Effects of polyamines and polyanions on a cyclic nucleotide-independent and a cyclic AMP-dependent

The phosphorylation of phosvitin in vitro by a cyclic nucleotide-independent protein kinase (phosvitin kinase) derived from rooster liver is markedly stimulated by the divalent cation, Mg²⁺. In addition, the activity is further stimulated by low concentrations of the polyamines putrescine, spermidine and spermine leading to higher rates of phosphate incorporation than could be obtained at any concentration of Mg²⁺. Spermine is inhibitory at higher concentrations. The polyamines shift the Mg²⁺ requirement for maximal activity to lower concentrations. The activity of a cyclic AMP-dependent histone kinase from beef heart is not altered by the presence of polyamines. Heparin is a potent inhibitor of phosvitin kinase but has no effect on histone kinase. Polyribonucleotides (polyadenylic acid and transfer RNA) inhibit both types of kinases, but the degree of inhibition of phosvitin kinase is variable and depends upon the type of the polyanion present. Spermidine and spermine, but not Mg²⁺, efficiently counteract the inhibitory action of heparin and tRNA. The results suggest that, also in vivo, naturally occurring polyamines and polyanions such ass tRNA may have a regulatory function on protein kinases.     

Calmodulin stimulates polyamine-responsive protein kinase in the absence of Ca2+

A cyclic nucleotide-independent, polyamine-responsive protein kinase from the cytosol of Morris hepatoma 3924A, which phosphorylated heat-stable endogenous substrates and casein in the presence of polyamines (Criss, W.E., Yamamoto, M., Takai, Y., Nishizuka, Y. and Morris, H.P. (1978) Cancer Res. 38, 3540–3545) was observed to be stimulated by an endogenous protein activator. This protein activator was identified to be calmodulin. the polyamine-responsive protein kinase was also stimulated by purified calmodulin, but only in the presence of polyamines such as polylysine. This action of cadmodulin did not require Ca²⁺ for activation of the enzyme; and activation occured in the presence of EGTA. DNA and RNA inhibited the polyamine-responsive protein kinase, either in the presence or absence of Ca²⁺. Purified calmodulin, in the presence of cyclic AMP or cyclic GMP, did not activate the protein kinase. Therefore, polyamines such as polylysine are an absolute requirement for this expression of calmodulin action. The increased enzyme activity by calmodulin was accompanied with an increased V_max and with no changes in the F_m (ATP). High levels of cation, up to 100 mM Mg²⁺, did not effect the action of cadmodulin. These results indicate that tumor cytosolic polyamine-responsive protein kinase is regulated by calmodulin, the latter being increased in the tumor tissue.     

Effects of polyamines on prostatic chromatin- and non-histone-protein-associated protein kinase reactions.

Studies are presented on the influence of polyamines on prostatic chromatin- and non-histone-protein-associated protein kinase reactions involving both exogenous and endogenous substrates. The activities toward the model acidic protein substrate, dephosphophosvitin, were maximal at 160--200mM-NaCl (or -KCl or -NH4Cl). Under these conditions, spermidine and spermine added in concentrations up to 2mM were essentially without effect. However, without addition of NaCl to the medium, marked stimulation of these reactions was elicited by these polyamines at 1--2mM concentrations. The stimulatory effects were not due to non-specific changes in the ionic strength or to substitution of spermine for Mg2+, as maximal stimulation by 1 mM-spermine was observed only at optimal (2--4mM) Mg2+ concentrations. Qualitatively similar effects of polyamines were observed with enzyme preparations from the prostates of castrated rats, and with chromatin and non-histone-protein preparations from other tissues besides ventral prostate. When phosphorylation of endogenous non-histone proteins of the chromatin was measured, spermine stimulated both the initial rates and the final extent of transphosphorylation, even in the presence of optimal concentration of NaCl. By contrast, spermine or spermidine had no effect on the chromatin- and non-histone-protein-associated protein kinase reactions determined with lysine-rich histones as substrates. Chemically NN-dimethylated dephosphophosvitin was a less active substrate for the chromatin-associated protein kinase, but its phosphorylation was more markedly stimulated by spermine in comparison with unmodified dephosphophosvitin. These observations hint that the polyamine stimulations of the various protein kinase reactions may be due to effects on the conformations of the non-histone protein substrates rather than on the kinases themselves.     

Monomolecular condensation of lambda-DNA induced by cobalt hexamine

Measurements of static and dynamic light scattering have been used to distinguish between monomolecular DNA condensation and aggregation of condensed molecules. In low salt, using Co³⁺(NH₃)₆ as the condensing agent, and at λ-DNA concentrations below 0.2 μg/mL, the transition curves for monomolecular condensation and aggregation are well separated for times of 16 h. In these conditions, the intensity of scattered light (90°) and also the diffusion coefficient of the condensed DNA show reasonable values for monomolecular condensation that are independent of DNA concentration and also of Na⁺ Co³⁺(NH₃)₆ concentrations for which monomolecular condensation is complete. At higher Co³⁺(NH₃)₆ concentrations, which produce aggregation (as judged by the intensity of scattered light), the diffusion coefficient decreases sharply. The transition curve for monomolecular condensation is independent of DNA concentration but shows a hysteresis loop. The kinetics of condensation are slow in the forward direction and fast in the reverse direction, indicating that the actual transition curve is measured closely by reversal experiments. Aggregation is blocked kinetically in both the forward and reverse directions when Co³⁺(NH₃)₆ is the condensing agent at low Na⁺ concentrations. When spermine or spermidine is the condensing agent and observations are made at 16 h, it is not possible to separate the transition curves for monomolecular condensation and for aggregation in conditions that are successful with Co³⁺(NH₃)₆. Some interesting properties of monomolecular condensation are noted. (1) The transition is not a two-state reaction, as judged by measurements of the diffusion coefficient through the transition zone. (2) The transition for monomolecular condensation is diffuse. (3) The dimensions of the monomolecular condensates have been calculated from the translational diffusion coefficient for an assumed toroidal shape by the formula derived by Allison and coworkers [(1981) Biopolymers 20 , 469–488]. These dimensions are in reasonable agreement with ones deduced from electron microscopy by Chattoraj and coworkers [(1978) J. Mol. Biol. 121 , 327–337]. (4) The phase diagram relating the Na⁺ to the Co³⁺(NH₃)₆ concentrations needed for condensation has a slope of 0.6 in a log–log plot. According to numerical solutions of Manning's theory for the atmospheric binding of competing cations to DNA, this means that condensation occurs at a late stage in the replacement of Na⁺ by Co³⁺(NH₃)₆ around the DNA. The fraction of DNA phosphate charge neutralized at condensation is computed to be in the neighborhood of 0.9, as found by Wilson and Bloomfield [(1979) Biochemistry 18 , 2192–2196], but to vary with the Na⁺ concentration.     

Inhibition of cation-induced DNA condensation by intercalating dyes

Several intercalating dyes are shown to inhibit the cation-induced condensation of λ-DNA when Co³⁺(NH₃)₆ is the condensing agent. The dyes that have been studied are ethidium, propidium, proflavin, quinacrine, and actinomycin D. Earlier work has shown that intercalating dyes inhibit ψ-DNA condensation. [Lerman, L. S. (1971) Prog. Mol. Subcell. Biol. 2 , 382–391; Cheng, S. & Mohr, S. C. (1975) Biopolymers 14 , 663–674.] Dye-induced decondensation of intramolecularly condensed DNA has been studied by making use of conditions in which Co³⁺(NH₃)₆ produces intramolecular condensation without significant aggregation. Some aggregation is caused, however, during dye-induced decondensation. Dye titration curves of DNA decondensation have been measured by excess light scattering to monitor decondensation and by fluorescence to monitor intercalation. All of the dyes studied act as competing cations in displacing the condensing cation Co³⁺(NH₃)₆ from the DNA. Competition occurs both in and below the transition zone for condensation. The effectiveness of a dye as a competing cation increases with its net positive charge. Before decondensation begins, no intercalated dye can be detected, suggesting that intercalation might be incompatible with the proper helix packing needed for cation-induced DNA condensation. To test this last point, methidium–spermine was synthesized: it contains an intercalating methidium head group combined with a polyamine tail. Methidium–spermine is found to cause λ-DNA condensation, but aggregation accompanies condensation, as has been found earlier for spermine and spermidine. Fluorescence and absorption spectra indicate that the methidium group is intercalated when the DNA is condensed, indicating that intercalation need not be incompatible with DNA condensation. The presence of aggregates among the condensed DNA molecules makes this last conclusion tentative.     

Partial purification of the protein system controlling the breakdown of trehalose in baker's yeast.

The inactive form of trehalase as well as its activating protein have been partially purified from resting cells of baker's yeast using (NH₄)₂SO₄ fractionation and subsequent DEAE- and CM-cellulose column chromatography. For its activation by cyclic 3′,5′-AMP the system appeared to be dependent on the presence of ATP and a divalent cation such as Mg²⁺, Mn²⁺ or Co²⁺. No sensitivity towards the pH was observed in the range 6.0 – 7.5. The amount of active trehalase formed was determined by the preincubation time and the concentration of the proteins involved. The activating protein partly lost its dependence on cyclic 3′,5′-AMP during purification. The results presented suggest that this protein may be a protein kinase and that activation of trehalase is associated with phosphorylation of the enzyme protein.     

The Ribosomal E Site at Low Mg2+: Coordinate Inactivation of Ribosomal Functions at Mg2+ Concentrations Below 10 mM and its Prevention by Polyamines

Abstract

Under standard conditions (Mg²⁺/150 mM NH₄ ⁺) ribosomes can quantitatively participate in tRNA binding at Mg²⁺ concentrations of 12 to 15 mM. The overall poly(U)-directed Phe incorporation and the extent of tRNA binding to either P,E or A sites decrease in a parallel manner when the Mg²⁺ concentration is lowered below 10 mM. At 4 mM the inactivation amounts to about 80%. The coordinate inactivation of all three binding sites is accompanied by an increasing impairment of the ability to translocate A-site bound AcPhe-tRNA to the P site. The translocation efficiency is already reduced at 10 mM Mg²⁺, and is completely blocked at 6—8 mM.

The severe inactivation seen at 6 mM Mg²⁺ vanishes when the polyamines spermine (0.6 mM) and spermidine (0.4 mM) are present in the assay; tRNA binding again becomes quantitative, the total Phe synthesis even exceeds that observed in the absence of polyamines by a factor of 4. In the presence of polyamines and low Mg²⁺ (3 and 6 mM) two essential features of the allosteric three-site model (Rheinberger and Nierhaus,J.Biol. Chem. 261, 9133 (1986)) are demonstrated. 1) Deacylated tRNA is not released from the P site, but moves to the E site during the course of translocation. 2) Occupation of the E site reduces the A site affinity and vice versa (allosteric interactions between E and A sites).

The quality of an in vitro system for protein synthesis can be assessed by two criteria. First, the incubation conditions must allow a near quantitative tRNA binding. Secondly, protein synthesis should proceed with near in vivo rate and accuracy. The 3 mM Mg²⁺/NH₄ ⁺/polyamine- system seems to be the best compromise at present between these two requirements.     

Effects of macrocyclic polyamines and polymethylenediamines on reactions influenced by polyamines

The effects of macrocyclic polyamines and polymethylenediamines on various reactions influenced by polyamines have been studied. Among the amines tested, 2,3,4,3- and 3,3,3,4-cyclic polyamines, NH₂(CH₂)₆NH₂ and NH₂(CH₂)₈NH₂ had some ability to stimulate polyphenylalanine synthesis, globin synthesis and rat liver isoleucyl-tRNA formation. The degree of stimulation was at most 40% of that obtained by polyamines. In the degradation of poly(C) by bovine pancreatic RNAase A, all tested amines stimulated the degradation. In the NADPH-dependent lipid peroxidation of rat liver microsomes, the degree of inhibition by 2,3,2,3- or 2,3,3,3-cyclic polyamine was greater than that by spermine. The hydrolysis of ATP by an oligomycin-sensitive ATPase was inhibited by 2,3,4,3- and 3,3,3,4-cyclic polyamines, NH₂(CH₂)₁₀NH₂ and spermine at somewhat comparable levels. None of the macrocyclic polyamines or polymethylenediamines stimulated the growth of a polyamine-requiring mutant of Escherichia coli. Possible explanations for the differences in the effects of amines on the various reactions are discussed.     

Characteristics of a nuclear protein kinase from rat epididymis

Purified rat epididymal nuclei possess a cyclic AMP-independent protein kinase activity that phosphorylates of casein. The enzymic activity was solubilized by treating intact nuclei with 1 M (NH4)₂SO₄. One major peak of kinase activity was obtained when the solubilized enzyme preparation was subjected to diethylaminoethyl-Sephadex chromatography. The activity of the kinase was dependent on a bivalent metal ion such as Mg²⁺, Co²⁺, Ca²⁺ or Mn²⁺. NaCl (0.3 M) caused a further activation (approx. 200%) of the metal (Co²⁺)-dependent enzyme. The apparentK _m values of the enzyme for casein, ATP and Co²⁺ are approx. 0.6 mg/ml, 10 ΜM and 2.2 mM respectively. The enzyme was maximally active at pH 5.5. The enzyme showed high specificity for phosphorylation of the acidic protein casein but did not phosphorylate basic proteins, such as histones and protamine. The properties of the nuclear protein kinase were clearly different from those of the cytosolic enzymes previously characterized.     

Effects of cyclic adenosine 3': 5'-monophosphate on phosphoprotein kinase and phosphatase fractions prepared from rat liver nuclei.

A soluble rat liver nuclear extract containing total RNA polymerase activities also exhibits appreciable amounts of protein kinase activity. This unfractionated protein kinase catalyzes the phosphorylation of both endogenous proteins and exogenous lysine-rich histone in the presence of [γ-³²P]ATP and Mg²⁺. The optimal concentration of Mg²⁺ is 5 mm for histone phosphorylation and 25 mm for the phosphorylation of endogenous proteins. Cyclic AMP has no effect on the phosphorylation of lysine-rich histone by this unfractionated nuclear protein kinase. However, addition of cyclic AMP causes a reduction in the ³²P-labeling of an endogenous protein (CAI) which can be characterized by its mobility during SDS-acrylamide gel electrophoresis and elution in the unbound fraction of a DEAESephadex column. If CAI is first labeled with ³²P and then incubated with 10^?6m cyclic AMP under conditions where protein kinase activity is inhibited, the presence of the cyclic nucleotide causes a loss of the ³²P-labeling of this protein, implying the activation of a substrate-specific protein phosphatase. When rat liver RNA polymerases are purified by DEAE-Sephadex chromatography, protein kinase activity is found in the unbound fraction and in those column fractions containing RNA polymerase I and II. The fractionated protein kinases exhibit different responses to cyclic AMP, the unbound protein kinase being stimulated and the RNA polymerase-associated protein kinases being dramatically inhibited. A second protein (CAII) whose phosphorylated state is modified by cyclic AMP is found within the DEAE-Sephadex column fractions containing RNA polymerase II. The cyclic nucleotide in this case appears to reduce labeling of CAII by inhibition of the protein kinase activity which co-chromatographs with both CAII and RNA polymerase II. Based on molecular weight estimates, neither CAI nor CAII appears to be an RNA polymerase subunit. The identity of CAI as a protein factor whose phosphorylated state influences nuclear RNA synthesis is suggested by the fact that addition of fractions containing CAI to purified RNA polymerase II inhibits the activity of this enzyme, but only if CAI has been previously incubated in the presence of cyclic AMP.     

Structural basis of polyamine-DNA recognition: spermidine and spermine interactions with genomic B-DNAs of different GC content probed by Raman spectroscopy

Four genomic DNAs of differing GC content (Micrococcus luteus, 72% GC; Escherichia coli, 50% GC; calf thymus, 42% GC; Clostridium perfringens, 27% GC) have been employed as targets of interaction by the cationic polyamines spermidine {[H₃N(CH₂)₃NH₂(CH₂)₄NH₃]³⁺} and spermine {[(CH₂)₄(NH₂(CH₂)₃NH₃)₂]⁴⁺}. In solutions containing 60 mM DNA phosphate (~20 mg DNA/ml) and either 1, 5 or 60 mM polyamine, only Raman bands associated with the phosphates exhibit large spectral changes, demonstrating that B-DNA phosphates are the primary targets of interaction. Phosphate perturbations, which are independent of base composition, are consistent with a model of non-specific cation binding in which delocalized polyamines diffuse along DNA while confined by the strong electrostatic potential gradient perpendicular to the helix axis. This finding provides experimental support for models in which polyamine-induced DNA condensation is driven by non-specific electrostatic binding. The Raman spectra also demonstrate that major groove sites (guanine N7 and thymine C5H₃) are less affected than phosphates by polyamine–DNA interactions. Modest dependence of polyamine binding on genome base composition suggests that sequence context plays only a secondary role in recognition. Importantly, the results demonstrate that polyamine binding has a negligible effect on the native B-form secondary structure. The capability of spermidine or spermine to bind and condense genomic B-DNA without disrupting the native structure must be taken into account when considering DNA organization within bacterial nucleoids or cell nuclei.     

Lysine-rich histone H1 kinase from soybean hypocotyl.

A protein kinase (ATP: histone phosphotransferase) with high specificity for the phosphorylation of the very lysine-rich histone H1 has been partially purified and characterized from soybean hypocotyl. The enzyme has a molecular weight of about 48,500. Its activity and sedimentation behavior are refractory to cyclic nucleoside monophosphates. No significant amount of cyclic AMP or cyclic GMP binding activity could be detected in the crude or partially purified enzyme preparations. Km for ATP and histone H1 are 0.4 μM and 0.7 μM, respectively. The enzyme requires Mg²⁺ or Mn²⁺ for activity, while addition of 0.5 mM Ca²⁺, Zn²⁺ or Hg²⁺ results in 50% inhibition. Arginine-rich histones H3 and H4 are inhibitory to histone H1 phosphorylation; these histones affect the Vmax of the enzyme, but not the Km for histone H1.     

High tyrosine-specific protein kinase activity in normal human peripheral blood lymphocytes

Tyrosine-specific protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) activity was measured in normal human nonadherent peripheral blood lymphocytes using synthetic peptide substrates having sequence homologies with either pp60^src or c-myc. A high level of tyrosine-specific protein kinase activity was found associated with the cell particulate fraction (100 000 × g pellet). High-pressure liquid chromatography and phosphoamino acid analysis of the synthetic peptide substrates substantiated the phosphorylation of tyrosine residues by the particulate fraction enzyme. The human enzyme was also capable of phosphorylating a synthetic random polymer of 80% glutamic acid and 20% tyrosine. Enzyme activity was half-maximal with 22 μM Mg·ATP and had apparent K_m values for the synthetic peptides from 1.9 to 7.1 mM. The enzyme preferred Mg²⁺ to Mn²⁺ for optimal activity and was stimulated 2–5-fold by low levels (0.05%) of some ionic as well as non-ionic detergents including deoxycholate, Nonidet P-40 and Triton X-100. The enzyme activity was not stimulated by N⁶;O^2′-dibutyryl cyclic AMP (100 μM), N⁶;O^2′-dibutyryl cyclic GMP (100 μM), Ca²⁺ (200 μM), insulin (1 μg/ml) or homogeneous human T-cell growth factor (3 μg/ml) under the conditions used. Alkaline-resistant phosphorylation of particulate proteins in vitro revealed protein bands with M_r 59 000 and 54 000 suggesting that there are endogenous substrates for the human lymphocyte tyrosine protein kinase.     

Spermine inhibits the phosphorylation of the 11,000- and 10,000-dalton nuclear proteins catalyzed by nuclear protein kinase NI in NB-15 mouse neuroblastoma cells

The nuclear protein kinase NI (NI kinase) was purified from NB-15 mouse neuroblastoma cells by phosphocellulose column and casein affinity column chromatography. The purified NI kinase exhibited (i) an apparent subunit molecular weight of about 37,000, (ii) autophosphorylation, and (iii) insensitivity to inhibition by heparin. When NI kinase was added to heat-treated neuroblastoma nuclei in the presence of [gamma-32P] ATP, two proteins with apparent subunit molecular weights of 11,000 and 10,000 were prominently phosphorylated. Other protein kinases tested including the nuclear protein kinase NII, Type I cAMP-dependent protein kinase, and protein kinase C did not catalyze the phosphorylation of these two proteins. The NI kinase-catalyzed phosphorylation of these two proteins was completely inhibited by 1 mM spermine. In contrast, 10 mM putrescine, 2 mM spermidine, 5 mM arginine, and 10 mM NH4Cl, had no inhibitory effect on this phosphorylation reaction. Our study also indicated that the phosphorylation of the 11,000- and 10,000-dalton proteins occurred in the nuclear matrix fraction but not in heterogeneous nuclear ribonucleoproteins, high mobility group proteins, or histone fractions. We have previously reported that spermine specifically inhibits the endogenous phosphorylation of an 11,000-dalton nuclear protein in various mammalian cell lines (Chen, K. Y., and Verma, R. (1984) Biochem. Biophys. Res. Commun. 118, 710-716). The present study suggests that the 11,000- and 10,000-dalton nuclear proteins may be native substrates of nuclear protein kinase NI and that their phosphorylation can be affected by physiological concentrations of spermine.     

Differential effects of spermine on phosphatidylinositol 3-kinase and phosphatidylinositol phosphate 5-kinase

The metabolism of phosphoinositides plays an important role in the signal transduction pathways. We report here that naturally occuring polyamines affect the activities of phosphatidylinositol (PI) 3-kinase and PI 4-phosphate (PIP) 5-kinase differently. While polyamines inhibited the PI 3-kinase activity, they stimulated the activity of PIP 5-kinase in the order of spermine > spermidine > putrescine. Spermine inhibited the PI 3-kinase activity in a concentration-dependent manner with an IC₅₀ of 100 μM. On the other hand, spermine (5 mH) stimulated the activity of PIP 5-kinase 2–3 fold. Kinetic studies of spermine-mediated inhibition of PI 3-kinase revealed that it was noncompetitive with respect to ATP. The effect of Mg²⁺ and PIP, concentration on kinase activity was sigmoidal, with spermine inhibiting PI 3-kinase activity at all PIP₂ concentrations. While 1 mH calcium stimulated PI 3-kinase activity at submaximal concentrations of Mg²⁺ (1.25 mH), inhibition was observed at optimal concentration of Mg²⁺(2 mM). We propose that spermine may modulate the cellular signal by virtue of its differential effects on phosphoinositide kinases.     

Catalytic effect of cis-diammineplatinum α-pyrrolidone tan adsorbed on a platinum electrode in electrochemical oxidation of water into molecular oxygen

Cyclic voltammograms of cis-diammineplatinum α-pyrrolidone-blue and -tan, [Pt₄(NH₃)₈(C₄H₆NO)₄]ⁿ⁺ (n = 5 and 6, respectively) show for either complex only one redox peak at 0.53 V (average potential of the anodic and cathodic peak potentials). Coulometry and UVVis spectra of bulk- electrolyzed solution indicated that the redox peak corresponds to the reaction [Pt₄(NH₃)₈(C₄H₆NO)₄]⁸⁺ + 4e ⇄ 2[Pt₂(NH₃)₄(C₄H₆NO)₂]²⁺. When cyclic voltammetry is carried out in a solution of [Pt₄(NH₃)₈(C₄H₆NO)₄]⁶⁺ or a platinum electrode adsorbed with [Pt₄(NH₃)₈(C₄H₆NO)₄]⁶⁺ is used in the presence of oxidizing agent in the solution, O₂ gas generates from the electrode surface with large catalytic cathodic current at potentials below ca. 0.8 V. The O₂ gas was confirmed to generate from water by GC-MS analysis. This abnormal O₂ generation phenomenon is explained with cyclic reactions of chemical surface oxide formation on the electrode by the oxidizing agent and electrochemical reduction of the surface oxide. Oxygen gas generates from the reaction of [Pt₄(NH₃)₈(C₄H₆NO)₄]⁸⁺ or [Pt₄(NH₃)₈(C₄H₆NO)₄]⁶⁺ with OH⁻ produced in the course of the electrochemical reduction of the electrode surface oxide. The ability of [Pt₄(NH₃)₈(C₄H₆NO)₄]⁸⁺ and [Pt₄(NH₃)₈(C₄H₆NO)₄]⁶⁺ to oxidize OH⁻ into O₂ has been reported previously.     

Cyclic AMP stimulation of membrane phosphorylation and Ca2+-activated,Mg2+-dependent ATPase in cardiac sarcoplasmic reticulum

Ca²⁺-activated ATPase (EC 3.6.1.15) in canine cardiac sarcoplasmic reticulum was stimulated 50–80% by cyclic adenosine 3′ : 5′-monophosphate. The relationship of this stimulation to cyclic AMP-dependent membrane phosphorylation with phosphoester bands was studied. Cyclic AMP stimulation of ATPase activity was specific for Ca²⁺-activated ATPase and was half-maximal at about 0.1 μM which is similar to the concentration required for half-maximal stimulation of membrane phosphorylation by endogenous cyclic AMP-stimulated protein kinase (EC 2.7.1.37). Cyclic AMP stimulation of Ca²⁺-activated ATPase was calcium dependent and maximal at calculated Ca²⁺ concentrations of 2.0 μM. Cyclic AMP-dependent Ca²⁺-activated ATPase correlated well with the cyclic AMP-dependent membrane phosphorylation of which 80% was 20 000 molecular weight protein identified by sodium dodecyl sulfate discontinuous polyacrylamide gel electrophoresis. In trypsin-treated microsomes, cyclic AMP did not stimulate Ca²⁺-activated ATPase or phosphorylation of the 20 000 molecular weight membrane protein. An endogenous calcium-stimulated protein kinase (probably phosphorylase b kinase) with an apparent K_m for ATP of 0.21–0.32 mM was present and appeared to be involved in the cyclic AMP-dependent phosphorylation of the 20 000 molecular weight protein which was calcium dependent. Cyclic guanosine 3′ : 5′-monophosphate did not inhibit any of the stimulatory effects of cyclic AMP. These data suggest that the cyclic AMP stimulation of Ca²⁺-activated ATPase in cardiac sarcoplasmic reticulum is mediated by the 20 000 molecular weight phosphoprotein product of a series of kinase reactions similar to those activating phosphorylase b.     

Hormone-induced changes in pyruvate kinase activity in isolated hepatocytes II. Relation to the hormonal regulation of gluconeogenesis gluconeogenesis

1. 1. Incubation of isolated hepatocytes with glucagon (10⁻⁶ M) or dibutyryl cyclic AMP (0.1 mM) causes a decrease in pyruvate kinase activity of 50%, measured at suboptimal substrate (phosphoenolpyruvate) concentrations and 1 mM Mg_free²⁺. The magnitude of the decrease in activity is not influenced by the applied extracellular concentrations of lactate (1 and 5 mM), glucose (5 and 30 mM) or fructose (10 and 25 mM). With all three substrates comparable inhibition percentages are induced by glucagon or dibutyryl cyclic AMP.

2. 2. The extent of inhibition of pyruvate kinase induced by incubation of hepatocytes with glucagon or dibutytyl cyclic AMP is not influenced by the extracellular Ca²⁺ concentration nor by the presence of 2 mM EGTA. The reactivation of pyruvate kinase seems to be inhibited by a high concentration of extracellular Ca²⁺ (2.6 mM) as compared to a low concentration of extracellular Ca²⁺ (0.26 mM).

3. 3. Incubation of hepatocytes in a Na⁺-free, high K⁺-concentration medium does not influence the magnitude of the pyruvate kinase inhibition induced by dibutyryl cyclic AMP. However, the reactivation reaction is stimulated under these incubation conditions.

4. 4. Incubation of hepatocytes with dibutyryl cyclic GMP (0.1 mM) leads to a 25% decrease in pyruvate kinase activity. The magnitude of the inhibition by dibutyryl cyclic (GMP) is not influenced by the presence of pyruvate (1 mM) or glucose (5 mM and 30 mM).

5. 5. The relative insensitivity of the pyruvate kinase inhibition induced by glucagon, dibutyryl cyclic AMP and dibutyryl cyclic GMP to the extracellular environment leads to the conclusion that the hormonal regulation of pyruvate kinase is not the only site of hormonal regulation of glycolysis and gluconeogenesis. It is concluded that hormonal regulation of pyruvate kinase activity is exerted by changes in the degree of (de)phosphorylation of the enzyme reflecting acute hormonal control as well as by changes in the concentration of the allosteric activator fructose 1,6-diphosphate. The latter depends at least in part on the hormonal control of the phosphofructokinase-fructose-1,6-phosphatase cycle.

The impact of spermine competition on the efficacy of DNA-binding Fe(II), Co(II), and Cu(II) complexes of dimeric chromomycin A3

Chromomycin (Chro) forms a 2:1 drug/metal complex through the chelation with Fe(II), Co(II), or Cu(II) ion. The effects of spermine on the interaction of Fe(II), Co(II), and Cu(II) complexes of dimeric Chro with DNA were studied. Circular dichroism (CD) measurements revealed that spermine strongly competed for the Fe(II) and Cu(II) cations in dimeric Chro-DNA complexes, and disrupted the structures of these complexes. However, the DNA-Co^II(Chro)₂ complex showed extreme resistance to spermine-mediated competition for the Co(II) cation. According to surface plasmon resonance (SPR) experiments, a 6 mM concentration of spermine completely abolished the DNA-binding activity of Fe^II(Chro)₂ and Cu^II(Chro)₂ and interfered with the associative binding of Co^II(Chro)₂ complexes to DNA duplexes, but only slightly affected dissociation. In DNA integrity assays, lower concentrations of spermine (1 and 2 mM) promoted DNA strand cleavage by Cu^II(Chro)₂, whereas various concentrations of spermine protected plasmid DNA from damage caused by either Co^II(Chro)₂ or Fe^II(Chro)₂. Additionally, DNA condensation was observed in the reactions of DNA, spermine, and Fe^II(Chro)₂. Despite the fact that Cu^II(Chro)₂ and Fe^II(Chro)₂ demonstrated lower DNA-binding activity than Co^II(Chro)₂ in the absence of spermine, while Cu^II(Chro)₂ and Fe^II(Chro)₂ exhibited greater cytoxicity against HepG2 cells than Co^II(Chro)₂, possibly due to competition of spermine for Fe(II) or Cu(II) in the dimeric Chro complex in the nucleus of the cancer cells. Our results should have significant relevance to future developments in metalloantibiotics for cancer therapy.