Studies on interaction of phosphorylase kinase from rabbit skeletal muscle with glycogen in the presence of ATP and ADP.

The influence of ATP on complex formation of phosphorylase kinase (PhK) with glycogen in the presence of Ca(2+) and Mg(2+) has been studied. The initial rate of complex formation decreases with increasing ATP concentration, the dependence of the initial rate on the concentration of ATP having a cooperative character. Formation of the complex of PhK with glycogen in the presence of ATP occurs after a lag period, which increases with increasing ATP concentration. The dependence of the initial rate of complex formation (v) on the concentration of non-hydrolyzed ATP analogue, beta,gamma-methylene-ATP, follows the hyperbolic law. A correlation between PhK-glycogen complex formation and (32)P incorporation catalyzed by PhK itself and by the catalytic subunit of cAMP-dependent protein kinase has been shown. For ADP (the product and allosteric effector of the PhK reaction) the dependence of v on ADP concentration has a complicated form, probably due to the sequential binding of ADP at two allosteric sites on the beta subunit and the active site on the gamma subunit.

Phosphorylation of the alpha-subunit of Na,K-ATPase from duck salt glands by cAMP-dependent protein kinase inhibits the enzyme activity.

Although it was shown earlier that phosphorylation of Na,K-ATPase by cAMP-dependent protein kinase (PKA) occurs in intact cells, the purified enzyme in vitro is phosphorylated by PKA only after treatment by detergent. This is accompanied by an unfortunate side effect of the detergent that results in complete loss of Na,K-ATPase activity. To reveal the effect of Na,K-ATPase phosphorylation by PKA on the enzyme activity in vitro, the effects of different detergents and ligands on the stoichiometry of the phosphorylation and activity of Na,K-ATPase from duck salt glands (alpha1beta1-isoenzyme) were comparatively studied. Chaps was shown to cause the least inhibition of the enzyme. In the presence of 0.4% Chaps at 1 : 10 protein/detergent ratio in medium containing 100 mM KCl and 0.3 mM ATP, PKA phosphorylates serine residue(s) of the Na,K-ATPase with stoichiometry 0.6 mol Pi/mol of alpha-subunit. Phosphorylation of Na,K-ATPase by PKA in the presence of the detergent inhibits the Na,K-ATPase. A correlation was found between the inclusion of P(i) into the alpha-subunit and the loss of activity of the Na,K-ATPase.

Phosphorylation of lactate dehydrogenase by protein kinases.

The influence of phosphorylation on the properties of lactate dehydrogenase (LDH) has been studied. Data obtained using the immobilization approach support the assumption that the autophosphorylation of LDH discovered previously in the presence of ATP has no relation to protein kinase activity of the enzyme. Phosphorylation of native LDH by tyrosine kinases was shown to be inefficient. However, the efficiency of the phosphorylation considerably increased after the dissociation of LDH into non-native forms of the enzyme. Ca2+/calmodulin-dependent protein kinase catalyzes incorporation of 0.8-0.9 mole phosphate per mole of LDH tetramer. The phosphorylation results in an increase in activity by 25-30% and increases markedly the stability of the enzyme during cold inactivation. Phosphorylation of LDH by Ca2+/calmodulin-dependent protein kinase, unlike the phosphorylation on tyrosine residues, is supposed to be of importance for the control of cell metabolism.

Antitumor activity of alpha fetoprotein and epidermal growth factor conjugates in vitro and in vivo.

Conjugates of carminomycin (Cm) with alpha-fetoprotein (AFP) and epidermal growth factor (EGF) were prepared and their cytotoxic activities were studied in vitro. Both conjugates showed cytotoxic activity which exceeded that of free Cm in tumor cell cultures of MCF-7, SKOV3, QOS, P388 and B16 cells. The antitumor effects of the conjugates were studied in vivo in mice with subcutaneous tumors of B16 and P388 cells. The Cm-AFP and Cm-EGF conjugates inhibited tumor growth and noticeably increased the mean life span in experimental animals. Our results suggest that the therapeutic activity of Cm can be significantly enhanced by conjugation to AFP or EGF.

Recognition of HIV-derived B and T cell epitopes displayed on filamentous phages.

The amino acid sequence of HIV reverse transcriptase (RT) from residue 248 to residue 262 was expressed on the surface of filamentous phage fd, fused to the major coat protein gVIIIp. The chimeric phage was used to assess the ability of anti-RT (248-262) human T cell lines and clones to become activated by the phage-displayed peptide. The RT peptide displayed on phage was recognized by the T-cells and induced production of Abs. However, not all T cells raised against the synthetic RT (248-262) peptide could respond. Lack of recognition did not depend on differences in the ability of different APCs to present the phage, but was apparently determined by the TCR specificity. The results presented here may be relevant to the design of recombinant protein-based subunit vaccines.

Phosphorylation of H,K-ATPase alpha-subunit in microsomes from rabbit gastric mucosa by cAMP-dependent protein kinase.

A 100-kDa protein that is a main component of the microsomal fraction from rabbit gastric mucosa is phosphorylated by cAMP-dependent protein kinase (PKA) in the presence of 0.2% Triton X-100. Microsomes from rabbit gastric mucosa possess activity of H,K-ATPase but not activity of Na,K-ATPase. Incubation of microsomes with 5 microM fluorescein 5'-isothiocyanate (FITC) results in both an inhibition of H,K-ATPase and labeling of a protein with an electrophoretic mobility corresponding to the mobility of the protein phosphorylated by PKA. The data suggest that the alpha-subunit of H,K-ATPase can be a potential target for PKA phosphorylation.

A nuclear receptor corepressor modulates transcriptional activity of antagonist-occupied steroid hormone receptor.

Synthetic steroid hormone antagonists are clinically important compounds that regulate physiological responses to steroid hormones. The antagonists bind to the hormone receptors, which are ligand-inducible transcription factors, and modulate their gene-regulatory activities. In most instances, a steroid receptor, such as progesterone receptor (PR) or estrogen receptor (ER), is transcriptionally inactive when complexed with an antagonist and competitively inhibits transactivation of a target steroid-responsive gene by the cognate hormone-occupied receptor. In certain cellular and promoter contexts, however, antagonist-occupied PR or ER acquires paradoxical agonist-like activity. The cellular mechanisms that determine the switch from the negative to the positive mode of transcriptional regulation by an antagonist-bound steroid receptor are unknown. We now provide strong evidence supporting the existence of a cellular inhibitory cofactor that interacts with the B form of human PR (PR-B) complexed with the antiprogestin RU486 to maintain it in a transcriptionally inactive state. In the presence of unliganded thyroid hormone receptor (TR) or ER complexed with the antiestrogen 4-hydroxytamoxifen, which presumably sequesters a limiting pool of the inhibitory cofactor, RU486-PR-B functions as a transcriptional activator of a progesterone-responsive gene even in the absence of hormone agonist. In contrast, hormone-occupied TR or ER fails to induce transactivation by RU486-PR-B. Recent studies revealed that a transcriptional corepressor, NCoR (nuclear receptor corepressor), interacts with unliganded TR but not with liganded TR. Interestingly, coexpression of NCoR efficiently suppresses the partial agonistic activity of antagonist-occupied PR-B but fails to affect transactivation by agonist-bound PR-B. We further demonstrate that RU486-PR-B interacts physically with NCoR in vitro. These novel observations suggest that the inhibitory cofactor that associates with RU486-PR-B and represses its transcriptional activity is either identical or structurally related to the corepressor NCoR. We propose that cellular mechanisms that determine the switch from the antagonistic to the agonistic activity of RU486-PR-B involve removal of the corepressor from the antagonist-bound receptor so that it can effect partial but significant gene activation.

Role of capsid structure and membrane protein processing in determining the size and copy number of peptides displayed on the major coat protein of filamentous bacteriophage.

Filamentous bacteriophage virions can be engineered to display small foreign peptides in the N-terminal regions of all 2700 copies of the major coat protein (pVIII), but larger peptides can be accommodated only in hybrid virions, in which modified and wild-type coat protein subunits are interspersed. The copy number of peptides accepted in hybrid virions is generally believed to be related to peptide size: the larger the insert, the lower the number of modified coat protein subunits in the assembled virion. However, we show here that some large peptides can be displayed at a much higher copy number than smaller ones and that some relatively small peptides are poorly displayed, if at all, in hybrid virions. X-ray diffraction studies of a recombinant virion together with model building experiments with peptide and protein epitopes of known structure demonstrated that it is feasible to accommodate much larger structures, without perturbation of the capsid protein packing, than it has proved possible to generate in vivo. We show further that the insertion of certain peptides greatly slowed or even prevented the processing of the pVIII pro-coat by leader peptidase at the inner membrane of the Escherichia coli cell. A good correlation was found between the effect of the insert on the rate of the processing of the pro-coat, an essential step in virus assembly, and the number of the mature but modified proteins in the subsequently assembled hybrid virion. These results have important implications for the design of peptide display systems based on filamentous bacteriophage.

Phosphorylation of the Na,K-ATPase by Ca,phospholipid-dependent and cAMP-dependent protein kinases. Mapping of the region phosphorylated by Ca,phospholipid-dependent protein kinase.

Ca,phospholipid-dependent (PKC) and cAMP-dependent (PKA) protein kinases phosphorylate the alpha-subunit of the Na,K-ATPase from duck salt gland with the incorporation of 0.3 and 0.5 mol 32P/mol of alpha-subunit, respectively. PKA (in contrast to PKC) phosphorylates the alpha-subunit only in the presence of detergents. Limited tryptic digestion of the Na,K-ATPase phosphorylated by PKC demonstrates that 32P is incorporated into the N-terminal 41-kDa fragment of the alpha-subunit. Selective chymotrypsin cleavage of phosphorylated enzyme yields a 35-kDa radioactive fragment derived from the central region of the alpha-subunit molecule. These findings suggest that PKC phosphorylates the alpha-subunit of the Na,K-ATPase within the region restricted by C3 and T1 cleavage sites.

[Tyrosine protein kinase from cattle cerebral cortex: purification, characteristics, protein substrates for phosphorylation and inhibitors of activity]. / Tirozinovaia proteinkinaza iz kory mozga byka: ochistka, kharakteristika svoistv, belkovye substraty fosforilirovaniia i ingibitory aktivnosti.

Tyrosine protein kinase present in the membrane fraction of bovine cerebral cortex were extracted and chromatographically fractionated. The activity associated with tyrosine protein kinases was fully extracted from the membranes by 1% sodium cholate and eluted in two peaks (I and II) during chromatography of protein extracts on DEAE-Toyopearl in the presence of sodium cholate. The predominant in cerebral cortex membrane tyrosine protein kinase of peak I (about 75% of the total activity) was purified 1930-fold by gel filtration on Sephacryl S-300, chromatography on hexyl- and phenyl-Sepharose and by rechromatography on DEAE-Toyopearl. The amount of the enzyme prepared from 250 g of bovine brain was 20 micrograms, the enzyme yield and specific activity being 3.8% and 3.9 nmol/mg protein/min, respectively. The purified protein kinase of peak I represents a protein with Mr of 62-63,000 (p62) capable of being autophosphorylated in the presence of [gamma-32P]. Protein kinase p62 phosphorylates enolase, tubulin and calpactin I as well as model substrates in the series: histone H5 greater than poly(G, T)n greater than or equal to histone H2A greater than poly(G, A, T)n, histone H4 greater than caseins, histones H1 and H2B, poly(G, A, L, T)n. The enzyme is specific for Mn2+ at the optimal concentration about 1 mM. The KmMn-ATP is 0.3 microM; Km for histone H5 and poly(G, T)n are 0.45 mg/ml and 0.06 mg/ml, respectively. The protein kinase p62 activity is inhibited by NaCl (IC50 approximately 75-100 mM) as well as by quercetin, adriamycin and lasalocid (IC50 approximately 14-34, 23 and 90 microM, respectively). It is concluded that protein kinase p62 is analogous to the c-src gene protein kinase.

[Properties of tyrosine-specific protein kinase from rat sarcoma cells]. / Izuchenie svoistv tirozinspetsificheskoi proteinkinazy iz kletok sarkomy krysy.

A procedure for measuring the activity of tyrosine-specific protein kinases was developed. The method is based on the fact that the phosphoryl groups of phosphotyrosine residues of phosphorylated protein substrates are not hydrolyzed in alkaline solutions, whereas the phosphoserine and phosphothreonine residues lose their phosphoryl groups upon heating in 2 N KOH. It was demonstrated that rat sarcoma XC cells in culture and in solid tumours contain tyrosine-specific protein kinase (TPK). The TPK is localized in the membrane fraction of the cells and is solubilized by 1% Triton X-100. Mn2+-ATP is a nucleotide substrate for TPK. Among other protein substrates, TPK strongly phosphorylates histones H5 and H2a, weakly phosphorylates histones H2b, H4 and H1 but does not phosphorylate protamine, casein, vinculin or angiotensin II. The optimal concentration of Mn2+ for the reaction is 2 mM; the Km values for ATP and histone H5 are 2-3 microM and 2.5 mg/ml, respectively. Tyrosine-specific protein kinases phosphorylating histone H5 were detected in the membrane fraction isolated from different mammalian tissues, e. g., spleen, heart, liver, brain and lungs, as well as from human intestinal mucosa and mucosal adenocarcinoma. These results suggest that tyrosine-specific protein kinases phosphorylating histone H5 are present in a vast majority of mammalian tissues.

[Effect of linear, impact and vibration accelerations on the accuracy of performance of force load programs by an operator]. / Vliianie lineinykh, udarnykh i vibratsionnykh uskorenii na tochnost' realizatsii operatorom programm silovykh nagruzok.

The factors that influence the accuracy with which motor commands are actualized were investigated. The study concentrated on linear acceleration of different sign (produced by rocking a heavy metal platform), vibration at a frequency of about 23 Hz, and impact acceleration resulting from the platform or the rotating chair being suddenly stopped. The force applied to hand sticks or foot levers was recorded by means of electromechanical sensors. It was demonstrated that smoothly varying and in particular impact acceleration caused normal men to subjectively underestimate their own muscle efforts. As a rule, this led to a greater than prescribed strength applied. It is very important to note that acceleration acting from right to left or from left to right (in the frontal plane) produced nonsymmetric pressure by the right and left limbs.

[Cyclic groups of nonlinear transformations in morphology and the conception of cyclogenesis]. / Tsiklicheskie gruppy nelineinykh preobrazovanii v morfologii i kontseptsiia tsiklogeneza.

When describing multiunit organic forms, it is expedient to generalize the classic science on biological symmetries of similarity at the expense of transition to symmetry operations from wider groups of transformations, first of all mebiusian and projective. A vast class of biological structures is successfully modelled on the base of cyclic groups not only the linear, but also non-linear transformations. This is evidently connected with existence of morphogenetically significant biochemical cycles. The suggested means of mathematical modelling for biological symmetries open new possibilities of morphometrical analysis and investigation of a known problem of morphogenetic parallelisms. The results obtained prove V. V. Vernadsky's hypothesis on biological significance of the non-Euclidian geometry.

[Changes in protein kinase activity of the skin in cancer]. / Izmenenie aktivnosti proteinkinaz v kozhe pri rake.

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.

[cAMP-dependent protein kinase from pigeon breast muscle. Isolation of regulatory subunits by affinity chromatography and study of the topography of the cAMP binding site using cAMP analogs]. / cAMP-zavisimaia proteinkinaza iz grudnoi myshtsy golubia. Vydelenie reguliatornoi sub"edinitsy metodom affinnoi khromatografii i izuchenie topografii cAMP-sviazyvaiushchego tsentra s pomoshch'iu analogov cAMP.

The cAMP-dependent protein kinase from the soluble fraction of pigeon breast muscle is represented by two forms, PK I and PK II. The ratio of the phosphotransferase activity of the two forms is 35-40% and 60-65% for PK I and PK II, respectively. The regulatory subunit of PK I was isolated in a homogeneous state by affinity chromatography on 8-(2-oxoethylthio)-cAMP immobilized on epoxy-activated Sepharose 4B. The molecular weight of the regulatory subunit of PK I as determined by SDS polyacrylamide gel electrophoresis is 45 000. The specific cAMP-binding activity is equal to 16 nmol of [3H]cAMP per mg of protein. The apparent dissociation constant (Kd') for cAMP equals to 380 nM. The preparation of the regulatory subunit of PK II obtained by affinity chromatography on the same adsorbent is made up of polypeptides with Mr 56 000, 39 000, 29 000, 17 000 and 11 000. The preparation possesses a cAMP-binding activity of 22 nmol of [3H]cAMP per mg of protein. The interaction of several analogs of cAMP containing substituents at different positions of the nucleotide molecule with the regulatory subunit of PK I was studied. Practically all the analogs with substituents at positions 8 and 6 of the adenine ring in the cAMP molecule had the affinity which was 2-9 times less than that of cAMP. The only exceptions were 8-carboxymethylamino- and 8-(2-oxyethyl)-amino-cAMP whose binding to the regulatory subunit was 100 and 53 times lower than that of cAMP. The substitutions in position N-1 of the cAMP molecule leads to a 30-50-fold decrease of the analogs affinity. beta-Bromoethyl ester of cAMP does not reveal the ability to bind to the regulatory subunit. The carboxymethyl ester of cAMP possesses the affinity for the cAMP-binding site that is 35 times less than that of cAMP. Modification of the 2'-hydroxyl of ribose (as in the case of 2'-amino-2'-deoxy-8-hydroxy-cAMP, 2'-deoxy-cAMP and 2'-O-acrylyl-cAMP) decreases the affinity of these compounds 125-, 313- and 126-fold as compared with cAMP. It was assumed that the cAMP molecule is bound to the regulatory subunit in the syn-conformation. A structural model of the cAMP-binding site in the regulatory subunit of cAMP-dependent protein kinase I from pigeon breast muscle is proposed.

[Kinetic mechanism of phosphotransferase reactions catalyzed by cAMP-dependent protein kinase type I and type II from rabbit skeletal muscle]. / Izuchenie kineticheskogo mekhanizma fosfotransferaznoi reaktsii, kataliziruemoi cAMP-zavisimymi proteinkinazami tipa I i tipa II iz skeletnykh myshts krolika.

The catalytic subunits of cAMP-dependent protein kinases I and II were isolated from rabbit skeletal muscles in a homogeneous state. The specific phosphotransferase activities of homogeneous preparations of catalytic subunits were 8 mumol/mg X min (type I) and 6 mumol/mg X min (type II). In order to elucidate the mechanisms of the phosphotransferase reaction, the steady-state kinetics method and an inhibitory analysis involving the phosphotransferase reaction products, ADP and phosphohistone H1, were used. It was shown that phosphorylation of histone H1 catalyzed both by protein kinases I and II occurs via a random "bi-bi" mechanism. The values of constants for kinetic equation of the phosphotransferase reaction coincide with those for the catalytic subunits of both protein kinase types and are equal to 11 microM (KmATP), 60 microM (KmH1), 5.0 microM (KSATP) and 27 microM (KSH1). The value of the competitive inhibition constant for Mg-ADP (KiADP) is also identical for the catalytic subunits of types I and II and is equal to 30 microM. In both cases, the phosphorylated histone H1 inhibits the phosphotransferase reaction; this inhibition is partly competitive with respect to histone H1.

[Isolation and study of the properties of the regulator subunit of cAMP-dependent protein kinase]. / Vydelenie i izuchenie svoistv reguliatornoi sub"edinitsy tsAMF-zavisimoi proteinkinazy.

The regulatory subunit of type II cAMP-dependent proteinkinase was isolated from cytosol of the rabbit small intestinal mucosa by affinity chromatography. The preparation contained 3 proteolytic enzymes and occurred in two forms differing as regards cAMP affinity. The cAMP-binding capacity of the preparation was equal to 17 nmol cAMP/mg protein. To study the topography of the cAMP-binding center, use was made of cAMP analogs. It was demonstrated that introduction of the substituents into the 8th position of the purine ring and substitution with respect to the N6-exoaminogroup affected insignificantly the analog affinity for the cAMP-binding center. At the same time the substituents introduced into the first position of the adenine base, into the area of the 2'-hydroxyl group of ribose and into the cyclophosphate part of the cAMP molecule considerably decreased the analog affinity for the regulatory center of type II cAMP-dependent proteinkinase.

[Isolation and study of the properties of the catalytic subunit of cAMP-dependent protein kinase of the small intestinal mucosa of the rabbit]. / Vydelenie i izuchenie svoistv kataliticheskoi sub"edinitsy tsAMF-zavisinoi proteinkinazy slizistoi obolochki tonkoi kishki krolika.

cAMP-dependent and casein proteinkinase were found in cytosol of the rabbit small intestine mucosa. cAMP-dependent proteinkinase of cytosol is represented by two forms of types I and II. The activity of enzymes of types I and II constitutes 10 and 90%, respectively. Casein proteinkinase is represented by a single form. The catalytic subunit of cAMP-dependent proteinkinase of type II was isolated in a homogenous state. The catalytic subunit phosphorylates histones H1, H2a, H2b and protamine and to a far less degree histones H3, H4 and casein (H2b greater than H1 greater than H2a greater than protamine much greater than H3 greater than casein). The Km value for histone H1 is equal to 65 mkM, and that for Mg-ATP 12 mkM. Chloromethylpyrophosphonate and adenosine p-fluorosulfobenzoate were studied as affine modifiers of the active center of the catalytic subunit from the small intestine mucosa. It was shown that only adenosine p-fluorosulfonate is an irreversible inhibitor of the catalytic subunit.

[Surgical tactics in hourglass tumors with intravertebral-mediastinal locations]. / Khirurgicheskaia taktika pri opukholiakh v forme pesochnykh chasov intravertebral'no-mediastinal'noi lokalizatsii.

The most expedient surgical tactics in hour-glass tumors of intravertebral-mediastinal localization are discussed. In 10 of 27 cases total removal of the tumor through a vertebral approach proved possible. Total removal of a rather large intrathoracic node by Guleke's method was conducted in 2 cases. In 10 cases with large intrathoracic nodes, total removal of the tumor was carried out by means of a combined intravertebral-thoracic (transpleural) approach suggested by the authors. It is emphasized that multi-stage operations and partial resection of the tumor are inexpedient and that attempts to remove it totally through a thoracic approach only are inadmissible.