Ca2+-independent stimulation of cyclic GMP-dependent protein kinase by calmodulin

Calmodulin purified from bovine brain markedly stimulated cyclic GMP-dependent protein kinase from pig lung in the presence of cyclic GMP. This stimulation by calmodulin did not require Ca²⁺ and was dose-dependent up to optimal amounts, but the extent of stimulation decreased at concentrations over the optimal condition. The concentrations of cyclic GMP and cyclic AMP producing half-maximal stimulation were 4.5 × 10^?8 M and 5.0 × 10^?6 M respectively, under optimal conditions. Calmodulin increased maximum velocity without altering the Km for ATP. These effects of calmodulin on cyclic GMP-dependent protein kinase were similar to those of the stimulatory modulator described by Kuo and Kuo (J. Biol. Chem. $\text{251}$, 4283–4286, 1976). Ouf findings indicate that calmodulin regulates enzyme activity both Ca²⁺-dependently and independently.     

Regulation of cardiac cyclic GMP-dependent protein kinase

An assay method based on the ability of high concentrations of Mg²⁺ to stimulate phosphorylation of histone in the presence of low concentrations of ATP was developed for the measurement of cyclic GMP-dependent protein kinase activity ratios (activity -cyclic GMP/activity + cyclic GMP). In tissues which contain only trace amounts of cyclic GMP-dependent protein kinase, the basal activity ratios were high due to interference from a cyclic nucleotide-independent protein kinase. In order to study the regulation of the cardica cyclic GMP-dependent protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0–4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0–4°C did not significantly alter the activity ratio of the enzyme under conditions of basal or elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMP-dependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2–3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated witth the acetylcholine-induced protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0–4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0–4°C did not significantly alter the activity ratio of the enzyme under conditions of basal elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMP-dependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2–3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated with the acetylcholine-induced increase in cyclic GMP and the cyclic GMP-dependent protein kinase activity ratio was a reduction in the force of contraction. In contrast, nitroprusside produced little or no increase in the cyclic GMP-dependent protein kinase activity ratio despite increasing the level of cyclic GMP 8–10-fold. Nitroprusside also had no effect on contractile force. In combination, nitroprusside and acetylcholine produced additive effects on cyclic GMP levels, but protein kinase activation and force of contraction were similar to those seen with acetylcholine alone. The results suggest that the cyclic GMP produced by acetylcholine in the rat heart is coupled to activation of the cyclic GMP-dependent protein kinase, while that produced by nitroprusside is not.     

Activation of cyclic AMP-dependent protein kinase by epinephrine in proliferating lymphoid cells

Activation of the cyclic AMP-dependent protein kinase in intact lymphosarcoma cells can be promoted by epinephrine. The lymphosarcoma protein kinase is approximately 90% Isozyme I. Using the synthetic peptide PK-1 (LeuArgArgAlaSerLeuGly) as substrate for the kinase, the cyclic AMP-dependent protein kinase activity was 95% of the total protein phosphotransferase activity in the cell extract. In control cells the optimum extraction buffer for preventing enzyme subunit dissociation or reassociation contained buffer (2(N-morpholino)ethanesulfonic acid), EDTA, 2-mercaptoethanol, and charcoal. The absence of charcoal or the presence of 0.14 m KCl in the buffer promoted enzyme dissociation in the extract. The phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine had no effect. In extracts from epinephrine-treated cells or extracts to which purified catalytic subunit of the cyclic AMP-dependent protein kinase was added, recovery of the total protein kinase activity was 25% of that predicted in experiments with control cells. Recovery of enzyme activity increased to 80–95% of the predicted value when 0.14 m KCl was included in the extraction buffer. Methods involving a two-buffer extraction procedure are presented as the optimum protocol for determining in vivo activation of the cyclic AMP-dependent protein kinase, Isozyme I. Using these methods, epinephrine (1 μm) dissociated the cyclic AMP-dependent protein kinase essentially 100% in intact lymphosarcoma cells. The dissociation was apparently maintained for up to 60 min. Approximately 10–15% of the dissociated enzyme may be specifically associated with particulate cell fractions. Collectively the data emphasize the experimental difficulty inherent in determination of the extent of in vivo dissociation of the cyclic AMP-dependent protein kinase.     

Interferon induction of polyamine-dependent protein kinase activity in Ehrlich ascites tumor cells

Treatment of Ehrlich ascites tumor cell cultures $\text{in}\text{vitro}$ with interferon induces a protein kinase activity that is activated by the polyamines, spermidine and spermine. Putrescine antagonizes the activation. The protein kinase yields a phosphorylated endogenous polypeptide of M_r 68,000–70,000. The polyamine-dependent protein kinase activity cofractionates with a double-stranded RNA-dependent protein kinase activity during affinity chromatography on poly (I) ·poly (C) - agarose or by chromatography on phosphocellulose. The double-stranded RNA-dependent protein kinase also phosphorylates an endogenous polypeptide of M_r 68,000–70,000. Unsuccessful attempts to discriminate between these two protein kinase activities on the bases of their respective capacities to be activated by either double-stranded RNA or spermidine/spermine, suggest that a single protein kinase enzyme may be activated by these strikingly dissimilar modifiers.     

Irreversible inhibition of folate transport in Lactobacillus casei by covalent modification of the binding protein with carbodiimide-activated folate

Activated folate formed by reaction of folic acid and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide irreversibly inhibits the folate transport system of Lactobacillus casei. Complete inhibition of both folate binding to the carrier protein and folate transport was achieved by pretreatment of the cells at low temperature (4 °C) and at neutral pH with 200 nm activated folate. Fifty percent inhibition of binding and transport occurred at 35 and 40 nm activated folate, respectively. Specificity was demonstrated by the fact that excess nonactivated folate added during the pretreatment step afforded complete protection of the binding protein against inhibition, and that activated folate had no effect on the binding or transport of thiamine. Rapid measurements at 4 °C were employed to show that, prior to the appearance of irreversible inhibition, activated folate (K_i = 15 nM) interacted reversibly with the binding site for folate (K_d = 0.8 nM). Cells treated with activated [³H]folate incorporated 1 mol of folate per mole of binding protein. Purification of the labeled protein followed by digestion with Pronase led to the isolation of a compound identified as ?-N-folyl lysine. The ?-amino group of a lysyl residue thus appears to be the nucleophilic group at the binding site that reacts with activated folate.     

Inactivation of cyclic GMP-dependent protein kinase by N alpha-tosyl-L-lysine chloromethylketone

Cyclic GMP-dependent protein kinase from bovine lung and cyclic AMP-dependent protein kinase from bovine heart are inactivated by N^α-tosyl-L-lysine chloromethylketone (TLCK) in the presence of cyclic GMP and cyclic AMP, respectively. The inactivation of both protein kinases is pseudo-first order, suggesting the rate limiting step is beyond the binding of TLCK. Cyclic GMP-dependent protein kinase is inactivated less than $\text{1}\text{4}$ as rapidly as cyclic AMP-dependent protein kinase, although it shows a higher apparent affinity for TLCK. Cyclic AMP stimulated the rate of inactivation of cyclic AMP-dependent protein kinase 10-fold but cyclic GMP stimulated the rate of inactivation of cyclic GMP-dependent protein kinase only 1.5-fold. The rate of inactivation of cyclic GMP-dependent protein kinase by TLCK is sufficiently rapid (half-time of about 30 min at 37°C with 2 mM TLCK) to account for the effects of TLCK on cell growth observed by others.     

Metal(II) complexes of 1-formylisoquinoline thiosemicarbazone: their preparation,characterization and antitumour activity

Complexes of Co(II), Ni(lI), Cu(II), Zn(II) and Pt(II) with 1-formylisoquinoline thiosemicarbazone (1-iqtsc-H) were prepared and characterized by elemental analyses, conductance measurement and spectral studies. On the basis of these studies a distorted octahedral structure for [Co(1-iqtsc)₂]·2H₂O, a distorted trigonal-bipyramidal structure for [Ni- (1-iqtsc-H)Cl₂], [Cu(1-iqtsc-H)Cl₂] and [Zn(1-iqtsc- H)(OAc)₂]·H₂O and a square-planar structure for [Pt(1-iqtsc)Cl] are suggested. All these metal(II) complexes were screened for their antitumour activity in the P388 lymphocytic leukaemia test system in mice. Except for Pt(Il), the complexes were found to possess significant activity; the Ni(II) complex showed a T/C value of 161 at the optimum dosage.     

Stoichiometry of phosphorylation of hepatic ATP-citrate lyase by protein kinase

Hepatic ATP-citrate lyase prepared with a fluoride-free step to allow endogenous phosphatases to dephosphorylate the enzyme was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase and [γ-³²P]ATP. After electrophoresis the radioactive phosphate was located predominantly in the gel slice containing the Coomassie blue stained protein corresponding to ATP-citrate lyase. The Stoichiometry of phosphorylation of hepatic ATP-citrate lyase in vitro by the catalytic subunit was such that 0.53 ± 0.02 molecules of phosphate were incorporated per subunit. The degree of phosphorylation was independent of the amount of ATP-citrate lyase present as substrate in the concentration range 1.2–6.4 μm. In the absence of catalytic subunit there was very little labeled phosphate incorporated into ATP-citrate lyase. Phosphorylation of ATP-citrate lyase by catalytic subunit was abolished by the specific protein inhibitor of cyclic AMP-dependent protein kinase. When ATP-citrate lyase was subjected to electrophoresis under nondenaturing conditions, lyase activity was recovered from the gel slice corresponding to the Coomassie blue staining phosphoprotein of a stained gel run in parallel.     

Purification and partial characterization of bovine heart phosphorylase kinase

Bovine heart phosphorylase kinase has been isolated by a procedure involving precipitation with polyethylene glycol, DEAE-Sephacel chromatography and calmodulin-Sepharose affinity chromatography. The isolated enzyme had a specific activity of 8.3 IU/mg of protein at pH 8.2 at 30 degrees C in the presence of 1% glycogen. The native enzyme had a sedimentation coefficient of 23 S and the Mr of the alpha', beta, gamma, and delta subunits, were 140,000, 130,000, 46,000, and 18,000, respectively. Activation of the phosphorylase kinase by the catalytic subunit of bovine heart cAMP-dependent protein kinase increases the pH 6.8/8.2 activity ratio from 0.01 to 0.32-0.38. Glycogen (1%) decreased the Km of the activated phosphorylase kinase at pH 6.8 for phosphorylase b from 5.5 to 1.25 mg/ml. Trypsin treatment increased the pH 6.8 activity but decreased the pH 8.2 activity. During this process the alpha' subunit was converted to a Mr 110,000 polypeptide and the enzyme activity was converted essentially to a 5.9 S species having an apparent Mr of 100,000 as determined by gel filtration. On extended trypsin treatment only one major polypeptide corresponding to the beta subunit remained. The same polypeptide was present in the active fractions following gel filtration of the trypsinized kinase.     

Inhibition of growth of C6 astrocytoma cells by inhibitors of calmodulin

We evaluated the effect of several classes of calmodulin inhibitors on the activity of calmodulin prepared from C6 astrocytoma cells and studied the activity of these drugs as inhibitors of the growth of C6 cells in tissue culture. There was a good correlation between the activity of the drugs as inhibitors of calmodulin and their activity as inhibitors of cell growth. The most potent compounds were calmidazolium and melittin as compared to the phenothiazines, trifluoperazine, chlorpromazine, chlorpromazine-sulfoxide or the diphenylbutylpiperidine, pimozide. The mechanism by which the inhibition of calmodulin leads to the death of cells could not be attributed entirely to inhibition of the calmodulin-sensitive cyclic nucleotide phosphodiesterase. Calmodulin is a heat stable, calcium-binding protein involved in numerous biological processes. Recent evidence indicates that calcium and calmodulin may be important for cellular proliferation. For example, this protein changes in concentration during the cell cycle; is involved in the disassembly of the mitotic apparatus; is increased in concentration in rapidly growing hepatomas and in transformed fibroblasts. Weiss and co-workers demonstrated that phenothiazines and structurally similar drugs are capable of binding to and inhibiting the activity of calmodulin. It has been recently observed that certain drugs that inhibit the activity of calmodulin also inhibit the growth of malignant cells in vitro and in vivo. In these studies, however, there was no direct correlation of the effect of the drugs on the calmodulin from the cell type under investigation with cytotoxicity. To learn more about the relationship between a drug's ability to inhibit calmodulin and its antiproliferative activity, we correlated the effect of drugs on the activity of calmodulin prepared from the C6 astrocytoma cell line with their effect on cellular proliferation. Since many inhibitors of calmodulin readily cross the blood-brain barrier and since no acceptable treatment for malignancies of the central nervous system exist, we chose this cell line as a model for elucidating the potential antineoplastic effects of calmodulin inhibitors.     

Metal(II) chelates of 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone: Their preparation,characterization and antitumour activity

The metal(II) complexes [M(4-Me-5-NH₂-1-iqtsc- H)Cl₂] (M = Co(II), Ni(II) or Cu(II) and 4-Me-5- NH₂-1-iqtsc-H = 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone), [Zn(4-Me-5-NH₂-1-iqtsc-H)- (OAc)₂]· H₂O and [Pt(4-Me-5-NH₂-1-iqtsc)Cl)] were isolated and characterized by elemental analysis, conductance measurement, magnetic moments (300- 78 K)and spectral studies. On the basis of these studies distorted trigonal-bipyramidal structures for the Co(II), Ni(II), Cu(II) and Zn(II) complexes and a square-planar structure for the Pt(II) complex are proposed. All these complexes were screened for their antitumour activity in the P388 lymphocytic leukaemia test system in mice. With the exception of the Pt(II) and Zn(II) complexes, the complexes showed no significant activity; the Zn(II) and Pt(II) complexes showed T/C (%) values of 150 and 144 at a much lesser extent [2].     

Photoaffinity labeling of a microtubule-associated cyclic AMP-binding protein

8-Azido cyclic AMP has been used as a photoaffinity probe to identify cyclic AMP-binding proteins in microtubule preparations. Bovine brain microtubule proteins and rabbit muscle protein kinase were incubated with the photoaffinity ligand in reduced light for 15 min, without additions or with 100-fold excess unlabeled cyclic AMP or 5′-AMP. Samples were then irradiated at 254 nm at a distance of 1 cm for 5 min, in ice. After irradiation aliquots were taken for electrophoresis in one or two dimensions. Polypeptides which bound the photoaffinity label were visualized by autoradiography. The apparent molecular weights of the most prominent 8-azido ³²P-cyclic AMP-binding proteins are in the same range as those of the RII of the muscle enzyme. Following two-dimensional electrophoresis the major microtubule-associated cyclic AMP-binding proteins resolve as two spots with about the same pI (~pH 5.0) but slightly different molecular weights. Both spots are in the molecular weight range of the tubulins but they are clearly resolved from the tubulins in the first dimension. Cyclic AMP, but not 5′-AMP blocks the labeling of these proteins. There are low levels of labeling of the tubulins, the high-molecular-weight MAPs and several polypeptides with molecular weights near tubulin but with more basic pI. The photoaffinity probe has demonstrated that the major microtubule-associated cyclic AMP-binding protein of bovine brain is distinct from other RII proteins and from tubulin isomorphs.     

GuHC1 induced unfolding-folding transition of a hinge-bending protein: horse muscle phosphoglycerate kinase

The antineoplastic compound N2-methyl-9-hydroxyellipticinium (9-OH-NME) is able to bind to different biological molecules after an oxidative activation by horseradish peroxidase and hydrogen peroxide. In this study, the efficient covalent binding in vitro of 9-OH-NME onto RNA and poly A is described. The phenomenon is analyzed by different HPLC methods and the yield of binding is determined using [3H]9-OH-NME. For an initial ratio drug per nucleotide of 0.07, the rb obtained (ratio of drug bound per nucleotide) of 0.026 for RNA and 0.044 for poly A, which represent respectively a yield of 40% and 60% for the drug fixation onto these macromolecules. These facts demonstrate the high electrophilicity of para-quinone-imine derivatives in ellipticinium series.     

Interaction of alpha adrenergic antagonists with calmodulin

Several alpha-adrenergic antagonists inhibited the activation of calmodulin-stimulated phosphodiesterase at concentrations that had little or no effect on basal phosphodiesterase activity. The most potent of these compounds were phenoxybenzamine and dibenamine (IC50 values of about 1 microM); the amino acid ergot alkaloids ergocryptine, ergocristine, ergotamine and their dihydrogenated derivatives were less potent calmodulin-inhibitors (IC50 values of 35-80 microM). The amino ergot alkaloids ergonovine and methysergide were essentially devoid of inhibitory activity. A variety of other alpha 1-antagonists (phentolamine, tolazoline and prazosin), an alpha 2-antagonist (yohimbine), alpha-agonists (norepinephrine, phenylephrine and clonidine), beta-adrenergic antagonists (propranolol and practolol) and the beta-adrenergic agonist methoxyphenamine displayed little or no anti-calmodulin activity (IC50 values greater than 300 microM). Similarly, the alkylating agents chlorambucil and mechlorethamine also failed to inhibit calmodulin activity. Phenoxybenzamine and dibenamine inhibited calmodulin activity irreversibly, whereas the inhibition caused by other alpha adrenergic blocking agents was reversible. Phenoxybenzamine inhibited calmodulin activity by binding directly to it. This binding was calcium-dependent and irreversible. The irreversible binding and inhibition of calmodulin activity by phenoxybenzamine (or dibenamine) may serve as a useful tool for studying the sites at which drugs bind to calmodulin and may also be useful for studying the distribution and turnover of calmodulin.     

Adenylate cyclase and cyclic AMP phosphodiesterase activity during the mitotic cycle of Physarum polycephalum.

Tyrosine, as well as small amounts of phenylalanine, were removed selectively and quantitatively from purified chick brain tubulin by enzymatic digestion with carboxypeptidase. The fraction of molecules containing hydrolyzable tyrosine changed with the stage of development and had the highest value (～0.5) around days 14–16 of the embryo. The increase in the fraction of tyrosinated molecules was found to be temporally correlated with an increased specific activity of the enzyme catalyzing the incorporation reaction. In addition, it was found that the availability of α-chain C-termini for $\text{in vitro}$ tyrosination also reached a maximum during the same period. Changes in the extent of modification of the C-terminus of tubulin may be relevant for the participation of the resultant microbubules in different developmental events.     

Purification of human skin tyrosinase and its protein inhibitor: properties of the enzyme and the mechanism of inhibition by protein

Tyrosinase from normal human skin was purified to high specific activity; 228 nmol of dopa formed/min/mg protein. The properties of the purified enzyme differ from those of the same enzyme in crude homogenates. The activity of the purified enzyme is not affected by dopa. It is not inhibited by excess tyrosine and exhibits no lag in its rate at 4 mm concentration of ascorbic acid. This preparation is free of peroxidase and yet will catalyze both hydroxylation of tyrosine to dopa and its further oxidation to dopa quinone with fourfold more activity with dopa as substrate suggesting that mammalian tyrosinase catalyzes both reactions rather than dopa oxidation alone as suggested by M. Okun, L. Edelstein, R. Patel, and B. Donnellan (1973, Yale J. Biol. Med.46, 535–540). A protein present in the cytosol and melanosomes that constitutes 30% of soluble epidermal proteins was purified and found to inhibit tyrosinase competitively with tyrosine. Its molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 66,000.     

Molecular dynamics of calmodulin as monitored by fluorescence anisotropy

Static and dynamic measurements of fluorescence anisotropy have been made for calmodulin, employing both the intrinsic fluorescence of Tyr-99 and Tyr-138 and the fluorescence of dansyl and fluorescein groups attached to Tyr-99, as well as AEDANS groups attached to methionines. All approaches indicate the presence of a significant internal mobility involving the probe for calmodulin in the absence of Ca²⁺. This is diminished in the presence of Ca²⁺.²     

Solubilization,purification, and properties of rabbit brain hexokinase

More than 90% of the total hexokinase activity in rabbit brain was found to be associated with the mitochondrial fraction. The participate enzyme was solubilized in a relatively specific way by glucose 6-phosphate and Triton X-100 and purified to apparent homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and affinity chromatography. The solubilized hexokinase activity has been purified 700-fold in 48% yield with a specific activity of 165 units/mg of protein. The molecular weight was found to be approximately 100,000 both for the native and the denatured enzyme. The isoelectric point, pI, was 6.3 pH units by isoelectric focusing and the enzyme was found to be able to phosphorylate several hexoses with different affinities. Mg · ATP, among the nucleotide substrates, was the most effective as a phosphate donor. The present results indicate considerable similarity between this enzyme and the other mammalian type I hexokinases.     

Increase in nuclear cyclic GMP-dependent protein kinase following partial hepatectomy

The level of cyclic GMP-dependent protein kinase in the nucleus of rat liver was shown to increase 80% at 3 hr following partial hepatectomy while cyclic AMP-binding activity decreased 28%. Subnuclear fractionation demonstrated that the increase in cyclic GMP-dependent protein kinase was localized to the nucleolus and nucleoplasm, with no change in the extranucleolar particulate material. Cyclic AMP-binding activity was decreased in all subnuclear fractions under these conditions. At 16 hr following partial hepatectomy, the level of cyclic GMP-dependent protein kinase was not changed in the nucleolus but was significantly increased in the nucleoplasm, while cyclic AMP-binding activity was slightly increased in the nucleolus and decreased in the nucleoplasm.