Involvement of metabotropic glutamate receptors in taurine release in the adult and developing mouse hippocampus

Summary The inhibitory amino acid taurine has been held to function as an osmoregulator and modulator of neural activity, being particularly important in the immature brain. lonotropic glutamate receptor agonists are known markedly to potentiate taurine release. The effects of different metabotropic glutamate receptor (mGluR) agonists and antagonists on the basal and K⁺-stimulated release of [³H]taurine from hippocampal slices from 3-month-old (adult) and 7-day-old mice were now investigated using a superfusion system. Of group I metabotropic glutamate receptor agonists, quisqualate potentiated basal taurine release in both age groups, more markedly in the immature hippocampus. This action was not antagonized by the specific antagonists of group I but by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX), which would suggest an involvement of ionotropic glutamate receptors. (S)-3,5-dihydroxyphenylglycine (DHPG) potentiated the basal release by a receptor-mediated mechanism in the immature hippocampus. The group II agonist (2S, 2R, 3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG IV) markedly potentiated basal taurine release at both ages. These effects were antagonized by dizocilpine, indicating again the participation of ionotropic receptors. Group III agonists slightly potentiated basal taurine release, as did several antagonists of the three metabotropic receptor groups. Potassium-stimulated (50 mM K⁺) taurine release was generally significantly reduced by mGluR agents, mainly by group I and II compounds. This may be harmful to neurons in hyperexcitatory states. On the other hand, the potentiation by mGluRs of basal taurine release, particularly in the immature hippocampus, together with the earlier demonstrated pronounced enhancement by activation of ionotropic glutamate receptors, may protect neurons against excitotoxicity.Abbreviations ACPD (1±)-1-aminocyclopentane-trans-1,3-dicarboxylate - AIDA (RS)-1-aminoindan-1,5-dicarboxylate - AMPA 2-amino-3-hydroxy05-methyl-4-isoxazolepropionate - CNQX 6-cyano-7-nitroquinoxaline-2,3-dione - CPPG (RS)-2-cyclopropyl-4-phosphonophenylglycine - DCG IV (2S,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine - DHPG (S)-3,5-dihydroxyphenylglycine - EGLU (2S)-2-ethylglutamate - L-AP3 L(+)-2-amino-3-phosphonopropionate - L-AP4 L(+)-2-amino-4-phosphonobutyrate - L-AP6 L(+)-2-amino-6-phosphonohexanoate - L-SOP O-phospho-L-serine - MPPG (RS)-2-methyl-4-phosphonophenylglycine - MSOP (RS)-2-methylserine-O-phosphate - MSOPPE (RS)-2-methylserine-O-phosphate monophenyl ester - MTPG (RS)-2-methyl-4-tetrazolylphenylglycine - NBQX 6-nitro-7-sulphamoyl[f]quinoxaline-2,3-dione - NMDA N-methyl-D-aspartate - QA quisqualate - S-3C4H-PG (S)-3-carboxy-4-hydroxyphenylglycine - S-4C-PG (S)-4-carboxyphenylglycine; - S-MCGP (S)-2-methyl-4-carboxyphenylglycine     

Enhanced taurine release in cultured cerebellar granule cells in cell-damaging conditions

Summary The release of taurine from cultured cerebellar granule neurons was studied in different cell-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress and in the presence of free radicals. The effects of both ionotropic and metabotropic glutamate receptor agonists on the release were likewise investigated. The release of [³H]taurine from the glutamatergic granule cells was increased by K⁺ (50mM) and veratridine (0.1 mM), the effect of veratridine being the greater. Hypoxia and ischemia produced an initial increase in release compared to normoxia but resulted in a diminished response to K. Hypoglycemia, oxidative stress and free radicals enhanced taurine release, and subsequent K^– treatment exhibited a correspondingly greater stimulation. A common feature of taurine release in all the bove conditions was a slow response to the stimulus evoked by K⁺ and particularly to that evoked by veratridine. All ionotropic glutamate receptor agonists potentiated taurine release, but only the action of kainate seemed to be receptor-mediated. Metabotropic receptor agonists of group I slightly stimulated the release. The prolonged taurine release seen in both normoxia and cell-damaging conditions may be of importance in maintaining homeostasis in the cerebellum and reducing excitability for a longer period than other neuroprotective mechanisms.Abbreviations AIDA (RS)-1-aminoindan-1,5-dicarboxylate - AMPA 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate - CNOX 6-cyano-7-nitroquinoxaline-2,3-dione - DCG IV (2S,2R,3R)-2-(2,3-dicarboxycyclo-propyl)glycine - DHPG (S)-3,5-dihydroxyphenylglycine - EGLU (2S)-2-ethylglutamate - L-AP3 L(+)-2-amino-3-phosphonopropionate - L-AP4 L(+)-2-amino-4-phosphonobutyrate - L-SOP o-phospho-l-serine - NBOX 6-nitro-7-sulphamoyl[f]quinoxaline-2,3-dione - NMDA n-methyl-d-aspartate - trans-ACPD (1S,3S)-1-aminocyclopentane-1,3-dicarboxylate     

Analysis of 5′-upstream non-coding region of FI-carboxymethyl cellulase gene from Aspergillus aculeatus

The 5-upstream non-coding region of an FI-carboxymethyl cellulase (CMCase) gene of Aspergillus aculeatus No. F-50 was obtained and sequenced up to –777 nucleotide in pCMG23. The 5-upstream region of different lengths were constructed and fused to a reporter gene (Escherichia coli lacZ) in pAN923-42BD, and the resulted constructs were introduced into the A. nidulans. The -galactosidase activities of the transformant with 5-upstream fragment of larger than 319 bp were expressed by the induction, but that of 109 bp drastically decreased to the basal level. This suggests that the region between –109 and –319 of the 5-upstream non-coding region is involved in the regulation of the FI-CMCase expression.     

Cation-activated ATPase activity of plasmalemma-enriched membrane preparations from maize coleoptiles

The ATPase activity present in plasmalemma-enriched preparations from maize coleoptiles shows an optimum at pH 6, a strong dependence on Mg²⁺, and is stimulated by K⁺ and other monovalent cations, both organic and inorganic. The activation of ATPase by K⁺ obeys Michaelis Menten kinetics, saturation being reached at 50 mM K⁺ concentration. K⁺, Mg²⁺-stimulated ATPase activity is strongly inhibited by N,N-dicyclohexylcarbodiimide and by diethylstilbestrol and, to a lesser extent, by octylguanidine.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - DTE dithioerythritol - Ellmans r 5-5 dithiobis (2 nitrobenzoic) acid - FC fusicoccin - NPA naphthylphthalamic acid - OG octylguanidine - PCMBS p-chloromercuribenzensulphonate     

Mineral catalysis of the formation of dimers of 5′-AMP in aqueous solution: The possible role of montmorillonite clays in the prebiotic synthesis of RNA

The reaction of the 5-AMP with water soluble carbodiimide (EDAC) in the presence of Na⁺-montmorillonite 22A results in the formation of 2,5-(pA)₂ (18.9%), 3,5-(pA)₂ (11%), and AppA (4.8%). When poly(U) is used in place of the clay the product yields are 2,5-(pA)₂ (15.5%), 3,5-(pA)₂ (3.7%) and AppA (14.9%). The 3,5-cyclic dinucleotide, 3,5-c(pA)₂, is also formed when poly(U) is used. AppA is the principal reaction product when neither clay nor poly(U) is present in the reaction mixture. Products which contain the phophodiester bond are formed at different ionic strengths, pH and temperatures using Na⁺-montmorillonite. Phosphodiester bond formation was not observed when Cu²⁺-montmorillonite was used or when DISN was used in the place of EDAC. The extent catalysis of phophodiester bond formation varied with the particular clay mineral used. Those Na⁺-clays which bind 5-AMP more strongly are better catalysts. Cu²⁺-montmorillonite, which binds 5-AMP strongly, exhibits no catalytic activity.     

IP3-and cAMP-induced responses in isolated olfactory receptor neurons from the channel catfish

Summary Olfactory receptor neurons enzymatically dissociated from channel catfish olfactory epithelium were depolarized transiently following dialysis of IP₃ or cAMP (added to the patch pipette) into the cytoplasm. Voltage and current responses to IP₃ were blocked by ruthenium red, a blocker of an IP₃-gated Ca²⁺-release channel in sarcoplasmic reticulum. In contrast, the responses to cAMP were not blocked by extracellularly applied ruthenium red, nor by l-cis-diltiazem or amiloride and two of its derivatives. The current elicited by cytoplasmic IP₃ in neurons under voltage clamp displayed a voltage dependence different from that of the cAMP response which showed marked outward rectification. A sustained depolarization was caused by increased cytoplasmic IP₃ or cAMP when the buffering capacity for Ca²⁺ of the pipette solution was increased, when extracellular Ca²⁺ was removed or after addition of 20–200 nm charibdotoxin to the bathing solution, indicating that the repolarization was caused by an increase in [Ca _i ] that opened Ca²⁺-activated K⁺ channels. The results suggest that different conductances modulated by either IP₃ or cAMP are involved in mediating olfactory transduction in catfish olfactory receptor neurons and that Ca²⁺-activated K⁺ channels contribute to the termination of the IP₃ and cAMP responses.Abbreviations ATP adenosine 5-triphosphate - BAPTA (bis-(o-aminophenoxy)-ethane-N-N-N-N)-tetraacetic acid - cAMP adenosine cyclic 3,5-monophosphate - cGMP guanosine cyclic 3,5-monophosphate - CTX charybdotoxin - DCB 3,4-dichlorobenzamil - EDTA ethylenediaminetetraacetic acid - EGTA ethylenglycol-bis-(b-aminoethyl)-N-N-N-N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IP₃ inositol-1,4,5-triphosphate - NMDG N-methyl-d-glucamine We would like to thank the Tanabe Seiyaku Co., Ltd., for their gift of l-cis-diltiazem. This work was supported by National Institutes of Health grants DC00566 and BRSG S07RR05825.     

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.     

Effects of Metabotropic Glutamate Receptor Agonists and Antagonists on D-Aspartate Release from Mouse Cerebral Cortical and Striatal Slices

The cytosolic release of L-glutamate has been held to be responsible for the increase in extracellular glutamate to toxic levels in the brain. The mechanism and regulation of this release was now studied in cerebral cortical and striatal slices with D-[³H]aspartate, a non-metabolized analogue of L-glutamate and a poor substrate for vesicular uptake. L-Glutamate and D-aspartate strongly stimulated the release in a concentration-dependent manner. Of the ionotropic glutamate receptor agonists, only kainate enhanced the basal release in the striatum. Of the metabotropic glutamate receptor ligands, the group I agonist (S)-3,5-dihydroxyphenylglycine (S-DHPG) failed to affect the basal release but inhibited the D-aspartate-evoked release in the striatum. The group I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) had no effect on the basal release in either preparation but enhanced the L-glutamate-evoked release and inhibited the D-aspartate-evoked release in the striatum, not however in the cerebral cortex. The group II agonist (2S,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG IV) and the group II antagonist (2S)-2-ethylglutamate (EGLU) were without effect on the basal, D-aspartate- and L-glutamate-evoked releases of D-[³H]aspartate in either preparation. The group III agonist L-serine-O-phosphate (L-SOP) failed to affect the basal release but reduced the D-aspartate-evoked release in the striatum. The group III antagonist (RS)-methylserine-O-phosphate (MSOP) failed to affect the basal release but increased the glutamate-evoked release and inhibited the D-aspartate-evoked release in the striatum. Both L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC) and (2S, 1S, 2R)-2-carboxycyclopropyl)glycine (L-CCG-III), transportable inhibitors of the high-affinity glutamate uptake, enhanced the basal release, more strongly in the striatum than in the cerebral cortex. L-CCG-III also increased the L-glutamate-evoked release in the striatum. Nontransportable dihydrokainate enhanced the basal release much less and failed to affect the glutamate-evoked release. The results indicate that the release of glutamate from cytosolic pools is carrier-mediated via homoexchange. This process is regulated in the striatum by metabotropic group I and group III receptors in a manner different from the regulation of the vesicular release of glutamate from presynaptic terminals.     

Binding of organic solvent molecules influences the P1′−P2′ stereo-and sequence-specificity of α-chymotrypsin in kinetically controlled peptide synthesis

In kinetically controlled peptide synthesis catalyzed by -chymotrypsin (EC 3.4.21.1, CT) the P₁-specificity and stereospecificity was found to increase for hydrophobic and decrease for positively charged nucleophiles in systems with hydrophobic organic solvent molecules. This was observed in the following systems: (i) buffer saturated with organic solvent molecules; (ii) the enzyme adsorbed on a hydrophobic adsorbent (Phenylbutylamine-Eupergit). These results show that organic molecules bound to the enzyme surface perturb the P₁-S₁ interactions and change the catalytic properties of the enzyme.     

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     

Taurine Release Is Enhanced in Cell-Damaging Conditions in Cultured Cerebral Cortical Astrocytes

The release of preloaded [³H]taurine from cultured cerebral cortical astrocytes was studied under various cell-damaging conditions, including hypoxia, ischemia, aglycemia and oxidative stress, and in the presence of free radicals. Astrocytic taurine release was enhanced by K⁺ (50 mM), veratridine (0.1 mM) and the ionotropic glutamate receptor agonist kainate (1.0 mM). Metabotropic glutamate receptor agonists had only weak effects on taurine release. Similarly to the swelling-induced taurine release the efflux in normoxia seems to be mediated mainly by DIDS-(diisothiocyanostilbene-2,2-disulphonate) and SITS-(4-acetamido-4-isothiocyanostilbene-2,2-disulphonate) sensitive CI^– channels, since these blockers were able to reduce both basal and K⁺ -stimulated release. The basal release of taurine was moderately enhanced in hypoxia and ischemia, whereas the potentiation in the presence of free radicals was marked. The small basal release from astrocytes signifies that taurine release from brain tissue in ischemia may originate from neurons rather than glial cells. On the other hand, the release evoked by K⁺ in hypoxia and ischemia was greater than in normoxia, with a very slow time-course. The enhanced release of the inhibitory amino acid taurine from astrocytes in ischemia may be beneficial to surrounding neurons, outlasting the initial stimulus and counteracting overexcitation.     

The catalysis of nucleotide polymerization by compounds of divalent lead

Summary Soluble lead salts and a number of lead-containing minerals catalyze the formation of oligonucleotides from nucleoside 5-phosphorimidazolides. The effectiveness of lead compounds correlates strongly with their solubility. Under optimal conditions we were able to obtain 18% of pentamer and higher oligomers from ImpA. Reactions involving ImpU gave smaller yields.Abbreviations A adenosine - U uridine - Im imidazole - MeIm 1-methyl-imidazole - EDTA ethylenediaminetetraacetic acid - pA adenosine 5-phosphate - pU uridine 5-phosphate - Ap adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - AppA P₁,P₂-diadenosine 5-diphosphate - pNp (N = A,U) nucleotide 2(3), 5-diphosphate - ImpA adenosine 5-phosphoreimidazolide - ImpU uridine 5-phosphorimidazolide - A ²pA adenylyl-[25]-adenosine - A ³pA adenylyl-[35]-adenosine - pA ²pA 5-phospho-adenylyl-[25]-adenosine - pA ³pA 5-phospho-adenylyl-[35]-adenosine - pUpU 5-phospho-uridylyl-uridine - pApU 5-phospho-adenylyl-uridine - pUpA 5-phospho-uridylyladenine - (pA)n (n, 2,3,4,) oligoadenylates with 5 terminal phosphate - ImpApA 5-phosphorimidazolide of adenylyl adenosine - (pA) ₅₊ pentamer and higher oligoadenylates with 5 terminal phosphate - (Ap)nA (n = 2,3,4) oligoadenylates without terminal phosphates In the following we do not specify the nature of the internucleotide linkageIn the following we do not specify the nature of the internucleotide linkage     

Purification and properties of the ATP: adenylylsulphate 3′-phosphotransferase from Chlamydomonas reinhardii

APS-kinase (ATP: adenylylsulphate 3-phosphotransferase, EC 2.7.1.25) has been purified from the alga Chlamydomonas reinhardii, strain CW 15 by means of chromatofocussing and affinity chromatography. The isolated protein showed an apparent molecular mass of 44,000 upon sodium dodecylsulphate polyacrylamide gel electrophoresis. The transfer of phosphate groups from ATP onto APS required a pH of 6.8, the presence of Mg²⁺ ions and a reducing thiol. Its catalytical activity was destroyed by sulphhydryl group inhibitors (phenyl-mercuri compounds, dithiopyridine) and alkylating reagents.The purified enzyme attained a V _max of 360 pkat under optimal reaction conditions declining to v _limit of 260 pkat in the presence of excess substrate APS. This sensitivity towards changes in substrate concentrations was parallelled by a high affinity and specificity: apparent K _m APS: 2 · 10^-6 mol · l^-1, and K _m ATP: 7 · 10^-6 mol · l^-1. The enzyme was found specific for ATP, d-ATP and CTP, while UTP, ITP and GTP showed marginal activity. The Hill coefficients suggested 4 binding sites for APS and 1 for ATP. Excessive APS resulted in a negative slope indicating 3 inhibiting sites of the substrate.Abbreviations APS Adenosine 5-phosphosulphate - dATP 2-deoxyadenosine 5-triphosphate - p-CMB p-chloromercuribenzoate - DTE dithioerythritol - DTT dithiothreitol - -MSH -mercaptoethanol - PAPS 3-phosphoadenosine 5-phosphosulphate - PAP 3-phosphoadenosine 5-phosphate - SDS sodium dodecyl sulphate This work is part of a dissertation submitted by H. G. J., Bochum 1982     

Heteropolynucleotides as templates for non-enzymatic polymerizations

Summary We have studied a number of condensation reactions involving ImpU, ImpT, ImpC, ImpA, ImpG, ImpUpG and ImpCpA as activated nucleotide donors and a variety of homo- and hetero-polynucleotides as templates. We did not obtain any evidence of a template effect with ImpU and ImpT, but observed some condensation of ImpC with GpG on appropriate templates. ImpA and ImpG take part in a number of more or less efficient template-directed reactions, as do ImpUpG and ImpCpA.Our results suggest that, on the primitive Earth, pyrimidine nucleotides could most easily have been incorporated into polymers as constituents of short oligomers, which contained one or more purine nucleotide. The linkage of the product depends strongly on the nature of the substrates; the percentage of the natural 3-5-linkage was, in some cases, less than 10% and, in others, as high as 70%. Wobble-pairing was often very effective in promoting condensations, suggesting that transition mutations would have been very frequent in prebiotic polynucleotide replication.Abbreviations and Conventions U uridine - T thymidine - C cytidine - A adenosine - G guanosine - pN nucleoside-5-phosphate - Np a mixture of 2- and 3-phosphates of a nucleoside - pNp a mixture of the 2-5-diphosphate and 3-5-diphosphate of a nucleoside - N₁ ² pN₂ a 2-5-linked dinucleoside monophosphate - N₁ ³ pN₂ a 3-5-linked dinucleoside monophosphate - N⁵ ppN a pyrophosphate derived from a nucleoside-5-phosphate. ImpN and ImpN₁pN₂ are 5-phosphorimidazolides of nucleosides and 3-5-linked dinucleoside monophosphates, respectively - poly(N) a homopolynucleotide - poly (U₁ C₂ A₄ G₃) a random copolymer derived from a substrate mixture containing U, C, A, G in ratio 1:2:4:3 - ODU optical density units measured at 260 nm     

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     

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)     

Sugar conformation of a stereospecific 2′-R or 2′-S deuterium-labeled DNA decamer studied with proton-proton J coupling constants

The sugar conformation of a DNA decamer was studied with proton-proton ³J coupling constants. Two samples, one comprising stereospecifically labeled 2-R-²H for all residues and the other 2-S-²H, were prepared by the method of Kawashima et al. [J. Org. Chem. (1995) 60, 6980–6986; Nucleosides Nucleotides (1995) 14, 333–336], the deuterium labeling being highly stereospecific 99% for all 2-²H, 98% for 2-²H of A, C, and T, and 93% for 2-²H of G). The ³J values of all H1-H2 and H1-H2 pairs, and several H2-H3 and H2-H3 pairs were determined by line fitting of 1D spectra with 0.1–0.2 Hz precision. The observed J coupling constants were explained by the rigid sugar conformation model, and the sugar conformations were found to be between C3-exo and C2-endo with _m values of 26° to 44°, except for the second and 3 terminal residues C2 and C10. For the C2 and C10 residues, the lower fraction of S-type conformation was estimated from J_H1H2 and J_H1H2 values. For C10, the N–S two-site jump model or Gaussian distribution of the torsion angle model could explain the observed J values, and 68% S-type conformation or C1-exo conformation with 27° distribution was obtained, respectively. The differences between these two motional models are discussed based on a simple simulation of J-coupling constants.     

The action of lipases on chloroplast membranes I. The release of plastocyanin from galactolipase-treated thylakoid membranes

Bean chloroplasts treated with galactolipase (lipolytic acyl hydrolase) isolated from bean leaves showed an inhibition of photosystem I activity as measured by methyl viologen-mediated oxygen uptake and NADP⁺ photoreduction. This inhibition was partially reversed by exogenous plastocyanin added to galactolipase-treated thylakoid membranes. Galactolipase released substantial amounts of endogenous plastocyanin (about 40%) from bean chloroplasts. The results are discussed with regard to the localization of plastocyanin in thylakoid membranes.Abbreviations chlf chlorophyll - DCMU 3-(2,4-dichlorophenyl)-1,1-dimethylurea - DGDG digalactosyldiacylglycerol - MGDG monogalactosyldiacylglycerol - MV methyl viologen - NADP⁺ nicotinamide dinucleotide phosphate - PC phosphatidylcholine - PG phosphatidylglycerol - PE phosphatidylethanolamine - PI phosphatidylinositol - SQDG sulphoquinovosyldiacylglycerol - SDS sodium dodecyl sulphate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - Tricine N-Tris-(hydroxymethyl)-methylglycine - Tris Tris-(hydroxymethyl)-aminomethane     

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