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We have investigated the effects of Pro-Met-Asp-Phe-NH2 (PMAP) on insulin and glucagon release from human fetal pancreatic microfragments in vitro. Four batches of precultured microfragments were incubated for 24 hrs in medium containing 5.5 mM glucose, 17 mM glucose, 1 microM PMAP or 1 microM PMAP plus 17 mM glucose. PMAP significantly enhanced both basal and glucose-stimulated insulin release (2.2- and 4.1-fold, respectively). Glucagon secretion was markedly inhibited by glucose (17 mM). PMAP neither affected the basal glucagon release nor potentiated the inhibitory action of glucose on glucagon release. Hence, PMAR selectively regulates insulin production in human fetal islet tissue without affecting glucagon production. Our results suggest that the substances similar or related to PMAP may prove to be of clinical value in drug correction of diabetes mellitus.

Administration of Ca-entry blockers with different chemical structure before the braining sessions produced the reduction of memory retention in mice and rats in the one-trial passive avoidance tests. This effect was absent in animals treated immediately after training test. Nootropic drugs piracetam and oxiracetam corrected the retention of memory when injected just after training test. Chronic treatment of rats with increasing doses of the nootropic drugs produced about two-fold tissue-specific elevation in the density of DHP-receptors, associated with L-type Ca-channels in synaptosomal membranes of rat cerebral cortex. Maximal effect was observed in a dose of 10 mg/kg. Diltiazem, administrated in a dose of 10 mg/kg, produced about two-fold decrease in the receptors density measured 24 hrs after the first injection. Oxiracetam (10 mg/kg) completely antagonized the effect of Ca-entry blocker. These data imply that nootropic action of piracetam and oxiracetam is mediated by L-type Ca-channels.

Ca2+ entry blockers riodipine, D-cis-diltiazem and verapamil, when administered i.p. to rats at a dose of 10 mg/kg, produced two-fold decreases in the density of 1,4-dihydropyridine (DHP) receptors in rat cerebral cortex, as revealed by Scatchard plot analysis of radioligand binding made 24 h after the first injection. Thereafter, the number of DHP binding sites increased up to the initial level on day 4 of the treatment. The nootropic drug oxiracetam, when injected simultaneously with Ca2+ channel blockers at a dose of 10 mg/kg, prevented this transient decrease in DHP receptor density in brain. These results can explain the opposite modulation of memory retention by calcium antagonists and nootropic drugs that has been observed previously.

The paper surveys the data obtained during the last years in the Laboratory of Regulation of Cellular Activity of the Institute of Molecular Biology. The data mostly concern the enzymes taking part in metabolism and in biological action of the secondary messengers, as well as the mechanisms of regulation of some metabolic pathways in the cultured cells. The results pertinent to the mechanisms of chromatin condensation and based on this genetic control processes are also discussed.

Ca2+-independent protein-modulator (BacM) was found in the culture medium of Staphylococcus aureus. BacM activated calmodulin-dependent cyclic nucleotide phosphodiesterase and Ca2+/Mg2+-ATPase in the same way as calmodulin. BacM was shown to be a proteolytic fragment of the exotoxin secreted by the S. aureus strain under study. The kinetic analyses of the ATPase activation by BacM and CaM were performed. These studies demonstrated that the enzyme molecule contains at least two activator-sensitive sites. Experiments on the ATPase activation by Ca2+ both in the presence and in the absence of BacM and CaM documented that CaM-ATPase and BacM-ATPase complexes can exist at low concentrations of calcium. Analysis of activation curves of ATPase by Ca2+ revealed three Ca2+-binding sites in the enzyme-activator complex.

The mechanisms of regulation of cyclic AMP phosphodiesterases were studied using the cytoplasmic fraction of PC-12 cells sensitive to the action of nerve growth factor. The cells contain phosphodiesterases of two types. One of them possesses a high affinity for cyclic AMP (Km = 2.46 mM), whereas the other has the affinity by an order worse (Km = 37.1 mM). PC-12 cell differentiation under the action of nerve growth factor is connected with the cyclic nucleotide elevation; however, activities of both phosphodiesterases remain unchanged. This indicates that the regulation of activity of these enzymes in PC-12 cells is mediated by second messenger effects. The main object of cell regulation is phosphodiesterase with low affinity for the substrate. Its activity is modulated by the calmodulin-Ca2+ complex, cyclic GMP and NAD+ at micromolar concentrations. The effect on the phosphodiesterase system of both a "quick" messenger, Ca2+ and "slow" messengers, cyclic GMP and NAD+, has the same consequences: the turnover number of the enzymic reaction increases that is accompanied by a proportional decrease in the enzyme affinity for cyclic AMP so that the ratio Vmax/Km remains constant. A possible explanation of functional significance of such an activity modulation may be the necessity to maintain the conditions for phosphodiesterase functioning when Km much greater than [cyclic AMP] and the reaction rate are directly proportional to the substrate concentration: v = Vmax/Km [cyclic AMP]. Then the cells are transferred into such a mode when autoregulation of the cyclic nucleotide level takes place. Besides the transient effects causing changes in phosphodiesterase activity, studies of PC-12 cells revealed a chronic effect of phosphodiesterase activity change under the action of staphylococcal enterotoxin A. This protein which induces differentiation of PC-12 cells and possesses a NAD+-glycohydrolase activity is translocated into cytoplasm of cells in the presence of NAD+ and accomplishes ADP-ribosylation of phosphodiesterase. As a result, the enzyme activity falls, cyclic AMP level increases and cell differentiation starts. The activity of soluble phosphodiesterase of PC-12 cells also decreases under the effect of two neurotoxins from bee venom, melittin and tertiapin. Both the toxins at concentration of 10 microM completely block calcium regulation of the enzyme. The mechanism of tertiapin action was investigated on a model system of calmodulin-bovine brain phosphodiesterase. It appeared that inhibition of Ca2+ action is achieved as the result of binding of two toxin molecules with Kd = 2 mM to the activated calmodulin molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies were conducted on the depolymerization of polyadenylic acid (poly (A)) by RNAse A (EC 3.1.4.22) depending on the pH (pH 5-8). The results showed that depending on the pH, the ratio Vmax/Km was analogous to that described earlier for nucleoside-2', 3'-cyclophosphates and dinucleoside phosphates. This indicates that depolymerization of poly (A), transesterification and hydrolysis of specific substrates is achieved by the same ionizing groups of the enzyme with pKa 5.4 and pKb 6.4. The rate of degradation of poly (A) is also influenced by the state of adenine ionization, the protonation of which leads to the formation of a double helical poly (A), and does not serve as a substrate for RNAse A. The low rate for the depolymerization of poly (A) in the presence of RNAse A is related to a decrease in the turnover number of the enzyme, and an increase in the molecular weight of the enzyme (RNAse dimer), leads to a decrease in the Km, and does not effect Vmax. This indicates that the rate of depolymerization of polynucleotides is determined by orientation of factors. On the basis of the comparison of the resultant kinetic data, and the structure of the enzyme inhibitory complexes of RNAse S, which were studied by the method of x-ray structural analysis, a conclusion was reached on the kinetic characteristics of RNAse A specificity with respect to polymeric substrates, which is determined by the orinetation of the ribose phosphate relative to the catalytic groups of the active site.

The plasma membrane of human embryonic fibroblasts was shown to contain receptor binding sites for 1,4-dihydropyridine (DHP) Ca2+ entry blockers. In a subconfluent culture grown in serum medium the content of the DHP receptor amounted to 1.2 +/- 0.3 pmol per 10(6) cells. With progression to confluency this value decreased up to 0.28 +/- 0.08 pmol per 10(6) cells. The DHP binding capacity was shown to be affected by the presence of growth factors in culture medium. In a subconfluent culture of serum-deprived cells the content of DHP binding sites increased 1.9 fold, the steady-state level being achieved within 3 days in culture. The serum gradually reversed this process, and the DHP receptor density approached the initial level within 3 days.

The physico-chemical properties have been studied of RNase A selectively modified at the E-NH2-group of Lys-7 and Lys-41 with pyridoxal-P. Modification did not affect conformational stability of the protein globule, thus all changes in the molecule of the modified RNase A were localised around the alkylated Lys residue. In the both cases pyridoxyl-P. The residue was shown to be localized in the active site region of the (P-Pxy)-Lys-7-RNase A and its chromophore parts was highly exposed to the solvent. (P-Pxy) E-Lys-7-RNase A and its chromophore parts was highly exposed to the solvent. In the Lys-41 derivative, pyridoxamine-P was situated exactly in the active site and is partially hidden in the protein grobule. The pH-dependence of absorption spectra indicates that the chromophore of pyridoxyl-P in modified proteins is quite sensible to the ionic state of its surrounding. The usefulness of pyridoxyl-P as a reporter group was proved in the study with (P-Pxy)-Lys-7-RNase A. Some conformational changes involving His-119 were shown to take place in the course of the enzyme-nucleotide complex formation.

The rate constants and Km for the hydrolysis of the optically active nonglycosidic analogues of the CpA and C greater than p catalysed by RNase A and RNase BS-I were measured. The rate of hydrolysis of the model substrates in 10(5) and 10(3) slower that for the appropriate dinucleoside phosphate and nucleoside cyclophosphate. However, substitution of the relatively rigid ribofuranose ring with flexible alifatic chains is accompanied by little variation in binding constants. The analyses based on the single substrate system indicate that the observed difference in rate constants must be accounted for by a difference between the binding of the substrates in the transition state to the RNase active site. Consequently, the "rigidity" of the ribose rings in RNA leads to large decreases in the free energy of activation for the reactions catalysed by RNases.

Steady state kinetics of DNA depolymerisation in the presence of the DNAase A and Mg2+ ions were investigated at pH 5.5 and wide region of the enzyme, substrate and metal ion concentrations. A model, which is consistent with experimental results obtained is suggested. According to the model catalytically active form of the DNAase A should be a metal-bound enzyme. That species reacts with the metal-free DNA to form the Michaelis complex. The kinetics observed can be described in terms of mechanism which involves covalent enzyme-substrate intermediate formation. It was shown that the second Mg2+ ion binding to the complex Mg2+ DNAase -- DNA (KD - 2.2 . 10(-3) M) enhances the kinetic parameters of the reaction. To rationalise the effect one has to assume that the rate of the intermediate formation was accelerated as a result of the second Mg2+ binding.

The primary structure of calmodulin from human brain has been determined. As compared to calmodulin from bovine brain, it shows three substitutions of the amide----acid type at positions 24, 60, 129, and two acid----amide changes at positions 104 and 135.

We have studied the spectral properties of RNAase A containing a phosphopyridoxyl residue at the epsilon-NH2 group of Lys-7 or Lys-14. The overall conformations of the native and modified enzymes were shown to be rather similar. All three proteins have similar circular dichroism spectra within the 220-300-nm region, and similar thermal transition temperatures. All the changes in the RNAase A molecule modified are located in close proximity to the alkylated lysine residue. The phosphopyridoxyl group of (P-Pxy)-epsilon-Lys-41-RNAase A is situated directly at the enzyme active site and is 25% butied in the protein globule. The P-pyridoxyl group of (P-Pxy)-epsilon-Lys-7-RNAase A was shown to be located in the vicinity of the active site and to be more exposed to the solvent. In the pyridoxyl phosphate absorption band, optical activity is induced in both proteins. Study of the pH dependence of the changes occurring in the circular dichroism and absorption spectra has shown that in the modified proteins, the pyridoxyl phosphate chromophore is rather sensitive to the ionic state of the surrounding medium and serves as a "reporter" group when the relationship between structure and function of the RNAase A active site is being investigated.

Isotermic unfolding of ribonuclease A, phosphopyridoxyl-8Lys41-RNAase A and complexes of the enzyme with cytidine, 2'-CMP, 3'-CMP, 3'-AMP and with the phosphoric ester of 1-(omega-oxypropyl)-cytosine in presence of urea has been studied. The stabilization of the protein structure resulting from the complex formation was shown to be determined by the ligand nucleobase binding. The comparison of the results obtained with those known from the literature suggests, that binding and catalytic zones of the enzyme active site form an integrated network system which is substained by multipoint contacts between the constituents. The change in the state of any part within the enzyme active state affects the energetics of the whole protein globule.

By using the radioactively labeled 1,4-dihydropyridine (DHP) probe, [3H]PMD, we have demonstrated that cultured human embryonic fibroblasts grown at a low density in Eagle's medium supplemented with serum contain a single class of non-interacting DHP binding sites (Bmax, 1.2 +/- 0.3 pmol/10(6) cells; Kd, 3.9 nM). After inhibition of the DHP receptor biosynthesis by cycloheximide, the number of [3H]PMD binding sites is reduced with a half-time of 12 h, which implies a turnover rate of 30,000 +/- 7500 receptors/h per cell. With progression to confluency, the Bmax value decreased up to 0.28 +/- 0.08 pmol/10(6) cells without significant change in Kd value. When cells were grown at a low density in serum-free conditions, the number of [3H]PMD binding sites gradually increased 1.9-fold within 3 days. Addition of serum reversed this effect with the same time course. These results imply that the DHP-sensitive Ca2+ channels are involved in the control of the proliferation of human embryonic fibroblasts.

The circular-dichroism and proton-magnetic-resonance spectra of complexes of ribonuclease A with dihydrouridine 3'-phosphate, 2'- and 3'-CMP, arabinosyl-3'-CMP, 1-(2-hydroxyethyl)cytosine 2'-phosphate and 1-(3-hydroxypropyl)cytosine 3'-phosphate were studied. Comparison of the results shows that non-additivity of the circular-dichroic spectrum of an enzyme-nucleotide complex may be due to: (a), alteration of the circular dichroic spectrum of the nucleotide under the influence of the asymmetric protein matrix (induced dichroism), and (b) a change in the nucleotide conformation. The contribution of each of the two factors was estimated to calculate the circular-dichoroic spectra of 2'-CMP and 3'-CMP in complex with ribonuclease A. 3'-CMP in this complex was characterized by negative circular dichroism in the long-wavelength absorption band of the nucleotide, whereas 2'-CMP was characterized by positive circular dichroism. Since both nucleotides in the complex are known to be in an anti conformation, it follows that even small changes in the conformation considerably modify the circular-dichroic spectrum of the nucleotide in complex with the enzyme.

Five Hybridoma clones producing monoclonal antibodies (MAT) to bovine nerve growth factor (NGF) were developed. The biological effects of antibodies were studied: the influence of MAT on neurit outgrowth induced by NGF in rat pheochromocytoma PC12 or spinal chicken ganglia was investigated. MAT fell into two groups. Two of them inhibited neurit induction by NGF, three others stimulated this process. The stimulation of the neurit outgrowth by MAT was observed at low concentration of NGF (3 ng/ml of culture medium). Mechanisms of antibodies effects are discussed.