Molecular properties of voltage-gated K+ channels

Subfamilies of voltage-activated K⁺ channels (Kv1-4) contribute to controlling neuron excitability and the underlying functional parameters. Genes encoding the multiple subunits from each of these protein groups have been cloned, expressed and the resultant distinct K⁺ currents characterized. The predicted amino acid sequences showed that each subunit contains six putative membrane-spanning -helical segments (S1-6), with one (S4) being deemed responsible for the channels' voltage sensing. Additionally, there is an H5 region, of incompletely defined structure, that traverses the membrane and forms the ion pore; residues therein responsible for K⁺ selectivity have been identified. Susceptibility of certain K⁺ currents produced by the Shaker-related subfamily (Kv1) to inhibition by -dendrotoxin has allowed purification of authentic K⁺ channels from mammalian brain. These are large (M_r 400 kD), octomeric sialoglycoproteins composed of and subunits in a stoichiometry of ()₄()₄, with subtypes being created by combinations of subunit isoforms. Subsequent cloning of the genes for ₁, ₂ and ₃ subunits revealed novel sequences for these hydrophilic proteins that are postulated to be associated with the subunits on the inner side of the membrane. Coexpression of ₁ and Kv1.4 subunits demonstrated that this auxiliary protein accelerates the inactivation of the K⁺ current, a striking effect mediated by an N-terminal moiety. Models are presented that indicate the functional domains pinpointed in the channel proteins.     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

Analogues of NADP+ as inhibitors and coenzymes for NADP+ malic enzyme from maize leaves

Structural analogues of the NADP⁺ were studied as potential coenzymes and inhibitors for NADP⁺ dependent malic enzyme from Zea mays L. leaves. Results showed that 1, N⁶-etheno-nicotinamide adenine dinucleotide phosphate ( NADP⁺), 3-acetylpyridine-adenine dinucleotide phosphate (APADP⁺), nicotinamide-hypoxanthine dinucleotide phosphate (NHDP⁺) and -nicotinamide adenine dinucleotide 2: 3-cyclic monophosphate (23NADPc⁺) act as alternate coenzymes for the enzyme and that there is little variation in the values of the Michaelis constants and only a threefold variation in V_max for the five nucleotides. On the other hand, thionicotinamide-adenine dinucleotide phosphate (SNADP⁺), 3-aminopyridine-adenine dinucleotide phosphate (AADP⁺), adenosine 2-monophosphate (2AMP) and adenosine 2: 3-cyclic monophosphate (23AMPc) were competitive inhibitors with respect to NADP⁺, while -nicotinamide adenine dinucleotide 3-phosphate (3NADP⁺), NAD⁺, adenosine 3-monophosphate (3AMP), adenosine 2: 5-cyclic monophosphate (25AMPc), 5AMP, 5ADP, 5ATP and adenosine act as non-competitive inhibitors. These results, together with results of semiempirical self-consistent field-molecular orbitals calculations, suggest that the 2-phosphate group is crucial for the nucleotide binding to the enzyme, whereas the charge density on the C₄ atom of the pyridine ring is the major factor that governs the coenzyme activity.Abbreviations NADP⁺ 1, N⁶-etheno-nicotinamide adenine dinucleotide phosphate - NHDP⁺ nicotinamide-hypoxanthine dinucleotide phosphate - APADP⁺ 3-acetylpyridine-adenine dinucleotide phosphate - SNADP⁺ thionicotinamide-adenine dinucleotide phosphate - AADP⁺ 3-aminopyridine-adenine dinucleotide phosphate - 23NADPc⁺ -nicotinamide adenine dinucleotide 2: 3-cyclic monophosphate - 3NADP⁺ -nicotinamide adenine dinucleotide 3-phosphate - 2AMP adenosine 2-monophosphate - 3AMP adenosine 3-monophosphate - 23AMPc adenosine 2: 3 monophosphate cyclic - A adenosine - RuBP ribulose 1,5-bisphosphate - SCF-MO Self-Consistent Field-Molecular Orbitals (method)     

Theoretical stability diagram of solvent-containing black lipid films

Recently, we have developed an analytical, semi-microscopic theory for the macroscopic behavior of a solvent-containing black lipid film subjected to an electric cross film voltage, . Here we employ the theoretical expressions derived for the disjoining pressure, _D, the film elasticity, _F, and the film tension, _F, to construct the stability diagram of the film, in the _D-. Depending on its state (_D, ), the film is stable or is prone to squeezing or bending deformations. For a monooleate film we show how the destruction of the plane film due to a periodic thickness fluctuation (squeezing) is facilitated by two mechanisms: i) lowering of _D at fixed ; ii) lowering of at fixed _D, provided that the film is in a stable state characterized by _D<–7.03×10³ dyne/cm² and >0 mV. Bending of a low tension film (single interface tension _s 0.025 dyne/cm¹) can be achieved only for >170 mV and _D > –8.7 × 10⁴ dyne/cm². Finally, we demonstrate the existence of a marginal state ( _D ⁰ , ⁰) where the film is predicted to exhibit strong fluctuations both in the squeezing and in the bending mode.     

Characteristics of Cl−-dependentl-[35S]cysteic acid transport into rat brain synaptic membrane vesicles

Uptake ofl-[³⁵S]cysteic acid (L-CA) in rat synaptic membrane vesicles was investigated. Preincubation with either 10 mMl-glutamic acid (L-Glu), 25 mM L-CA, 10 mMdl-homocysteic acid, or 25 mMdl-2-amino-4-phosphonobutyrate on membrane vesicles enhanced L-[³⁵S]CA and L-[³H]Glu uptake. Na⁺ (5 mM) and omission of Cl^– from the assay medium decreased L-[³⁵S]CA uptake into both 10 mM L-Glu-loaded and non-loaded membrane vesicles. The anion transport blockers, 4-acetamide-4-isothiocyano-2,2-disulfonic acid stibene (SITS) and 4,4-diisothiocyano-2,2-disulfonic acid stilbene (DIDS), inhibited L-[³⁵S]CA uptake in a dose-dependent manner. The maximal uptake rate for L-[³⁵S]CA was decreased by 50 M SITS, while the apparent Km value of L-CA was not changed. SITS increased the EC₅₀ value of Cl^– for L-[³⁵S]CA uptake from 5 mM to 10 mM with reduction of the maximal effect. These results suggested that L-[³⁵S]CA uptake into synaptic membrane vesicles was mediated by a SITS-sensitive hetero-exchange transport with non-labeled substrates.Abbreviations SITS 4-Acetamide-4-isothiocyano-2,2-disulfonic acid stilbene - DIDS 4,4-Diisothiocyano-2,2-disulfonic acid stilbene - CA Cysteic acid - APB 2-Amino-4-phosphonobutyrate - CSA Cysteine sulfinic acid - EGTA Ethyleneglycol bis(aminoethylether) tetraacetate - GABA -Aminobutyric acid     

Aminoglycoside blockade of Ca2+-activated K+ channel from rat brain synaptosomal membranes incorporated into planar bilayers

Summary Ca²⁺-activated K⁺ channels from rat brain synaptosomal membranes were incorporated into planar lipid bilayers, and the effects of aminoglycoside antibiotics on the single channel conductance (258±13 pS at 100mm K⁺) were investigated. Aminoglycosides reduced the single channel conductance from the cis (cytoplasmic) side in a dose- and voltage-dependent manner. Voltage dependence of the blockade indicated an interaction between positively charged amino residues of aminoglycoside antibiotics and a binding site located within the electric field of the ion-conducting pathway. The order of blocking potency was consistent with that of the number of amino residues of aminoglycosides (neomycin (6)>dibekacin (5)>ribostamycin (4)=kanamycin (4)), while the electrical distance (z=0.46–0.49) of the binding site kept almost constant for each drug. Thesezs were almost the same with those (0.46–0.51) of alkyldiamine blockers with two amino residues (total net charge of +2) and approximately twice of those (0.25–0.26) of alkylmonoamine blockers (total net charge of +1). Assuming that amino residues of aminoglycosides and alkylamines shared the same binding site located at 25% voltage drop from the cytoplasmic surface of the channel, the site would have to be at least large enough to accommodate one diamino sugar residue of the aminoglycoside in order to simultaneously interact with two positively charged amino groups. Dose- and voltage-dependent blockade of the channel by gallamine, an extremely bulky trivalent organic cation, supported the picture that the channel has a wide mouth on the cytoplasmic side and its pore region, where voltage drop occurs, may also be quite wide and nonselective, suddenly tapering to a constriction where most charged cations block the channel by occluding the K⁺-conducting pathway.     

Comparative Analysis of the Mechanisms Underlying Nifedipine-Induced Blockade of Three Subtypes of T-Type Ca2+ Channels

We analyzed the effects of nifedipine on a family of recombinant low-threshold Ca²⁺ channels functionally expressed in Xenopus oocytes and formed by three different subunits (1G, 1H, and 1I). The 1G and 1I channels demonstrated a low sensitivity to nifedipine even in high concentrations (IC₅₀ = 98 and 243 M, maximum blocking intensity A_max = 25 and 47%, respectively). At the same time, the above agent effectively blocked channels formed by the 1H-subunit (IC₅₀ = 5 M and A_max = 41%). The nifedipine-caused effects were voltage-dependent, and their changes depended on the initial state of the channel. In the case of 1G-subunits, the blockade was determined mostly by binding of nifedipine with closed channels, whereas in the cases of 1H- and 1I-subunits this resulted from binding of nifedipine with channels in the activated and inactivated states. The obtained data allow us to obtain estimates of the pharmacological properties of the above three subtypes of recombinant channels and, in the future, to compare these characteristics with the properties of low-threshold Ca²⁺ channels in native cells.     

Expression of Bacillus thuringiensis full-length and N-terminally truncated vip184 gene in an acrystalliferous strain of subspecies kurstaki

Vip3A is an 89-kDa protein secreted by Bacillus thuringiensis during vegetative growth. The 3.5 kb full-length vip184 gene was cloned from a wild-type isolate of B. thuringiensis, and the vip184S gene was constructed by deletion of the putative signal peptide encoding sequence. Both genes were expressed in the acrystalliferous strain Cry^–B of B. thuringiensis. Vip184 protein was observed mainly in the centrifuged pellets of B. thuringiensis Cry^–B(pHPT3), which contains the vip184 gene, and was less abundant in the concentrated supernatant. However, Vip184S proteins were not detected in the concentrated supernatant, but only in the pellets of Cry^–B(pHPT3S), which contains vip184S gene. This indicated that Vip184S proteins were not secreted into the culture medium and that the putative signal peptides were essential for the secretion of Vip184. The toxicity of Cry^–B(pHPT3) and Cry^–B(pHPT3S) were demonstrated against the neonate larvae of Spodoptera exigua and S. litura. Pellets and concentrated supernatant of Cry^–B(pHPT3) showed high activity against S. exigua and S. litura, but the Cry^–B(pHPT3S) strain was not toxic to either because of the deletion of N-terminal putative signal peptides. Therefore, this may suggest that the putative signal peptides are required for lethality.     

A novel PH-CT-COSY methodology for measuring JPH coupling constants in unlabeled nucleic acids. Application to HIV-2 TAR RNA

A quantitative analysis of J_PH scalar couplings in nucleic acids is difficult due to small couplings to phosphorus, the extreme overlap of the sugar protons and the fast relaxation of the spins involved in the magnetization transfer. Here we present a new methodology that relies on heteronuclear Constant Time Correlation Spectroscopy (CT-COSY). The three vicinal ³J_PH3, ³J_PH5 and ³J_PH5 scalar couplings can be obtained by monitoring the intensity decay of the P_i-H3_{i – 1} peak as a function of the constant time T in a 2D correlation map. The advantage of the new method resides in the possibility of measuring the two ³J_PH5 and ³J_PH5 scalar couplings even in the presence of overlapped H5/H5 resonances, since the quantitative information is extracted from the intensity decay of the P-H3 peak. Moreover, the relaxation of the H3 proton is considerably slower than that of the H5/H5 geminal protons and the commonly populated conformations of the phosphate backbone are associated with large ³J_PH3 couplings and relatively small ³J_{PH5 / H5}. These two facts lead to optimal signal-to-noise ratio for the P-H3 correlation compared to the P-H5/H5 correlation.The heteronuclear CT-COSY experiment is suitable for oligonucleotides in the 10–15 kDa molecular mass range and has been applied to the 30mer HIV-2 TAR RNA. The methodology presented here can be used to measure P-H dipolar couplings (D_PH) as well. We will present qualitative results for the measurement of P-H_base and P-H2 dipolar couplings in the HIV-2 TAR RNA and will discuss the reasons that so far precluded the quantification of the D_PHs for the 30mer RNA.     

Facile synthesis of 2′-substituted lactoses

Isopropylidenation of lactose with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid gave two products, which were identified by¹H- and¹³C-NMR as 2,35,63,4-tri-O-isopropylidenelactose dimethyl acetal (1) and its 6-O-(2-methoxy)-isopropyl derivative (2). These products were used for the synthesis of 2-O-methyllactose (7), 2,6-di-O-methyllactose (9) and 2-O-benzyllactose (13).     

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.     

Is haemoglobin Gα Philadelphia linked to α-thalassaemia?

Summary The question, Is Hb G Philadelphia linked to -thalassaemia? was first posed because the abnormal haemoglobin is found in heterozygotes at a concentration greater than 25%, the proportion predicted from a 4 -chain gene model. Globin chain biosynthesis was studied in a West Indian family in which one parent had ⁺ thalassaemia and the other was heterozygous for the G Philadelphia chain gene. The former had a globin chain production ratio / well above 1, while the latter had a ratio significantly less than 1. One child of the marriage had inherited the ⁺ thallassaemia from one parent and the G Philadelphia chain gene from the other and showed the typical picture of /-thalassaemia (/ ratio slightly above normal). It is explained in the discussion that the evidence favours a close linkage of 2 -chain genes.     

Conformation of the circular dumbbell d〈pCGC-TT-GCG-TT〉: Structure determination and molecular dynamics

Summary The circular DNA decamer 5-dpCGC-TT-GCG-TT-3 was studied in solution by means of NMR spectroscopy and molecular dynamics in H₂O. At a temperature of 269 K, a 50/50 mixture of two dumbbell structures (denoted L2L2 and L2L4) is present. The L2L2 form contains three Watson-Crick C-G base pairs and two two-residue loops in opposite parts of the molecule. On raising the temperature from 269 K to 314 K, the L2L4 conformer becomes increasingly dominant (95% at 314 K). This conformer has a partially disrupted G(anti)-C(syn) closing base pair in the 5-GTTC-3 loop with only one remaining (solvent-accessible) hydrogen bond between NH of the cytosine dC(1) and O6 of the guanine dG(8). The opposite 5-CTTG-3 loop remains stable. The two conformers occur in slow equilibrium (rate constant 2–20 s^–1). Structure determination of the L2L2 and L2L4 forms was performed with the aid of a full relaxation matrix approach (IRMA) in combination with restrained MD. Torsional information was obtained from coupling constants. Coupling constant analysis (³J_HH, ³J_HP, ³J_CP) gave detailed information about the local geometry around backbone torsion angles , , and , revealing a relatively high flexibility of the 5-GTTC-3 loop. The values of the coupling constants are virtually temperature-independent. Weakly constrained molecular dynamics in solvent was used to sample the conformational space of the dumbbell. The relaxation matrices from the MD simulation were averaged over r^–3 to predict dynamic NOE volumes. In order to account for the 1:1 conformational mixture of L2L2 and L2L4 present at 271 K, we also included S² factors and r^–6 averaging of the r^–3-averaged relaxation matrices. On matrix averaging, the agreement of NOE volumes with experiment improved significantly for protons located in the thermodynamically less stable 5-GTTC-3 loop. The difference in stability of the 5-CTTG-3 and 5-GTTC-3 loops is mainly caused by differences in the number of potential hydrogen bonds in the minor groove and differences in stacking overlap of the base pairs closing the minihairpin loops. The syn conformation for dC(1), favored at high temperature, is stabilized by solvation in the major groove. However, the conformational properties of the dC(1) base, as deduced from R-factor analysis and MD simulations, include a large flexibility about torsion angle .     

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.     

DeoxyNAD and deoxyADP-ribosylation of proteins

Recently, two deoxyribose analogs of NAD⁺ (2-deoxy and 3-deoxyNAD⁺) have been synthesized and purified in this laboratory. Whereas 2-deoxyNAD⁺ was an efficient substrate for arg-specific mon(ADP-ribosyl) transferases, it was not a substrate for poly(ADP-ribose) polymerase (PARP). Instead, it was a non-competitive inhibitor of NAD⁺ in the ADP-ribose polymerization reaction catalyzed by PARP. Thus, 2-deoxyNAD⁺ has been utilized to distinguish between mono(ADP-ribose) and poly(ADP-ribose) acceptor proteins. 2-deoxyNAD⁺ has also been used to characterize the arg-specific mono(2-deoxyADP-ribosyl)ation reaction of PARP with cholera toxin or avian mono(ADP-ribosyl)transferase. By contrast, 3-deoxyNAD⁺ can effectively be utilized as a substrate by PARP. However, while the estimated Km and Kcat of polymerization with 3-deoxyNAD⁺ can were 20 M and 0.11 moles/sec, the Km and Kcat with NAD⁺ as a substrate were 59 M and 1.29 moles/sec, respectively. Determination of the average size of 3-deoxyADP-ribose polymers indicated that chains no larger than four residues are synthesized with this substrate. Thus, the utilization of 3-deoxyNAD⁺ has facilitated the electrophoretic identification of poly(ADP-ribose) acceptor proteins in mammalian chromatin.     

Prevalence and molecular heterogeneity of alfa+thalassemia in two tribal populations from Andhra Pradesh,India

Summary We describe here the screening of a small group of apparently healthy individuals belonging to the tribal communities of Koya Dora and Konda Reddi. A remarkably high incidence of deletion and nondeletion ⁺ thalassemia mutants has been found with allele frequencies and distributions characteristic to each tribe. We have confirmed the strict relationship between Hb S levels and the number of globin genes in double heterozygotes for the S gene and thalassemia. In this population sample we did not find either heterozygous carriers of ⁰ thalassemia (deletion of both alpha genes in cis) or individuals showing hemolytic anemia due to inactivation of three -globin genes (Hb H disease). Selection by malaria is most probably responsible for the prevalence of the various ⁺ thalassemia haplotypes among the two tribal populations of Andhra Pradesh.     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

The activity of CK2 in the extracts of COS-7 cells transfected with wild type and mutant subunits of protein kinase CK2

Protein kinase CK2 is ubiquitous in eukaryotes and is known to phosphorylate many protein substrates. The enzyme is normally a heterotetramer composed of catalytic ( and ) and regulatory () subunits. The physiological regulation of the enzyme is still unknown but one of the factors that may play an important role in this regulation is the ratio of the catalytic and regulatory subunits present in cells. The possible existence of free CK2 subunits, not forming part of the holoenzyme, may be relevant to the physiological function of the enzyme in substrate selection or in the interaction of the subunits with other partners. The objective of this work was to study in COS-7 cells the effects of transient expression of CK2 subunits and mutants of the catalytic subunit on the CK2 phosphorylating activity of the extracts of these cells. Using pCEFL vectors that introduce hemaggutinin (HA) or a heptapeptide (AU5) tags in the expressed proteins, COS-7 cells were transfected with and subunits of Xenopus CK2, with the subunit of D. rerio, and with Xl CK2A¹⁵⁶, which although inactive can bind tightly to CK2, and with Xl CK2E⁷⁵E⁷⁶, which is resistant to heparin and polyanion inhibition. The efficiency of transient transfection was of 10–20% of treated cells.Expression of CK2 or CK2E⁷⁵E⁷⁶ in COS-7 cells caused an increase of 5–7-fold of the CK2 activity in the soluble cell extracts. If these catalytic subunits were cotransfected with CK2, the activity increased further to 15–20-fold of the controls. Transfection of CK2 alone also increase the activity of the extracts about 2-fold. Transfection with the inactive CK2A¹⁵⁶ yielded extracts with CK2 activities not significantly different from those transfected with the empty vectors. However, cotransfection of CK2 or CK2E⁷⁵E⁷⁶ with CK2A¹⁵⁶ caused a 60–70% decrease in the CK2 activity as compared to those of cells transfected with only the active CK2 subunits. These results can be interpreted as meaning that CK2A¹⁵⁶ is a dominant negative mutant that can compete with the other catalytic subunits for the CK2 subunit. Addition of recombinant CK2 to the assay system of extracts of cells transfected with catalytic subunits causes a very significant increase in their CK2 activity, demonstrating that CK2 subunit is limiting in the extracts and that an excess of free CK2 has been produced in the transfected cells. Transfection of cells with CK2E⁷⁵E⁷⁶ results in a CK2 activity of extracts that is 90% resistant to heparin demonstrating that a very large proportion of the CK2 activity is derived from the expression of the exogenous mutant. In both the in vivo and in vitro systems, the sensitivity of CK2E⁷⁵E⁷⁶ to heparin increases considerably when it forms part of the holoenzyme CK2₂₂.     

Stable isotope and radiocarbon compositions of methane emitted from tropical rice paddies and swamps in Southern Thailand

Stable isotopes (¹³C, D) and radiocarbon weremeasured in methane bubbles emitted from rice paddies and swamps in southernThailand. Methane emitted from the Thai rice paddies was enriched in¹³C (mean ¹³C; –51.5 ±7.1 and–56.5 ± 4.6 for mineral soil and peat soil paddies,respectively)relative to the reported mean value of methane from temperate rice paddies(– 63 ± 5). Large seasonal variation was observed in¹³C(32) in the rice paddies, whereas variationinD was much more smaller (20), indicating that variation in¹³C is due mainly to changes in methane production pathways.Values of ¹³C were lower in swamps (–66.1 ±5.1)than in rice paddies. The calculated contribution of acetate fermentation from¹³C value was greater in rice paddies (mineral soils:62–81%, peat soils: 57–73%) than in swamps (27–42%). Din methane from Thai rice paddies (–324± 7 (n=46)) isrelativelyhigher than those from 14 stations in Japanese rice paddies ranging from–362 ± 5 (Mito: n=2) to –322 ± 8(Okinawa: n=3), due tohigher D in floodwaters. ¹⁴C content in methane produced fromThai rice paddies (127±1 pMC) show higher ¹⁴Cactivity compared with previous work in paddy fields and those from Thai swamps(110±2 pMC).     

Inhibition of DNA-ethidium bromide intercalation due to free radical attack upon DNA

Summary The fluorescent intercalation complex of ethidium bromide (ETB) with DNA was used as a probe to compare the effects of various radicals with respect to impairment of the DNA base-pair region.OH radicals inhibit up to 0.7 dye intercalations perOH at low salt concentration, and for various oxidizing species the effect decreases in the orderOH > Br ₂ ^– > N ₃ > $$ " align="middle" border="0"> (SCN) ₂ ^–. DNA impairment by theOH product of Met-Gly is comparable to that of N ₃ , but no effect was found due to the interaction between DNA and Lys-Tyr-Lys phenoxyl radicals. The reducing speciese _aq ^– , H, O ₂ ^–, and CO ₂ ^– hardly affect the DNA-ETB intercalation.