBP-hydrolyzing activity is attributable to IgG-antibodies. Thus, the presence of catalytically active antibodies (protabzymes) in the blood serum of patients with SLE has been demonstrated. Their origination and biological role are discussed.

[Catalytically active antibodies (abzymes) in human milk].

The review is focused on the analysis of published data and the results obtained by the authors about the catalytic activity of antibodies (abzymes) of human colostrum and milk. Possible mechanisms of origination of these abzymes and their potential role in the regulation of biological activity of human milk compounds are considered. A hypothesis about the role of secretoty abzymes in non-specific humoral defense for the epithelial cells against viral infections is proposed.

[Protein kinase activity of immunoglobulins in human blood plasma].

Protein kinase activity of the immunoglobulins (Ig) fractions from blood plasma of clinically healthy humans has been studied. IgA, IgG and IgM preparations have been obtained using column chromatography on sorbents with rabbit antibody to H-chains of human Ig. The level of 32P incorporation in casein in the presence of [gamma-32P]ATP was used to determine protein kinase activity of the Ig-fractions. The protein kinase activity of the preparation of IgA (but not IgG or IgM) was defined. The high-purified preparation of IgA for studing the protein kinase activity has been obtained. Three stages of purifications were used--the separation of plasma proteins by polyethylenglycol 6000, gel-filtration on the column with Toyopearl HW-60 Fine and affinity chromatography on the column containing rabbit antibody to H-chains of human IgA. It was revealed that the fraction of IgA possesses the casein phosphorylation activity. Heparin and trifluoperazine completely and partially inhibited protein kinase activity of IgA while spermidine did not render essential influence. On the basis of the obtained results the conclusion is made that the blood of clinical by healthy humans contains IgA possessing the protein kinase activity.

[Interaction of oligonucleotides and ATP with preparations of sIgA possessing protein kinase activity]. / Vzaimodeistvie oligonukleotidov i ATP s preparatami sIgA, obladaiushchimi proteinkinaznoi aktivnost'iu.

Interaction of secretory immunoglobulins A of a varying degree of purity with oligonucleotides and ATP has been studied by the method of affinity modification. For this aim we used reactive derivatives of 32P-labeled deoxyoligonucleotide (ClRCH2NHp(T)14) and [gamma-32P]ATP (ClR-32PppA) or ATP (ClR-pppA) bearing a 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine residue. Preparations of sIgA were obtained from human milk by sequential chromatography on protein A-sepharose (P1), DEAE-fractogel (P2) and by gel-filtration in 50 mM NaOH (P3). It was revealed, that the H- and L-chains of sIgA P1; H-, L-chains and secretory component (SC) in sIgA P2 and only SC in sIgA P3 were exposed to modification after incubation with ClRCH2NHp(T)14. LPS, DNA, tRNA, heparin, sufficiently inhibited the modification of chains of sIgA P1. These competitors did not influence the modification of H- and L-chains of sIgA P2, but DNA, tRNA, heparin, inhibited binding of SC with the modifier. Suppressing affect of binding of ClRCH2NHp(T)14 with secretory component of sIgA P3 by d(T)14 has been observed as well. The research of ClR-32PppA interaction with sIgA P3 has shown that H- and L-chains of sIgA are exposed to modification. ATP inhibited the reaction. Study of the influence of modification on the protein kinase activity of sIgA P3 has revealed, that the preliminary incubation of sIgA P3 with ClR-pppA leads to inhibition of protein kinase activity. We suggest that sIgA, possessing the protein kinase activity (sIgA-abzymes) has an ATP-binding center (catalytic center) and has an oligonucleotide-binding center as well.

[Method of detection of protein kinase activity in polyacrylamide gel using two-dimensional protein separating system]. / Metod vyznachennia proteïnkinaznoï aktyvnosti v poliakrylamidnomu heli z vykorystanniam systemy dvovymirnoho rozdilennia bilkiv.

Method for detection of protein kinase activity in polyacrylamide gel have been developed. After separation of proteins by isoelectric focusing in non-denaturing condition, gel was incubated in a reaction buffer containing [gamma-32P]ATP. 32P-labeled proteins were separated by subsequent SDS/PAGE electrophoresis in second dimension. The proposed method was used for detection of protein kinase activity in human blood serum and triton X-100 soluble proteins of heads of Drosophila melanogaster.

[Study of expression of desoxyribonucleases in mammalian cells by a protein renaturation method in polyacrylamide gel]. / Doslidzhennia ekspresiï dezoksyrybonukleaz u klitynakh ssavtsiv metodom renaturatsiï bilkiv u poliakrylamidnomu heli.

Analysis of enzymatic activity in polyacrylamide gel is based on highly effective separation of proteins by SDS-electrophoresis with their subsequent renaturation and detection of enzymatic activity. This method was used to study an expression of DNAases in culturing of cells HEK293, NIH 3T3, U937. We have found that in HEK293 cells the nucleases with molecular weights 47 and 45 kDa were expressed. The localization of DNAases in the cell nuclei was shown as well. Induction of apoptosis in HEK293 cells increase the level of p47 DNAase and causes the expression of novel 50 kDa DNAase. We suggested that those discovered DNAases could take part in apoptotic DNA degradation.

[Do catalytically active antibodies exist in healthy people? (Protein kinase activity of sIgA antibodies from human milk)]. / Sushchestvuiut li kataliticheski aktivnye antitela u zdorovykh liudei? (Proteinkinaznaia aktivnost' sIgA antitel iz moloka cheloveka).

The fraction of secretory immunoglobulin A (sIgA) from the milk of healthy mothers was purified by sequential affinity chromatography on protein A-sepharose (in the presence of 1% triton x100), adsorbent Toyopearl HW-55 (gel-filtration), DEAE cellulose (separation of IgG and IgA antibodies), affinity sorbents with immobilized ATP and casein. The protein obtained corresponded to sIgA antibodies according to all known criteria and did not contain any protein contaminations. The ability of sIgA to phosphorylate selectively serine residues of casein (not histones) in the presence of [gamma-32P]ATP was shown. Purified kinase activity was stable at acid shock (pH 2.3), strongly interacted with immobilized antibodies against H-chain of sIgA and eluted from the sorbent with the peak corresponding to sIgA antibodies. The complex of sIgA and ATP was stable enough at the conditions of gel-filtration. Affinity modification of sIgA by chemically reactive analogs of ATP resulted in preferential modification of its light chain (2-3 mole reagent per mole of dimer form). In the condition of oligomer dissociation ATP-sepharose sorbed only the light chains of sIgA. sIgA have optimal conditions for phosphorylating activity different from those of known protein kinases. We suppose that sIgA antibodies with kinase activity are a first example of sIgA antibodies with catalytic activity. For the first time the possibility of existence of natural abzymes in a healthy human was shown. These abzymes catalyze the reaction of synthesis but not substrate degradation reaction.