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Here, we report on the evaluation of critical packing parameters, PC, of l-tyrosine and ethylene glycol (PEG) amphiphilic block copolymers. The copolymers were synthesized via l-tyrosine-N-carboxyanhydride ring-opening polymerization using different amino-terminated PEGs as macroinitiators. The size and shape of their associates formed in water were investigated by transmission electron microscopy and dynamic and static light scatterings. The copolymers containing 20–30 wt. % of Tyr formed rod-like micelles. The copolymers with 30–50 wt. % of Tyr formed spherical vesicles, while those with 50–80 wt. % Tyr formed irregularly shaped polymeric nanoparticles. PC of the copolymers was estimated as the ratio of the van der Waals volume of the hydrophobic block (V) to its length (L) and the cross-sectional area of the polar block (SH). The values of SH for each copolymer were calculated according to the equation obtained from the correlation analysis of the published data. The hydrophobic block length L was calculated in three ways, i.e., assuming that polytyrosine adopts (1) stretched, (2) amyloid hairpin, or (3) Gaussian coil conformation. In the latter case, the best match of the calculated PC values with the morphology of the copolymer assemblies was observed.

Here we report formation of gold nanoparticles (GNPs) in micelles of polytyrosine-PEG copolymers that combine the properties of a reducer and a stabilizer. The size and properties of the GNPs were tailored by the excess chloroaurate over the copolymer. The latter quickly formed non-covalent complexes with HAuCl4 and then slowly reduced it to form GNPs. 3 Tyr residues are consumed by reduction of one mole of chloroaurate. The size of the GNPs was controlled by the [Tyr]/[Au(iii)] molar ratio. Small GNPs with D ≅ 8 nm were formed at [Tyr]/[Au(iii)] = 0.5-1.5. 90% of these small GNPs remained bound to the copolymer and could be stored in a lyophilized state. Such polypeptide-gold hybrid materials produced at [Tyr]/[Au(iii)] = 0.5 demonstrated high activity in the catalytic reduction of 4-nitrophenol by sodium borohydride. [Tyr]/[Au(iii)] = 5 led to the formation of large nanoplates (D ≅ 30-60 nm). Thus, in the polymer-based system the GNP size grew in line with the excess of the reducing agent in contrast to Turkevich synthesis of GNPs with citric acid, which also combines the functions of a stabilizer and a reducer. The difference results from the reduction of HAuCl4 in solution according to the Turkevich method and in the micelles of the amphiphilic polymer where the seed growth is limited by the amount of neighboring reducer.

We report here the first evidence for the interaction of poly(ethylene glycol) (PEG) with hyaluronan (HA) in aqueous solutions. PEG-HA complexes (Kapp = 45,000 ± 8000 M-1) contained about 3.3 ± 0.1 of ethylene glycol units per disaccharide of HA. The carboxyl of the D-glucuronic acid and the amide of the N-acetyl-D-glucosamine did not participate in PEG binding. Similar experiments performed with dextran and monosaccharides showed that multiple free primary hydroxyls regularly distributed along the polysaccharide chain are necessary for PEG binding. Another novelty of our study is contraction of HA upon PEG binding. The effect was observed with HA in solution or adsorbed on positively charged liposomes. The thickness of the HA layer on the liposomes decreased 2-fold upon PEG addition. HA compaction induced by PEG may underlie the changes in the plasma membrane properties and resealing of mechanical injuries induced by Pluronics.

The influence of synthetic water-soluble uncharged block copolymers on the multiplication of cells in a culture is studied. Their hydrophilic blocks are represented by linear poly(ethylene glycol) or branched polyglycerol, and hydrophobic blocks, by poly(propylene oxide) or polydimethylsiloxane. MCF-7/Adr cells are incubated with the copolymers in a serum-free culture medium for one hour, and their number after three days of cultivation without a copolymer is determined. The number of cells increases only under the action of polymers containing a poly(ethylene glycol) block, i.e., the longer this block, the lower the concentration of the polymer sufficient for the increase. The replacement of poly(ethylene glycol) by a hydrophilic block with a different structure, branched polyglycerol, fully cancels such an effect, thus indicating the defining role of poly(ethylene glycol) in the ability of block copolymers to maintain the multiplication of cells.

Glutathione-capped gold nanoclusters display polyanionic properties that are manifested in the interaction with cationic species. The nanoclusters form complexes with doxorubicin with effective dissociation constant about 10 µM that increased by an order of magnitude in the presence of 0.15 M NaCl confirming electrostatic character of binding. Adsorption of polylysine arose an increase in the fluorescence of gold nanoclusters due to aggregation induced emission enhancement effect. Fluorescence of the complexes increased several-fold upon addition of up to 1 M of KCl suggesting contribution of non-Coulombic forces in the stabilization of aggregates of gold nanoclusters.

The overproduction of hydrogen peroxide is an inherent feature of some tumour cells and inflamed tissues. We took advantage of this peculiarity to eliminate cells using chemiluminescent peroxyoxalate reaction. We designed dispersions containing polyoxalate and tetramethylhematoporhyrin (TMHP) in dimethylphthalate droplets stabilized with Pluronic L64. The porphyrin plays the dual role. On the one hand, it serves as an activator of the peroxyoxalate reaction of polyoxalate with intracellular hydrogen peroxide and experiences excitation as a result of the reaction. The light emitted in the reaction in the model system without cells was used to optimize the dispersion’s composition. On the other hand, TMHP acts as a photosensitizer (PS) causing cell damage. The formation of singlet oxygen led to cell elimination if the dispersions were used in combination with inducers of oxidative stress: hydrogen peroxide, paraquat, antitumour drug doxorubicin, or a nutritional additive menadione. The PS-induced cytotoxicity correlated with the level of intracellular ROS. The developed approach targeted to endogenous ROS is orthogonal to the classical chemotherapy and can be applied to increase its efficiency.

N-hydroxysuccinimide ester of monomethoxy polyethylene glycol hemisuccinate was synthesized. It acylated amino groups in a molecule of recombinant L-asparaginase from Erwinia carotovora. A method of L-asparaginase modification by the obtained activated polyethylene glycol derivative was developed. The best results were produced by modification of the enzyme with a 25-fold excess of reagent relative to the enzyme tetramer. The modified L-asparaginase was isolated from the reaction mixture by gel filtration on Sepharose CL-6B. The purified bioconjugate did not contain PEG unbound to the protein, demonstrated high catalytic activity, and exhibited antiproliferative action on cell cultures.

A novel strategy is described for preparing pH-sensitive liposomes which releases the encapsulated drug in response to the change in pH of surrounding solution. The liposomes, composed of conventional zwitter-ionic egg yolk lecithin (EL), additionally contains a pH-sensitive "activator" (AMS), a derivative of lithocholic acid with anionic and cationic groups attached to the opposite ends of the steroid core. AMS changes its orientation in the liposomal membrane thus adapting to acidity/basicity of the outer solution. The rotation of AMS induces disordering of the membrane and a fast release of the bioactive cargo. In particular, 50-60 % of the encapsulated antitumor drug, doxorubicin and cisplatin, leaks from the liposomes within the first minute after acidification of the surrounding solution. Low-toxic EL-AMS liposomes, loaded with doxorubicin, show themselves active towards multidrug resistant cells. Fast-acting and low-toxic EL-AMS liposomes can be used in the design of smart liposomal containers in the drug delivery field.

The comparison study of interaction of linear poly(2-dimethyl amino)ethyl methacrylate and its cationic nanogels of various cross-linking with both DNA and sodium poly(styrene sulfonate) has been performed. Although all amino groups of the nanogels proved to be susceptible for protonation, their accessibility for ion pairing with the polyanions was controlled and impaired with the cross-linking. The investigation of nanogels complexes with cells in culture that was accomplished by using of calcein pH-sensitive probe revealed a successive increase in the cytoplasmic fluorescence upon the growth in the cross-linking due to calceine leakage from acidic compartments to cytosol. This regularity implies that amino groups which are buried presumably inside the nanogel are protected against the ion-pairing with polyanions of plasma membrane and hence are able to manifest buffer properties while captured into acidic endosomes, i.e. possess lyso/endosomolytic capacity. These findings suggest that network architecture makes an important contribution to proton sponge properties of weak polycations.

Positively charged linear chitosan molecules were cross-linked with sulfate-anions to form chitosan nanoparticles which were used as a scaffold of negatively charged cardiolipin/egg lecithin liposomes loaded with doxorubicin (DOX). Thus formed multi-liposomal complexes (MLCs) containing 55 liposomes/chitosan and bearing a slight positive net charge effectively transmitted DOX into the cytoplasm of cells in culture. The efficiency of DOX delivery increased 4-5-fold upon drug incorporation in MLCs. Inside the cells, the penetrated MLCs released DOX thus enabling its accumulation in the nuclei and interaction with its intracellular target DNA leading to a decrease in cell survival. The effects were demonstrated on 3T3 line of mouse fibroblasts, drug sensitive tumor MCF-7 cells and drug resistant human ovarian carcinoma OVCAR-8 cells (formerly called MCF-7/ADR). Thus, MLC particles can be used for effective delivery of payloads in cells of different origin.

Purpose: Pluronics are known as inhibitors of multidrug resistance thus making tumor cells sensitive to therapeutic doses of drugs. The purpose of our study consists in revealing molecular targets of the hydrophobic poly(propylene oxide) block of pluronics in living cells and the dependence of the polymers chemosensitizing efficiency upon targeting. Methods: A photo sensitive tracer was attached to the hydrophobic poly(propylene oxide) block of 3H-labeled tert-Bu-EO-PO copolymer. The conjugate was used for treatment cells in culture. We searched for its complexes with cellular lipids or proteins using RP TLC and SDS-electrophoresis, respectively. The chemosensitizing efficiency of pluronics was evaluated by their least concentrations sufficient for MDR reversion (CMDR). Results: The poly(propylene oxide) block inserts in the lipid core of plasma membrane. No preferential binding of the conjugate with any cellular protein, particularly P-gp, has been detected. FITC-labeled pluronic L61 bound to alcohol insoluble cellular targets did not participate in MDR reversion. CMDR values of 13 block copolymers have been determined. These values inversely correlated with the polymers affinity toward lipids and the ability to accelerate flip-flop. Conclusion: Insertion of the hydrophobic poly(propylene oxide) block of amphiphiles in the lipid core of plasma membrane and acceleration of flip-flop of lipids underlie the mechanism of MDR reversion.

N-hydroxysuccinimide ester of monomethoxy polyethylene glycol hemisuccinate was synthesized. It acylated amino groups in a molecule of recombinant L-asparaginase from Erwinia carotovora. A method of L-asparaginase modification by the obtained activated polyethylene glycol derivative was developed. The best results were produced by modification of the enzyme with a 25-fold excess of reagent relative to the enzyme tetramer. The modified L-asparaginase was isolated from the reaction mixture by gel filtration on Sepharose CL-6B. The purified bioconjugate did not contain PEG unbound to the protein, demonstrated high catalytic activity, and exhibited antiproliferative action on cell cultures.

The purpose of this study is to ascertain the relationship between the structure of an amphiphilic nonionic polymer and its toxicity for cells (cytotoxicity) growing in a culture. To this end, 16 polymers of different architectures and chemical structures are tested, namely, linear triblock copolymers of poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (Pluronics); diblock copolymers of propylene oxide, ethylene oxide, and hyperbranched polyglycerol; alternating and diblock copolymers of ethylene oxide and dimethylsiloxane; and two surfactants containing linear (Brij-35) or branched (Triton X-100) aliphatic chains. Polymer-cell interaction is assayed in a culture medium in the absence of serum. Effective concentrations of the polymers causing 50% cell death, EC50, vary within three orders of magnitude. Toxic concentrations of the alternating copolymer, Triton X-100, and Brij-35 are lower than their CMC values. In contrast, all block copolymers, regardless of their chemical structures, become toxic at concentrations above the CMC; that is, they acquire cytotoxicity only in the micellar form. The EC50 values of the copolymers depend on their hydrophilic-liphophilic balance (HLB) through the following empirical formula: EC50 × 106 = 8.71 × HLB2.1. This relationship makes it possible to predict the cytotoxic concentration region of a block copolymer of a known structure.

Partition coefficients of ethylene oxide-propylene oxide block copolymers between the lipid phase and water have been estimated via equilibrium dialysis. It has been shown that for the triblock copolymer (Pluronic L61), the partition coefficient is 45 ± 9, while for the diblock copolymer (REP), this parameter is as high as 78 ± 17. The effect of the copolymers on the permeation of the charged organic ion carboxyfluorescein across the lecithin bilayer membrane changes in the same direction. Even though the triblock copolymer binding is weaker, it shows a stronger effect on the rate of transbilayer migration of lipids and on the permeation of the uncharged substance (doxorubicin). The incorporation of cholesterol into the membrane decreases its sensitivity to the action of copolymers; however, the character of changes induced by both copolymers remains invariable. The experimental data of this study indicate that the triblock structure of amphiphilic macromolecules is responsible for their higher ability to disturb lipid bilayer membranes.

All polymeric chemosensitizers proposed thus far have a linear poly(ethylene glycol) (PEG) hydrophilic block. To testify whether precisely this chemical structure and architecture of the hydrophilic block is a prerequisite for chemosensitization, we tested a series of novel block copolymers containing a hyperbranched polyglycerol segment as a hydrophilic block (PPO-NG copolymers) on multi-drug-resistant (MDR) tumor cells in culture. PPO-NG copolymers inhibited MDR of three cell lines, indicating that the linear PEG can be substituted for a hyperbranched polyglycerol block without loss of the polymers' chemosensitizing activity. The extent of MDR reversal increased with the polymers affinity toward the cells and the expression level of P-glycoprotein. In contrast with Pluronic L61, which increases viability of tumor cells in the absence of drugs, PPO-NG chemosensitizers are completely devoid of this property undesired in cancer therapy, making them promising candidates for application as novel MDR reversal agents.

PC12 cells permeabilized with a low concentration of digitonin (5 μM) under controlled conditions were loaded with monoclonal antibodies (MoAb) against the regulatory subunit type II (RII) of cAMP-dependent protein kinase. After digitonin removal from the nutrient medium (DMEM) the loaded cells repaired within 20–30 min and recontinued growth. The inserted MoAb stayed in the repaired cells at least for several hours. MoAb inhibiting the cAMP binding activity of neural RII [Grozdova et al. (1992) Biochem. Int. 27, 811–822; Sveshnikova et al. (1996) Biochem. Int. 39, 1063–1070] were shown to bind the target antigen inside the cells and influence the properties of intracellular protein kinases.

Triblock copolymers of ethylene oxide (EO) and propylene oxide (PO) of EOn/2POmEOn/2 type (Pluronics) demonstrate a variety of biological effects that are mainly due to their interaction with cell membranes. Previously, we have shown that Pluronics can bind to artificial lipid membranes and enhance accumulation of the anti-tumor drug doxorubicin (DOX) inside the pH-gradient liposomes and transmembrane migration (flip-flop) of NBD-labeled phosphatidylethanolamine in the liposomes composed from one component—lecithin. Here, we describe the effects caused by insertion of other natural lipids in lecithin liposomes and the significance of the lipid composition for interaction of Pluronic L61 with the membrane. We used binary liposomes consisting of lecithin and one of the following lipids: cholesterol, phosphatidylethanolamine, ganglioside GM1, sphingomyelin, cardiolipin or phosphatidic acid. The influence of the additives on (1) membrane microviscosity; (2) binding of Pluronic L61; (3) the copolymer effect on lipid flip-flop and membrane permeability towards DOX was studied. The results showed that insertion of sphingomyelin and cardiolipin did not influence membrane microviscosity and effects of Pluronic on the membrane permeability. Addition of phosphatidic acid led to a decrease in microviscosity of the bilayer and provoked its destabilization by the copolymer. On the contrary, cholesterol increased microviscosity of the membrane and decreased binding of Pluronic and its capacity to enhance flip-flop and DOX accumulation. Analogous tendencies were revealed upon incorporation of egg phosphatidylethanolamine or bovine brain ganglioside GM1. Thus, a reverse dependence between the microviscosity of membranes and their sensitivity to Pluronic effects was demonstrated. The described data may be relevant to mechanisms of Pluronic L61 interaction with normal and tumor cells.

Alterations of DNA structure, of cAMP content, of cAMP-dependent histokinases (HK) activity and cAMP-independent casein kinases (CK) activity were studied during transformation of resting cells to proliferation. These patterns were studied in human lymphocytes from peripheral blood immediately after their isolation and after cultivation within 3 days in presence of concanavalin A (ConA) or without the mitogen as well as in cultivated cells of human T-lymphoma Jurkat. Increase in content of alkaline labile sites in DNA, in activity of CK as well as distinct increase in content of cAMP were detected within the first 18 hrs of lymphocytes cultivation both in presence of ConA or without it. Early steps of cell transformation from G0 phase to G1 may be related to these alterations observed. Only slight increase in content of the alkaline labile sites in DNA and decrease in cAMP content were found in both these cell cultures. Activity of CK in the lymphocytes culture not containing ConA was decreased down to initial level, while in presence of the mitogen the enzymatic activity was increased and within 3 days it reached the level of CK activity in Jurkat cells. The rate of CK relative activity, calculated as CK/cAMP or CK/HK/cAMP ratios for each cell preparation, correlated with DNA biosynthesis rate measured by 3H-thymidine incorporation. The data obtained suggest that these patterns, used in differential diagnosis of human large intestine and gastric tumors, demonstrated also the intensity of tissue proliferation.

Immunochemical analysis of the cAMP-dependent protein kinase regulatory subunit type II was performed with the use of two rabbit antisera elicited to a free R-subunit from pig brain and to a RcAMP complex. Quantitative precipitation of the homogeneous antigen revealed six determinants on the R-molecule. Of these at least one is localized in the R-fragment (37 kD), the others--in the N-terminal part of the R-molecule. The antigenic determinants seem to be remoted from the cAMP-binding centers, since the attachment of the affinity purified antibody Fab-fragments to the R-subunit did not influence the cAMP-binding activity of the latter. The antibodies to RcAMP caused dissociation of the holoenzyme. The antibody Fab-fragment binding to the R-subunit prevented its association with the catalytic subunit. The results of immunochemical analysis suggest that the R-subunit adopts different conformations when bound to cAMP or to the catalytic subunit.

Localization of the regulatory subunit of cAMP-dependent protein kinase type II was studied in proliferating and quiescent fibroblasts 3T3 and in a cell line of neural origin pheochromocytoma PC12. In actively proliferating PCl2 cells the regulatory subunit was found to be localized in the nucleus. Transition of these cells into a quiescent state was accompanied by a regulatory subunit translocation to the cytoplasm. In 3T3 cells the regulatory subunit was localized in the cytoplasm both in the quiescent and proliferating (though less actively than PC12 cells) states. Similar results were obtained both with monoclonal antibodies and with rabbit monospecific antiserum raised against the regulatory subunit type II from pig brain.

The activity of cAMP-dependent histone kinases (HK) and cAMP-independent casein kinases (CK) as well as cAMP level were assessed in gastric mucosa affected by various forms of gastritis as well as in gastric tumor and adjacent normal tissue. CK activity was higher whereas that of HK and cAMP level were lower in tumor and at a distance of 5-20 cm from its edge as compared to gastritis. CK/HK cAMP ratio in adenocarcinoma was 6 times that in normal tissue and 2-2.5 times that in gastritis. The data obtained and those published earlier suggest the relative increase in cAMP-independent protein phosphorylation with respect to its cAMP-dependent counterpart to be characteristic of human tumors.

This paper describes the wide variety of functions of cyclic nucleotides in living organisms. Modifications in cyclic AMP function during the process of evolution from primitive unicellular organisms to highly organized multicellular systems are considered. Studies with a synchronous eukaryotic cell culture permit us to estimate changes in the cyclic nucleotide content throughout the cell cycle as well as in the activity of the enzymes related to cyclic nucleotides and their metabolism. The antimitogenic role of cyclic nucleotides during the period prior to mitosis is elucidated, and the cyclic nucleotide-dependent processes, in particular phosphorylation of nuclear proteins, are shown to be characteristic only of early stages of the cell cycle. Other studies on the role and significance of cyclic nucleotides in systems of multiple biological mediators are reviewed.

The inhibition of rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase by specific antibodies produced in rabbits has been studied. The results suggest that no influence on the enzyme active site is caused by the interaction with antibody, the inhibition being due entirely to the restricted accessibility for substrates of a part of dehydrogenase molecules included in the immune precipitate. Soluble complexes of the enzyme with monovalent Fab antibody fragments retain full catalytic activity. Modification of 8 -SH groups per mole of glyceraldehyde-3-phosphate dehydrogenase with p-chloromercuribenzoate results in no alterations in the quantitative precipitin curve, thus supporting the conclusion about the different localization of species-specific antigenic determinants of the enzyme and its active center. Interaction with monovalent Fab fragments of antibody stabilizes the structure of the dehydrogenase. Eight molar equivalents of Fab fragments almost completely protect the enzyme from cold inactivation in the presence of 0.15 M NaCl. Complex formation with Fab fragments does not prevent, however, the ADP-induced inactivation of the enzyme.

The content of cAMP and cGMP as well as the levels of phosphodiesterase and protein kinase activities were studied throughout the cell cycle of synchronous Physarum polycephalum. The increase in cAMP content was observed in S phase of the cell cycle. The analogous increases in cGMP were found in late S and early G2 phase. The appearance of cyclic nucleotide-dependent protein kinases paralleled the corresponding increase in cyclic nucleotide concentration. The cAMP phosphodiesterase activity was comparatively high throughout the cell cycle except for mitosis when it decreased. Histone H1 (P1) was shown to be phosphorylated in vivo in all the cell cycle, the degree of phosphorylation gradually increasing on approaching mitosis. Prior to mitosis the number of phosphate groups attached to a molecule of H1 increased to as high as 7. Effects of phosphorylation are discussed in terms of histone-DNA interactions.

Inactivation of apo-glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase(phosphorylating) (EC 1.2.1.12) from rat skeletal muscle at 4 degrees C in 0.15 M NaC1, 5 mM EDTA, 4 mM 2-mercaptoethanol pH 7.2 is a first-order reaction. The rate constant of inactivation depends on protein concentration. With one molecule of NAD bound per tetrameric enzyme, a 50 per cent loss in activity is observed and the rate constant of inactivation becomes independent of the protein concentration over a 30-fold range. Two moles of NAD bound per mole of enzyme fully protect it against inactivation. NADH affords a cooperative effect on enzyme structure similar to that of NAD. Inactivation of 7.8 S apoenzyme is reflected in its dissociation into 4.8-S dimers. In the case of enzyme-NAD1 complex, no direct relationship between the extent of inactivation and dissociation is observed, suggesting that these two processes do not occur simultaneously; we may say that dissociation is slower than inactivation. A mechanism in which the rate-limiting step for inactivation is a conformational change in the tetramer occurring prior to dissociation and affecting only the structure of the non-liganded dimer, is consistent with the experimental observations. Inorganic phosphate protects apoenzyme against inactivation. Its effect is shown to be due to the anion binding at specific sites on the protein with a dissociation constant of 2.6 plus or minus 0.4 mM. The NaC1-induced cold inactivation of glyceraldehyde-phosphate dehydrogenase is fully reversible at 25 degrees C in the presence of 20 mM dithiothreitol and 50 mM inorganic phosphate. The rate of reactivation is independent of protein concentration. Inactivated enzyme retains the ability to bind specific antibodies produced in rabbits, but diminishes its precipitating capability.

Antibodies were prepared in chickens against glyceraldehyde-phosphate dehydrogenase (d-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating) EC 1.2.1.12) isolated from rabbit and rat skeletal muscle; antibodies to the rat muscle enzyme were produced in rabbits. The data obtained in gel-diffusion and enzyme-inhibition experiments carried out with different antisera demonstrate the presence of two types of antigenic determinants on the dehydrogenase molecule-interspecific, cross-reactive, and species-specific determinants.

The rate constants of 1H--3H exchange between water and C8H-groups of purinic residues of DNA in solution and in nuclei of synchronous Physarum polycephalum at different phases of the cell cycle were measured. The nuclear membranes and nonhistone proteins were shown not to create additional hindrances and not to diminish the availability of DNA in nuclei for solvent molecules. A small increase of the rate of the 1H--3H exchange between water and the DNA of nuclei upon transition from the S-phase to the late G2-phase seems to reflect the process of chromatin decondensation connected with activation of the transcription and local changes of the secondary structure of DNA at the late G2-phase of the cell cycle.

The phosphorylating enzymes of human skin were studied in bioptic samples of normal tissue (18 samples) and tumors (21 cases), melanomas and basaliomas, which developed in different regions of head and face. The activity of cAMP-dependent and cAMP-independent protein kinases was tested in skin extracts using histone HI and casein as substrates of phosphorylation, respectively. In most tumors the casein kinase activity was found to be significantly elevated (about 10-fold) as compared with normal counterparts. The histone kinase activity was only slightly elevated in tumors (by 30%). For each bioptic sample the ratio of histone kinase activity versus casein kinase activity was calculated. In normal skin this ratio constituted from 1 to 3.7, while in 90% of samples it was higher than 1.5. In all tumors except one the ratio was decreased down to 0.2-0.9. The effect did not depend upon the type of malignancy and tumor location. The change in the protein kinase ratio is considered to be a specific feature of transformed tissue.

Activity of cAMP-dependent and -independent protein kinases as well as content of cAMP were studied in biopsy preparations of gastric mucosal membranes obtained from patients with superficial gastritis, erosion, ulcer, adenomatous polyps and ulcer after treatment with laser irradiation. All the patterns studied were similar in preparations of adenomatous polyps and normal mucosal membrane. Activity of cAMP-independent protein kinases was increased under conditions of erosion, ulcer, gastroduodenal reflux and, especially, in ulcer tissue treated with laser. However, the ratio between cAMP-independent and -dependent phosphorylation was increased only in 50% of ulcerous diseases and in gastroduodenal reflux. The equilibrium of cAMP-dependent and -independent phosphorylation was not altered in the other impairments studied even though benign proliferation was stimulated using laser irradiation.

Immunochemical study of D-glyceraldehyde-3-phosphate dehydrogenase from rat skeletal muscle was done using antibodies, raised in rabbit. One molecule of the enzyme binds three antibody molecules at the equivalence point and eight molecules at full saturation of the antibody binding sites. Modification of SH-groups of the dehydrogenase with pCMB, as well as ADP-induced inactivation of the enzyme do not cause any alterations in the quantitative precipitation curve. This suggests that antigenic determinants and the active site are situated in different loci on the enzyme molecule. A quantitative relationship between the dehydrogenase concentration and the percentage decrease of its catalytic activity in the presence of antibody excess are established, and a mechanism of apparent inhibition in the insoluble immune complex is proposed.

In recent years great attention has been devoted to the investigation of the biological role of cyclic AMP-dependent protein kinases. Intensive studies have been carried out on the effect of cyclic nucleotides and protein phosphorylation on the processes of proliferation and differentiation of cells. In this connection the role of protein kinases in transcription, biosynthesis of proteins, and regulation of various enzymes is being currently investigated. In this paper some new data on the mechanism of action of cAMP-dependent pig brain protein kinase are presented and the biological role of phosphorylation in the cell cycle is discussed.

The true slime mould Physarum polycephalum was treated with various agents by spraying them upon the cell surface 4 hrs before the second synchronous mitosis. The onset of mitosis was considerably approximated after the plasmodium treatment with protein kinases from rat hepatoma or Ph. polycephalum at the late G2 phase. The catalytic and regulatory subunits of cAMP-dependent pig brain protein kinase caused retardation of mitosis, while the holoenzyme, casein kinase and alkaline phosphatase did not affect the timing of mitosis. The cyclic nucleotides and inhibitors of their metabolic enzymes were used to investigate the role of phosphorylation processes in the mitotic cycle.

The binding of antibodies to enzymes-antigens markedly affects the activity and structure of the latters. The mechanisms of activation and inhibition of enzymes by antibodies and the use of immunochemical methods for the study of the active sites structure, conformational changes during interaction with substrates and other ligands and the quaternary structure of enzymes are discussed.

Regulatory subunits type II (RII) purified from human, pig and bovine brains were compared using II monoclonal antibodies (MoAb) against bovine brain RII. Bovine RII has at least 5 antigenic sites located in the N-terminal 1-110 residues. Immunochemical difference detected between human and animal RII was more pronounced than between pig and bovine RII. Certain MoAb influenced R-cAMP binding and holoenzyme formation. RII of the three species responded to MoAb attachment in a similar fashion. The results suggest that anchoring of neural protein kinase via the N-terminal part of RII may influence the enzyme activity.

The regulatory subunit type II (RII) of cAMP-dependent protein kinase purified from human brain was represented by two proteins with apparent molecular masses of 51-52 kD and 54 kD. Dephosphorylation of human RII containing 3 mol phosphate/mol protein did not change the electrophoretic pattern. One-dimensional peptide mapping of 51-52 kD and 54 kD proteins after digestion with St. aureus V8 protease evidenced to their being distinct proteins. The data obtained permit to assume that human RII of neural type is represented by two isoforms.

Protein kinases help regulate eukaryotic cell division. We investigated the regulation of cAMP-dependent protein kinase A (PKA) and casein kinase (CK) type I activity in normal cells and in cancer. To assess this activity in biopsies we suggest a new parameter--the ratio of CK activity and total PKA activity divided by cAMP concentration: CK/PKA/cAMP. In 98 samples of colon mucosa in normal, inflamed, polyp, and adenocarcinoma cells, we found this parameter to be fairly constant in normal conditions and increased 10-fold in colon cancer; the ratio does not depend on the place of biopsy or the patient's age or sex. Experiments with model systems of concanavalin A-stimulated lymphocytes and regenerating rat liver showed that in normal cell proliferation the parameter increases 2-3-fold, as compared to a 30-fold increase in cancer. Unlike normal cells, malignant cells show CK activation and decrease of cAMP; therefore, PKA activity decreases. This suggests a correlation of CK and PKA activity and significant damage to their regulation at pathological changes of tissue proliferation. To further study concerted CK and PKA regulation we used monoclonal antibodies (mAbs) against cAMP-dependent protein kinase regulatory subunit RKII beta. We produced 11 antibodies in three groups: inhibiting, which block cAMP binding with RII beta and inhibit holoenzyme formation (RS6); activating, which enhance cAMP binding and do not affect holoenzyme formation (RS28); and neutral (RS17). To investigate mAb influence on protein kinase regulation in live cells we permeabilized pheochromocytoma PC12 by digitonin. When used at 5-microM concentration for 5 min, digitonin allowed us to deliver mAb into PC12 cells at 30-34-nM concentration, leaving 68-75% viable cells. Protein kinase activity was measured within 0.5 and 4 h after incorporation of mAbs into cells. After 30 min incorporation, mAb RS6 blocked PKA activation in PC12 cells under the influence of cAMP; other mAbs showed no effect. mAb RS6 caused a 4-fold increase of free C subunit activity 4 h after incorporation. mAb RS38 decreased R2C2 activity and did not influence C subunit activity. The change of free C subunit activity caused by mAb incorporation was followed by a synchronized, well-balanced change of CK type I activity, which suggests a correlation between the two phosphorylation systems of cell proteins.