Phorbol ester and diacylglycerol activation of native protein kinase C species from various tissues

The characteristics of PKC activation induced by a number of compounds were investigated using PKCs, partially-purified from sources with a naturally high abundance of certain Ca²⁺ dependent PKC isoforms. Native isoforms were used rather than PKC isoforms expressed from a baculovirus system to assess the effect of tissue specific factors on activity. However, some data using recombinant PKC were included for comparison.The presence of specific PKC isoforms in different tissues was determined using Western blot analysis. Protein kinase C , ₁, , , and / were all present in rat midbrain cytosolic extract, PKC , ₁, , and / were present in spleen cytosol, and PKC and / were present in COS 7 cell cytosol. The predominance of and activities in COS 7 and spleen extracts respectively was confirmed by enzymic assay.The PKC activity assay was configured such that the Ca²⁺ dependence of the PKC activity induced by different PKC activators could be determined. Phorbol 12,13-dibutyrate (PDBu) was virtually equipotent on the Ca²⁺-dependent PKC activity from midbrain and spleen and slightly less potent on that from COS 7 cells. In the absence of Ca²⁺, PDBu was considerably less potent overall (as, indeed, were the other PKC activators) and was less potent on COS 7 cell PKC than on those from midbrain or spleen. Mezerein was more potent than PDBu at inducing PKC activity in COS 7 cell extracts in either the absence or presence of Ca²⁺ whereas in the presence of Ca²⁺, mezerein was slightly less potent on midbrain and spleen than PDBu and equipotent in the absence of Ca²⁺. Maximum values for Ca²⁺-independent activation by mezerein indicated that this activator was particularly effective in recruiting Ca²⁺-dependent PKC isoform activity in a Ca²⁺ free environment. The greater potency of mezerein on PKC was confirmed using PKC and further purified from rat spleen by hydroxylapatite (HAP) chromatography. The effects of both PDBu and mezerein were investigated using anterior pituitary tissue where a particularly high potency of mezerein in the absence of Ca²⁺ was noted. The diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DOG), appeared to cause little or no activation of native Ca²⁺-dependent isoforms in Ca²⁺ free conditions unlike another longer chain diacylglycerol, 1,2-dioleoyl-sn-glycerol. Also DOG activated midbrain PKCs more potently than PKCs from spleen or COS 7 cells (or lung and pituitary tissue) in the presence of Ca²⁺. The concentration-dependence of DOG was examined on PKC and PKC further purified from brain by HAP chromatography, revealing that DOG was equally potent on both of these isoforms derived from brain and on recombinant PKC . However, [³H]PDBu binding data using PKC purified from several sources gave very different IC₅₀ values when DOG was used as a displacer, and in general these values correlated with the EC₅₀ values recorded from the activity assay.The data presented here indicate that there are distinct differences in the activator pharmacology of different native PKC isoforms and between the same isoform expressed in different tissues, either because of post-translational modifications or some other tissue specific factor.     

Phosphorylation of cardiac junctional and free sarcoplasmic reticulum by PKCα, PKCβ, PKA and the Ca2+/calmodulin-dependent protein kinase

Phosphorylation of cardiac junctional and free sarcoplasmic reticulum (SR) by protein kinase C (PKC) isoforms and was investigated. Both SR and PKC were isolated from canine heart. Junctional and free SR vesicles were prepared by calcium-phosphate-loading. The substrate specificities of PKC and PKC were found to be similar in both SR fractions. A high molecular weight junctionally-associated protein was phosphorylated by PKA, PKC and an endogenous Ca²⁺/calmodulin-dependent protein kinase activity: the highest levels of phosphate incorporation being catalysed by the latter kinase. In addition to this high molecular weight junctionally-associated protein, PKC induced phosphorylation of 45, 96 kDa and several proteins of greater than 200 kDa in junctional SR. A protein of 96 kDa was phosphorylated by both isoforms in junctional and free SR. The major substrate for PKA, PKC, PKC and the Ca²⁺/calmodulin-dependent protein kinase, in both junctional and free SR, was phospholamban. Although the phosphorylation of phospholamban by PKC was activated by Ca²⁺, a component of this activity appeared to be independent of Ca²⁺. PKC-mediated phosphorylation of phospholamban was fully activated by 1 M Ca²⁺ whereas the Ca²⁺/calmodulin dependent kinase required concentrations in excess of 5 M Ca²⁺. In the in vitro system employed in these studies, the concentrations of either PKC or the catalytic subunit of PKA required to phosphorylate phospholamban were found to be similar. In addition, in the presence of a 15 kDa sarcolemmal-associated protein, which becomes phosphorylated upon activation of PKC in vivo, phosphorylation of phospholamban by PKC was unaffected. These results demonstrate that, although substrates for both subtypes are found in both junctional and free SR, PKC and PKC do not show differences in selectivity towards these substrates.Abbreviations Ca²⁺ free calcium - CaM kinase Ca²⁺/calmodulin-dependent protein kinase - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis(b-aminoethylether)-N,N,N,N-tetraacetic acid - FSR free sarcoplasmic reticulum - JSR junctional sarcoplasmic reticulum - PKC protein kinase C - PS phosphatidylserine - SDS sodium dodecyl sulfate - SAG 1-stearoyl-2-arachidonylglycerol - TPCK L-1-tosylamido-2-phenylethyl chloromethyl ketone - Tris/HCI tris(hydroxymethyl)aminomethane hydrochloride This work was supported by a grant (to S.K.) from the Heart and Stroke Foundation of B.C. and Yukon. The costs of publication of this article were defrayed in part by the payment of page charges This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.Recipient of a Studentship form the Heart and Stroke Foundation of Canada.     

Optimized Binding of [35S]GTPγS to Gq-Like Proteins Stimulated with Dopamine D1-Like Receptor Agonists

Subtypes of dopamine D₁-like receptors are coupled through the G proteins Gs or Gq to stimulate either adenylate cyclase or phospholipase C signaling cascades. In the present study, we have uncovered the marked enhancement by sodium deoxycholate of D1-like agonist-stimulated [³⁵S]GTPS binding to Gq-like G proteins in brain membranes, and determined the optimal experimental conditions for assessing agonist effects on [³⁵S]GTPS binding in the presence of the detergent. Factors and their optimal levels that were found to significantly enhance the sensitivity and robustness of the agonist-stimulated [³⁵S]GTPS binding reaction include protein concentration at 40 g/ml, cationic concentrations of 120 mM Na⁺, 1.8 mM K⁺, and 20 mM Mg²⁺, a molar guanine nucleotide ratio of 100,000 GDP to [³⁵S]GTPS, the presence of 1 mM deoxycholate, and an overall incubation duration of 30–120 min. Under the optimized conditions, the D₁-like agonist SKF38393 induced potent and highly efficacious (up to 1000%) stimulation of [³⁵S]GTPS binding in membrane preparations from the striatum and other rat brain regions. In striatal membranes incubated with drug for 2 h, immunoprecipitation of the [³⁵S]GTPS-bound proteins with specific G antibodies showed that at least 70% of SKF38393-stimulated [³⁵S]GTPS binding was to Gq. The present reaction parameters are consistent with conditions previously found to support dopaminergic stimulation of phospholipase C-mediated signaling in brain slice preparations. These results imply that different but equally physiologically relevant conditions can be obtained under which subtypes of dopaminergic receptors may couple preferentially to Gs and the adenylate cyclase pathway or to Gq and the phospholipase C pathway.     

Regulation of electrical coupling between Arabidopsis root hairs

Voltage clamp was used to measure the voltage dependence of cell-to-cell coupling via plasmodesmata between higher-plant cells (root hairs of Arabidopsis thaliana (L.) Heynh.). In addition, ionophoresis was used to introduce a variety of ions [Ca²⁺, inositol-trisphosphate, Li⁺, K⁺, Mg²⁺, ethylene glycol-bis(-aminoethyl ether)-N,N,N, N-tetraacetic acid (EGTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), H⁺, and OH^–] to examine whether they regulate cell-to-cell coupling. Electrical coupling showed high variability in this single cell type at the same developmental stage; the coupling ratio ranged from near 0% to about 90% with a mean value of 32%. It was voltage independent for intracellular voltage gradients (transplasmodesmatal) of -163 to 212 mV. While Ca²⁺ closes the plasmodesmatal connections (at concentrations higher than those causing cessation of cytoplasmic streaming), inositol-trisphosphate and lithium are without effect. Apparently, inositol-trisphosphate may not cause increased cytosolic Ca²⁺ in root hairs. Alkalinization by OH ionophoresis caused a modest decline in cell-to-cell coupling, as did acidification by H⁺ ionophoresis (to an extent causing the cell to become flacid). Increases in cytosolic K⁺, Mg²⁺, and the calcium chelator BAPTA by ionophoresis had no effect on cell-to-cell coupling. The regulation (and lack thereof) reported here for plant plasmodesmata is quite similar to that of gap junctions.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N, N-tetraacetic acid     

Manganese accumulation in yeast cells

Summary Manganese accumulation was studied by room-temperature electron spin resonance (ESR) spectroscopy inSaccharomyces cerevisiae grown in the presence of increasing amounts of MnSO₄. Mn²⁺ retention was nearly linear in intact cells for fractions related to both low-molecular-mass and macromolecular complexes (free and bound Mn²⁺, respectively). A deviation from linearity was observed in cell extracts between the control value and 0.1 mM Mn²⁺, indicating more efficient accumulation at low Mn²⁺ concentrations. The difference in slopes between the two straight lines describing Mn²⁺ retention at concentrations lower and higher than 0.1 mM, respectively, was quite large for the free Mn²⁺ fraction. Furthermore it was unaffected by subsequent dialyses of the extracts, showing stable retention in the form of low-molecular-mass complexes. In contrast, the slope of the line describing retention of bound Mn²⁺ at concentrations higher than 0.1 mM became less steep after subsequent dialyses of the cell extracts. This result indicates that the macromolecule-bound Mn²⁺ was essentially associated with particulate structures. In contrast to Cu²⁺, Mn²⁺ had no effect on the major enzyme activities involved in oxygen metabolism except for a slight increase of cyanide-resistant Mn-superoxide dismutase activity, due to dialyzable Mn²⁺ complexes.     

Ethylene biosynthesis in Fusarium oxysporum f. sp. tulipae proceeds from glutamate/2-oxoglutarate and requires oxygen and ferrous ions in vivo

Liquid cultures of the deuteromycete, Fusarium oxysporum f. sp. tulipae, a tulip pathogen, produced high amounts of ethylene during stationary phase. 1-Aminocyclopropane-1-carboxylic acid, the direct precursor of ethylene in plants, was not present in the fungus. Radioactivity from [3,4-³H]glutamate as well as [U-¹⁴C]glutamate was incorporated into ethylene, indicating that it was derived from C3 and C4 of glutamate or 2-oxoglutarate. Ferrous ions markedly stimulated the rate of ethylene formation in vivo, whereas Fe³⁺, Cu²⁺ or Zn²⁺ had little or no effect. Ethylene biosynthesis was strongly inhibited by the heavy metal chelator ,-dipyridine. The effect of ,-dipyridine was fully reversed by Fe²⁺ ions and partially by Cu²⁺ and Zn²⁺ ions but not by the supply of glutamate or 2-oxoglutarate, suggesting that a step in the ethylene biosynthetic pathway downstream of 2-oxoglutarate is dependent on Fe²⁺. When stationary phase cultures were supplied with arginine, ornithine, or proline, ethylene production increased dramatically while addition of glutamate or 2-oxoglutarate had little effect. Tracer studies were performed to test the possibility that an intermediate in the catabolism of arginine to glutamate was the direct precursor of ethylene. In cultures supplied with [U-¹⁴C]arginine or [U-¹⁴C]glutamate, the specific radioactivity of ethylene was closely similar to the specific radioactivity of the endogenous glutamate pool, indicating that glutamate was on the pathway between arginine and ethylene. An enzyme system converting 2-oxoglutarate to ethylene in a reaction dependent on oxygen, ferrous ions and arginine has previously been described in extracts from Penicillium digitatum (Fukuda et al. 1986). The present results suggest that a similar enzyme system catalyzes the final step of ethylene biosynthesis in F. oxysporum.Non-standard abbreviations AdoMet S-adenosyl methionine - ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene forming enzyme     

Heteronuclear relayed E.COSY applied to the determination of accurate 3J(HN,C′) and 3J(Hβ,C′) coupling constants in Desulfovibrio vulgaris flavodoxin

Summary A simple constant-time 3D heteronuclear NMR pulse sequence has been developed to quantitatively determine the heteronuclear three-bond couplings ³J(H^N,C) and ³J(H,C) in uniformly ¹³C-enriched proteins. The protocols for measuring accurate coupling constants are based on ¹H,¹³C-heteronuclear relayed E.COSY [Schmidt, J.M., Ernst, R.R., Aimoto, S. and Kainosho, M. (1995) J. Biomol. NMR, 6, 95–105] in combination with numerical least-squares spectrum evaluation. Accurate coupling constants are extracted from 2D spectrum projections using 2D multiplet simulation. Confidence intervals for the obtained three-bond coupling constants are calculated from F-statistics. The three-bond couplings are relevant to the determination of and X ₁ dihedral-angle conformations in the amino acid backbone and side chain. The methods are demonstrated on the recombinant ¹³C, ¹⁵N-doubly enriched 147-amino acid protein Desulfovibrio vulgaris flavodoxin with bound flavin mononucleotide in its oxidized form. In total, 109 ³J(H^N,C) and 100 ³J(H,C) coupling constants are obtained from a single spectrum.Abbreviations ANOVA analysis of variances - COSY correlated spectroscopy - E.COSY exclusive correlation spectroscopy - FMN flavin mononucleotide - HMQC heteronuclear multiple-quantum coherence - HSQC heteronuclear single-quantum coherence     

Modulation of Phosphoenolpyruvate Carboxylase Phosphorylation in Leaves of Amaranthus hypochondriacus,a NAD-ME Type of C4 Plant

PEP carboxylase (PEPC) in leaves of C₄ plants is activated by phosphorylation of enzyme by a PEPC-protein kinase (PEPC-PK). We reevaluated the pattern of PEPC phosphorylation in leaf extracts of Amaranthus hypochondriacus. It was dependent on Ca²⁺, the optimum concentration of which for stimulation was 10 mM. The extent of stimulation was inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), a Ca²⁺ chelator. The inhibition by BAPTA was relieved by the addition of Ca²⁺ but not by the addition of Mg²⁺. The stimulation by Ca²⁺ of PEPC phosphorylation was marginally enhanced by calmodulin (CaM), but not by diacylglycerol (DAG). Phosphorylation was strongly restricted by Ca²⁺ or Ca²⁺-CaM-dependent protein kinase inhibitors. Thus phosphorylation of PEPC is Ca²⁺-dependent in leaves of A. hypochondriacus and a calcium-dependent protein kinase (CDPK) may modulate PEPC-PK and subsequently the phosphorylation status of PEPC.     

Structural Specificity for the Inhibitory Effect of Calmodulin on Specific 125I-Omega-Conotoxin GVIA Binding

To clear the structural specificity of calmodulin (CaM) on the specific ¹²⁵I--CTX binding to crude membranes from whole chick brain, the following experiments were investigated in this study: (i) the attenuating effect of semisynthetic tetrahydroisoquinoline derivatives on the inhibitory effect of Ca²⁺/CaM, (ii) the effects of chimeras of yeast and chicken Ca²⁺/CaM, and (iii) the effects of Ca²⁺-binding proteins (such as troponin c, S 100 a and b, and annexin I, III–V). The inhibitory effect of Ca²⁺/CaM was attenuated by isoquinoline derivatives (PX 28, 34, 216, 224, and CPU57) and a CaM antagonist W-7. PX 34, a typical synthesized isoquinoline derivative, showed the attenuating effect in a dose-dependent manner. The ED₅₀ value for the attenuating effect of PX 34 was about 20 M, which is similar to that of W-7 reported previously. Some chimeric CaMs such as YC 51–53 (which are close to the properties of vertebrate CaM) showed a significant inhibitory effect on the specific ¹²⁵I--CTX binding, but YC 129 and 130 (which retain the properties of yeast CaM), troponin c, S100 a, b, and annexin I, III–V had no effect on the specific ¹²⁵I--CTX binding. These results suggest that the characteristic structure containing the EF-hand structure of CaM itself is needed to cause the inhibitory effect on the specific ¹²⁵I--CTX binding.     

The peptidase activity of human serum butyrylcholinesterase: Studies using monoclonal antibodies and characterization of the peptidase

Purified human serum butyrylcholinesterase, which exhibits cholinesterase, aryl acylamidase, and peptidase activities, was cross-reacted with two different monoclonal antibodies raised against human serum butyrylcholinesterase. All three activities were immunoprecipitable at different dilutions of the two monoclonal antibodies. At the highest concentration of the antibodies used, nearly 100% of all three activities were precipitated, and could be recovered to 90–95% in the immunoprecipitate. The peptidase activity exhibited by the purified butyryl-cholinesterase was further characterized using both Phe-Leu and Leu-enkephalin as substrates. ThepH optimum of the peptidase was in the range of 7.5–9.5 and the divalent cations Co²⁺, Mn²⁺, and Zn²⁺ stimulated its activity. EDTA and other metal complexing agents inhibited its activity. Thiol agents and -SH group modifiers had no effect. The serine protease inhibitors, diisopropylfluorophosphate and phenyl methyl sulfonyl fluoride, did not inhibit. When histidine residues in the enzyme were modified by diethylpyrocarbonate, the peptidase activity was not affected, but the stimulatory effect of Co²⁺, Mn²⁺, and Zn²⁺ disappeared, suggesting the involvement of histidine residues in metal ion binding. These general characteristics of the peptidase activity were also exhibited by a 50 kD fragment obtained by limited -chymotrypsin digestion of purified butyrylcholinesterase. Under all assay conditions, the peptidase released the two amino acids, leucine and phenylalanine, from the carboxy terminus of Leu-enkephalin as verified by paper chromatography and HPLC analysis. The results suggested that the peptidase behaved like a serine, cysteine, thiol-independent metallopeptidase.     

Mitochondria in Ca2+ Signaling and Apoptosis

Cellular Ca²⁺ signals are crucial in the control of most physiological processes, cell injuryand programmed cell death; mitochondria play a pivotal role in the regulation of such cytosolicCa²⁺ ([Ca²⁺]_c) signals. Mitochondria are endowed with multiple Ca²⁺ transport mechanismsby which they take up and release Ca²⁺ across their inner membrane. These transport processesfunction to regulate local and global [Ca²⁺]_c, thereby regulating a number of Ca²⁺-sensitivecellular mechanisms. The permeability transition pore (PTP) forms the major Ca²⁺ effluxpathway from mitochondria. In addition, Ca²⁺ efflux from the mitochondrial matrix occursby the reversal of the uniporter and through the inner membrane Na⁺/Ca²⁺ exchanger. Duringcellular Ca²⁺ overload, mitochondria take up [Ca²⁺]_c, which, in turn, induces opening of PTP,disruption of mitochondrial membrane potential (_m) and cell death. In apoptosis signaling,collapse of ;_m and cytochrome c release from mitochondria occur followed by activationof caspases, DNA fragmentation, and cell death. Translocation of Bax, an apoptotic signalingprotein from the cytosol to the mitochondrial membrane, is another step during thisapoptosis-signaling pathway. The role of permeability transition in the context of cell death in relationto Bcl-2 family of proteins is discussed.     

Determination of 13Cα relaxation times in uniformly 13C/15N-enriched proteins

Summary Relaxation times of ¹³C carbons of uniformly ¹³C/¹⁵N-enriched probes have been investigated. The relaxation behaviour was analyzed in terms of a multispin system. Pulse sequences for the determination of T₁, T₂ and the heteronuclear NOE of ¹³C in uniformly ¹³C/¹⁵N-enriched ribonuclease T1 are presented. The experiments performed in order to obtain T₁ and the heteronuclear NOE were similar to those of the corresponding ¹⁵N experiments published previously. The determination of T₂ for the C-carbon in a completely labeled protein is more complicated, since the magnetization transfer during the T₂ evolution period owing to the scalar coupling of C–C must be suppressed. Various different pulse sequences for the T₂ evolution period were simulated in order to optimize the bandwidth for which reliable T₂ relaxation times can be obtained. A proof for the quality of these pulse sequences is given by fitting the intensity decay of individual ¹H–¹³C cross peaks, in a series of (¹H, ¹³C)-ct-HSQC spectra with a modified CPMG sequence as well as a T_1p sequence for the transverse relaxation time, to a single exponential using a simplex algorithm.     

Characterization of NO adducts of the diiron center in protein R2 of<Emphasis Type="Italic"> Escherichia coli</Emphasis> ribonucleotide reductase and site-directed variants; implications for the O<Subscript>2 </Subscript>activation mechanism*

The R2 subunit of Escherichia coli ribonucleotide reductase contains a diiron site that reacts with O₂ to produce a tyrosine radical (Y122·). In wild-type R2 (R2-wt), the first observable reaction intermediate is a high-valent [Fe^III-Fe^IV] state called compound X, but in related diiron proteins such as methane monooxygenase, ⁹-desaturase, and ferritin, peroxodiiron(III) complexes have been characterized. Substitution of iron ligand D84 by E within the active site of R2 allows an intermediate (-1,2-peroxo)diiron species to accumulate. To investigate the possible involvement of a bridging peroxo species within the O₂ activation sequence of R2-wt, we have characterized the iron-nitrosyl species that form at the diiron sites in R2-wt, R2-D84E, and R2-W48F/D84E by using vibrational spectroscopy. Previous work has shown that the diiron center in R2-wt binds one NO per iron to form an antiferromagnetically coupled [{FeNO}⁷]₂ center. In the wt and variant proteins, we also observe that both irons bind one NO to form a {FeNO}⁷ dimer where both Fe–N–O units share a common vibrational signature. In the wt protein, (Fe–NO), (Fe–N–O), and (N–O) bands are observed at 445, 434 and 1742 cm^–1, respectively, while in the variant proteins the (Fe–NO) and (Fe–N–O) bands are observed ~10 cm^–1 higher and the (N–O) ~10 cm^–1 lower at 1735 cm^–1. These results demonstrate that all three proteins accommodate fully symmetric [{FeNO}⁷]₂ species with two identical Fe–N–O units. The formation of equivalent NO adducts in the wt and variant proteins strongly favors the formation of a symmetric bridging peroxo intermediate during the O₂ activation process in R2-wt.     

Internal motions of apo-neocarzinostatin as studied by 13C NMR methine relaxation at natural abundance

Summary Dynamics of the backbone and some side chains of apo-neocarzinostatin, a 10.7 kDa carrier protein, have been studied from ¹³C relaxation rates R1, R2 and steady-state ¹³C-{¹H} NOEs, measured at natural abundance. Relaxation data were obtained for 79 nonoverlapping C resonances and for 11 threonine C single resonances. Except for three C relaxation rates, all data were analysed from a simple two-parameter spectral density function using the model-free approach of Lipari and Szabo. The corresponding C–H fragments exhibit fast (_e < 40 ps) restricted libration motions (S²=0.73 to 0.95). Global examination of the microdynamical parameters S² and _e along the amino acid sequence gives no immediate correlation with structural elements. However, different trends for the three loops involved in the binding site are revealed. The -ribbon comprising residues 37 to 47 is spatially restricted, with relatively large _e values in its hairpin region. The other -ribbon (residues 72 to 87) and the large disordered loop ranging between residues 97–107 experience small-amplitude motions on a much faster (picosecond) time scale. The two N-terminal residues, Ala¹ and Ala², and the C-terminal residue Asn¹¹³, exhibit an additional slow motion on a subnanosecond time scale (400–500 ps). Similarly, the relaxation data for eight threonine side-chain C must be interpreted in terms of a three-parameter spectral density function. They exhibit slower motions, on the nanosecond time scale (500–3000 ps). Three threonine (Thr⁶⁵, Thr⁶⁸, Thr⁸¹) side chains do not display a slow component, but an exchange contribution to the observed transverse relaxation rate R2 could not be excluded at these sites. The microdynamical parameters (S², _e and R2_ex) or (S _infslow ^sup2 , S _inffast ^sup2 and _slow) were obtained from a straightforward solution of the equations describing the relaxation data. They were calculated assuming an overall isotropic rotational correlation time _e for the protein of 5.7 ns, determined using standard procedures from R2/R1 ratios. However, it is shown that the product (1–S²)× _e is nearly independent of _e for residues not exhibiting slow motions on the nanosecond time scale. In addition, this parameter very closely follows the heteronuclear NOEs, which therefore could be good indices for local fast motions on the picosecond time scale.     

Measurement of the protein backbone dihedral angle φ based on quantification of remote CSA/DD interference in inter-residue 13C′(i − 1)-13Cα(i) multiple-quantum coherences

A novel triple-resonance NMR method is presented for the measurement of the protein backbone dihedral angle based on differential multiple-quantum relaxation induced by relaxation interference between ¹H(i)-¹³C(i) dipolar and ¹³C(i–1) (carbonyl) chemical shift anisotropy mechanisms. The method employs a simultaneous transfer of ¹⁵N magnetization to the inter- and intra-residue ¹³C carbons as well as the directly attached carbonyl carbon ¹³C. Results obtained on ¹³C,¹⁵N-labeled ubiquitin demonstrate the potential of the method.     

Characterization of vanadium-binding sites of the vanadium-binding protein Vanabin2 by site-directed mutagenesis

Background

Vanabins are a unique protein family of vanadium-binding proteins with nine disulfide bonds. Possible binding sites for VO²⁺ in Vanabin2 from a vanadium-rich ascidian Ascidia sydneiensis samea have been detected by nuclear magnetic resonance study, but the metal selectivity and metal-binding ability of each site was not examined.

Methods

In order to reveal functional contribution of each binding site, we prepared several mutants of Vanabin2 by in vitro site-directed mutagenesis and analyzed their metal selectivity and affinity by immobilized metal-ion affinity chromatography and Hummel Dreyer method.

Results

Mutation at K10/R60 (site 1) markedly reduced the affinity for VO²⁺. Mutation at K24/K38/R41/R42 (site 2) decreased the maximum binding number, but only slightly increased the overall affinity for VO²⁺. Secondary structure of both mutants was the same as that of the wild type as assessed by circular dichroism spectroscopy. Mutation in disulfide bonds near the site 1 did not affect its high affinity binding capacity, while those near the site 2 decreased the overall affinity for VO²⁺.

General significance

These results suggested that the site 1 is a high affinity binding site for VO²⁺, while the site 2 composes a moderate affinity site for multiple VO²⁺.     

Oligomerization reactions of ribonucleotides: The reaction of the 5′ -phosphorimidazolide of adenosine with diadenosine pyrophosphate on montmorillonite and other minerals

The reaction of the 5 -phosphorimidazolide of adenosine (5-ImpA) with diadenosine pyrophosphate (A⁵ppA) in the presence of Na⁺-montmorillonite in aqueous, pH 8 solution results in the regiospecific formation of A⁵ppA³pA and A⁵ppA³pA³ pA. The formation of oligomers of general structure (pA)n decreases in the presence of A⁵ppA. A⁵ppA³pA is the principal reaction product when a 1:1 ratio of ImpA and A⁵ppA is used. The yield of A⁵ppA³pA³pA is optimal when 9:1 or 4:1 ratios of ImpA: A⁵ppA are used. The overall regiospecificity of formation of 3,5-links is about 80%. The reaction between ImpA and A⁵ppA on montmorillonite differs from the self-condensation of ImpA in that it proceeds in the absence of Mg²⁺ and there are only small differences in oligomer yields when Na⁺, Li⁺ Ca²⁺, and NH ₄ ⁺ are the exchangeable cations on the montmorillonite. The reaction is inhibited by 0.4 M imidazole but the inhibition is suppressed with 0.4 M Mg²⁺. Little or no phosphodiester bond formation was observed with Mg²⁺- or Al³⁺-montmorillonite. Montmorillonites other than 22A and Volclay exhibited no catalysis for the formation of adducts between ImpA and A⁵ppA and no catalysis was exhibited in ferrugenous smectite, nontronite, allophane, or sepiolite.     

Turgor regulation and cytoplasmic free Ca2+ in the algaLamprothamnium

Summary In internodal cells ofLamprothamnium succinctum, turgor regulation in response to hypotonie treatment is inhibited by lowering external Ca²⁺ concentration ([Ca²⁺]_e) from 3.9 (normal) to 0.01 (low) mM. In order to clarify whether a change in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_c) is involved in turgor regulation, the Ca²⁺ sensitive protein aequorin was injected into the cytoplasm of internodal cells. A large transient light emission was observed upon hypotonic treatment under normal [Ca²⁺]_e but not under low [Ca²⁺]_e. Thus hypotonic treatment induces a transient increase in [Ca²⁺]_c under normal [Ca²⁺]_e but not under low [Ca²⁺]_e.Abbreviations ASW artificial sea water - _i cellular osmotic pressure - [Ca²⁺]_c cytoplasmic free Ca²⁺ concentration - EDTA ethylenediamine-tetraacetic acid - EGTA ethylenglycol-bis(-aminoethyl ether(N,N-tetraacetic acid - [Ca²⁺]_e external Ca²⁺ concentration - _e external osmotic pressure - GM glass micropipette - GP glass plate - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid - MS microscope stage - OL objective lens - PIPES piperazine-N-N-bis(2-ethanesulfonic acid) - W Weight     

Calcium-dependent protein kinase from apple fruit membranes is calmodulin-independent but has calmodulin-like properties

Crude Ca²⁺-activated protein kinase from membranes of apple (Malus domestica L. Borkh., Cox's Orange Pippin) fruit can be partially purified to yield a Ca²⁺-dependent protein kinase whose activity is apparently not regulated by calmodulin. The autophosphorylating catalytic subunit of this protein kinase shows a Ca²⁺-dependent mobility shift of approx. 10 kilodaltons (kDa) on sodium dodecyl sulphate-polyacrylamide gel electrophoresis; in the absence of added Ca²⁺ or ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) its apparent molecular mass is approx. 50 kDa. The Ca²⁺-dependent protein kinase is inhibited by the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide and trifluoperazine with IC₅₀ values of approx. 45 M and 15 M, respectively. These similarities between the protein kinase and calmodulin indicate that the kinase may be a calmodulin-like protein.Abbreviations DEAE diethylaminoethyl - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(-2-hydroxyethyl)-1-piperazineethanesulphonic acid - kDa kilodalton - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - W7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide - W5 N-(6-aminohexyl)-naphthalenesulphonamide