Saturation-transfer electron spin resonance studies on the mobility of spin-labeled sodium and potassium ion activated adenosinetriphosphatase in membranes from Squalus acanthias

The sodium and potassium ion activated adenosinetriphosphatase [(Na+,K+)-ATPase] in membranous preparations from Squalus acanthias has been spin-labeled on sulfhydryl groups after prelabeling with N-ethylmaleimide. Saturation-transfer electron spin resonance spectroscopy has been used to study the rotational motions of the labeled protein on the microsecond time scale. Effective rotational correlation times deduced from the diagnostic line-height ratios in the second-harmonic, 90 degrees out-of-phase (V2') spectra are much larger than those deduced from the spectral integrals, indicating the presence of large-scale segmental motions, in addition to rotation of the protein as a whole. Experiments involving controlled cross-linking of the protein by glutaraldehyde, as well as measurements of the line broadening of the conventional electron spin resonance spectra, support this interpretation. Both the spectral integrals and diagnostic line-height ratios are found to increase irreversibly with time on incubation at temperatures greater than 20 degrees C, corresponding to a decrease in the segmental motion of the protein and probably also in the overall protein rotation. The native enzyme displays a marked nonlinearity in the Arrhenius temperature dependence of the activity at temperatures above 20 degrees C, and the activity decreases with a half-life of ca. 70 min on incubation at 37 degrees C (but not on incubation at low temperature), paralleling the time- and temperature-dependent changes in the saturation-transfer spectra of the labeled protein. Both of these observations suggest that the changes observed in the molecular dynamics could correspond to functional properties of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

A study on the role of protein kinase C and intracellular calcium in the activation of superoxide generation

Accumulating evidence indicates that protein kinase C plays an essential role in the activation of NADPH oxidase. In the present study, the correlation between superoxide generation, intracellular calcium, activation of purified protein kinase C and stabilized membrane-bound protein kinase C was studied. Phorbol 12-myristate 13-acetate (PMA) and 1-deacyl-2-acetyl-rac-glycerol (OAG) were found to induce equal activation of purified protein kinase C and translocation of protein kinase C to the membrane fraction, but differed significantly in their ability to induce superoxide generation. Intracellular calcium was varied using calcium ionophores and increasing the intracellular calcium concentration to more than 1 microM was found to induce increased superoxide generation in maximally OAG-stimulated cells; this contrasted to maximally PMA-stimulated leukocytes. Ionomycin and A23187 were both found to induce a translocation of protein kinase C to the membrane fraction. This translocation was highly dependent upon extracellular calcium. In contrast, PMA- and OAG-induced translocation of protein kinase C was not dependent upon extracellular calcium. In conclusion, our results indicate that although PMA, OAG and calcium ionophores seem to activate protein kinase C in human polymorphonuclear leukocytes these activators differ in their ability to induce superoxide generation.     

Solubilized (Na+ + K+)-ATPase from shark rectal gland and ox kidney--an inactivation study

The bi-exponential time-course of detergent inactivation at 37 degrees C of C12E8-solubilized (Na+ + K+)-ATPase from shark rectal glands and ox kidney was investigated. The data for shark enzyme, obtained at detergent/protein weight ratios between 2 and 16, are interpreted in terms of a simple model where the membrane bound enzyme is solubilized predominantly as (alpha-beta)2 diprotomers at low detergent concentrations and as alpha-beta protomers at high C12E8 (octaethyleneglycoldodecylmonoether) concentrations. It is observed that the protomers are inactivated 15-fold more rapidly than the diprotomers, and that the rate of inactivation of both oligomers is proportional to the detergent/protein ratio. Inactivation of kidney enzyme was biexponential with a very rapid inactivation of up to 40% of the enzyme activity. The observed rate of inactivation of the slower phase varied with the detergent/protein ratio, but the inactivation pattern for the kidney enzyme could not readily be accommodated within the model for inactivation of the shark enzyme. The rates of inactivation at 37 degrees C were about the same in KCl and NaCl, i.e., in the E2(K) and E1 X Na forms, for both enzymes.     

Phosphorylation of glycogen synthase in a homogenate of human polymorphonuclear leukocytes

Summary Glycogen synthase I in a homogenate of human polymorphonuclear leukocytes was phosphorylated under imitated physiological conditions utilizing the endogenous protein kinases. At subsequent steps of phosphorylation the³²P-labelled synthase was purified and characterized. Limited tryptic hydrolysis of the³²P-labelled synthase released four phosphopeptides (t-A, t-B, t-C, t-D) and subsequent chymotrypsinization of the trypsin resistant core released three phosphopeptides (c-A, c-B, c-C). One P_i/subunit was incorporated within 8–10 min and 2.2 P_i/subunit within 60 min increasing the K_c for Gle-6-P to 4–6 mM. The initial phosphorylation up to 0.8 P_i/subunit occurred mainly in peptide c-A and a linear relation between ratio of independence (RI) of glycogen synthase in the interval RI 0.85 to RI 0.05 and phosphorylation of this peptide to 0.5 P_i was observed. Phosphorylation of this peptide is responsible for the decrease in ratio of independence. From experiments with inhibitors and activators, the initial phosphorylation was found predominantly catalysed by the endogenous cAMP independent synthase kinase, however, the endogenous cAMP dependent protein kinase and phosphorylase kinase also phosphorylate endogenous glycogen synthase I to a minor degree. Circumstantial evidence for a Ca-dependent synthase kinase different from phosphorylase kinase is presented. The endogenous Gle-6-P dependent glycogen synthase occurring in a homogenate of leukocytes disrupted in the presence of NaF incorporated 1.07 P_i/subunit and K_c for Glc-6-P was increased from 6–8 mM to 20 mM. From the present and previous experiments [7] a total of 8 major phosphorylatable sites have been defined, one on each of the peptides t-A, t-B, t-C, c-B and c-C and two on peptide c-A, which in addition may contain a third site for phosphorylase kinase. Assuming identical subunits, only 13 out of 32 sites are thus covalently modified at maximum phosphorylation. The operational defined synthase R (K_c for Glc-6-P 0.5 mM) and D (K_c for Glc-6-P 2–8 mM) activities correspond to synthase with about 0.8 P_i and 1.8–2.3 P_i/subunit, respectively.     

Sulphydryl groups of (Na+ + K+)-ATPase from rectal glands of Squalus acanthias: Detection of ligand-induced conformational changes

1. Modification of the Class II sulphydryl groups on the (Na⁺ + K⁺)-ATPase from rectal glands of Squalus acanthias with N-ethylmaleimide has been used to detect conformational changes in the protein. The rates of inactivation of the enzyme and the incorporation of N-ethylmaleimide depend on the ligands present in the incubation medium. With 150 mM K⁺ the rate of inactivation is largest (k₁ = 1.73 mM^?1 · min^?1) and four SH groups per α-subunit are modified. The rate of inactivation in the presence of 150 mM Na⁺ is smaller (k₁ = 1.08 mM^?1 · min^-1) but the incorporation of N-ethylmaleimide is the same as with K⁺. 2. ATP in micromolar concentrations protects the Class II groups in the presence of Na⁺ (k₁ = 0.08 mM^?1 · min^?1 at saturating ATP) and the incorporation id drastically reduced. ATP in millimolar concentrations protects the Class II groups partially in the presence of K⁺ (k₁ = 1.08 mM^?1 · min^?1) and three SH groups are labelled per α subunit. 3. The K⁺ -dependent phosphatase is inhibited in parallel to the (Na⁺ + K⁺)-ATPase under all conditions, and the ligand-dependent incorporation of N-ethylmaleimide was on the α-subunit only. 4. It is shown that the difference between the Na⁺ and K⁺ conformations sensed with N-ethylmaleimide depends on the pH of the incubation medium. At pH 6 there is a very small difference between the rates of inactivation in the presence of Na⁺ and K⁺, but at higher pH the difference increases. It is also shown that the rate of inactivation has a minimum at pH 6.9, which suggests that the conformation of the enzyme changes with pH. 5. Modification of the Class III groups with N-ethylmaleimide-whereby the enzyme activity is reduced from about 16% to zero-shows that these groups are also sensitive to conformational changes. As with the Class II groups, ATP in micromolar concentrations protects in the presence of Na⁺ relative to Na⁺ or K⁺ alone. ATP in millimolar concentrations with K⁺ present increases the rate of inactivation relative to K⁺ alone, in contrast to the effect on the Class II groups. 6. Modification of the Class II groups with a maleimide spin label shows a difference between Class II groups labelled in the presence of Na⁺ (or K⁺) and Class II groups labelled in the presence of K + ATP, in agreement with the difference in incorporation of N-ethylmaleimide. The spectra suggest that the SH group protected by ATP in the presence of K⁺ is buried in the protein. 7. The results suggest that at least four different conformations of the (Na⁺ + K⁺)-ATPase can be sensed with N-ethylmaleimide: (i) a Na⁺ form of the enzyme with ATP bound to a high-affinity site (E₁-Na-ATP); (ii) a Na⁺ form without ATP bound (E₁-Na); (iii) a K⁺ form without ATP bound (E₂-K); and (iv) an enzyme form with ATP bound to a low-affinity site in the presence of K⁺, probably and E₁-K-ATP form.     

Preparation of membrane-bound and of solubilized (Na+ + K+)-ATPase from rectal glands of Squalus acanthias. The effect of preparative procedures on purity, specific and molar activity.

The effect of spectrin on the polymerization of muscle actin has been investigated by hydrodynamic methods and electron microscopy. Spectrin markedly accelerated polymerization of actin. The effect was more easily observed in lower concentrations of KCl (e.g. 24 mM) where spontaneous polymerization was negligibly small. Similarly large acceleration was observed for polymerization in MgCl2 or CaCl2. The rate of polymerization of actin was proportionally increased with the concentration of spectrin added to a fixed concentration of action. The stationary level of specific viscosity also increased with the spectrin concentration, but at larger concentrations it became smaller. The flow birefringence and electron microscope measurements indicated that actin polymers formed under the influence of spectrin were shorter than those of control F-actin filaments. The structural viscosity and electron microscope observations suggested that the interaction between F-actin fibers was not increased by spectrin. These data strongly suggest a seeding role of spectrin in the polymerization of actin. Spectrin accelerates formation of the nuclei for polymerization. The more the nuclei are formed, the larger the number of the grown polymers are and this leads to rapid formation of shorter polymers since the amount of actin is limited. The acceleration activity was found only in freshly prepared spectrin from fresh ghosts taken from freshly drawn blood.     

Solubilization and molecular weight determination of the (Na+ + K+)-ATPase from rectal glands of Squalus acanthias.

The membrane-bound (Na+ + K+)-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) system was treated with the nonionic detergent octaethylene-glycoldodecyl ether, yielding a transparent supernatant after centrifugation. The supernatant was highly active with both ATPase and p-nitrophenylphosphatase, with initial specific activities of 2300 mumol Pi released . mg-1 protein. h-1 and 350 mumol p-nitrophenol released.mg-1 protein.h-1, respectively. The supernatant was purified to 95--100%, with respect to the 96 000 dalton and the 56 000 dalton peptides. The solubilized enzyme was gel filtered in Sepharose 4B-Cl and displayed 2 peaks, both with catalytic activity. The low molecular weight particles eluted at Kav = 0.54, corresponding to a molecular weight of approximately 500 000 daltons and the particles had a specific activity of 2100 mumol Pi.mg-1 protein.h-1. Both peaks contained phospholipid with 60 mol phospholipid bound per 300 000 g protein. The low molecular weight particles had a molecular weight of 276 000 as determined by sedimentation equilibrium analysis.     

Purification and properties of cAMP dependent and independent histone kinases from human leukocytes

Summary Histone kinase activity was purified from human polymorphonuclear leukocytes by ammonium sulphate precipitation of a 180 000 × g supernatant, followed by DEAF-cellulose chromatography and gelfiltration. On DEAE-cellulose cAMP dependent kinase activity eluted in two peaks, I and III, at 1.2 mmho and 6.5 mmho, respectively. Catalytic subunit (C) from both peaks had M_r 33 000, 3.0S. Regulatory subunit (R) from peak I and III both had M_r 33 000 upon gelfiltration, but sedimented at 2.8–3.0S and 3.0–3.2S, respectively. R₂ and R₄ subunits were identified. The R-C dimer from peak I and III sedimented at 4.8S and (4.8)–5.1S, respectively. The holoenzyme from peak I had M_r 165 000, 6.7S, which suggest a R₂C₂ structure, while that of peak III sedimented at 6.7S, but eluted at M_r 330 000 (2R₂C₂) by gelfiltration.The K _m ^app for peak I and III enzymes were, respectively: histone IIA 0.5 mg/ml (both forms), ATP 18 m and 23 m, and cAMP 5 × 10^–8 m and 6.3 × 10^–8 m. Both enzymes had pH optimum 6.7–6.9 and were equally sensitive to Ca²⁺ temperature and protein kinase inhibitor. The substrate specificity was histone VS histone IIA = histone VIS casein > phosvitin. Peak I enzyme, but not peak III enzyme, was dissociated by histone and high ionic strength and reassociation of R and C subunits were facilitated by ATP-Mg. It is concluded that peak I and III enzymes represent type I and II cAMP dependent protein kinases, respectively. Type I comprises 20–30% of cAMP dependent protein kinase activity and is absent from the 180 000 × g supernatant of gently disrupted cells.Purified catalytic subunit had K _m ^app (ATP) 20 m with rabbit muscle glycogen synthase I as substrates. Synthase I from rabbit muscle and human leukocytes were phosphorylated by catalytic subunit to synthase D (ratio of independence less than 0.07).cAMP independent histone kinase activity eluted in one peak (Peak II) at 3 mmho. The enzymatic activity sedimented at 3.4S and eluted from gelfiltration with M_r 78 000. K _m ^app for ATP was 78 m and for histone IIA 0.5 mg/ml. The enzyme was sensitive to temperature, but less sensitive than cAMP dependent protein kinase to Ca²⁺, and insensitive to protein kinase inhibitor. The substrate specificity was histone IIA > histone VS = histone VIS, while casein and phosvitin were poor substrates. Glycogen synthase I was not phosphorylated. The cAMP independent histone kinase activity comprised 15% of the total histone kinase activity in a crude homogenate of leukocytes. Its physiological substrate is unknown.Abbreviations AR activity ratio for cAMP dependent protein kinase - cAMP adenosine cyclic 3:5-monophosphate - cIMP inosine cyclic 3:5-monophosphate - cGMP guanosine cyclic 3:5-monophosphate - Glucose-6-P glucose-6-phosphate - DDT dithiothreitol - EGTA ethylene glycol-bis-(-aminoethylether)-N, N-tetraacetic acid - PMSF phenylmethylsulfonylfluoride - PKI protein kinase inhibitor - RI ratio of independence for glycogen synthase - SDS sodium dodecyl sulphate     

Sequential dimerization of human zipcode-binding protein IMP1 on RNA: a cooperative mechanism providing RNP stability

Active cytoplasmic RNA localization depends on the attachment of RNA-binding proteins that dictate the destination of the RNA molecule. In this study, we used an electrophoretic mobility-shift assay in combination with equilibrium and kinetic analyses to characterize the assembly of the human zipcode-binding protein IMP1 on targets in the 3′-UTR from Igf-II mRNA and in H19 RNA. In both cases, two molecules of IMP1 bound to RNA by a sequential, cooperative mechanism, characterized by an initial fast step, followed by a slow second step. The first step created an obligatory assembly intermediate of low stability, whereas the second step was the discriminatory event that converted a putative RNA target into a ‘locked’ stable RNP. The ability to dimerize was also observed between members of the IMP family of zipcode-binding proteins, providing a multitude of further interaction possibilities within RNP granules and with the localization apparatus.     

Nucleotide Binding to Na,K-ATPase: The Role of Electrostatic Interactions.

The contribution of electrostatic forces to the interaction of Na,K-ATPase with adenine nucleotides was investigated by studying the effect of ionic strength on nucleotide binding. At pH 7.0 and 20 degrees C, there was a qualitative correlation between the equilibrium dissociation constant (K(d)) values for ATP, ADP, and MgADP and their total charges. All K(d) values increased with increasing ionic strength. According to the Debye-Hückel theory, this suggests that the nucleotide binding site and its ligands have "effective" charges of opposite signs. However, quantitative analysis of the dependence on ionic strength shows that the product of the effective electrostatic charges on the ligand and the binding site is the same for all nucleotides, and is therefore independent of the total charge of the nucleotide. The data suggest that association of nucleotides with Na,K-ATPase is governed by a partial charge rather than the total charge of the nucleotide. This charge, interacting with positive charges on the protein, is probably the one corresponding to the alpha-phosphate of the nucleotide. Dissociation rate constants measured in complementary transient kinetic experiments were 13 s(-1) for ATP and 27 s(-1) for ADP, independent of the ionic strength in the range 0.1-0.5 M. This implies similar association rate constants for the two nucleotides (about 40 x 10(6) M(-1) s(-1) at I = 0.1 M). The results suggest that long-range Coulombic forces, affecting association rates, are not the main contributors to the observed differences in affinities, and that local interactions, affecting dissociation rates, may play an even greater role.