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.     

One-bond carbon-proton coupling constants: Angular dependence in β-linked oligosaccharides

Summary The angular dependence of¹J_C,H in model compounds related to -linked oligosaccharides has been established by FPT INDO quantum chemical calculations. Values calculated for models of (1 1)-, (1 2)-, (1 3)- and (1 4)-linked disaccharides were compared, and the effect of the orientation of HO-2 elucidated. The angular dependence of¹J_C,H on the torsional angles ^H and ^H and the solvent dielectric constant (s) was characterized in the form:¹J_C,H = A cos2+B cos + C sin2 + D since + E + Fe. The¹J_C,H values, measured by DEPT methods for C-1-H-1 and C-X-H-X in cellobiose, cyclic trisaccharide and hexopyranoses were used to adjust the calculated angular dependences. Based on the occurrence of the conformers for agarobiose, neoagarobiose, mannobiose and methyl -xylobioside, the thermodynamically averaged <¹J_C,H > values were calculated. The results obtained (<¹J_C-1,H-1 > 162.4, <¹J_{C-4, H-4} > 147.6 Hz for methyl -xylobioside; <¹J_C-1,H-1 > 162.4 and <¹J_C-4,H-4] > 147.6 Hz for mannobiose; <¹J_C-1,H-1 > 162.8 Hz for neo agarobiose and <¹J_C-1,H-1 > 163.2 Hz for agarobiose) agree well with the experimental values of 162.7, 147.5, 160.4, 147.2, 160.9 and 165.7 Hz, respectively.     

β-Amyloid Peptide Binds Equivalently to Binary and Ternary α<Subscript>2</Subscript>-Macroglobulin–protease Complexes

₂-Macroglobulin (₂M) is a protease inhibitor that has separate binding sites for transforming growth factor- (TGF-) and -amyloid peptide (A), both of which have been identified in the ₂M sequence. In the 3D-structure of ₂M, TGF- occupies the ₂M central cavity, overlapping with the space that can accommodate up to two molecules of protease. As a result, ternary ₂M–protease complexes (2 mol protease/mol ₂M) have been reported to not bind TGF-. The goal of the present study was to test whether binding of A to ₂M is controlled by steric constraints imposed by associated proteases, similarly to TGF-. We confirmed that binary ₂M–trypsin complex (1 mol trypsin/mol ₂M) binds increased amounts of TGF-1, compared with native ₂M, while ternary ₂M–trypsin complex binds substantially decreased amounts of TGF-1. By contrast, A-binding to binary and ternary ₂M–trypsin complex was equivalent. In both cases, binding was substantially increased compared with the negligible level observed with native ₂M. Plasmin is a large protease (Mr ~82,000) that substantially occupies the ₂M central cavity; however, ₂M–plasmin complex also bound increased amounts of A, compared with native ₂M. We conclude that A accesses its binding site, in ₂M, from outside the ₂M central cavity. The TGF--and A-binding sites are spatially separated not only in the primary sequence of ₂M, but also in the 3D-structure.     

Enzymic synthesis of the trisaccharide core region of the carbohydrate chain ofN-glycoprotein

Transmannosylation from mannotriose (Man1-4Man1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue ofN,N-diacetylchitobiose was regioselectively induced through the use of -d-mannanase fromAspergillus niger. The enzyme formed the trisaccharide Man1-4GlcNAc1-4GlcNAc (3.7% of the enzyme-catalysed net decrease ofN,N-diacetylchitobiose) from mannotriose as a donor andN,N-diacetylchitobiose as an acceptor. Mannobiose (Man1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.Abbreviations Man d-mannose - (M _n) (n=1–5) -linkedn-mer of mannose - GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1–4)-2-acetamido-2-deoxy-d-glucose     

Alzheimer’s Disease: Soluble Oligomeric Aβ(1–40) Peptide in Membrane Mimic Environment from Solution NMR and Circular Dichroism Studies

Amyloid beta peptide (A) is a small peptide present in normal cells and aggregated A is the main constituent of the extracellular amyloid plaques found in Alzheimers disease (AD) brain. Recent studies suggest that soluble A oligomers are neurotoxic rather than amyloid fibrils found in amyloid plaques. This study using multidimensional NMR spectroscopy and circular dichroism (CD) provides the first direct evidence that alterations in membrane structure can trigger the conversion of soluble -helical monomeric A into oligomeric A in a -sheet conformation.     

Mode of uptake of sterol by Arthrobacter simplex

The mechanism of uptake of water-insoluble -sitosterol by a newly isolated strain of Arthrobacter simplex SS-7 was studied. The production of an extracellular sterol-pseudosolubilizing protein during growth of A. simplex on -sitosterol was demonstrated by isolating the factor from the cell-free supernatant and its subsequent purification by Sephadex G-150 column chromatography. The M _r of the purified sterol-pseudosolubilizing protein determined by SDS–PAGE was 19kDa. The rate of sterol pseudosolubilization (5.2×10^–3g l^–1h^–1) could not adequately account for the rate of sterol uptake (72×10^–3g l^–1h^–1) and the specific growth rate (56×10^–3 h^–1). However in the unfavourable growth condition, when the cells were treated with sodium azide at the level of 30–60% of MIC, the sterol pseudosolubilization accounted for nearly 74% of the total growth containing 96% free cells. Cellular adherence to substrate particles was found to play an active role in the normal growth of the strain on -sitosterol. Unlike sodium acetate-grown cells, whose surface activity was negligible (60mNm^–1), the sterol-grown cells had strong surface activity (40mNm^–1). The high lipid content and long chain fatty acids in the cell-wall of -sitosterol-grown cells probably contribute to the high sterol adherence activity of the cells.     

Purification and characterization of the human protein tyrosine phosphatase,PTPμ, from a baculovirus expression system

The receptor like PTPase, PTP, displays structural similarity in its extracellular segment to members of the immunoglobulin superfamily of cell adhesion molecules. The full length form of PTP (200 kD) and a construct expressing only the intracellular PTPase domain-containing segment *80 kD) were expressed in the baculovirus/Sf9 cell system, purified and characterized. Full length PTP was membrane associated while the truncated form was recovered in the soluble fraction. PTP preferentially dephosphorylated a reduced carboxamidomethylated and maleylated derivative of lysozyme (RCML) over other tyrosine phosphorylated substrates such as myelin basic protein (MBP) or the synthetic peptide EDNDYINASL. The enzymatic properties of the soluble, truncated form of the enzyme were examined in detail. The pH optimum was 7.5. It dephosphorylated RCML with a K_m of 400 nM and a V_max of 725 nmol/min/mg. This form of the enzyme was 2 fold more active than full length PTP. Trypsinization of the full length form inhibited activity. Vanadate and molybdate, potent tyrosine phosphatase inhibitors, abolished activity of the enzyme. Zn⁺⁺ and Mn⁺⁺ ions, polylysine, poly-glu/tyr, and spermine were also inhibitory.     

Evidence of β-carotene 7,8(7′,8′) oxygenase (β-cyclocitral,crocetindial generating) in Microcystis

A -carotene oxygenase is described which occurs in the Cyanobacterium Microcystis. It cleaves -carotene and zeaxanthin specifically at the positions 7,8 and 7,8, while echinenone and myxoxanthophyll are not affected. The oxidative cleavage of -carotene leads to the formation of -cyclocitral and crocetindial and that of zeaxanthin to hydroxy--cyclocitral and crocetindial in nearly stoichiometric amounts. Oxidant is dioxygen as has been demonstrated by high incroporation (86%) of ¹⁸O₂ into -cyclocitral. -Carotene oxygenase is membrane bound, sensitive to sulfhydryl reagents, antioxidants and chelating agents. Iron seems to be an essential part of the enzyme activity. Cofactors necessary for the reaction could not be detected.Abbreviations TLC thin layer-chromatography - PIPES piperazine-N,N-bis-(2-ethanesulfonate) Na - TES 2{[tris-(hydroxymethyl)-methyl]-amino} ethanesulfonic acid Dedicated to Professor G. Drews on occasion of his 60th birthday     

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.     

Expression,purification, characterization,and deletion mutations of phosphorylase kinase γ subunit: identification of an inhibitory domain in the γ subunit

A catalytic fragment, _1-298, derived from limited chymotryptic digestion of phosphorylaseb kinase (Harris, W.R.et al., J. Biol. Chem., 265: 11740–11745, 1990), is reported to have about six-fold greater specific activity than does the subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the subunit, full-length wild-type and seven truncated forms of were expressed inE. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar ( _1-353 and _1-341) or less ( _1-331 and _1-276) specific activity than does the full-length wild-type , _1-386. Three truncated forms, _1-316, _1-300, and _1-290 have molar specific activities approximately twice as great as those of the full-length wild-type and the nonactivated holoenzyme. All recombinant s exhibit similarK _m values for both substrates, i.e., about 18M for phosphorylaseb and about 75 M for MgATP. Three truncated s, _1-316, _1-300, and _1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (V _max/K _m) than that of the full-length wild-type and a 3.5- to 4.5-fold greater efficiency than that of the truncated _1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of , which is located at _301-331· _1-290, but not _1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the subunit to exhibit phosphotransferase activity. Both _1-290 and _1-300 have several properties similar to full-length wild-type , including metal ion responses (activation by free Mg²⁺ and inhibition by free Mn²⁺) pH dependency, and substrate specificities.     

ATP-dependent activation of phospholipase C by antigen,NECA, Na3VO4, and GTP-γ-S in permeabilized RBL cell ghosts: Differential augmentation by ATP,phosphoenolpyruvate and phosphocreatine

Ghosts prepared from rat basophilic leukemia cells (RBL cell ghosts) and permeabilized with -toxin fromS. aureus are a simplified system for the study of FcRI-mediated activation of phospholipase C (PLC). This activity is dependent upon ATP and magnesium, and is enhanced by the addition of another compound containing an energetic phosphate group, either phosphoenolpyruvate (PEP) or phosphocreatine (PCr). This effect appears to be specific for PEP and PCr in that other compounds with energetic phosphate bonds including fructose 1,6-bisphosphate and additional ATP are not effective. On the contrary, GTP--S, an activator of G proteins, activates PLC in the presence of ATP alone and this is not further enhanced by the addition of PEP. In addition to FcRI and GTP--S, two other stimuli lead to enhanced activity of PLC in permeabilized RBL cell ghosts: 1) an inhibitor of tyrosine phosphatases (Na₃VO₄) and 2) an analog of adenosine (NECA). Data presented here extend previous results to show that activation of PLC by GTP--S is not enhanced either by the addition of PCr or by the addition of a more MgATP. Further new findings include the observations that activation of PLC by Na₃VO₄ is augmented by PEP and PCr in a fashion similar to that observed for FcRI-mediated activation of PLC and that activation of PLC by NECA shows even more marked dependency on PEP than does activation by FcRI or Na₃VO₄. Together, these experiments demonstrate that antigen, Na₃VO₄, GTP--S, and NECA can all activate PLC in permeabilized RBL cell ghosts in the presence of ATP and that these activities are differentially affected by the addition of a second compound with an energetic phosphate bond.Abbreviations DNP₂₅BSA bovine serum albumin derivitized with 25 dinitrophenyl groups per mole of BSA - FcRI high affinity receptor for IgE - GTP--S guanosine-5-O-(3-thiotriphosphate) - IPs inositol phosphate(s) - K₂ Pipes dipotassium Pipes - Na₃VO₄ sodium vanadate - NECA 5-(N-Ethyl)carboxamidoadenosine - PCr phosphocreatine - PEP phosphoenolpyruvate - PLC phospholipase C specific for phosphoinositides - RBL rat basophilic leukemia     

Excitation energy transfer between β-carotene and chlorophyll-a in various systems

The excitation energy transfer from -carotene to chlorophyll-a in several seminatural systems such as liposomes, lipid layers and PSI complex has been studied at room and liquid nitrogen temperature. Only in a case of PSI complex an efficient energy transfer (about 30%) from -carotene to chlorophyll-a has been observed. The results of energy transfer were discussed on the ground of Dexter's mechanism by taking into account the recently discovered energy level (¹Ag) of -carotene.Abbreviations chl-a chlorophyll-a - -car -carotene - R_DA mean donor-acceptor distance - PSI photosystem I - _exe excitation wavelength - _e emission wavelength - d optical pathlength     

Inhibition of ADP-ribosylation of histone H1 by analogs of diadenosine 5′, 5‴-p1,p4-tetraphosphate

The effects of analogs of diadenosine 5,5-p¹,p⁴-tetraphosphate (Ap4A) were examined on the ADP-ribosylation reaction of histone Hl catalysed by purified bovine thymus poly(ADP-ribose)transferase. Among the compounds tested, Ap4A and ApCH₂PPPA were shown to be the most efficient inhibitors of the enzyme. From kinetic studies of their action, it appears that Ap4A and ApCH₂pppA might be mixed type inhibitors.Abbreviations ADP-ribose adenosine diphosphate ribose - ADPRT poly-(ADP-ribose)transferase - Ap4A diadenosine 5,5-p₁,p₄-tertraphosphate - Ap4A diadenosine 5,5-p¹,p⁴(-1,N⁶-ethenyl-)tetra-phosphate - ApAA diadenosine 5,5-p¹,p⁴(-N⁶(-1,N⁶-)bisethenyl-)tetraphosphate - ApCH₂pppA diadenosine 5,5-p¹,p⁴(-p¹,p²-methylene-)tetraphosphate - AppCH₂ppA diadenosine 5,5-p¹,p⁴(-p²,p³methylene-)tetraphosphate - AppNHppA diadenosine 5,5-p¹,p⁴(-p²,p³-amino-)tetraphosphate - AppCHBrppA diadenosine 5,5-p¹,p⁴(-p²,p³-bromine methyno-)tetraphosphate - CpCH₂ppCH₂PC dicytidine 5,5-p¹,p⁴(-p¹,p²-p³,p⁴-bismethylene-)tetraphosphate - ApCH₂ppCH₂pA diadenosine 5,5-p¹,p⁴(-p¹,p²-p³,p⁴-bismethylene-)tetraphosphate.     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

Differences in N-acetyllactosamine synthesis between β-1,4-galactosyltransferases I and V

Unlike classical -1,4-galactosyltransferase (-1,4-GalT I), -1,4-GalT V (formerly IV*) has little activity towards 1 mM N-acetylglucosamine [Sato et al. (1998) Proc Natl Acad Sci USA 95: 472-477]. The human -1,4-GalTs I and V were expressed individually in Sf-9 cells by transfection of the full coding sequences, and their N-acetyllactosamine synthetase activities were determined towards different N-acetylglucosamine concentrations. Kinetic studies using the cell homogenates as an enzyme source revealed that -1,4-GalTs I and V possess Km values of 0.6 mM and 33 mM towards N-acetylglucosamine, and of 48 µM and 41 µM towards UDPGal, respectively. No significant inhibition of N-acetyllactosamine synthesis with -lactalbumin was observed for -1,4-GalT V but the significant inhibition with -lactalbumin was observed for -1,4-GalT I.     

Purification, properties and possible gene assignment of an α1,3-fucosyltransferase expressed in human liver

1,3-Fucosyltransferase solubilized from human liver has been purified 40 000-fold to apparent homogeneity by a multistage process involving cation exchange chromatography on CM-Sephadex, hydrophobic interaction chromatography on Phenyl Sepharose, affinity chromatography on GDP-hexanolamine Sepharose and HPLC gel exclusion chromatography. The final step gave a major protein peak that co-chromatographed with 1,3-fucosyltransferase activity and had a specific activity of 5–6 µmol min^–1 mg^–1 and anM _r 44 000 deduced from SDS-PAGE and HPLC analysis. The purified enzyme readily utilized Gal1-4GlcNAc, NeuAc2-3Gal1-4GlcNAc and Fuc1-2Gal1-4GlcNAc, with a preference for sialylated and fucosylated Type 2 acceptors. Fuc1-2Gal1-4Glc and the Type 1 compound Gal1-3GlcNAc were very poor acceptors and no incorporation was observed with NeuAc2-6Gal1-4GlcNAc. A polyclonal antibody raised against the liver preparation reacted with the homologous enzyme and also with the blood group Lewis gene-associated 1,3/1,4-fucosyltransferase purified from the human A431 epidermoid carcinoma cell line. No cross reactivity was found with 1,3-fucosyltransferase(s) isolated from myeloid cells. Examination by Northern blot analysis of mRNA from normal liver and from the HepG2 cell line, together with a comparison of the specificity pattern of the purified enzyme with that reported for the enzyme expressed in mammalian cells transfected with theFuc-TVI cDNA, suggests a provisional identification ofFuc-TVI as the major 1,3-fucosyltransferase gene expressed in human liver.Died June, 1991     

Biodiversity ‘hotspots’, patterns of richness and endemism, and taxonomic affinities of tropical Australian sponges (Porifera)

Hotspots of biodiversity (taxonomic richness, endemism, taxonomic affinities between communities) at small (), medium () and larger () scales of diversity were examined for marine sponge populations throughout tropical and subtropical Australia, with the faunas of Vanuatu, Palau and Thailand used as outgroups for comparison. Spatial and numerical (ordination) models and hierarchic classifications delineated 37 and 13 scale faunas from 1343 investigated localities using a pool of 2324 species. The Australian taxonomic literature was ignored completely to avoid the many still unresolved taxonomic problems and to allow equal treatment of collecting localities. Richness and endemism varied considerably between marine areas, for species and genera at all spatial scales, with gradients strongly corroborated by hierarchic taxonomic relationships between faunas. Richness and endemism were equally effective indicators of biodiversity hotspots, whereas species-level vs. genus-level data produced differing patterns, with the latter substantially underestimating biodiversity and marine area relationships, and consequently a poor 'surrogate for species data. Patterns of taxa shared between adjacent areas were more informative than richness and endemism data alone, as they more accurately reflect the processes in these areas. Latitudinal gradients in sponge diversity were not evident, whereas various environmental factors were prominent at scales and biogeographic factors were prominent at and scales of diversity. An example of a small () scale diversity fauna revealed substantial spatial heterogeneity (mean of 41 spp/locality, 33% apparently endemic, and a total fauna of 226 spp) containing few ubiquitous species (40% or 78 spp), with adjacent reefs having relatively low faunal similarity (mean 33%). Faunas at the medium () scale of diversity were less heterogeneous (mean 127 spp/region, 27% apparently endemic to a particular region, with a total fauna of 2324 spp), containing a significantly larger dataset (829 spp) found in >1 region to assess taxonomic affinities. At the larger () scale of diversity faunas were far more heterogeneous (mean 263 spp/region, 47% apparently endemic to a particular region) containing a smaller dataset (only 588 spp or 26% of the fauna with >1 species/region) to assess taxonomic affinities. Consequently, sponge faunas at the and scales of diversity are ineffective and inappropriate as biodiversity models, respectively, with scale diversity also less relevant as a practical tool for marine resource management and marine area conservation.     

Alpha2-adrenoreceptor stimulation does not inhibit L-type calcium channels in mouse pancreatic β-cells

The effects of ₂-adrenergic stimulation on the Ca²⁺-current in mouse pancreatic -cells were investigated using the patch-clamp technique. When using the conventional whole-cell recording configuration (dialysis of cell interior with pipette solution), addition of adrenaline (1 M) or the ₂-adrenergic agonist clonidine (5 M) failed to reduce the Ca²⁺-current, irrespective of whether intracellular GTP (or GTP S) was present or not and at both physiological (1.3 mM) and elevated (10.2 mM) Ca²⁺-concentrations. In fact, in the absence of added guanine nucleotides, the agonists tended toincrease the Ca²⁺-current amplitude in the presence of the higher Ca²⁺-concentration. Ca²⁺-channel activation measured at 1.3 mM Ca²⁺ was not affected by clonidine. Half-maximal activation was observed at –20 mV. In addition, when Ca²⁺-currents were recorded from intact -cells, using the perforated patch whole-cell configuration, clonidine (1 M) also failed to detectably affect the Ca²⁺-current. It is therefore suggested that the inhibition of -cell electrical activity and insulin-secretion produced by ₂-adrenoreceptor stimulation does not result from suppression of the L-type Ca²⁺-current.     

Phorbol ester stimulation of phosphatidylcholine synthesis in four cultured neural cell lines: Correlations with expression of protein kinase C isoforms

Phosphatidylcholine (PtdCho) can provide lipid second messengers involved in signal transduction pathways. As a measure of phospholipid turnover in response to extracellular stimulation, we investigated differential enhancement of [³H]choline incorporation into PtdCho by phorbol esters. In C6 rat glioma and SK-N-SH human neuroblastoma cells, [³H]PtdCho synthesis was 2–4 fold stimulated by -12-O-tetradecanoylphorbol-13-acetate (-TPA) when [³H]choline was incubated simultaneously with, or 15 min prior to, -TPA treatment. By contrast, in N1E-115 mouse and SK-N-MC human neuroblastoma cells, phorbol esters had no appreciable effect on [³H]choline incorporation; however, in all cells, 200 M oleic acid enhanced PtdCho synthesis, indicating a stimulable process. Alterations by thymeleatoxin (TMT), an activator of conventional PKC isoforms (, and ), were similar to -TPA. We investigated whether expression of specific PKC isoforms might correlate with these effects of phorbol esters on PtdCho synthesis. All cell lines bound phorbol esters, had PKC activity that was translocated by phorbol esters and differentially expressed isoforms of PKC. Northern and western blot analyses, using specific cDNA and antibodies for PKC-,-,-,-,-, and-, revealed that expression of -isoform predominated in C6 and SK-N-SH cells. In contrast, TPA-responsive -isoform predominated in SK-N-MC cells. -PKC was not detected in any cells and only in C6 cells was PKC- present and translocated by -TPA treatment. PKC- was not detected in SK-N-MC cell lines but translocated with TPA treatment in the other three cell lines. PKC- was present in all cells but was unaltered by TPA treatment. Accordingly, stimulation of PtdCho turnover by phorbol esters correlated only with expression of PKC-; presence of PKC- alone was insufficient for a TPA response.Abbreviations DAG diacylglycerol - DMEM Dulbecco's modified Eagle's medium - dPPA 12-deoxyphorbol-13-phenylacetate-20-acetate - PKC protein kinase C - cPKC conventional PKC - PtdCho phosphatidylcholine - TPA 12-O-tetradecanoylphorbol-13-acetate - TMT thymeleatoxin     

Genetic characterization of a new splice variant of the β2 subunit of the voltage-dependent calcium channel

This study reports a novel splice variant form of the voltage-dependent calcium channel ₂ subunit (_2g). This variant is composed of the conserved amino-terminal sequences of the _2a subunit, but lacks the -subunit interaction domain (BID), which is thought essential for interactions with the ₁ subunit. Gene structure analysis revealed that this gene was composed of 13 translated exons spread over 107 kb of the genome. The gene structure of the ₂ subunit was similar in exon-intron organization to the murine ₃ and human ₄ subunits. Electrophysiological evaluation revealed that _2a and _2g affected channel properties in different ways. The _2a subunit increased the peak amplitude, but failed to increase channel inactivation, while _2g had no significant effects on either the peak current amplitude or channel inactivation. Other subunits, such as ₃ and ₄, significantly increased the peak current and accelerated current inactivation.