Characterization of a half-molecular fragment obtained by reduction of human α2-macroglobulin with dithiothreitol

A half-molecular fragment of ₂-macroglobulin has been prepared by reducing and alkylating the inter-subunit disulfide bonds in the tetrameric ₂-macroglobulin molecule with 1 mM dithiothreitol (40 min) and 3 mM iodoacetamide (40 min). Further purification was made by gel chromatography and the homogeneous population of halfmolecules has been characterized by the techniques of small-angle X-ray and neutron scattering. The radii of gyration found by the two methods are 57.0 and 58.0 Å, respectively. The match point, obtained by neutron scattering from solutions with different H₂O/D₂O rations, is at 43% D₂O; the data are consistent with a particle having a higher scattering density at large distances from the particle centre. From the X-ray and neutron intensities scattered at zero angle, the specific volume was determined to be 0.73 cm³/g at+5°C and the molecular weight to be 390,000; the latter value is associated with a relatively large error due to the uncertainty in the concentration determination. Shape analysis indicates that the best-fitting scattering-equivalent threeaxial bodies are oblate shaped, with two of their axial dimensions about three to four times larger than the third one. From the volume of the best-fitting scattering-equivalent three-axial bodies, 0.72×10⁶ ų, we obtain a water content equal to 0.38 g H₂O/g protein (dry weight).Abbreviations SANS small-angle neutron scattering - SAXS small-angle X-ray scattering - ₂ M ₂-macroglobulin - DTT dithiothreitol - Tris tris(hydroxymethyl)aminomethane     

Enzymatic formation of prostaglandin F2α in human brain

Prostaglandin (PG)E₂ 9-ketoreductase, which catalyzes the conversion of PGE₂ to PGF₂, was purified from human brain to apparent homogeneity. The molecular weight, isoelectric point, optimum pH, Km value for PGE₂, and turnover number were 34,000, 8.2, 6.5–7.5, 1.0 mM, and 7.6 min^–1, respectively. Among PGs tested, the enzyme also catalyzed the reduction of other PGs such as PGA₂, PGE₁, and 13,14-dihydro-15-keto PGF₂, but not that of PGD₂, 11-PGE₂, PGH₂, PGJ₂, or ¹²-PGJ₂. The reaction product formed from PGE₂ was identified as PGF₂, by TLC combined with HPLC. This enzyme, as is the case for carbonyl reductase, was NADPH-dependent, preferred carbonyl compounds such as 9,10-phenanthrenequinone and menadione as substrates, and was sensitive to indomethacin, ethacrynic acid, and Cibacron blue 3G-A. The reduction of PGE₂ was competitively inhibited by 9,10-phenanthrenequinone, which is a good substrate of this enzyme, indicating that the enzyme catalyzed the reduction of both substrates at the same active site. These results suggest that PGE₂ 9-ketoreductase, which belongs to the family of carbonyl reductases, contributes to the enzymatic formation of PGF₂ in human brain.Special issue dedicated to Dr. Sidney Udenfriend.     

Molecular cloning,sequencing and expression of an α2-adrenergic receptor complementary DNA from rat brain

We have isolated a cDNA clone from rat brain using a human platelet ₂-adrenergic receptor genomic clone as a probe. Comparison of the deduced amino acid sequence (450 residues) corresponding to the rat brain cDNA with that of the human platelet and human kidney ₂-adrenergic receptors showed 84% and 44% sequence similarity, respectively. The major sequence difference between the rat brain and human platelet proteins, was a stretch of 48 amino acids within the third cytosolic loop in which the similarity was only 42%. Analysis of the 48 amino acid-region indicated that the two receptors significantly differ in terms of their primary amino acid sequence and the predicted secondary and tertiary structural features. There was no sequence similarity between the human platelet and rat brain clone over the 177 bases of 3-noncoding sequence and a less than 50% similarity over a stretch of 210 nucleotides in the 5-untranslated region. Southern-blot analysis with a human platelet ₂-adrenergic receptor probe revealed the existence of a single 5.2 kb restriction fragment (KpnI/SacI) in both human and rat genomic DNA; the rat brain ₂-receptor probe, however, hybridized to a single 1.9 kb band in rat DNA. Northern-blot analysis of rat brain poly(A⁺) RNA with the rat brain cDNA probe under stringent hybridization conditions revealed a single 4.5 kb mRNA; none was detected by the human platelet receptor probe. The rat brain 4.5 kb mRNA was not detected in any (other than brain) tested rat tissues utilizing either rat brain or human platelet DNA probes. The rat brain cDNA was expressed in a mammalian cell line (COS-2A) and found to bind the ₂-adrenergic antagonist [³H]yohimbine; based on the binding-affinity for prazosin, the presently cloned receptor was pharmacologically closer to the _2A subclass. We conclude that the rat brain cDNA encodes a new ₂-adrenergic receptor subtype that may be brain-specific.Abbreviations G protein guanine nucleotide-binding proteins - cA2-47 ₂-adrenergic receptor cDNA from rat brain - SSC (1X SSC contains 0.15 M NaCl, 15 mM Na₃citrate, pH 7.0)     

Expression pattern of voltage-dependent calcium channel α1 and β subunits in adrenal gland of N-type Ca2+ channel α1B subunit gene-deficient mice

The Ca²⁺ channel _1B subunit is a pore-forming component capable of generating N-type Ca²⁺ channel activity. Although N-type Ca²⁺ channel plays a role in a variety of neuronal functions, _1B-deficient mice exhibit normal life span without apparent abnormalities of behavior, histology or plasma norepinephrine level, presumably owing to compensation by some other Ca²⁺ channel ₁ or subunit. In this study, we studied the levels of _1A, _1C, _1D, _1E, ₁, ₂, ₃ and ₄ mRNAs in adrenal gland of _1B-deficient mice. The _1A mRNA in homozygous mice was expressed at higher level than in wild or heterozygous mice, but no difference in the expression levels of _1C, _1D, _1E, ₁, ₂, ₃ and ₄ was found among wild, heterozygous and homozygous mice. The protein level of _1A in homozygous mice was also expressed at higher level than in wild or heterozygous mice. To examine whether increased expression is induced by cis-regulatory element within 5-upstream region of _1A gene, we examined lacZ expression in _1B-deficient × _1A6.3-lacZ mice (carrying a 6.3-kb 5-upstream fragment of _1A gene fused to E. coli lacZ reporter gene), which express lacZ in medullar chromaffin cells, but not in cortex. The levels of lacZ expression in homozygous _1B-deficient × _1A6.3-lacZ mice were higher than in wild or heterozygous mice. Therefore, a possible explanation of the normal behavior and plasma norepinephrine level of _1B-deficient mice is that compensation by _1A subunit occurs and that 6.3-kb 5-upstream region of _1A gene contains enhancer cis-element(s) for compensation in adrenal medulla chromaffin cells. (Mol Cell Biochem 271: 91–99, 2005)     

Functional and neurochemical evidence that neurotensin-induced release of acetylcholine from Auerbach's plexus of guinea-pig ileum is presynaptically controlled via α2-adrenoceptors

The effect of neurotensin (NT) on [³H]acetylcholine release and contraction from isolated longitudinal muscle strip of guinea-pig ileum was examined. Neurotensin dose-dependently enhanced the release of [³H]-acetylcholine. This effect of neurotensin was inhibited by stimulation of ₂-adrenoceptors: noradrenaline, clonidine, xylazine or dexmedetomidine (₂-adrenoceptor agonists) inhibited neurotensin-induced release of acetycholine (ACh) as well as the contractions, while CH-38083 or yohimbine (₂-adrenoceptor antagonist) prevented this inhibitory effect. Our findings suggest that neurotensin may play a neuromodulatory role in the regulation of cholinergic neuronal activity in the gut and this modulatory effect is continuously controlled by the tonic activity of the sympathetic nervous system: endogenous noradrenaline release is capable of reducing the release of ACh and the consequent contraction of the gut enhanced by neurotensin.     

Interaction between Duodenase and α1-Proteinase Inhibitor

The interaction between duodenase, a newly recognized serine proteinase belonging to the small group of Janusfaced proteinases, and ₁-proteinase inhibitor (₁-PI) from human serum was investigated. The stoichiometry of the inhibition was 1.2 mol/mol. The presence of a stable enzyme–inhibitor complex was shown by SDS-PAGE. The mechanism of interaction between duodenase and ₁-PI was shown to be of the suicide type. The equilibrium and inhibition constants are 13 ± 3 nM and (1.9 ± 0.3)·10⁵ M^–1·sec^–1, respectively. Based on the association rate constant of the enzyme–inhibitor complex and localization of duodenase and ₁-PI in identical compartments, ₁-PI is suggested to be a duodenase inhibitor in vivo.     

C-Terminal exchange between Gα o and Gα i3 proteins differently modulates α2A-adrenoceptor activation

Chimeric G proteins, obtained by exchanging their C-terminal portion for that of a G protein from an unrelated class, drive the receptor selectivity to that corresponding to the introduced G protein domain. The _2A-adrenoceptor (_2AAR), which yielded an efficacious and weak [³⁵S]GTPS binding response by respectively G _o and G _i3 protein, was investigated in CHO-K1 cells co-expressing chimeric G proteins for which the six last C-terminal amino acids between G _o and G _i3 proteins, and reciprocally, were permuted. Activation of the chimeric G _{o / i3} protein was highly efficient whereas the G _{i3 / o} protein yielded a weak stimulation. These [³⁵S]GTPS binding responses were not different from their parental wild-type G _o and G _i3 proteins. Similar results were obtained with an _2AAR carrying a facilitating Thr³⁷³Lys mutation in a putative G protein interaction domain. These data indicate that the six terminal G _o protein amino acids do not constitute a major _2AAR interaction domain for G protein activation.     

Abnormal expression ofα-L-fucosidase in lymphoid cell lines of fucosidosis patients

Fucosidosis is an autosomal recessive lysosomal storage disease due to a deficiency of-L-fucosidase activity in tissues and body fluids. Exponentially growing lymphoid cell cultures from four fucosidosis patients had 2.7-fold to 15.6-fold less extracellular-L-fucosidase protein and 28.8-fold to 144.0-fold less intracellular-L-fucosidase protein with negligible catalytic activity, compared to the mean of 19 control cultures. The percentage of total-L-fucosidase protein released extracellularly by cultures from the four patients was 64 to 85%, compared to 35±9% for control cultures. Intracellular and extracellular enzyme forms in fucosidosis and control cell lines were glycoproteins containing polypeptide chains ofM _r=52,000. During a 1.5-hr pulse-label with³⁵S-methionine,-L-fucosidase was synthesized by control cells and two fucosidosis cell lines as an intracellular form withM _r=58,000. During a subsequent 21-hr chase with unlabeled methionine, mutant enzyme was almost entirely processed to an extracellular form withM _r=62,000. In contrast, only 25–30% of control enzyme was processed to an extracellular form (M _r=62,000), with the remainder retained intracellularly (M _r=60,000). In the other two fucosidosis cell lines,-L-fucosidase was synthesized as an intracellular form withM _r=56,000 that was processed to an extracellular form withM _r=60,000. In summary, the fucosidosis mutation(s) affected the catalytic activity, quantity, and extracellular release of-L-fucosidase as expressed by lymphoid cells.This work was funded by NIH Grants DK 32161 to R. A. DiCioccio and GM 28428 to J. K. Darby.     

Photoaffinity labeling of the DDT1 MF-2 cell α1-adrenergic receptor

Summary In this study, we have used an ₁-adrenergic receptor photoaffinity ligand, 2-[4-(4-azido-3-iodo-benzoyl)-piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline (¹²⁵I-APD), to label covalently the ₁-adrenergic receptor in a smooth muscle cell line. Our results indicate that in the absence of light, (¹²⁵I)APD binds reversibly to a site in the DDT₁ MF-2 cell membranes having pharmacological characteristics of an ₁-adrenergic receptor. Following incorporation of (¹²⁵I)ADP into partially purified membranes a single labeled band of protein with a M_r of 81 000 was visualized by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incorporation of (¹²⁵I)-APD into this band was affected by adrenergic agonists and antagonists in a manner consistent with an ₁-adrenergic interaction. Prazosin (₁-selective) blocked incorporation of the label into the Mr = 81 000 protein while yohimbine (₂-selective) did not. Of the adrenergic agonists, (–)-epinephrine and (–)-norepinephrine but not (–)-isoproterenol blocked labeling of the M_r – 81 000 protein. We conclude that the ligand binding site of the DDT₁ MF-2 cell ₁-adrenergic receptor resides in a M_r = 81 000 protein.     

Endothelin-1 and insulin induce cellular inactivation of protein kinase FA/glycogen synthase kinase-3α in a common signaling pathway

In this study, we investigate the effects of endothelin-1 (ET-1) and insulin on the cellular activity of protein kinase F_A/glycogen synthase kinase-3 (kinase F_A/GSK-3) in rat adipocytes. The cellular activity of kinase F_A/GSK-3 is inhibited to 50% of control within 30 min when cells are treated with 1 nM ET-1 at 37°C; in addition, significant inhibition to 60% of control is observed at as low as 1 pM ET-1. Conversely, ET-1 at concentrations up to 1 nM has no direct effect on purified kinase F_A/GSK-3 in vitro. Immunoblotting analysis further reveals that the protein level of this kinase is not significantly changed when treated with 1 nM ET-1 for 30 min. Similar to ET-1, insulin as low as 10 nM can also induce inactivation of kinase F_A/GSK-3 to 50% of control in adipocytes when processed under identical conditions. Most importantly, when treated with both insulin and ET-1, the activity of kinase F_A/GSK-3 can be decreased only to 50% of control. Taken together, the results provide initial evidence that ET-1 and insulin may regulate this important multisubstrate/multifunctional protein kinase in a common signaling pathway in cells.     

Symmetric interspecies hybrids of mouse and human hemoglobin: Molecular basis of their abnormal oxygen affinity

Interspecies hybrids of HbA and Hb from mouse C57BL/10 [ ₂ ^M ₂ ^H and ₂ ^H ₂ ^M (H=human, M=mouse)], representing 19 and 27 sequence differences per dimers (as compared with human dimer) have been generatedin vitro. The efficiency of the assembly of the interspecies hybrids by the alloplex intermediate pathway is about twofold higher than the low-pH-mediated subunit approach. The interspecies hybrids exhibit a cooperative O₂ binding. The intrinsic O₂ affinity of mouse Hb is slightly lower than HbA, while the 2,3-diphosphoglycerate (DPG) effect is comparable. Interestingly, the interspecies hybrid ₂ ^M ₂ ^H has high O₂ affinity (compared to either human or mouse Hb), while the interspecies hybrid ₂ ^H ₂ ^M exhibits a very low O₂ affinity. These results suggest that the mouse chain generates a tetramer with very low oxygen affinity. However, the complementarity of the mouse and chains generates a set of unique interactions that compensate for the low-oxygen-affinity propensity of the mouse chain. DPG binds the tetramer in the central cavity formed by the two subunits, hence the DPG effects on the interspecies hybrids should be as in the parent molecule. However, the results of the present study demonstrate that the DPG binding pocket is influenced by the nature of the chain present in the tetramer. The mouse chain reduces considerably the DPG right shift of the O₂ affinity of the human-chain containing hybrid. Sequence analysis suggest that perturbations of the _{1 1} (not the _{1 2}) are communicated to the DPG binding pocket in the presence of the alien subunit, and are the primary determinant of the ligand binding properties. The results have implications for the design of Hb-based blood substitutes and understanding of the inhibitory potential of mouse chains in transgenic mouse expressing human ^S chains.     

Properties and synthesis de novo of auxin-induced α-amylase in pea cotyledons

Analysis of starch-degrading enzymes in a crude extract of detached cotyledons of Pisum sativum L. by polyacrylamide gel electrophoresis (PAGE) demonstrated the presence of one band of -amylase (EC 3.2.1.1) activity. The activity of only this amylase was promoted in cotyledons incubated with 2,4-dichlorophenoxyacetic acid (2,4-D). The auxin-induced -amylase from pea cotyledons was purified to homogeneity, as judged by the criterion of a single band after PAGE. The relative molecular mass (M_r), estimated by gel filtration, was approx. 42 000 and the enzyme contained no carbohydrate moiety. Sodium dodecylsulfate-PAGE yielded a single band that corresponded to an M_r of 41 000. The isoelectric point was 5.85 and the aminoacid composition was similar to that of -amylase from other plants. When [³H]leucine was fed to detached dry cotyledons prior to incubation, the radioactivity in -amylase from cotyledons incubated in the presence of 2,4-D was found to be approx. 10-fold higher than that from cotyledons incubated in distilled water. When -amylase from cotyledons incubated with ²H₂O that contained 2,4-D and the tritiated amylase were centrifuged together in a CsCl density gradient, the peak of enzymatic activity of deuterated -amylase was shifted to a denser fraction than the peak of radioactivity of the tritiated enzyme. These results show that auxin-induced -amylase in pea cotyledons is synthesized de novo.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PAS periodic acid-Schiff - pI isoelectric point - SDS sodium dodecyl sulfate We are very grateful to Mr. Kazuo Itoh and Mrs. Matsumi Doe for carrying out the analysis of amino-acid composition.     

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.     

Glial α1-receptors probably inhibit the high-affinity uptake of noradrenaline into astrocytes in the rat brain in vivo

The effect of ₂-receptor blockage on the extraneuronal turnover of noradrenaline (NA) has been studied in the intact rat brain. Tropolone and yohimbine, along with reserpine or desmethylimipramine, were given 30 min after intracerebroventricular injection of [7-³H]NA, i.e. after the tracer had been stored or inactivated. Tropolone given alone did not change the fractions of³H-activity recovered as [³H]NA from hypothalamus, septum, striatum and pons-medulla, but in the presence of yohimbine improved the [³H]NA recovery in all areas except pons-medulla. The maximum effect was seen in the hypothalamus of reserpine-treated rats. Since the ₂-autoreceptors were blocked, the increased [³H]NA recovery does not reflect a down-regulated neuronal NA turnover. Instead it seems to show that a fraction greater than normal of neuronally released NA had been taken up into astrocytes and remained unmetabolized if catechol-O-methyltransferase was inactive. It is assumed that yohimbine enabled the protective tropolone effect by blocking astrocytic ₂-receptors that otherwise, either by itself or by antagonizing -receptor-induced hyperpolarization or cAMP formation, had impaired parameters that stimulate the high-affinity NA Uptake₁ of astrocytes (e.g. membrane potential, Na⁺, K⁺-ATPase) or control the gap junction permeability in the glial syncytium.     

Genetic evidence for α2-adrenergic receptor subtypes in rat brain,heart and adrenal gland

Summary A complementary DNA (cDNA) clone - cA₂-47 - corresponding to a new ₂-adrenergic receptor subtype has been isolated from a rat brain cDNA library and used as a hybridization probe to scrutinize the ₂-receptor poly(A⁺) RNAs in rat brain, heart and adrenal gland. Hybridization of the 5 half of the coding region of this cDNA at 37°C to rat brain poly(A+) RNA revealed a single band at 5.8 kb as the size of its corresponding mRNA. Under identical hybridization conditions, a human platelet ₂-receptor genomic probe failed to hybridize to any rat brain mRNAs.Under lower stringency conditions, hybridization of the full-length cDNA, cA₂-47, to selected rat tissue poly(A⁺) RNA showed the presence of four different sized mRNAs in brain and three in both heart and adrenal gland. Messages of 1.3 kb and 2.1 kb were common in all three tissues (although the band at 2.1 kb was slightly higher in the heart and adrenal gland). A 5.8 kb mRNA was unique to the brain and a slightly higher band at 6.0 kb was consistently present in heart and adrenal gland but was absent in the brain. A fourth message at 3.4 kb was found predominantly in the brain and was either absent or present at very low levels in the other tissues examined. Under the same conditions, a human platelet ₂-receptor probe hybridized to similar sized messages of 2.1 and 5.8 kb in rat brain and 2.2 and 6.0 kb in rat heart and adrenal gland. This probe, however, failed to detect the abundant 1.3 kb mRNA common to all tissues or the 3.4 kb message in rat brain. The extent of homology of these messages with cA₂-47 is not confined to limited regions of the cDNA since similar hybridization patterns were observed using either 5-noncoding or 5-coding regions of the probe.These results provide the first direct evidence of a surprisingly large range of mRNA sizes for members of the ₂-receptor family in brain, heart, and adrenal gland. The unique nature of certain members of the family in each of the tissues examined raises the curious possibility that these members might contribute to some of the individualized functions of the brain, cardiovasculature and adrenal gland.     

Changes in soil organic matter with cropping as measured by organic carbon fractions and 13C natural isotope abundance

The decline in soil organic matter with cropping is a major factor affecting the sustainability of cropping systems. Changes in total C levels are relativelyinsensitive as a sustainability measure. Oxidation with different strength KMnO₄ has been shown to be a more sensitive indicator of change. The relative size of soil C fractions oxidised by 333 mM KMnO₄ declined with cropping, whilst the relative size of the unoxidised fraction increased. Changes in ¹³C ratio have been used to measure C turnover in systems which include C3 and C4 species.     

Regulation of α-amylase activity in Amaranthus caudatus seeds by methyl jasmonate, gibberellin A3, benzyladenine and ethylene

Methyl jasmonate (JA-Me) at 10^–3 Minhibited Amaranthus caudatus seed germination anddecreased -amylase activity. Exogenous gibberellin A₃(GA₃) and ethylene, but not benzyladenine (BA), increased activity ofthe enzyme in the presence of JA-Me, with ethylene being the most effective. Theinhibitor of ethylene action, 2,5-norbornadiene (NBD) inhibited seed germinationand decreased -amylase activity. The inhibitory action of JA-Me onAmaranthus caudatus seed germination is associated with theinhibition of -amylase activity. Exogenous GA₃ and ethylenecontrol both -amylase activity and seed germination in the presence of JA-Me.     

α-adrenergic receptor regulation of Ca2+/Mg2+-ATPase in brain synaptic membranes

The effects of ₁ and ₂ receptor ligands on Ca²⁺/Mg²⁺-ATPase have been studied using synaptosomal plasma membranes isolated from rat brain cortex. Both phenylephrine and clonidine inhibited Ca²⁺/Mg²⁺-ATPase, in a concentration-dependent fashion. IC₅₀ values for half-maximal inhibition for phenylephrine and clonidine were 29 M and 18 M, respectively. The inhibitory effect of phenylephrine was reversed by the alpha antagonist prazosin while yohimbine and rauwolscine reversed the inhibition of enzyme activity by clonidine. The two antagonist subtypes were effective only against the respective agonist subtypes, demonstrating distinct subtype preferences. Analysis of the kinetics of enzyme inhibition indicate both agonists to be noncompetitive. Some evidence suggests that yohimbine may exhibit mixed agonist/antagonist properties which depend on [Ca²⁺]. The present study provides biochemical evidence to support auto receptor adrenergic receptor regulation of neurotransmitter release.     

HNCCH-TOCSY,a triple resonance experiment for the correlation of backbone 13Cα and 15N resonances with aliphatic side-chain proton resonances and for measuring vicinal 13CO, 1Hβ coupling constants in proteins

Summary A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and -carbons in uniformly ¹³C¹⁵N-labeled proteins. In-phase ¹³C magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from ¹³C to ¹H leads to E.COSY-type cross peaks, from which the ³J_{H co} coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.     

Comparative study of the effects of cromakalim (BRL 34915) and diazoxide on membrane potential, [Ca2+] i and ATP-sensitive potassium currents in insulin-secreting cells

Summary Patch-clamp and single cell [Ca²⁺] _i measurements have been used to investigate the effects of the potassium channel modulators cromakalim, diazoxide and tolbutamide on the insulin-secreting cell line RINm5F. In intact cells, with an average cellular transmembrane potential of –62±2 mV (n=42) and an average basal [Ca²⁺] _i of 102±6nm (n=37), glucose (2.5–10mm): (i) depolarized the membrane, through a decrease in the outward K_ATP current, (ii) evoked Ca²⁺ spike potentials, and (iii) caused a sharp rise in [Ca²⁺] _i . In the continued presence of glucose both cromakalim (100–200 m) and diazoxide (100 m) repolarized the membrane, terminated Ca²⁺ spike potentials and attenuated the secretagogue-induced rise in [Ca²⁺] _i . In whole cells (voltage-clamp records) and excised outside-out membrane patches, both cromakalim and diazoxide enhanced the current by opening ATP-sensitive K⁺ channels. Diazoxide was consistently found to be more potent than cromakalim. Tolbutamide, a specific inhibitor of ATP-sensitive K⁺ channels, reversed the effects of cromakalim on membrane potential and K_ATP currents.