Hormonal effects on fat cell adenosine 3′,5′-monophosphate dependent protein kinase

Fat cell extracts were electrophoresed on polyacrylamide gels to separate the regulatory subunit and holoenzyme species of protein kinase. Gels were incubated with cyclic [³H]AMP ([³H]cAMP) and washed, and the bound [³H]cAMP was estimated. The band of [³H]cAMP found closest to the origin (Peak I) was associated with cAMP-dependent protamine kinase activity. A seond [³H]cAMP peak (Peak II) also contained protamine kinase activity. Although the kinase activity of Peak II was much less than Peak I, more [³H]-cAMP was bound in Peak II than in Peak I. The [³H]cAMP peak furthest from the origin (Peak III) was devoid of kinase activity.Incubation of extracts with cAMP prior to electrophoresis diminished or abolished kinase activity in Peaks I and II. This incubation also decreased [³H]cAMP binding in Peaks I and II, and increased binding in Peak III. When extracts were incubated with [³H]cAMP before electrophoresis, essentially all of the radioactivity was found in Peak III. It was concluded that Peak I represents a holoenzyme form and that Peak III is composed of the regulatory subunits of this enzyme. Peak II may represent a relatively inactive holoenzyme form not previously described.Incubation of adipocytes with epinephrine resulted in a dose- and time-dependent decrease in Peak I and increase in Peak III, and insulin opposed these effects of epinephrine. After 1-min incubations with epinephrine, the decreases in Peak I or increases in Peak III correlated with increases in phosphorylase a activity, decreases in glycogen synthase I activity and changes in cAMP, both in the presence and absence of insulin. However, after incubation with epinephrine for more than 2 min in the presence of insulin, phosphorylase a activity did not correlate with cAMP, suggesting that factors other than the cyclic nucleotide mediate the effects of epinephrine and insulin.     

Phosphorylation of immunopurified rat liver glucocorticoid receptor by the catalytic subunit of cAMP-dependent protein kinase

We have examined phosphorylation of the rat liver glucocorticoid receptor (GR) and GR-associated protein kinase (PK) activity in the immunopurified receptor preparations. Affinity labeling of hepatic cytosol with [³H]dexamethasone 21-mesylate showed a covalent association of the steroid with a 94 kDa protein. GR was immunopurified with antireceptor monoclonal antibody BuGR2 (Gametchu & Harrison, Endocrinology 114: 274–279, 1984) to near homogeneity. A 23° C incubation of the immunoprecipitated protein A-Sepharose adsorbed GR with [-³²P]ATP, Mg²⁺ and the catalytic subunit of cAMP-dependent PK (cAMP-PK) from bovine heart, led to an incorporation of radioactivity in the 94 kDa protein. Phosphorylation of GR was not evident in the absence of the added kinase. Of the radioinert nucleotides (ATP, GTP, UTP or CTP) tested, only ATP successfully competed with [-³²P]ATP demonstrating a nucleotide specific requirement for the phosphorylation of GR. Other divalent cations, such as Mn²⁺ or Ca²⁺, could not be substituted for Mg²⁺ during the phosphorylation reaction. Phosphorylation of GR was sensitive to the presence of the protein kinase inhibitor, H-8, an isoquinoline sulfonamide derivative. In addition, the incorporation of radioactivity into GR was both time- and temperature-dependent. The phosphorylation of GR by cAMP-PK was independent of the presence of hsp-90 and transformation state of the receptor. The results of this study demonstrate that GR is an effective substrate for action of cAMP-PK and that the immunopurified protein A-Sepharose adsorbed GR lacks intrinsic kinase activity but can be conveniently used for the characterization of the phosphorylation reaction in the presence of an exogenous kinase.Abbreviations BUGR2 anti-GR monoclonal antibody - cAMP-PK cAMP-dependent protein kinase - DMSO dimethyl sulfoxide - EDTA ethylenediamine tetra acetic acid - GR glucocorticoid receptor - H-8 Isoquinoline sulfonamide derivative - hsp-90 90 kDa heat-shock protein - PMSF phenylmethylsulfonyl fluoride - PR progesterone receptor - NaF sodium fluoride - SDS sodium dodecyl sulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis - SR steroid receptor - TA triamcinolone acetonide     

The effect of GTP on the aluminum fluoride- and forskolin-activated adenylyl cyclase from human embryonic kidney 293 cells

GTP has been shown to inhibit AlF₄⁻-stimulated, and to activate forskolin-stimulated adenylyl cyclase activity in the presence of Mg²⁺ in cell membranes from human embryonic kidney 293 cells. The maximal inhibitory response of AlF₄⁻-stimulated adenylyl cyclase activity by GTP was not dependent on the concentration of Mg²⁺, but was so in the case of forskolin-activated activity at all forskolin concentrations assayed. Mn²⁺ ions stimulated AlF₄⁻- or forskolin-activated adenylyl cyclase activity to a greater extent than Mg²⁺. The inhibition of AlF₄⁻-stimulated cyclase by GTP was still observed with Mn²⁺, but the activation of forskolin-stimulated cyclase by GTP was not. When assayed together, Mn²⁺ and Mg²⁺ showed non-additive behaviours with respect to the amount of cyclic AMP formed after AlF₄⁻-stimulation of adenylyl cyclase. The temperature dependence of the activation of adenylyl cyclase by forskolin, AlF₄⁻ or under basal conditions was observed to be somehow different in the presence of Mn²⁺ than in the presence of Mg²⁺ ions. Cholera toxin treatment produced a markedly increased cyclase activity, specially when assayed with AlF₄⁻. In the case of forskolin-activated adenylyl cyclase, UTP and CTP were unable to reproduce the cyclase activation detected with GTP. However, in the case of AlF₄⁻-stimulated adenylyl cyclase, UTP was as good as GTP at inhibiting cyclase activity, and CTP virtually eliminated the activation of the cyclase with AlF₄⁻.     

Inhibition of membrane transport ATPases by halenaquinol,a natural cardioactive pentacyclic hydroquinone from the sponge Petrosia seriata

Halenaquinol, a natural cardioactive pentacyclic hydroquinone from the sponge Petrosia seriata, was found to be a powerful inhibitor of the rat brainstem and of the rat brain cortex Na⁺, K⁺-ATPases and the rabbit muscle sarcoplasmic reticulum Ca²⁺-ATPase with I₅₀ values of 7.0×10⁻⁷, 1.3×10⁻⁶ and 2.5×10⁻⁶ M, respectively. Halenaquinol also inhibited K⁺-phosphatase activity of the rat brain cortex Na⁺, K⁺-ATPase with an I₅₀ value of 3×10⁻⁶ M. Ouabain-insensitive Mg²⁺-ATPase activity of the microsomal fraction of the rat brain cortex was weakly inhibited by halenaquinol. Inhibition was irreversible, dose- and time-dependent. Naphthohydroquinone fragment in structures of halenaquinol, related natural and model compounds was very important for an inhibiting effect.     

Properties of spinach chloroplast fructose-1,6-diphosphatase

Photosynthetic fructose-1,6-diphosphatase (FDPase) fractions I and II, earlier purified from spinach leaves, show a similar amino acid composition, with the exception of a higher glutamic acid content in the latter. In both fractions glutamic and aspartic acids are the main amino acids. pH activity profiles of fractions I and II are similar, with optima at 8·65–8·70, both showing a high specificity for fructose- 1,6-diphosphate. These two fractions are Mg²⁺-dependent for activity, with an Optimum Mg²⁺ concentration of 10 mM in standard conditions, which shifts to 5 mM when the MG²⁺/EDTA ratio is increased to 10; Mn²⁺ and Co²⁺ are slightly active. EDTA enhances FDPase activity slightly, with an optimum at 0·4–0·8 mM. Cysteine has no activating effect, and acts as an inhibitor above 10 mM. Both I and II have an optimum substrate concentration of 4 mM, and the substrate inhibits at concns above this value. Kinetic velocity curves are sigmoidal, with the concave zone located in the range of physiological substrate concns. (Hill coefficient 1·75 for both). This suggests a strong regulatory role of fructose-1,6-diphosphate. K_m values are 1·4 × 10⁻³ M (fraction I) and 1·1 × 10⁻³ M (fraction II). The highest activity rate occurs at 60°, in accordance with the high thermostability of both fractions; the activation energies are 14·3 kcal/mol (fraction I) and 13·0 kcal/mol (fraction II).     

A specific alkaline phosphatase from Saccharomyces cerevisiae with protein phosphatase activity

In this paper, specific PHO13 alkaline phosphatase from Saccharomyces cerevisiae was demonstrated to possess phosphoprotein phosphatase activity on the phosphoseryl proteins histone II-A and casein. The enzyme is a monomeric protein with molecular mass of 60 kDa and hydrolyzes p-nitrophenyl phosphate with maximal activity at pH 8.2 with strong dependence on Mg²⁺ ions and an apparent K_m of 3.6×10⁻⁵ M. No other substrates tested except phosphorylated histone II-A and casein were hydrolyzed at any significant rate. These data suggest that the physiological role of the p-nitrophenyl phosphate-specific phosphatase may involve participation in reversible protein phosphorylation.     

Protein phosphorylation in the larval epidermis of Locusta migratoria: Role of cyclic AMP and 20-hydroxyecdysone

Cyclic nucleotide-dependent and cyclic nucleotide-independent protein kinase (PK) activities were quantified in the wing-pad epidermis of Locusta migratoria during the fifth larval instar. A low level of cyclic nucleotide-independent PK activity was detected in this tissue, whereas no PK-C (Ca²⁺- and phospholipid-dependent enzyme) activity was found. The main cyclic nucleotide-PK activity was cAMP-PK and large changes were observed during the intermoult. Concomitant increases in cAMP-PK activity and cAMP-dependent phosphorylation of several endogenous polypeptide substrates occurred during the last part of the intermoult cycle. The most marked ³²P-incorporation was observed in a 43 kDa polypeptide. These changes could be correlated with the peak of 20-OH-ecdysone in both hemolymph and integument.In vitro incubation of wing-pads with 20-OH-ecdysone (48 h) did not change cAMP-PK activity, but there were major changes in the pattern of endogenous phosphoproteins in particular the appearance of 43 and 45 kDa phosphoproteins. Two-dimensional analysis revealed several groups of phosphorylated spots. The results indicate a close relationship between certain cAMP-dependent phosphorylated polypeptides and the action of 20-OH-ecdysone.     

Resolution and characterization of two soluble calcium-dependent protein kinases from silver beet leaves

Two soluble Ca²⁺-dependent protein kinases (enzymes I and II) have been extensively purified from silver beet leaf tissue by means of a protocol involving batch-wise elution from DEAE-cellulose, Ca²⁺-dependent binding to phenyl-Sepharose, gradient elution from DEAE-Sephacel, gel filtration and binding to Cibacron F3GA-Sepharose CL-6B. Protein kinases I and II are resolved on gradient elution from DEAE-Sephacel and are further distinguished by their different K_m values for ATP and large differences in relative rates of phosphorylation of histone H1, casein and bovine serum albumin (the latter two proteins are relatively poor substrates for enzyme II but not enzyme I). Both enzymes have similar molecular weights as determined from gel filtration (56000 ± 2000 and 57000 ± 3000 for enzymes I and II, respectively). Both enzymes are absolutely dependent on free Ca²⁺ for activity with maximal histone H1 kinase activity being obtained at 0.5 μM free Ca²⁺. A millimolar concentration of Mg²⁺ is required in addition to a micromolar concentration Ca²⁺ for maximal activity. Both enzymes specifically phosphorylate serine residues of histone H1, are thiol activated and are inhibited by lanthanides and a range of calmodulin antagonists and inhibitors of protein kinase C.     

Isolation and divalent-metal activation of a β-xylosidase,RUM630-BX

The gene encoding RUM630-BX, a β-xylosidase/arabinofuranosidase, was identified from activity-based screening of a cow rumen metagenomic library. The recombinant enzyme is activated as much as 14-fold (k_cat) by divalent metals Mg²⁺, Mn²⁺ and Co²⁺ but not by Ca²⁺, Ni²⁺, and Zn²⁺. Activation of RUM630-BX by Mg²⁺ (t_0.5 144 s) is slowed two-fold by prior incubation with substrate, consistent with the X-ray structure of closely related xylosidase RS223-BX that shows the divalent-metal activator is at the back of the active-site pocket so that bound substrate could block its entrance. The enzyme is considerably more active on natural substrates than artificial substrates, with activity (k_cat/K_m) of 299 s⁻¹ mM⁻¹ on xylotetraose being the highest reported.     

Kinetics of the reversible association of ribosomal subunits: Stopped-flow studies of the rate law and of the effect of Mg2+

The kinetics of association and dissociation of Escherichia coli 30 S and 50 S ribosomal subunits appear to fit the simple scheme

over a wide range of Mg²⁺ and ribosome concentrations, for the preparations studied (which have a sharp [Mg²⁺]-dependence on the equilibrium degree of association, e.g. 10% to 90% for 1.5 mm to 3.5 mm). Both rate constants depend strongly upon magnesium ion concentration (k₂ goes from 0.04 × 10⁶ to 21 × 10⁶m⁻¹ s⁻¹ as [Mg²⁺] goes from 1.5 mm to 8 mm; k₁ goes from 150 to 2 s⁻¹ in the interval 1.0 to 3.0 mm), but k₁ may level off above 3.0 mm and k₂ increases slowly at high [Mg²⁺]. (The highest rate may not be far from the diffusion-controlled limit.) The primary effect of Mg²⁺, as calculated from the rather large changes in binding as a function of [Mg²⁺], is to decrease the contribution of electrostatic repulsion to the free energy of activation; specific, or class-specific, interactions of di- and multivalent cations with unknown ribosomal substructures may modulate this effect.     

Second messengers and ion channels in acetylcholine-induced chloride secretion in hen trachea

1. Hen tracheal epithelium can be stimulated by serosal application of acetylcholine (ACh) to secrete Cl⁻ equal to ~ 60–90 μA/cm².2. Radio-ligand-displacement for IP₃, cAMP and cGMP and ion channel selective drugs in voltage clamp setups were employed to characterize second messengers and Cl⁻, K⁺ and Ca²⁺ channels involved in the ACh response.3. ACh induced a significant rise in IP, in isolated tracheocytes, while ACh did not influence the production of cAMP in whole tissue, isolated tracheocytes or basolateral cell membrane vesicles. Further ACh desensitization did not effect cAMP level in tracheocytes. In addition neither ACh stimulation nor desensitization interfered with cAMP production in presence of 4.5 μM forskolin in tracheocytes, a level of forskolin rising base level cAMP by around five fold.4. Around 35% of ACh Cl⁻ secretion depends on Ca²⁺ mobilization from internal stores and about 65% on Ca²⁺ influx over basolateral membrane. The activated Ca²⁺ channel is insensitive to class I, II, III and IV Ca²⁺ antagonists.5. A 23187 can mimic the ACh effect although 30% is indomethacin-sensitive demonstrating a prostaglandin activated adenylyl cyclase.6. Two K⁺ channels are involved in ACh secretion, one sensitive to Ba²⁺ and quinine and both insensitive to 4-aminopyridine, apamin, charybdotoxin and TEA.7. Flufenamate and triaminopyrimidine block a non-selective ion channel likely involved in the ACh response. An ACh activated apical Cl⁻ channel is NPPB-sensitive.     

ATPases in rabbit erythrocytes: stimulation by HCO3-anion and by Na-cation-plus-K-cation.

A Mg²⁺Na⁺K⁺ATPase was found in a ghost preparation from rabbit erythrocytes, a finding in conflict with previous reports, but in agreement with the known kinetics of cation movements in these cells. However the Mg²⁺Na⁺K⁺ATPase was not inhibited by 10⁻⁴M ouabain, nor by 10⁻⁴M Ca²⁺. The physiological status of this enzyme is discussed. The basic Mg²⁺-ATPase activity in this preparation is also stimulated by HCO₃⁻; it is suggested that the HCO₃⁻-stimulated ATPases reported in a variety of other preparations are not necessarily due to mitochondrial contamination but could well originate from the plasma membrane.     

Purification and characterization of an adenosine cyclic 3':5' monophosphate-dependent protein kinase from human erythrocyte membrane.

A cAMP dependent protein kinase was extracted from human erythrocyte membrane with hydrosoluble fraction and partially purified by ammonium sulfate-precipitation and DEAE-cellulose chromatography. The pH of optimal activity is 6.5; the enzyme has an absolute requirement of Mg²⁺ ions at the concentration of 10 mM and is strongly inhibited by Ca²⁺. It uses ATP as phosphate donor with a Km of 3.7 × 10^?6 M. Cyclic AMP stimulates the activity with an apparent Ka of 5 × 10^?8 M; cIMP and cGMP also acts as activators. Enzyme activity is thermolabile and not protected by Mg ATP complex. The enzyme purified from erythrocyte membrane is a type I protein-kinase as proven by DEAE cellulose chromatography and dissociation of the subunits in presence of NaCl 0.5 M and histone.     

Regulation by Magnesium of Potato Tuber Mitochondrial Respiratory Activities

Dehydrogenase activities of potato tuber mitochondria and corresponding phosphorylation rates were measured for the dependence on external and mitochondrial matrix Mg²⁺. Magnesium stimulated state 3 and state 4 respiration, with significantly different concentrations of matrix Mg²⁺ required for optimal activities of the several substrates. Maximal stimulation of respiration with all substrates was obtained at 2-mM external Mg²⁺. However, respiration of malate, citrate, and -ketoglutarate requires at least 4-mM Mg²⁺ inside mitochondria for maximization of dehydrogenase activities. The phosphorylation system, requires a low level of internal Mg²⁺ (0.25 mM) to reach high activity, as judged by succinate-dependent respiration. However, mitochondria respiring on citrate or -ketoglutarate only sustain high levels of phosphorylation with at least 4-mM matrix Mg²⁺. Respiration of succinate is active without external and matrix Mg²⁺, although stimulated by the cation. Respiration of -ketoglutarate was strictly dependent on external Mg²⁺ required for substrate transport into mitochondria, and internal Mg²⁺ is required for dehydrogenase activity. Respiration of citrate and malate also depend on internal Mg²⁺ but, unlike -ketoglutarate, some activity still remains without external Mg²⁺. All the substrates revealed insensitive to external and internal mitochondrial Ca²⁺, except the exogenous NADH dehydrogenase, which requires either external Ca²⁺ or Mg²⁺ for detectable activity. Calcium is more efficient than Mg²⁺, both having cumulative stimulation. Unlike Ca²⁺, Mn²⁺ could substitute for Mg²⁺, before and after addition of A23, showing its ability to regulate phosphorylation and succinate dehydrogenase activities, with almost the same efficiency as Mg²⁺.     

Spectrofluorimetric determination of magnesium ions in water,ampoule, and suspension samples using a fluorescent azothiazol-benzenesulfonamide derivative

In this study, a fluorescence azothiazol-benzenesulfonamide derivative (M-sensor) was prepared for the determination of Mg²⁺ ions in different samples. The utilized M-sensor exhibited an emission fluorescence activity at 587 nm upon excitation at 537 nm. The developed method was based on the quenching effect of Mg²⁺ ions on the fluorescence intensity of the M-sensor with the above-mentioned fluorescence features. Furthermore, the utilized M-sensor was complexed with Mg²⁺ ions in the molar ratio of 1:1 (Mg²⁺ to M-sensor) and the selectivity of M-sensor toward Mg²⁺ against other metals ions, and the reversibility and reusability of the sensor were studied and verified. After optimization of the fluorometric detection, the quenching effect was directly proportional to the increase in the concentration of Mg²⁺ in the linear range 100–600 ng ml⁻¹ with a limit of detection value of 18 ng ml⁻¹. The fluorescence sensor was successfully applied with good recovery for the determination of Mg²⁺ in water samples and different pharmaceutical samples (ampoules and suspension) without any interference from aluminium.     

Significant impact of divalent metal ions on the fidelity,sugar selectivity,and drug incorporation efficiency of human PrimPol

Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn²⁺-dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn²⁺, rather than Mg²⁺, as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979 nM in the presence of Mn²⁺ and Mg²⁺, respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn²⁺ than with Mg²⁺. Notably, the substitution fidelity of PrimPol in the presence of Mn²⁺ was determined to be in the range of 3.4 × 10⁻² to 3.8 × 10⁻¹, indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57–1800 with Mn²⁺ and 150–4500 with Mg²⁺, and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine).     

Regulation of glucose metabolism in rat lung: Subcellular distribution,isozyme pattern,and kinetic properties of hexokinase

The subcellular distribution and isozyme pattern of hexokinase in rat lung were studied. Of the total hexokinase activity of lung, one-third was bound to mitochondria and one-third of the mitochondrial activity was in a latent form. The overt-bound mitochondrial hexokinase was specifically solubilized by physiological concentrations of glucose 6-phosphate and ATP. Inorganic phosphate partially prevented the solubilization by glucose 6-phosphate (Glc 6-P), whereas Mg²⁺ ions promoted rebinding of the solubilized enzyme to mitochondria. Thus, the distribution of hexokinase between soluble and particulate forms in vivo is expected to be controlled by the relative concentrations of Glc 6-P, ATP, P_i, and Mg²⁺. Study of the isozyme pattern showed that hexokinase types I, II, and III constitute the cell-sap enzyme of lung. The overt and latent hexokinase activities could be separately isolated by successive treatments of mitochondria with Glc 6-P and Triton X-100. The overt-bound activity consisted primarily of hexokinase type I, with a small proportion of type II isozyme. The latent activity, on the other hand, exclusively consisted of type I isozyme. Type I hexokinase, the predominant isozyme in lung, was strongly inhibited by intracellular concentration of Glc 6-P and this inhibition was counteracted by P_i. The bound form of hexokinase exhibited a significantly higher apparent K_i for Glc 6-P inhibition and a lower apparent K_m for ATP as compared to the soluble form. Thus, the particulate form of hexokinase is expected to promote glycolysis and may provide a mechanism for the high rate of aerobic glycolysis in lung.     

An inducible hydrolase from Mortierella isabellina (basidiomycetes). The deacylation of (+)-usnic acid

An inducible enzyme catalysing the hydrolysis of (+)-usnic acid to (+)-2-desacetylusnic acid and acetic acid has been purified 150-fold from the mycelium of Mortierella isabellina grown in the presence of (+)-usnic acid. Purification was achieved by treatment with protamine sulfate, (NH₄)₂SO₄ fractionation, negative adsorption on alumina Cγ gel and hydroxylapatite followed by chromatography on DEAE-cellulose and Sephadex G-200. The elution pattern from a Sephadex G-200 column indicated a MW of ca 7.6 × 10⁴ for the enzyme. The apparent K_m value for (+)-usnic acid at the pH optimum (pH 7) was 4.0 × 10^?5 M. The enzyme was specific for (+)-usnic acid and inactive towards (?)-usnic acid, (+)-isousnic acid or certain phloracetophenone derivatives. Its activity was enhanced in the presence of divalent metal ions such as Co²⁺, Ni²⁺, Mn²⁺, Mg²⁺ and Zn²⁺.