Synthesis of oligoguanylates on oligocytidylate templates

Summary Short oligocytidylates can act as templates for the self-condensation of guanosine 5-phosphorimidazolide. In the absence of a catalytic metal ion or in the presence of Pb²⁺ a noticeable template effect is already observed with the dimer and the yield of long oligomers reaches a plateau with a hexamer template. Short templates give oligomers longers than the template length. The products are predominantly 2-5 linked for the Pb²⁺-catalyzed reaction while mixed linkages are observed in the uncatalyzed reaction.In the presence of Zn²⁺, a template effect is first observed with the pentamer and is maximal by the heptamer. The products are predominantly 3-5 linked. Oligomers shorter than or as long as the template are obtained in substantial yield, and longer products in much lower yields.Abbreviations G Guanosine - Gp guanosine 2(3)-phosphate - pG guanosine 5-phosphate - Gp! guanosine cyclic 2,3-phosphate - ImpG guanosine 5-phosphorimidazolide - ImpG^* [8-¹⁴C]-guanosine 5-phosphorimidazolide - pGp 5-phosphoguanosine 2(3)-phosphate - G²pG guanylyl-[2-5]-guanosine - G³pG guanylyl-[3-5]-guanosine - ImpGpG 5-phosphorimidazolide of GpG - (pG)_n (n = 2,3) oligomers of pG - GppG P₁, P₂-diguanosine 5-diphosphate - GppGpG 5-[guanosine 5-pyrophosphate] of GpG - NH₂pG guanosine 5-phosphoramidate - (pG)₄₊ tetramer and higher oligoguanylates with 5 terminal phosphate - oligo(G) oligoguanylate - Cp cytidine 2(3)-phosphate - Cp! cytidine cyclic 2,3-phosphate - (Cp)_n–1 Cp! (n= 2,3,4) oligocytidylates terminated by 5-OH groups and 2,3-cyclic phosphates - oligo(C) oligocytidylate - poly(C) polycytidylic acid - poly(U) polyuridylic acid - poly(C,G) random copolymer of C and G - BAP bacterial alkaline phosphatase (E. coli) - EDTA ethylenediaminetetraacetic acid - R_f chromatographic mobility     

Transmembrane Ca2+ gradient is essential for high anion transport activity of human erythrocytes

The role of a transmembrane Ca²⁺ gradient in anion transport by Band 3 of human resealed erythrocyte ghosts has been studied. The results show that a transmembrane Ca²⁺ gradient is essential for the conformation of erythrocyte Band 3 with higher anion transport activity. The dissipation of the transmembrane Ca²⁺ gradient by the ionophore A23187 inhibits the anion transport activity. The extent of this inhibition approaches 90% as the Ca²⁺ concentration on both sides of the ghost membrane is increased to 1.0 mM and half-maximum inhibition is observed at 0.25 mM Ca²⁺. Addition of ATP (0.4 mM) to the resealing medium can partly reestablish the transmembrane Ca²⁺ gradient by activation of Ca²⁺-ATPase and alleviate the inhibition to some extent. N-ethylmaleimide, an inhibitor of erythrocyte Ca²⁺-ATPase, prevents such restoration. Electron micrographs reveal that numerous larger intramembranous particles can be observed on the P-faces of freeze-fractured resealed ghosts in the absence of a transmembrane Ca²⁺ gradient.Abbreviations DPA dipicolinic acid - EITC eosin 5-isothiocyanate - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonate - TES N-Tris-(hydroxymethyl)methyl-2-aminoethane sulfonic acid - PMSF phenylmethyl-sulfonylfluoride - NEM N-ethylamaleimide - BSA bovine serum albumin - EGTA ethyleneglycol-bis (aminoethylether)-tetra-acetic acid - EITC-Band 3 Band 3 labeled with EITC - Ca_i Ca²⁺ inside resealed ghosts - Ca_o Ca²⁺ outside resealed ghosts     

Influence of isolation media on synaptosomal properties: Intracellular pH,pCa, and Ca2+ uptake

Preparations of synaptosomes isolated in sucrose or in Na⁺-rich media were compared with respect to internal pH (pH₁), internal Ca²⁺ concentration ([Ca²⁺]_i), membrane potential and⁴⁵Ca²⁺ uptake due to K⁺ depolarization and Na⁺/Ca²⁺ exchange. We found that synaptosomes isolated in sucrose media have a pH_i of 6.77±0.04 and a [Ca²⁺]_i of about 260 nM, whereas synaptosomes isolated in Na⁺-rich ionic media have a pH_i of 6.96±0.07 and a [Ca²⁺]_i of 463 nM, but both types of preparations have similar membrane potentials of about –50 mV when placed in choline media. The sucrose preparation takes up Ca²⁺ only by voltage sensitive calcium channels (VSCC'S) when K⁺-depolarized, while the Na⁺-rich synaptosomes take up⁴⁵Ca²⁺ both by VSCC'S and by Na⁺/Ca²⁺ exchange. The amiloride derivative 2, 4 dimethylbenzamil (DMB), at 30 M, inhibits both mechanisms of Ca²⁺ influx, but 5-(N-4-chlorobenzyl)-2, 4 dimethylbenzamil (CBZ-DMB), at 30 M, inhibits the Ca²⁺ uptake by VSCC'S, but not by Na⁺/Ca²⁺ exchange. Thus, DMB and CBZ-DMB permit distinguishing between Ca²⁺ flux through channels and through Na⁺/Ca²⁺ exchange. We point out that the different properties of the two types of synaptosomes studied account for some of the discrepancies in results reported in the literature for studies of Ca²⁺ fluxes and neurotransmitter release by different types of preparations of synaptosomes.Abbreviations used BCECF 2,7-Biscarboxyethyl-5(6)-carboxyfluorescein - BCECF/AM acetoxymethyl ester of BCECF - [Ca²⁺]_i Internal free calcium ion concentration - CBZ-DMB 5-(N-4-chlorobenzyl)-2,4-dimethylbenzamil - DMB 2, 4-dimethylbenzamil - DMSO dimethyl sulfoxide - Indo-1/AM acetoxymethyl ester of Indo-1 - MES 2-|N-Morpholino|ethanesulfonic acid - NMG N-methyl-D-glucamine - pH_i internal pH - TPP⁺ tetraphenylphosphonium - _p plasma membrane potential     

Inhibitory ca2+-control of movement of beads coated withphysarum myosin along actin-cables inChara internodal cells

Summary The actin-activated ATPase activityPhysarum myosin was shown to be inhibited of M levels of Ca²⁺. To determine if Ca²⁺ regulates ATP-dependent movement ofPhysarum myosin on actin, latex beads coated withPhysarum myosin were introduced intoChara cells by intracellular perfusion. In perfusion solution containing EGTA, the beads moved along the parallel arrays ofChara actin filaments at a rate of 1.0–1.8 m/sec; however, in perfusion solution containing Ca²⁺, the rate reduced to 0.0–0.7 m/sec. The movement of beads coated with scallop myosin, whose actin-activated ATPase activity is activated by Ca²⁺, was observed only in the perfusion solution containing Ca²⁺, indicating that myosin is responsible for the inhibitory effect of Ca²⁺ onPhysarum myosin movement. The involvement of this myosin-linked regulation in the inhibitory effect of Ca²⁺ on the cytoplasmic streaming observed inChara internodal cell andPhysarum plasmodium was discussed.Abbreviations ATP adenosine 5-triphosphate - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis(-aminoethylether) N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

Purification and characterization of cyclic 3′5′-nucleotide phosphodiesterase D3 from Sinorhizobium fredii MAR-1

The cyclic 35-nucleotide phosphodiesterase D3 was purified from Sinorhizobium fredii MAR-1. The native enzyme had a molecular weight of approximately 44.5kDa and a subunit molecular weight of approximately 21kDa as judged by SDS-gel electrophoresis. The pH optimum of the enzyme for the hydrolysis of cyclic AMP was approximately 6.0 with both acetate and Tris-maleate buffers. The optimum temperature for hydrolysing cyclic AMP was approximately 50C. No metal ion was required for activity and EDTA up to 2.5mM did not markedly affect the enzyme. However, methylxanthines, adenine and adenosine as well as 5-AMP, ATP, ADP and metal ions like Zn²⁺, Fe²⁺, Pb²⁺, Al³⁺ and Fe³⁺, were strongly inhibitory at 2.5mM.The D3 enzyme could hydrolyse both cyclic AMP and cyclic GMP with the apparent K _m for cyclic AMP of approximately 0.23M.     

Oligoadenylates formation on an oligouridylate template in the presence of a lead catalyst

Summary Oligouridylates with more than eight chain units can serve as a template for the template-directed condensation of ImpA catalyzed by Pb²⁺ ion. The templates and the Pb²⁺ ion catalyst facilitate the formation of longer oligoadenylates with five or more units. The ratio of 3–5 linked oligomers to the 2–5 isomers increases with increasing chain length of the oligouridylate template. Short oligouridylates up to a hexamer tend to decrease the yield of oligoadenylates, and do not affect the selectivity of internucleotide linkage.Abbreviations EDTA ethylenediaminetetracetic acid - Tris tris(hydroxymethyl)aminomethane - A adenosine - ImpA adenosine 5-phosphorimidazolide - pA adenosine 5-phosphate - Ap adenosine 2(3)-phosphate - poly A polyadenylic acid - AppA P₁,P₂-diadenosine 5-diphosphate - pAp adenosine 2(3),5-diphosphate - ApA adenylyl adenosine - (pA)_n (n = 2,3,) oligomers of pA - ImpApA 5-phosphorimidazolide of ApA - U uridine - pU uridine 5-phosphate - Up uridine 2(3)-phosphate - poly U polyuridylic acid - pUp uridine 2(3),5-diphosphate - (pU)_n (n = 2,3,) oligomers of pU - (pU)_n – (pA)_m cooligomers composed of (pU)_n and (pA)_m units - AppUpUpUpUp pyrophosphate derived from pA and (pU)₄ - AppUp P₁-(adenosine 5)-P₂-(uridine 2(3)-phosphate 5) -pyrophosphate - BAP bacterial alkaline phosphatase - VPD venom phosphodiesterase - N.P₁ nuclease P₁ - RNase A pancreatic ribonuclease - A^* radioactive adenosine     

Ca2+ oscillation in the homogenate ofPhysarum plasmodium

Summary Using aequorin luminescence, we observed a distinct oscillation in Ca²⁺ levels in the supernatant of the homogenate ofPhysarum plasmodium. Ca²⁺ oscillation continued for 10–120 minutes, with a period coinciding with that of the contraction rhythm of a plasmodium.Abbreviations EDTA Ethylenediaminetetraacetic acid - EGTA Ethyleneglycol-bis-(-aminoethylether)-N,N-tetraacetic acid - PIPES Piperazine-N,N-bis-(2-ethane sulfonic acid) - DTT Dithiothreitol The present work was supported by Grants-in Aid from the Ministry of Education, Science and Culture, Japan.     

A non-specific Ca2+ (or Mg2+)-stimulated ATPase in rat heart sarcoplasmic reticulum

ATPase activity in rat heart sarcoplasmic reticulum was stimulated in a concentration-dependent manner by both Ca²⁺ and Mg²⁺ in the complete absence of the other cation. Increasing concentrations of Mg²⁺ produced an apparent inhibition of the Ca²⁺-dependent ATP hydrolysis. CDTA (trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate) had no effect on these responses. The results indicate the presence of a low affinity non-specific divalent cation-stimulated ATPase in rat heart sarcoplasmic reticulum. However, sarcoplasmic reticulum vesicles transported Ca²⁺ with a high affinity (K_0.5 Ca²⁺ = 0.41 M) suggesting the presence of a high affinity Ca²⁺-transporting ATPase. Calmodulin did not stimulate rat heart sarcoplasmic reticulum ATPase activity over a range of Ca²⁺ and Mg²⁺ concentrations and failed to stimulate membrane phosphorylation and Ca²⁺ transport into sarcoplasmic reticulum vesicles. Calmodulin antagonists trifluoperazine and compound 48180 did not affect the ATPase activity. Catalytic subunit of cAMP-dependent protein kinase was also ineffective in stimulating the ATPase activity. These results suggest the presence of an ATPase activity in rat heart sarcoplasmic reticulum with different properties from the high affinity Ca²⁺-pumping ATPase previously characterized in dog heart and other species.Abbreviations cAMP adenosine 3,5-monophosphate - CaM calmodulin - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate - EDTA ethylene-diaminetetraacetate - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate - PLB phospholamban - SR sarcoplasmic reticulum - TFP trifluoperazine     

In vitro analysis of ion channels in periaxolemmal-myelin and white matter clathrin coated vesicles: Modulation by calcium and GTPγS

This study reports the analysis of K⁺ channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K⁺ channels were observed with amplitudes of 25–30, 45–55, and 80–100 pS, all of which exhibited mean open-times of 1–2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K⁺ channel activity was regulated by Ca²⁺. Little or no change in activity was observed when Ca²⁺ was added to thecis side of the bilayer. Addition of 10 M total Ca²⁺ also resulted in little change in K⁺ channel activity. However, at 80 M total Ca²⁺ all K⁺ channel activity was suppressed along with the activation of a 100 pS Cl^– channel. The K⁺ channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTPS to thetrans side of the bilayer resulted in a restriction of activity to the 45–50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K⁺ channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the subunits of G₀, G_s, and G_i and the low molecular weight G protein,ras. As with periaxolemmal-myelin treated with GTPS, the vesicle membrane exhibited only the 50 pS channel. The channel was active at all holding potentials and had open times of 1–6 msec. Addition of GTPS to the bilayer fused with vesicle membrane appeared to suppress this channel activity at low voltages yet induced a hyperactive state at holding potentials of 45 mV or greater. The vesicle 50 pS K⁺ channel was also activated by the 6-methyl-dihydropyron-2-one (20 M).Abbreviations CNPase 2–3 cyclic nucleotide phosphohydrolase - EDTA ethylenediamine N,N,N,N-tetraacetic acid - G-protein GTP(guanosine triphosphate) binding protein - GTPS guanosine 5-O-(3-thiotriphosphate) - MAG myelin associated glycoprotein - Na⁺ K⁺ ATPase, Na⁺ and K⁺ stimulated adenosine triphosphatase - PLP myelin proteolipid protein Special issue dedicated to Dr. Majorie B. Lees.     

Oligonucleotide formation catalyzed by divalent metal ions. The uniqueness of the ribosyl system

Summary Polymerization of various nucleoside-5-phosphorimidazolides has been conducted in neutral aqueous solution using divalent metal ions as catalysts. Oligonucleotide formation took place from each of the ribonucleoside-5-phosphorimidazolides, ImpC, ImpU, ImpA, ImpG, and ImpI. The yields and distributions of the resulting oligonucleotides varied depending on the difference of the nucleic acid base and the metal ions used. The catalytic effect of divalent metal ions on the formation of oligocytidylates occurred in the following order: Pb²⁺>Zn²⁺>Co²⁺, Mn²⁺>Cd²⁺>Cu²⁺>Ni²⁺>Ca²⁺, Mg²⁺, none >Hg²⁺. The order changes slightly for other types of oligoribonucleotide formation. Oligoribonucleotides up to hexamers were obtained in 35–55% overall yield, when Pb²⁺ ion was used as a catalyst. Zn²⁺ ions yielded oligoribonucleotides up to tetramers in 10–20% overall yield. The resulting oligonucleotides contained mainly 2–5 internucleotide linkages.Little or no oligonucleotide was obtained from nucleoside-5-phosphorimidazolides modified in the sugars, Imp(3-dA), Imp(2-dA), Imp(Ara), Imp(Aris), and Imp(Nep). The results indicate that a ribosyl system is required for the metal ion-catalyzed synthesis of oligonucleotides. Abbreviations. EDTA, ethylenediaminetetraacetic acid; Versenol,N-hydroxyethylethylenediaminetriacetic acid; Tris, tris-(hydroxymethyl)aminomethane; pN (N is A, C, G, U, I, 3-dA, 2-dA, AraA, Aris, or Nep), nucleoside-5-phosphate; Np, nucleoside-2(3)-phosphate; I, inosine; 3-dA, 3-deoxyadenosine; 2-dA, 2-deoxyadenosine; AraA, arabinosyladenine; Aris, aristeromycin; Nep, neplanocin A; ImpN, nucleoside-5-phosphorimidazolide; NppN, P₁,P₂-dinucleoside-5,5-pyrophosphate; (pN)_n (n=2, 3, ...), oligomers of pN, numbers given between a nucleoside and a phosphate indicate the type of internucleotide linkage, e.g., pC₂ p₅C is 5-phosphorylcytidyl-(2–5)-cytidine; , cyclic dimers of pN; BAP, bacterial alkaline phosphatase; N.Pl, nuclease Pl; VPDase, venom phosphodiesterase; HPLC, high pressure liquid chromatography     

Cytoplasmic streaming in the cell model ofNitella

Summary The plasmalemma ofNitella internode was made freely permeable to solutes by treating the cell with detergent and EGTA under plasmolysis. After the treatment, the cytoplasmic streaming was stopped by bathing the cell in a medium lacking ATP. The streaming was reactivated by perfusing the exterior of the permeabilized cell with a medium containing both Mg²⁺ and ATP. The reactivated streaming could be reversibly stopped by depletion of ATP. However, depletion of Mg²⁺ irreversibly inhibited the streaming.Cytochalasin B at 5 g/ml irreversibly inhibited the reactivated streaming within a minute, showing that microfilaments are involved in the streaming.Abbreviations ATP adenosine-5-triphosphoric acid - CB cytochalasin B - CyDTA cyclohexanediamine-N,N-tetraacetic acid - DMSO dimethylsulfooxide - DTT dithiothreitol - EGTA ethyleneglycol-bis(-aminoethylether)-N,N tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - PMSF phenylmethyl-sulfonylfluoride     

5-O-β-d-galactopyranosyl-7-methoxy-3′,4′-dihydroxy-4-phenylcoumarin,an inhibitor of photophosphorylation in spinach chloroplasts

5-O--d-galactopyranosyl-7-methoxy-3,4-dihydroxy-4-phenylcoumarin isolated from Exostema caribaeum (Rubiaceae) has been found to act as an energy-transfer inhibitor in spinach chloroplasts. ATP synthesis and phosphorylating (coupled) electron flow were inhibited by 89 and 72%, respectively, at a concentration of 400 M. H⁺-uptake, basal and uncoupled electron transport were not affected by the coumarin. The light-activated Mg⁺²-ATPase activity from bound membrane thylakoid chloroplasts was slightly inhibited by the coumarin. Also, the heat-activated Ca⁺²-ATPase activity of the isolated coupling factor protein was insensitive to this compound. In chloroplasts partially stripped of coupling factor 1 by an EDTA treatment, the coumarin showed a restoration of the proton uptake process. These results suggest that the 4-phenylcoumarin under investigation inhibited phosphorylation in chloroplasts by specifically blocking the transport of protons through a membrane-bound component or a carrier channel (CF_O) located in a hydrophobic region at or near the functional binding site for the coupling factor 1.Abbreviations CF₁ chloroplast coupling factor 1 - CF_O coupling factor zero - DCCD dicyclohexylcarbodiimide - DTT dithiothreitol - EDTA ethylene-diaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - MES 2-(N-morpholino) ethanesulphonic acid - TCA trichloroacetic acid Taken in part from PhD thesis of M.R. Calera.     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1     

Application of H(N)CA,CO-E.COSY experiments for calibrating the φ angular dependences of vicinal couplings J(C′i−1,Hi α), J(C′i−1,Ci β) and J(C′i−1,C′i) in proteins

A triple-resonance NMR technique suitable for the determination ofcarbonyl-related couplings in polypeptide systems is introduced. Theapplication of three novel pulse sequences to uniformly¹³C/¹⁵N-enriched proteins yields E.COSY-likemultiplet patterns exhibiting either one of the³J(C_i–1,H_i ), ³J(C_i–1,C_i ) and³J(C_i–1,C_i)coupling constants in the indirectly detected ¹³Cdimension, depending on the passive spin selected. The experiments aredemonstrated with oxidized flavodoxin from Desulfovibrio vulgaris. On thebasis of the J-values measured and the backbone -angles derived from ahigh-resolution X-ray structure of the protein, the three associated Karplusequations were reparametrized. The root-mean-square differences between theexperimental coupling constants and those predicted by the optimized Karpluscurves are 0.41, 0.33 and 0.32 Hz for³J(C_i–1,H_i ),³J(C_i–1,C_i ) and³J(C_i–1,C_i),respectively. The results are compared with the Karplus parameters previouslypublished for the same couplings.     

Metals are directly involved in the redox interconversion of Saccharomyces cerevisiae glutathione reductase

Summary Redox inactivation of glutathione reductase involves metal cations, since chelators protected against NADPH-inactivation, 3 µM EDTA or 10 µM DETAPAC yielding full protection. Ag⁺, Zn²⁺ and Cd²⁺ potentiated the redox inactivation promoted by NADPH alone, while Cr³⁺, Fe²⁺, Fe³⁺, Cu⁺, and Cu²⁺ protected the enzyme. The Zn²⁺ and Cd²⁺ effect was time-dependent, unlike conventional inhibition. Glutathione reductase interconversion did not require dioxygen, excluding participation of active oxygen species produced by NADPH and metal cations. One Zn²⁺ ion was required per enzyme subunit to yield full NADPH-inactivation, the enzyme being reactivated by EDTA. Redox inactivation of glutathione reductase could arise from the blocking of the dithiol formed at the active site of the reduced enzyme by metal cations, like Zn²⁺ or Cd²⁺.The glutathione reductase activity of yeast cell-free extracts was rapidly inactivated by low NADPH or moderate NADH concentrations; NADP⁺ also promoted rapid inactivation in fresh extracts, probably after reduction to NADPH. Full inactivation was obtained in cell-free extracts incubated with glucose-6-phosphate or 6-phosphogluconate; the inactivating efficiency of several oxidizable substrates was directly proportional to the specific activities of the corresponding dehydrogenases, confirming that redox inactivation derives from NADPH formed in vitro.Abbreviations DETAPAC diethylenetriaminepentaacetic acid - 2,5-ADP-Sepharose-N⁶-(6-aminohexyl) adenosine 2,5-bisphosphateSepharose     

Purification and characterization of bovine brain calmodulin-dependent protein kinase II. The significance of autophosphorylation in the regulation of 63 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme

Bovine brain contains two calmodulin-dependent phosphodiesterase kinases which are separated on Sephacryl S-300 column. One of these kinases has been purified to homogeneity and shown to belong to the calmodulin-dependent protein kinase II family. Phosphorylation of the 63 kDa phosphodiesterase by this purified protein kinase results in the incorporation of 1.0 mol phosphate per mol subunit and an accompanying increase in Ca²⁺ concentrations required for the phosphodiesterase activation by calmodulin. The protein kinase undergoes autophosphorylation to incorporate 1.0 mol phosphate per mol of subunit of the enzyme and the autophosphorylated enzyme is active, independent of the presence of Ca²⁺. The autophosphorylation reaction as well as the protein kinase reaction are rendered Ca²⁺ independent in less than 15 seconds when approximately one mol phosphate per mol protein kinase is incorporated. The result suggests that activation of phosphodiesterase phosphorylation reaction may occur prior to the activation of phosphodiesterase and phosphatase during a cell Ca²⁺ flux via the protein kinase autophosphorylation mechanism.Abbreviations SDS sodium dodecyl sulfate - EGTA ethylene glycol bis (-aminoethyl ether) - N,N,N,N tetra acetic acid - EDTA ethylenediamine-tetraacetic acid - cAMP cyclic adenosine 35 monophosphate This work is supported by grants from the Medical Research Council of Canada (JHW), the Heart and Stroke Foundation of Alberta (JHW and RKS) and the Heart and Stroke Foundation of Saskatchewan (RKS)     

Pollen tube extract supports the movement of actin filaments in vitro

Summary Muscle actin filaments labeled with rhodamine-phalloidin were observed to move on the surface coated with a crude extract of pollen tubes ofLilium longiflorum with an average velocity of 1.99±0.55 m/sec. The movement required both Mg²⁺ and ATP. These results indicate that the extract of pollen tubes contains a myosin-like translocatorAbbreviations ATP adenosine-5-triphosphate - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - PMSF phenylmethylsulfonyl fluoride     

Further studies on cell adhesion based on Lex-Lex interaction,with new approaches: embryoglycan aggregation of F9 teratocarcinoma cells,and adhesion of various tumour cells based on Lex expression

We previously proposed specific interaction of Le^x (Gal1 4[Fuc1 3]-GlcNAc1 3Gal) with Le^x as a basis of cell adhesion in pre-implantation embryos and in aggregation of F9 teratocarcinoma cells, based on several lines of evidence (Eggenset al., J Biol Chem (1989)264:9476–9484). We now present additional evidence for this concept, based on autoaggregation studies of plastic beads coated with glycosphingolipids (GSLs) bearing Le^x or other epitopes, and affinity chromatography on Le^x-columns of multivalent lactofucopentaose III (Le^x oligosaccharide) conjugated with lysyllysine. Comparative adhesion studies of Le^x-expressing tumour cellsvs their Le^x-non-expressing variants showed that only Le^x-expressing cells adhere to Le^x-coated plates and are involved in tumour cell aggregation, in analogy to F9 cell aggregation. The major carrier of Le^x determinant in F9 cells is not GSL but rather polylactosaminoglycan (embryoglycan), and we demonstrated autoaggregation of purified embryoglycan in the presence of Ca²⁺, and reversible dissociation in the absence of Ca²⁺ (addition of EDTA). Defucosylated embryoglycan did not show autoaggregation under the same conditions. Thus, Le^x-Le^x interaction has been demonstrated on a lactosaminoglycan basis as well as a GSL basis. A molecular model of Le^x-Le^x interaction based on minimum energy conformation with involvement of Ca²⁺ is presented.Abbreviations BSA bovine serum albumin - CHO carbohydrate - DMEM Dulbecco's modified Eagle's medium - EDTA ethylenediaminetetraacetic acid - GP glycopeptide - GSL glycosphingolipid - LAG lactosaminoglycan - Le^x Gal1 4[Fuc-1 3]GlcNAc1 R - LFP lacto-N-fucopentaose - LysLys-OH lysyllysinol - Mr relative molecular weight - PBS phosphate-buffered saline - PG paragloboside (Gal1 4GlcNAc1 3Gal1 4Glc1 1Cer) - TBS Tris-buffered saline (10mM Tris-HCl, pH 7.4, containing 0.15M NaCl) - TC tumour cell     

Calcium and tip growth inNeurospora crassa

Summary We examined the ionic regulation of tip growth inNeurospora crassa by a combination of electrophysiology and confocal microscopy. To determine if transmembrane ionic fluxes are required for tip growth, we voltage clamped the membrane from –200 to +50 mV. In this voltage range, transmembrane ionic fluxes would either reverse (e.g., K⁺) or change dramatically (e.g., Ca²⁺ influx) but had no effect on hyphal growth rates. Therefore, ionic fluxes (including Ca²⁺ influx) may not be required for tip growth. However, intracellular Ca²⁺ may still play an obligatory role in tip growth. To assess this possibility, we first increased cytosolic Ca²⁺ directly by ionophoresis. Elevated Ca²⁺ induced subapical branch initiation, often multiple tips. At hyphal tips, fluorescence ratio imaging using fluo-3 and fura-red revealed a pronounced tip-high Ca²⁺ gradient within 10 m of the tip in growing hyphae which was not observed in nongrowing hyphae. Injection of the Ca²⁺ chelator 1,2-bis(ortho-aminophenoxy)ethane-N,N,N,N-tetrapotassium acetate consistently inhibited growth concomitantly with a depletion of intracellular Ca²⁺ and dissipation of the tip-high gradient. We conclude that Ca²⁺ plays a regulatory role in tip initiation and the maintenance of tip growth. Because plasma membrane ionic fluxes do not play a role in tip growth, we suggest that the tip-high Ca²⁺ gradient is generated from intracellular Ca²⁺ stores in the ascomyceteN. crassa.Abbreviations BAPTA 1,2-bis(ortho-aminophenoxy)ethane-N,N,N,N-tetrapotassium acetate - [Ca²⁺]_i intracellular Ca²⁺ concentration - fluo-3 2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl-4-methyl-2,2-(ethylenedioxy)dianiline-N,N,N,N-tetraacetic acid     

Stereochemistry of protected ornithine side chains in gramicidin S derivatives and their resistance to N-methylation

Derivatives of gramicidin S (GS) and its mono- and di-d-cyclohexylalanine (d-Cha) analogs possessing various protecting groups on Orn side chains were prepared. ¹H NMR spectra of the unsymmetrically protected analogs [Orn(X)²,Orn(X)²,d-Cha⁴]GS were similar to the composites of the spectra of the symmetrical derivatives [Orn(X)²,2,d-Cha⁴,4]GS and [Orn(X)²,2]GS, revealing the proximity of the protecting groups of NH of Orn residues at the 2 and 2 positions to the side chains of d-Phe (or d-Cha) residues at the 4 and 4 positions, respectively. The results indicated the presence of H-bonds between the NH of Orn and the carbonyl of d-Phe residues in the i i + 2 sense and not in i i – 3, which was also supported by the ROESY analysis. The substantially strong H-bonds can explain the observed resistance of the urethane NH of the Orn side chains in the GS derivatives to the N-methylation with CH³I–Ag²O in DMF.