The status of cysteine residues in the extrinsic 33 kDa protein of spinach photosystem II complexes

Two cysteine residues of the extrinsic 33 kDa protein in the oxygen-evolving photosystemII (PS II) complexes were found to exist as cystine residues in situ. The 33 kDa protein, when reduced by 2-mercaptoethanol in either the presence or the absence of 6 M guanidine-HCl (Gdn-HCl), could not rebind with the CaCl₂-treated PS II complexes, from which the 33 kDa protein was removed, and evolve any oxygen. Two sulfhydryl (SH) groups of the 33 kDa protein were easily reoxidized to a disulfide (S-S) bond by stirring under aerobic conditions with the concomitant regaining of both the binding ability to the CaCl₂-treated PS II complexes and the oxygen-evolving activity.The molecular conformation of the 33 kDa protein was examined by circular dichroic (CD) spectrometry in the UV regions to reveal that the conformation in the reduced state was completely different from those of the untreated and reoxidized states. The disulfide (S-S) bond of the 33 kDa protein is thus essential to maintain the molecular conformation required to function.Abbreviations CD circular dichroism - Chl chlorophyll - DMQ 2,5-dimethyl-p-benzoquinone - DTNB 5,5-dithio-bis (2-nitrobenzoic acid) - EDTA ethylendiamine-tetraacetic acid - Gdn-HCl guanidine-hydrochloric acid - PS II photosystem II - SDS sodium dodecylsulfate This paper was presented at the U.S.-Japan Binational Seminar on Solar Energy Conversion, Okazaki, Japan, March 17–21, 1987     

Restriction in the conformational flexibility of apoproteins in the presence of organic cosolvents: a consequence of the formation of "native-like conformation".

The influence of n-propanol on the overall -helical conformation of -globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of -helical conformation is induced into these proteins atpH 6.0 and 4°C in the presence of relatively low concentrations of n-propanol. The induction of -helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of -globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The -helical conformation induced into - and -globin by n-propanol decreased as the temperature is raised from 4 to 24°C. In contrast, the -helical conformation of both - and -chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced -helical conformation of globins and the -helical conformation of - and -chains. A cross-correlation of the n-propanol induced increase in the fluorescence of -globin with the corresponding increase in the -helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the -helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a native-like structure. The induction of -helical conformation into these proteins in the presence of n-propanol and the consequent generation of native-like conformation is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an -helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases. Thus, the high sensitivity of apoproteins and the protein domains to assume -helical conformation in the presence of low concentration of n-propanol with a concomitant restriction of the proteolytic susceptibility of their polypeptide chain appears to be unique to those proteins that exhibit high -helical propensities. Apparently, this phenomenon of helix induction and the restriction of proteolysis reflects the formation of rudimentary tertiary interaction of the native protein and is unique to apoproteins or structural domains of -helical proteins. Consistent with this concept, the induction of -helical conformation into shorter polypeptide fragments of 30 residues, (e.g., _1-30, which exists in an -helical conformation in hemoglobin) is very low. Besides, this peptide exhibited neither the high sensitivity to the low concentrations of n-propanol seen with the apoproteins/protein domains nor the resistance toward proteolysis. The results suggest that the organic cosolvent induced decrease in the conformational flexibility of the apoprotein, and the consequent restriction of their proteolytic cleavage provides an opportunity to develop new strategies for protease catalyzed segment condensation reactions.     

A possible calcium binding site in D1 protein: A fluorescence and FTIR study of the interaction between lanthanides and a synthetic peptide

A peptide ranging from residue 229 to 240 of the D₁ protein of Photosystem (PS) II was synthesized and lanthanides were used as candidates of calcium. Fluorescence and FTIR spectroscopy were used to test the conformational adaptation after lanthanide additions. Fluorescence spectroscopy showed that the synthetic peptide provides lanthanide binding site, and that glutamic acids are involved in lanthanide binding. Resolution enhancement techniques were combined with band curve-fitting procedures to quantitate the FTIR spectral information from the amide 1 bands. The relative areas of these component bands indicate that lanthanide induced a substantial decrease in the amount of unordered structure and turns, while a corresponding increase in the amount of -helix and open loop was also observed. This indicates that a relatively compact structure of the synthetic peptide is formed if lanthanides are applied. The results may reflect on the physiological and biochemical function of calcium in PS II, including preventing D₁ from trypsin digestion.Abbreviations DCMU 3-(3,4-Dichlorophenyl)-1,1-dimethylurea - FTIR Fourier transform infrared - FSD Fourier self-deconvolution - PS Photosystem - Q_B Secondary plastoquinone electron acceptor of PS II     

Sialidase of swine influenza A viruses: variation of the recognition specificities for sialyl linkages and for the molecular species of sialic acid with the year of isolation

The sialidase of swine influenza A viruses of N1 and N2 subtypes, isolated from 1930 to 1992, was studied for substrate specificity with ganglio-series, lacto-series type II and GM3 gangliosides containing Neu5Ac2-3Gal, Neu5Gc2-3Gal and Neu5Ac2-6Gal linkages. All viral sialidases tested showed that the activity for hydrolysing substrates with Neu5Ac2-3Gal was higher than the activities with Neu5Gc2-3Gal and Neu5Ac2-6Gal linkages. When GM1b, GM3 and sialylparagloboside were used as substrates, the earliest strain (A/Wisconsin/15/30 H1N1, isolated in 1930) showed the activity ratio of Neu5Ac2-6Gal to Neu5Ac2-3Gal to be 0.13:0.2, and the ratio Neu5Gc2-3Gal/Neu5Ac2-3Gal to be 0.19:0.37, while those strains isolated from 1978 to 1992 exhibited ratios of 0.29:0.58 for Neu5Ac2-6Gal/Neu5Ac2-3Gal and 0.51:0.76 for Neu5Gc2-3Gal/Neu5Ac2-3Gal. The above results indicate that the substrate specificities of sialidases from swine influenza A viruses towards sialyl linkages and the molecular species of sialic acid are related to the year of isolation, i.e. strains isolated after 1978 exhibited higher activity towards Neu5Ac2-6Gal and Neu5Gc2-3Gal linkages when compared with strains isolated in an earlier year, 1930.Abbreviation Neu5Ac 5-N-acetylneuraminic acid - Neu5Gc 5-N-glycolyneuraminic acid - Gal d-galactose - Glc d-glucose - Cer Ceramide - II³(Neu5Ac)Lac Neu5Ac2-3Gal1-4Glc - GM3(Neu5Ac2-3Gal) Neu5Ac2-3Gal1-4Glc1-Cer - GM3(Neu5Gc2-3Gal) Neu5Gc2-3Gal1-4Glc1-Cer - GM1b(Neu5Ac2-3Gal) Neu5Ac2-3Gal1-3GalNac1-4Gal1-4Glc1-Cer - GMlb(Neu5Gc2-3Gal) Neu5Gc2-3Gal1-3GalNAc1-4Gal1-4Glc1-Cer - IV³(Neu5Ac)nLc4Cer Neu5Ac2-3Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - IV³(Neu5Gc)nLc4Cer Neu5Gc2-3Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - IV⁶(Neu5Ac)nLc4Cer Neu5Ac2-6Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - TDC taurodeoxycholate.     

Differential accumulation of mRNAs encoding extracellular and intracellular PR proteins in tomato induced by virulent and avirulent races of Cladosporium fulvum

Tomato leaves infected by the fungal pathogen Cladosporium fulvum contain several types of intracellular and extracellular pathogenesis-related (PR) proteins. Previously, we reported the purification and serological characterization of five extracellular PR proteins: P2, P4, P6, a chitinase and a -1,3-glucanase [22, 23]. Here we describe the purification of a basic intracellular 33 kDa -1,3-glucanase and the isolation and characterization of cDNA clones encoding the two extracellular P14 isomers P4 and P6, the extracellular acidic -1,3-glucanase and a basic 35 kDa -1,3-glucanase, different from the purified 33 kDa protein. Southern blot analysis demonstrated that tomato PR proteins are not encoded by large gene families, as is the case in tobacco. The number of genes corresponding to each protein was estimated to vary between one and three. A northern blot analysis indicated that the mRNAs for the extracellular PR proteins (P4, P6 and acidic -1,3-glucanase) accumulate to similar levels in compatible and incompatible tomato-C. fulvum interactions, although the maximum level of expression is reached much faster in the incompatible interaction. On the other hand, the mRNA for the basic 35 kDa -1,3-glucanase is induced rapidly to high levels in both interactions, but declines in time to background levels only in the incompatible interaction. The relevance of this difference in relation to plant defence is discussed.     

Composition of poly-β-hydroxyalkanoate from Syntrophomonas wolfei grown on unsaturated fatty acid substrates

Poly--hydroxyalkanoate (PHA) from crotonate-grown cultures of Syntrophomonas wolfei contained only the d-isomer of -hydroxybutyrate. The PHA from cultures grown with trans-2-pentenoate or one of several hexenoates as the substrate also contained small amounts (5%) of -hydroxypentanoate or -hydroxyhexanoate, respectively. Thus, some PHA was synthesized without cleavage of the carbon skeleton of the substrate, but the predominant route for PHA synthesis was by the condensation and subsequent reduction of acetyl-coenzyme A (CoA). The ratio of the -hydroxypentanoate to the -hydroxybutyrate in PHA in pentenoate-grown cultures increased immediately after inoculation and then decreased as the amount of the -hydroxybutyrate in PHA increased. The amount of -hydroxypentanoate in the PHA did not markedly change throughout the remainder of growth. These data indicated that the unbroken carbon-chain was used for polymer production only in the early stages of growth and, later, polymer synthesis occurred by the condensation and reduction of acetyl-CoA molecules.     

Intracellular sites of the synthesis of sweet potato mitochondrial F1ATPase subunits

Summary Five subunits (-, -, -, - and -subunits) of the six -and -subunits) in the F₁ portion (F₁ATPase) of sweet potato (Ipomoea batatas) mitochondrial adenosine triphosphatase were isolated by an electrophoretic method. The - and -subunits were not distinguishable immunologically but showed completely different tryptic peptide maps, indicating that they were different molecular species. In vitro protein synthesis with isolated sweet potato root mitochondria produced only the -subunit when analyzed with anti-sweet potato F₁ATPase antibody reacting with all the subunits except the -subunit. Sweet potato root poly(A)⁺RNA directed the synthesis of six polypeptides which were immunoprecipitated by the antibody: two of them immunologically related to the -subunit and the others to the - and -subunits. We conclude that the -subunit of the F₁ATPase is synthesized only in the mitochondria and the -, - and -subunits are in the cytoplasm.     

Adenylate effects on protein phosphorylation in the interenvelope lumen of pea chloroplasts

A 64-kilodalton (kDa) protein, situated in the lumen between the inner and outer envelopes of pea (Pisum sativum L.) chloroplasts (Soll and Bennett 1988, Eur. J. Biochem., 175, 301–307) is shown to undergo reversible phosphorylation in isolated mixed envelope vesicles. It is the most conspicuously labelled protein after incubation of envelopes with 33 nmol·1^-1 [-³²P]ATP whereas incubation with 50 mol·1^-1 [-³²P]ATP labels most prominently two outer envelope proteins (86 and 23 kDa). Half-maximum velocity for phosphorylation of the 64-kDa protein occurs with 200 nmol·1^-1 ATP, and around 40 mol·1^-1 ATP for phosphorylation of the 86- and 23-kDa proteins, indicating the operation of two distinct kinases. GGuanosine-, uridine-, cytidine 5-triphosphate and AMP are poor inhibitors of the labelling of the 64-kDa protein with [-³²P]ATP. On the other hand, ADP has a potent influence on the extent of labelling (half-maximal inhibition at 1–5 mol·1^-1). The ADP-dependent appearance of ³²P in ATP indicates that ADP acts by reversal of kinase activity and not as a competitive inhibitor. However, the most rapid loss of ³²P from pre-labelled 64-kDa protein occurs when envelope vesicles are incubated with ATP t1/2=15 s at 20 molsd1^-1 ATP). This induced turnover of phosphate appears to be responsible for the rapid phosphoryl turnover seen in situ.Abbreviations LHCP ligh-harvesting chlorophyll-a/b-binding protein - S_0.5 concentration giving half-maximal phosphorylation - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

1H, 15N and 13C NMR resonance assignment, secondary structure and global fold of the FMN-binding domain of human cytochrome P450

The FMN-binding domain of human NADPH-cytochrome P450 reductase,corresponding to exons 3-;7, has been expressed at high level in anactive form and labelled with ¹³C and ¹⁵N. Mostof the backbone and aliphatic side-chain ¹H, ¹⁵Nand ¹³C resonances have been assigned using heteronucleardouble- and triple-resonance methods, together with a semiautomaticassignment strategy. The secondary structure as estimated from the chemicalshift index and NOE connectivities consists of six -helices and five-strands. The global fold was deduced from the long-range NOEsunambiguously assigned in a 4D ¹³C-resolved HMQC-NOESY-HMQCspectrum. The fold is of the alternating / type, with the five-strands arranged into a parallel -sheet. The secondarystructure and global fold are very similar to those of the bacterialflavodoxins, but the FMN-binding domain has an extra short helix in place ofa loop, and an extra helix at the N-terminus (leading to the membrane anchordomain in the intact P450 reductase). The experimental constraints werecombined with homology modelling to obtain a structure of the FMN-bindingdomain satisfying the observed NOE constraints. Chemical shift comparisonsshowed that the effects of FMN binding and of FMN reduction are largelylocalised at the binding site.     

Ecto-ATPase/phosphatase activity in the olfactory sensilla of the spiny lobster,Panulirus argus: localization and characterization

Summary Electrophysiological studies have shown that the olfactory organ (antennule) of the spiny lobster, Panulirus argus, has chemoreceptors that are selectively excited by adenine nucleotides in seawater. Biochemical studies have revealed that these same nucleotides can be rapidly dephosphorylated by ectoenzymes associated with the olfactory sensilla (aesthetascs). In this study the distribution of ecto-ATPase/phosphatase activity within aesthetascs was determined cytochemically and the nature of the adenine-nucleotide dephosphorylating activity was dissected biochemically. Cytochemically, the distribution of ATP-dephosphorylating activity was similar to that shown previously for AMP and -glycerol phosphate; i.e., cerium phosphate reaction product was specifically localized to the transitional zone where the sensory dendrites develop cilia and branch to form the outer dendritic segments. Unlike the dephosphorylation of AMP and -glycerol phosphate, Mg²⁺ or Ca²⁺ was required for ecto-ATPase/phosphatase activity. Biochemical measures of both AMP-and ATP-dephosphorylating activity within aesthetascs corroborated the cytochemical evidence that these activities are localized to the transitional zone. A major portion of the AMP dephosphorylation (about 67%) derives from nonspecific alkaline phosphatase activity that is insensitive to levamisole and L-bromotetramisole. In contrast, nonspecific phosphatase activity accounted for a much smaller part of the ATP dephosphorylation (about 15%). Ectoenzymatic activity in the transitional zone may be an important means of removing excitatory/inhibitory nucleotides from this region.Abbreviations ADP Adenosine 5-diphosphate - AMP adenosine 5-monophosphate - AMPCP , -methylene ADP - ASW artificial seawater - ATP adenosine 5-triphosphate - -GP -glycerol phosphate - EM electron microscopy     

A simple brassinolide analogue 2α, 3α-dihydroxy-17β-(3-methyl-butyryloxy)-7-oxa-B-homo-5α-androstan-6-one which induces bean second internode splitting

A new synthetic brassinolide analogue, 2,3-dihydroxy-17-(3-methylbutyryloxy)-7-oxa-B-homo-5-androstan-6-one (11), has been shown to exhibit typical brassinolide activity characterised by elongation, swelling, twisting and splitting of the bean second internode. It was prepared from the known lactone 2,3,17-trihydroxy-7-oxa-B-homo-5-androstan-6-one (4) which was transformed to an isopropylidenedioxy derivative. After protection of the 2- and 3-hydroxy groups it yielded the 2,3-isopropylidenedioxy-17-(3-methyl-butyryloxy)-7-oxa-B-homo-5-androstan-6-one (7) on treating with 3-methylbutyryl chloride in pyridine. The analogue with a 2-methylbutyric moiety (10, 2,3-dihydroxy-17-(2-methyl-butyryloxy)-7-oxa-B-homo-5-androstan-6-one) in position 17 stimulated only elongation and swelling of the bean second internode. However, in this bioassay 100 times more 10 or 11 compared to 24-epibrassinolide is required to obtain the same effects. Analogues with -oriented hydroxyl groups at C-2 and C-3 (14,15), a 6-ketone (17,18) or 6-oxa-7-oxo-lactone system (12,13) in ring B lack the typical brassinolide activity. In addition, the active brassinosteroids applied to the second internode stimulated a similar, but 30% lower elongation of the first internode. From data presented here we conclude that the presence of two hydroxy groups in the positions 22 and 23 of the brassinolide side chain, which are considered as a key structural requirement, is not absolutely necessary for a compound to exhibit typical brassinosteroid activity. Nevertheless, these compounds have generally 2–10 times lower activity than that having 22,23-vicinal diol in the side chain.     

Expression of a chimeric stilbene synthase gene in transgenic wheat lines

A chimeric stilbene synthase (sts)gene was transferred into wheat. Stilbene synthases play a role in the defence against fungal diseases in some plant species (e.g. groundnut or grapevine) by producing stilbenetype phytoalexins like resveratrol. Resveratrol is also claimed to have positive effects to human health. Embryogenic scutellar calli derived from immature embryos of the two commercial German spring wheat cultivars Combi and Hanno were used as target tissue for cotransformation by microprojectile delivery. The selectable marker/reporter gene constructs contained the bargene either driven by the ubiquitinpromoter from maize (pAHC 25, also containing the uidAgene driven by the ubiquitinpromoter), or by the actinpromoter (pDM 302) from rice. The cotransferred plasmid pStil 2 consisted of a grapevine stscoding region driven by the ubiquitin promoter. Eight transgenic Combi and one Hanno T_Oplant were obtained and, except one Combi T_Oplant, found to be cotransformants due to the integration of both the stsgene and the selectable marker or reporter genes. Expression of the stsgene was proven by RTPCR, and, for the first time, by detection of the stilbene synthase product resveratrol by HPLC and mass spectrometry. The stsgene was expressed in four of the seven transgenic Combi T_{_o}plants. Two of the respective T₁progenies segregated in a Mendelian manner were still expressing the gene. Investigations into methylation of the stsgene showed that in three nonexpressing progenies inactivation was paralleled by methylation.     

Cloned β1,4N-acetylgalactosaminyltransferase: subcellular localization and formation of disulfide bonded species

Cloned human 1,4N-acetylgalactosaminyltransferase (GalNAcT) catalyses the synthesis of the glycosphingolipids GM2, GD2, and gangliotriosylceramide. To determine the subcellular location of this enzyme and whether it exists in intermolecular disulfide bonded species, we stably transfected Chinese hamster ovary (CHO) cells with three myc epitope-tagged forms of the GalNAcT gene: the native enzyme; the lumenal domain of GalNAcT fused to the cytoplasmic and transmembrane domains ofN-acetylglucosaminyltransferase I (GNT); and the transmembrane and lumenal domains of GalNAcT fused to the cytoplasmic domain of the Iip33 form of human invariant chain in order to retain the enzyme in the endoplasmic reticulum (ER). Immunoelectron microscopic analysis with anti-myc revealed that GalNAcT/myc was present throughout the Golgi stack, the GNT/GalNAcT/myc form was restricted primarily to the medial Golgi cisternae, and the Iip33/GalNAcT/myc form was restricted to the ER. Cells transfected with each of the three constructs contained high levels of GM2 synthase activityin vitro, but only the GalNAcT/myc form and the GNT/GalNAcT/myc forms were able to synthesize the GM2 productin vivo. The enzyme produced by all three constructs was present in the transfected cells in a disulfide bonded form having a molecular size consistent with that of a homodimer or higher aggregate.Abbreviations GSL glycosphingolipid(s) - CHO Chinese hamster ovary - GSL structures: GM2 GalNAc1,4(NeuAc2,3)Gal1,4GlcCer - GD2 GalNac1,4(NeuAc2,8NeuAc2,3)Gal1,4GlcCer - GM3 NeuAc2,3Gal1,4GlcCer - Gg₃ GalNAc1,4Gal1,4GlcCer - LacCer Gal1,4GlcCer - GlcCer glucosylceramide - PBS-BSA phosphate buffered saline pH 7.4 containing 1% bovine serum albumin - GalNAcT N-acetylgalactosaminyltransferase - GNT N-acetylglucosaminyltransferase I - Iip33 p33 form of human invariant chain - HPTLC high performance thin layer chromatography - PCR polymerase chain reaction - BFA Brefeldin A This paper is dedicated to Professor Sen-itiroh Hakomori on the occasion of his 65th birthday.     

Pheromone-induced stimulation of inositol-trisphosphate formation in insect antennae is mediated by G-proteins

Summary Phospholipase C in the antennae of Periplaneta americana was stimulated by pheromones in a GTP-dependent manner. The enzyme activity was potentiated by hydrolysis-resistant analogs of GTP and decreased by GDP analogs. Guanine nucleotide binding regulatory proteins (G-proteins) in antennal preparations were identified by bacterial toxin-catalyzed ADP-ribosylation and immunoreactivity with antibodies of designed specificity. The stimulatory effect of pheromones on inositol phosphate formation was completely blocked by pertussis toxin suggesting that the pheromone action was mediated via specific G-proteins.Abbreviations G-protein guanine nucleotide-binding regulatory protein - IP ₃ inositol 1,4,5-trisphosphate - GTPS guanine-5-[thio]-triphosphate - GDPS guanine-5-[-thio]-diphosphate - NAD nicotinic acid adenine dinucleotide - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - DTT dithiothreitol - MOPS 3-(N-morpholino)propanesulfonic acid - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - DMSO dimethyl-sulfoxide     

Specific in vitro phosphorylation of plant eIF2α by eukaryotic eIF2α kinases

Phosphorylation of the subunit of eukaryotic initiation factor 2 (eIF2) is known to be an important translational control mechanism in all eukaryotes with the major exception of plants. Regulation of mammalian and yeast eIF2 activity is directly governed by specific phosphorylation on Ser-51. We now demonstrate that recombinant wheat wild-type (51S) but not mutant 51-Ala (51A) protein is phosphorylated by human PKR and yeast GCN2, which are defined eIF2 kinases. Further, only wheat wild-type eIF2 is a substrate for plant-encoded, double-stranded RNA-dependent kinase (pPKR) activity. Plant PKR and GCN2 phosphorylate recombinant yeast eIF2 51S but not the 51A mutant demonstrating that pPKR has recognition site capability similar to established eIF2 kinases. A truncated version of wild-type wheat eIF2 containing 51S but not the KGYID motif is not phosphorylated by either hPKR or pPKR suggesting that this putative eIF2 kinase docking domain is essential for phosphorylation. Taken together, these results demonstrate the homology among eukaryotic eIF2 species and eIF2 kinases and support the presence of a plant eIF2 phosphorylation pathway.     

ADP-ribosyltransferase activity in myelin membranes isolated from human brain

An ADP-ribosyltransferase has been identified in compact myelin and in several white matter fractions which contain less compact myelin, fractionated on the basis of increasing protein/lipid ratios. One fraction the P₃A contained the greatest activity although the activity in compact myelin was only slightly less. The ADP-ribosyltransferase activity of solubilized myelin was stimulated by increasing amounts of GTPS and was specific for the -isomer of NAD. Although ADP-ribosylation was demonstrated with the heterotrimeric G proteins in the 40–50 kDa range, the substrate for the ADP-ribosyltransferase in the 20 kDa range was identified as MBP. ADP-ribosyltransferase; myelin basic protein; signal transduction.Abbreviations ADP-ribose adenosine diphosphate ribose - APAD 3-acetylpyridine adenine dinucleotide - ATP adenosine triphosphate - C-1, 2, 3 etc MBP components isolated by CM52 chromatography - EDTA ethylenediaminetetraacetic acid - GTP guanosine triphosphate - GTPS guanosine 5-(3-0-thio)triphosphate - INH isonicotinic acid hydrazide - MBP myelin basic protein - NAD nicotinamide adenine dinucleotide - PMSF phenylmethylsulfonyl fluoride - PLP proteolipid protein Special issue dedicated to Dr. Leon S. Wolfe.     

The effect of different growing conditions on water relations parameters of leaf epidermal cells of Tradescantia virginiana L.

Summary Tradescantia virginiana L. plants were cultivated under contrasting conditions of temperature, humidity, light quality and intensity, and nutrient status in order to investigate the effect of growth conditions on the water relations parameters of the leaf epidermal cells. Turgor pressure (P), volumetric elastic modulus (), half-time of water potential equilibration (T _1/2), hydraulic conductivity (L _p ) were measured with the miniaturized pressure probe in single cells of the upper and lower epidermis of leaves. Turgor differed (range: 0.1 bar to 7.2 bar) between treatments with lowest values under warm and humid conditions and additional supply of fertilizer, and highest values under conditions of low air humidity and low nutrient supply. The volumetric elastic modulus changed by 2 orders of magnitude (range: 3.0 bar to 350 bar, 158 cells), but was only affected by the treatments, in as much as it was dependent on turgor. The turgor dependence of , measured on intact leaves of T. virginiana, was similar to that for cells of the isolated (peeled) lower epidermis, where as a function of turgor was linear over the whole range of turgors. This result has implications for the discussion of pressure/volume curves as measured by the pressure bomb where changes in bulk leaf are frequently discussed as adaptations to certain treatments. The measurements of the hydraulic conductivity indicate that this parameter varies between treatments (range of means: 2.4×10^-6 cm s^-1 bar^-1 to 13.4×10^-6 cm s^-1 bar^-1). There was a negative correlation for L _p in cells of intact leaves as a function of turgor which was altered by the growing conditions. However, a correlation with turgor could not be found for cells from isolated epidermis or cells from a uniform population of plants. The large variation in L _p from cell to cell observed in the present and in previous studies was accounted for in a study of 100 cells from a uniform population of plants by the propagation of measurement errors in calculating L _p . The results suggest that in T. virginiana cellular water relations are changed mainly by the turgor dependence of .     

Reaction centers from three herbicide resistant mutants of Rhodobacter sphaeroides 2.4.1: Kinetics of electron transfer reactions

Electron transfer rates were measured in RCs from three herbicide-resistant mutants with known amino acid changes to elucidate the structural requirements for last electron transfer. The three herbicide resistant mutants were IM(L229) (Ile-L229 Met), SP(L223) (Ser-L223 Pro) and YG(L222) (Tyr-L222 Gly). The electron transfer rate D⁺Q_A ^-Q_BD⁺Q_AQ_B (k _AB) is slowed 3 fold in the IM(L229) and YG(L222) RCs (pH 8). The stabilization of D⁺Q_AQ_B ^- with respect to D⁺Q_AQ_B ^- (pH 8) was found to be eliminated in the IM(L229) mutant RCs (G⁰ 0 meV), was partially reduced in the SP(L223) mutant RCs (G⁰=–30 meV), and was unaltered in the YG(L222) mutant RCs (G⁰=–60 meV), compared to that observed in the native RCs (G⁰=–60 meV). The pH dependences of the charge recombination rate D⁺Q_AQ_B ^-DQ_AQ_B (k _BD) and the electron transfer from Q_A ^- (k _QA ^-Q_A) suggest that the mutations do not affect the protonation state of Glu-L212 nor the electrostatic interactions of Q_B and Q_B ^- with Glu-L212. The binding affinities of UQ₁₀ for the Q_B site were found in order of decreasing values to be native IM(L229) > YG(L222) SP(L223). The altered properties of the mutant RCs are used to deduce possible structural changes caused by the mutations and are dicscussed in terms of photosynthetic efficiency of the herbicide resistant strains.Abbreviations Bchl bacteriochlorophyll - Bphe bacteriopheophytin - cholate 3,7,12-trihydroxycholanic acid - D donor (bacteriochlorophyll dimer) - EDTA ethylenediamine tetraacetic acid - Fe²⁺ non-heme iron atom - LDAO lauryl dimethylamine oxide - PS II photosystem II - Q_A and Q_B primary and secondary quinone acceptors - RC bacterial reaction center - Tris tris(hydroxymethyl)aminomethane - UQ₀ 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50     

Availability of oncogene activated production system for mass production of light chain of human antibody in CHO cells

We previously established a ras-oncogene amplified Chinesehamster ovary (CHO) cell line, named ras clone I, as anuniversal host cell line for oncogene activated production(OAP) system to mass-produce recombinant protein by activationof the cytomegalovirus immediate early (CMV) promoter with ras protein. The light chain(C5) of human monoclonal antibody HB4C5 is expected tobe potentially useful for lung cancer targeting. We generated aC5 hyper-producing cell line by transfecting ras cloneI with the C5 gene expression plasmid regulated by theCMV promoter, of which productivity was 5.3 times greater thanthe hyper productive CHO cell line generated by using conventional CHO cells. Introduction of the adenovirus E1A geneinto the hyper-producing cell line derived from ras clone I resulted in further 9.5 times enhancement of the productivity,suggesting the synergistic effect of E1A and ras oncogenes on the recombinant protein production driven by the CMV promoter. In addition, intracellular accumulation of C5 andupregulation of BiP was found in hyper-producing cell lineswhich were introduced E1A and ras oncogene. This resultsuggests that excessive intracellular accumulation ofC5 protein, which might be caused by that the amount of produced C5 in ER is beyond the ability of CHO cells to secrete, might signal the BiP promoter. Our data imply that ras clone I is available as a general host cell for establishing the recombinant protein hyper-producing CHOcells by the OAP system, and suggest that further mass production of recombinant proteins in the OAP system can be possible by clarifying the accurate role of upregulated BiP protein.