Identification and characterization of a divalent cation-dependent glycosaminoglycan-binding protein from rat liver endothelium

Rat liver endothelial cells (LECs) express a membrane-associatedCa²⁺-dependent hyaluronan-binding activity (CaHA-BP) which isdistinct from the Ca²⁺-dependent, endocytic LEC HA receptor(Yannariello-Brown et al., J. Cell Biochem., 48, 73–80,1992). The CaHA-BP is specific for a subset of glycosaminoglycans,since Ca²⁺-dependent binding of ¹²⁵I-HA ({small tilde}80kDa)to LECs was competed with a 100-fold excess (w/w) of HA, chondroitinsulfate, and heparin, but not with chondroitin. The CaHA-BPactivity on intact LECs was pH-dependent. Optimal binding occurredat pH 6.0; no binding was detected at pH values 5 or 9. ¹²⁵I-HA,pre-bound in the presence of Ca²⁺ could also be dissociatedwith an acidic buffer (pH 5.0), as well as the divalent cationchelators EDTA and EGTA. ¹²⁵I-HA binding was stimulated by divalentcations other than Ca²⁺ such as Mg²⁺, Mn²⁺ and Ba²⁺; with theexception of Zn²⁺. A photoaffinity crosslinking reagent (¹²⁵I-ASD-HA)was used to identify specifically crosslinked polypeptides onLECs. In the absence of Ca²⁺ and in the presence of EGTA, onlybands at 175/166 kDa were consistently crosslinked. These bandshave been previously identified as the LEC Ca²⁺-independentendocytic HA receptor (Yannariello-Brown et al., J. Biol. Chem.,267, 20451/20455, 1992). In the presence of Ca²⁺, crosslinkingwas consistently seen to a 68 kDa polypeptide. Crosslinkingwas competed with a 100-fold excess (w/w) of HA. These and otherdata suggest that a 68 kDa protein is the most likely candidatefor the CaHA-BP in LECs. photoaffinity crosslinking hyaluronan calcium lectin     

Purification of Ribulose 5-phosphate Kinase and Minor Polypeptides of Pyrenoid from the Green Alga Bryopsis maxima

Ribulose 5-phosphate (Ru5P) kinase (ATP:D-ribulose 5-phosphate1-phosphotrans- ferase; EC 2.7.1.19 [EC] ), an enzyme in the reductivepentose phosphate cycle, was purified from the green alga Bryopsismaxima and its activity and peptide composition were studied.The specific activity of purified Ru5P kinase was 20 µmoleRuBP formed (mg protein)^–1 min^–1 corresponding toa 490-fold purification from the supernatant of chloroplasts.The K_m values of Ru5P kinase for ATP and Ru5P were 69 µMand 330 µM, respectively. The molecular size of Ru5P kinase was estimated as 90 kDa bygel filtration and that of its polypeptide as 41 kDa by SDS-polyacrylamidegel electrophoresis. A small portion of the Ru5P kinase wasfound in a large molecular state (500 kDa) which was consideredto be an inactive form of the enzyme. Ru5P kinase activity has been reported in the pyrenoid of Eremosphaeraviridis as well as ribulose 1,5-bisphosphate carboxylase-oxygenase(RuBisCO) and ribose 5-phosphate isomerase activity (Holdsworth1971). In Bryopsis maxima, among the pyrenoid polypeptides otherthan that of RuBisCO, we found a polypeptide of 42 kDa, similarto that of Ru5P kinase in molecular size and ratio to RuBisCO.A peptide map of the 42 kDa pyrenoid polypeptide, however, showedthat it differed from that of Ru5P kinase. In conclusion, Ru5Pkinase may be not involved in the pyrenoid of this alga. (Received January 19, 1985; Accepted May 15, 1985)     

Phosphoproteins and Protein Kinase Activities Intrinsic to Inner Membranes of Potato Tuber Mitochondria

Inside-out submitochondrial particles (IO-SMP) were isolatedand purified from potato (Solanum tuberosum L. cv.) tubers.When these IO-SMP were incubated with [ ³²P]ATP more then 20proteins became labelled as a result of phosphorylation. The³²P incorporation was stimulated by the oxidising reagent ferricyanide.Except for a 17 kDa protein which was phosphorylated only inthe absence of divalent cations, the protein phosphorylationrequired Mg²⁺. The time for half-maximum ³²P incorporation was4 mm for the 22 kDa phospho-F₁ -subunit and 2 min for the 28kDa phospho-F₀ b-subunit of the proton-ATPase. The K_m for ATPfor the detected phosphoproteins was between 65 µM and110 µM. The pH optimum for protein phosphorylation ininner membranes was between pH 6 and 8, and for the F₁ -subunitand the F₀ b-subunit the pH optima were 6.5–8 and pH 8,respectively. A 37 kDa phosphoprotein was phosphorylated ona histidine residue while the remainder of the inner membraneproteins were phosphorylated on serine or threonine residues.Two autophosphorylated putative kinases were identified: oneat 16.5 kDa required divalent cations for autophosphorylation,while another at 30 kDa did not. A 110 kDa protein was labelledonly with [-³²P] suggesting adenylylation. ³ Present address; Novartis Seeds AB, Box 302, S-261 23 Landskrona,Sweden.     

A Protein from Immature Raspberry Fruits which Inhibits Endopolygalacturonases from Botrytis cinerea and other Micro-organisms

A polygalacturonase-inhibiting protein (PGIP) was purified fromimmature raspberry fruits using ion exchange chromatography.The protein was composed of a single polypeptide chain withM_r of 38·5 kDa and a pI residing above pH 10. Kineticstudies suggested that the inhibition was of a non-competitivenature. The PGIP inhibited two endopolygalacturonases (endo-PG)purified from Botrytis cinerea and an endo-PG produced by Aspergillusniger to varying degrees but did not inhibit two exo-PGs purifiedfrom B. cinerea, bacterial endopectate lyases and bacterialendo-PGs. The concentration of PGIP at various stages of flowerand fruit development was determined. The inhibitor was notdetected in the flower, but reached a maximum of 69 units g^–1in the immature green fruit decreasing to 9 units g^–1as fruits matured. The N-terminal amino-acid sequence was determined. Key words: Polygalacturonase-inhibiting protein, Rubus idaeus, red raspberry, Botrytis cinerea, pectinases     

Two Polypeptides Inducible by Low Levels of CO2 in Soluble Protein Fractions from Chlorella regularis Grown at Low or High pH

Previous studies suggested that certain protein(s) other thancarbonic anhydrase might play an important role in the facilitatedtransport of dissolved inorganic carbon (DIC) from the mediumto the site of CO₂ fixation by ribulose-1,5-bisphosphate carboxylase/oxygenasein the unicellular green alga Chlorella regularis adapted tolow-CO₂ (ordinary air) conditions [Shiraiwa et al. (1991) Jpn.J. Phycol. 39: 355; Satoh and Shiraiwa (1992) Research in Photosynthesis,Vol. III, p. 779]. The proteins that might be involved in thisfacilitated transport of DIC were investigated by pulse-labelingof induced proteins with ³⁵S-sulfate during adaptation of cellsgrown under high-CO₂ conditions to low CO₂. Analysis by SDS-PAGErevealed that synthesis of two polypeptides, with molecularmasses of 98 and 24 kDa, respectively, was induced under low-CO₂conditions. The 24-kDa polypeptide was induced at pH 5.5 butnot at pH 8.0, whereas the 98-kDa polypeptide was induced atboth pH 5.5 and pH 8.0. The possible role of these polypeptidesin the facilitated transport of DIC in Chlorella regularis isdiscussed. (Received October 30, 1995; Accepted February 26, 1996)     

Purification and properties of DNA topoisomerase II from Daucus carota cells

Topoisomerase II was partially purified from Daucus carota cellsby a procedure including ammonium sulphate fractionation, ion-exchange,and affinity chromatography steps. The type II enzyme, identifiedfor its ability to unknot knotted P4 DNA and decatenate Trypanosomacruzi kDNA, requires ATP and Mg²⁺ for activity. The unknottingactivity was sensitive to an inhibitor of the mammalian typeII enzyme, the drug VP16 (IC₅₀ 32 mmol m^–3), whereas inhibitorsof DNA gyrase showed a limited effect on activity. The SDS-PAGEanalysis of the dsDNA cellulose fraction revealed the presenceof four polypeptides of apparent molecular masses of 72, 71,34, and 33 kDa among which only a polypeptide of about 70 kDacrossreacted with antibodies against yeast topoisomerase II.Immunoprecipitation experiments with monoclonal antibodies tothe and ß isoforms of the human enzyme confirmedthe recognition of a polypeptide of 70 kDa. The sedimentationcoefficient (S) of the topoisomerase II in the phosphocellulosefraction, calculated by analytical glycerol gradient, was 6.1corresponding to a molecular mass of about 123 kDa. Resultssuggest the presence in carrot of a protein of molecular massof 70 kDa having the typical properties of an eukaryotic topoisomeraseII and carrying epitopes recognized by MoAbs to both human and ß enzymes. The 70 kDa polypeptide might then representthe monomer of a homodimer enzyme of 123 kDa. Key words: Daucus carota, topoisomerase II, immunoprecipitation     

A Nitrate-Inducible Plasma Membrane Protein of a Marine Alga, Heterosigma akashiwo

The rate of nitrate uptake by Heterosigma akashiwo cells thathad been cultured in medium with nitrate or ammonium ions asthe source of nitrogen was measured using¹⁵NO_3– The ratioof ¹⁵N/¹⁴N increased dramatically in nitrate-grown cells. Inammonium-grown cells, the ratio of ¹⁵N/¹⁴N did not increasefor 3 h but then it began to increase. Even when nitrate reductaseactivity was inhibited by tungstate, nitrate-grown cells couldtake up nitrate. Plasma membranes from nitrate-grown and ammonium-grown cellswere purified by the silica-microbead method, and polypeptidesassociated with the membranes were analyzed by SDS-PAGE andimmunostaining. A major polypeptide with a molecular mass of26 kDa appeared 3 h after the transfer of ammonium-grown cellsto nitrate-containing medium, and it disappeared 2 d after thetransfer of nitrate-grown cells to ammonium-containing medium.The 26 kDa polypeptide also appeared when cell growth shiftedfrom the logarithmic phase to the stationary phase and the ammoniumcontent of the medium decreased, even when the cells were culturedin ammonium-containing medium. (Received April 10, 1992; Accepted July 30, 1992)     

Characterization of a Maize Ca2+-Dependent Protein Kinase Phosphorylating Phosphoenolpyruvate Carboxylase

Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31 [EC] ] of plantsundergoes regulatory phosphorylation in response to light ornutritional conditions. However, the nature of protein kinase(s)for this phosphorylation has not yet been fully elucidated.We separated a Ca²⁺-requiring protein kinase from Ca²⁺-independentone, both of which can phosphorylate maize leaf PEPC and characterizedthe former kinase after partial purification. Several linesof evidence indicated that the kinase is one of the characteristicCa²⁺-dependent but calmodulin-independent protein kinase (CDPK).Although the M_r, of native CDPK was estimated to be about 100kDa by gel permeation chromatography, in situ phosphorylationassay of CDPK in a SDS-polyacrylamide gel revealed that thesubunit has an M_r of about 50 kDa suggesting dimer formationor association with other protein(s). Several kinetic parameterswere also obtained using PEPC as a substrate. Although the CDPKshowed an ability of regulatory phosphorylation (Ser-15 in maizePEPC), no significant desensitization to feedback inhibitor,malate, could be observed presumably due to low extent of phosphorylation.The kinase was not specific to PEPC but phosphorylated a varietyof synthetic peptides. The possible physiological role of thiskinase was discussed. ¹Present address: NEOS Central Research Laboratory, 1-1 Ohike-machi,Kosei-cho, Shiga, 520-3213 Japan. ²Present address: Chugai Pharmaceutical Co., Ltd., 1-135 Komakado,Gotemba, 412-0038 Japan. ⁴N.O. and N.Y. contributed equally to this work.     

Mutagenesis of the N-glycosylation site of hNaSi-1 reduces transport activity

The human Na⁺-sulfate cotransporter (hNaSi-1) belongs to the SLC13 gene family, which also includes the high-affinity Na⁺-sulfate cotransporter (hSUT-1) and the Na⁺-dicarboxylate cotransporters (NaDC). In this study, the location and functional role of the N-glycosylation site of hNaSi-1 were studied using antifusion protein antibodies. Polyclonal antibodies against a glutathione S-transferase fusion protein containing a 65-amino acid peptide of hNaSi-1 (GST-Si65) were raised in rabbits, purified, and then used in Western blotting and immunofluorescence experiments. The antibodies recognized native NaSi-1 proteins in pig and rat brush-border membrane vesicles as well as the recombinant proteins expressed in Xenopus oocytes. Wild-type hNaSi-1 and two N-glycosylation site mutant proteins, N591Y and N591A, were functionally expressed and studied in Xenopus oocytes. The apparent mass of N591Y was not affected by treatment with peptide-N-glycosylase F, in contrast to the mass of wild-type hNaSi-1, which was reduced by up to 15 kDa, indicating that Asn⁵⁹¹ is the N-glycosylation site. Although the cell surface abundance of the two glycosylation site mutants, N591Y and N591A, was greater than that of wild-type hNaSi-1, both mutants had greatly reduced V_max, with no change in K_m. These results suggest that Asn⁵⁹¹ and/or N-glycosylation is critical for transport activity in NaSi-1. antifusion protein antibodies; Xenopus oocytes; sulfate; immunofluorescence     

Purification and Characterization of a Ca2+-Dependent Protein Kinase from the Halotolerant Green Alga Dunaliella tertiolecta

A Ca²⁺-dependent protein kinase (CDPK) that has been partiallypurified and characterized previously [Yuasa and Muto (1992)Arch. Biochem. Biophys. 296: 175] was further purified to about20,000-fold from the soluble fraction of Dunaliella tertiolecta.The enzyme preparation contained 60- and 52-kDa polypeptidesboth of which phosphorylated casein as a substrate. Both polypeptidesshowed a Ca²⁺-dependent increase in mobility during SDS-PAGEand ⁴⁵Ca²⁺-binding activity after SDS-PAGE and electroblottingonto a nitrocellulose membrane, suggesting that both the 60-and 52-kDa CDPKs directly bind Ca²⁺. The protein kinase inhibitors,K-252a and staurosporine, inhibited the CDPK competitively withrespect to ATP. An antibody raised against the 60-kDa CDPK crossreactedwith both the 60- and 52-kDa polypeptides. Both molecular specieswere autophosphorylated in the presence of Ca²⁺, and a highlyphosphorylated 80-kDa band appeared in addition to these phosphorylatedbands at 60 and 52 kDa in SDS-PAGE. However, the specific activityof CDPK was not changed by prior autophosphorylation when theautophosphorylated enzyme was assayed as a mixture of thesephosphorylated molecular species. Only the 60-kDa polypeptidewas immunodetected in subcellular fractions of Dunaliella cells.The 52-kDa polypeptide increased during storage of the enzyme.These results suggest that the 52-kDa polypeptide is a proteolyticartifact produced during purification. Immunoreactive bandsof 60-kDa were detected in extracts of several green algae butnot in extracts of higher plants or a brown alga. ¹This research was partly supported by Grants-in-Aid from theMinistry of Education, Science and Culture, Japan (No. 06454013and 06304023) and Research Fellowship of the Japan Society forthe Promotion of Science for Young Sciencists. ²Research Fellow (PD) of the Japan Society for the Promotionof Science.     

The Isolation and Characterization of a Cytochrome b6f Complex from the Cyanobacterium Spirulina sp.

A cytochrome b₆f complex was isolated and purified from Spirulinasp. The complex was solubilized with n-heptyl ß-D-thioglucosideand chromatographed on a DEAE-Toyopearl 650M column. The purifiedcomplex contained a small amount of chlorophyll and carotenoid.At least four polypeptides were present in the complex: cytochromef (29 kDa), cytochrome b₆(23 kDa), iron-sulfur protein (ISP,23 kDa), and a 17 kDa polypeptide. Each polypeptide was separatedfrom the complex treated with 2-mercaptoethanol or urea. Theabsorption spectra of cytochrome b₆ and cytochrome f were similarto those of Anabaena and spinach as expected. The complex wasactive in supporting ubiquinol-cytochrome c oxidoreductase activity.Fifty percent inhibition of the activity was accomplished by1 µM dibromothymoquinone (DBMIB). The K_m values for ubiquinol-2and cytochrome c (horse heart) were 5.7 µM and 7.4 µM,respectively. (Received August 15, 1988; Accepted November 14, 1988)     

Isolation of the Photoactive Reaction Center Complex that Contains Three Types of Fe-S Centers and a Cytochrome c Subunit from the Green Sulfur Bacterium Chlorobium limicola f. thiosulfatophilum, Strain Larsen

The photoactive reaction center (RC) complex from the greensulfur bacterium Chlorobium limicola f. thiosulfatophilum, strainLarsen, was isolated after solubilization and ammonium sulfatefractionation followed by ion-exchange chromatography. The spectrumof the complex was almost identical with that of the similarRC complex isolated by Feiler et al. [(1992) Biochemistry 31:2608–2614] except for the presence of cytochrome c₅₅₁instead of c₅₅₃ in the latter study. A molecular ratio of BChla to P840 of the isolated RC complex was assayed to be 25–35.SDSPAGE analysis revealed that the isolated complex containedthree major polypeptides with apparent molecular masses of 68,41 and 21 kDa, respectively. The 21-kDa polypeptide was identifiedto be a heme-binding protein by staining the gel for peroxidaseactivity. The cytochrome c₅₅₁ was oxidized by flash light ina biphasic manner with half times of 90 and 390 µs, respectively,that coincided with the reduction half times of P840⁺. Threedistinct iron-sulfur centers assigned to F_A, F_B and F_x, respectively,from their g-values were detected by EPR spectroscopy at cryogenictemperature. These results suggest that the present preparationcontains a minimal functional unit of the RC of this bacterium,and that this complex appears to lie on a evolutionary linebetween RC's of purple bacteria and photosystem I. (Received August 18, 1992; Accepted October 28, 1992)     

Voltage-dependent stimulation of the Na(+)-K(+) pump by insulin in rabbit cardiac myocytes

Insulin enhancesNa⁺-K⁺ pump activity in various noncardiactissues. We examined whether insulin exposure in vitro regulates Na⁺-K⁺ pump function in rabbit ventricularmyocytes. Pump current (I_p) was measured using thewhole-cell patch-clamp technique at test potentials(V_ms) from 100 to +60 mV. When theNa⁺ concentration in the patch pipette([Na]_pip) was 10 mM, insulin caused aV_m-dependent increase in I_p.The increase was ~70% when V_m was at nearphysiological diastolic potentials. This effect persisted afterelimination of extracellular voltage-dependent steps and whenK⁺ and K⁺-congeners were excluded from thepatch pipettes. When [Na]_pip was 80 mM, causingnear-maximal pump stimulation, insulin had no effect, suggesting thatit did not cause an increase in membrane pump density. Effects oftyrphostin A25, wortmannin, okadaic acid, or bisindolylmaleimide I inpipette solutions suggested that the insulin-induced increase inI_p involved activation of tyrosine kinase,phosphatidylinositol 3-kinase, and protein phosphatase 1, whereasprotein phosphatase 2A and protein kinase C were not involved.

     

Identification of G proteins mediating fungal elicitor-induced dephosphorylation of host plasma membrane H+ -ATPase

Tomato cells (with the Cf-5 resistance gene) were treated withelicitor preparations containing the avr5 gene product fromtwo Cf-5 incompatible races of the fungal pathogen Cladosporiumfulvum (race 2.3 and race 4), or with elicitor preparationscontaining no avr5 gene product from two Cf-5 compatible races(race 5 and race 2.4.5.9 [EC] .11). Elicitor preparations from race2.3 or race 4 caused dephosphorylation of host plasma membraneH⁺ -ATPase in isolated plasma membranes, while the preparationsfrom race 5 or race 2.4.5.9 [EC] .11 did not. GTP()S, AlF₄^–and cholera toxin (CTX) each induced similar dephosphorylationin the absence of active elicitors. The elicitor-induced dephosphorylationof the H⁺ -ATPase was blocked by preincubation of membraneswith an antibody raised against a stimulatory G protein -subunit(anti-G_s This antibody cross-reacted with a 42 kDa polypeptidefrom tomato plasma membranes. A 42 kDa polypeptide was alsoADP-ribosylated by CTX. When plasma membranes were treated withelicitor preparations from race 4 and separated on non-dissociatingPAGE, two proteins were detected on Western blots with the antibodyraised against the -subunit, suggesting the dissociation ofthe trimeric complex. No dissociation of the complex was detectedwith antibodies raised against either the - or ß-subunitswhen the plasma membranes were treated with elicitor preparationsfrom race 5. The results provide evidence for the activationof a stimulatory subtype of trimeric G proteins in the stimulationof elicitor-induced host defences to fungal pathogens. Key words: G protein, dephosphorylation, H⁺ -ATPase, fungal elicitor, tomato     

Effect of Calcium on Cell Wall Structure, Protein Phosphorylation and Protein Profile in Senescing Apples

The effect of Ca⁺⁺ on various parameters of apple fruit senescencewas investigated. Distinct and specific changes in polypeptideand phosphoprotein patterns were observed in Ca⁺⁺ treated ascompared to control fruits. A 70 kDa salt-extracted polypeptidebecame apparent in control fruits after 8 months of cold storagewhich was not apparent in Ca⁺⁺-treated fruits until 12 months.The soluble protein profile of Ca⁺⁺-treated fruits showed anaccumulation of a 30 kDa polypeptide while the control fruitsaccumulated a 60 kDa polypeptide. Autoradiographs of phosphorylatedpolypeptides revealed a 60 kDa membrane polypeptide becomingphosphorylated in the Ca⁺⁺-treated and not in the control fruitprotein fractions. Transmission electron micrographs of thecell showed Ca⁺⁺ to be effective in maintaining the cell wallstructure, particularly the middle lamella. Furthermore, increasein fruit Ca⁺⁺ reduced CO₂ and C₂H₂ evolution and altered chlorophyllcontent, ascorbic acid level and hydraulic permeability. ¹Scientific Paper No: 7930, College of Agriculture and HomeEconomics Research Center, Washington State University, Pullman,Washington, Project 0321. ²Supported by grants from the National Science Foundation CB-8502215and Washington State Tree Fruit Research Commission to BWP. (Received September 3, 1987; Accepted March 3, 1988)     

Leaf Development in Lolium temulentum: Photosynthesis and Photosynthetic Proteins in Leaves Senescing under Different Irradiances

Changes in carbon fixation rate and the levels of photosyntheticproteins were measured in fourth leaves of Lolium temulentumgrown until full expansion at 360 µmol quanta m^–2s^–1 and subsequently at the same irradiance or shadedto 90 µmol m^–2 s^–1. Ribulose-1,5-bisphosphatecarboxylase/oxygenase (Rubisco), light-harvesting chlorophylla/b protein of photosystem II (LHCII), 65 kDa protein of photosystemI (PSI), cytochrome f (Cytf) and coupling factor 1 (CF₁) declinedsteadily in amount throughout senescence in unshaded leaves.In shaded leaves, however, the decrease in LHCII and the 65kDa protein was delayed until later in senescence whereas theamount of Cyt f protein decreased rapidly following transferto shade and was lower than that of unshaded leaves at the earlyand middle stages of senescence. Decreases in the Rubisco andCF₁ of shaded leaves occurred at slightly reduced rates comparedwith unshaded leaves. These results indicate that chloroplastproteins in fully-expanded leaves are controlled individually,in a direction appropriate to acclimate photosynthesis to agiven irradiance during senescence. (Received August 20, 1992; Accepted January 5, 1993)     

Localization and Characterization of a Novel 20 kDa Polypeptide in the Chloroplast of the Green Alga Dunaliella salina

Recent work with the green alga Dunaliella salina showed thepresence of a {small tilde}20 kDa chloroplast protein that wasrecognized by polyclonal antibodies raised against the isolatedLHC-II [Webb M.R. and Melis A. (1995) Plant Physiol. 107: 885].In this report, a characterization of the {small tilde}20 kDapolypeptide is presented. It is shown that it is localized inthe chloroplast envelope membrane of D. salina. The abundanceof this protein is constant on a per cell basis and independentof the light regime during cell growth. The {small tilde}20kDa polypeptide is easily degraded to a {small tilde}19 kDaproduct during sample preparation. A limited amino acid sequenceof 21 residues from the free N-terminus of the {small tilde}19kDa product was obtained. On the basis of this partial sequence,it was concluded that the {small tilde}20 kDa polypeptide isnot a degradation product of a known LHC-II but rather a novelprotein. The {small tilde}20kDa polypeptide did not cross-reactwith antibodies raised against the Cbr (carotene biosynthesis-related)gene product and showed a different electrophoretic mobilityfrom the latter. Light-shift experiments suggest that the {smalltilde}20 kDa polypeptide is not an ELIP (early light-inducibleprotein). Possible functions of the {small tilde}20 kDa proteinare discussed. ¹Permanent address: Department of Biochemistry, University oflund, PO Box 124, S-221 00 Lund, Sweden     

Structural and Kinetic Properties of NADP-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L.

NADP-malic enzyme (EC 1.1.1.40 [EC] ), which is involved in Crassulaceanacid metabolism (CAM), was purified to electrophoretic homogeneityfrom the leaves of the inducible CAM plant Mesembryanthemumcrystallinum. The NADP-malic enzyme, which was purified 1,146-fold,has a specific activity of 68.8 µmol (mg protein)^–1min^–1. The molecular weight of the subunits of the enzymewas 64 kDa. The native molecular weight of the enzyme was determinedby gel-filtration to be 390 kDa, indicating that the purifiedNADP-malic enzyme is a hexamer of identical subunits. The optimalpH for activity of the enzyme was around 7.2. Double-reciprocalplots of the enzymatic activity as a function of the concentrationof L-malate yielded straight lines both at pH 7.2 and at pH7.8 and did not reveal any evidence for cooperativity of bindingof L-malate. The K_m value for L-malate was 0.35 mM. Hill plotsof the activity as a function of the concentration of NADP⁺indicated positive cooperativity in the binding of NADP⁺ tothe enzyme with a Hill coefficient (nH) of 2.0. An S_0.5 value(the concentration giving half-maximal activity) of 9.9 µMfor NADP⁺ was obtained. Oxaloacetate inhibited the activityof the NADP-malic enzyme. Effects of succinate and NaHCO₃ onthe activity of NADP-malic enzyme were small. (Received October 30, 1991; Accepted May 1, 1992)     

Characterization of ganglioside associated with the thyrotrophin receptor

The receptor protein for thyrotrophin (thyroid-stimulating hormone;TSH) is associated with a glycosphingolipid moiety. The proteinbelongs to the family of receptors that couple to guanine nucleotidebinding proteins; the glycosphingolipid contains sialic acidand belongs to the family of gangliosides. This report definesthe structure of the receptor ganglioside in the Fisher ratthyroid cell line (FRTL-5). Receptor protein was purified byTSH affinity chromatography from FRTL-5 cells, biosyntheticallylabelled with [³H]galactose and [³H]glucosamine, and resolvedby SDS-PAGE. A single radiolabelled band of M_r     

Rat cerebellar granule cells are protected from glutamate-induced excitotoxicity by S-nitrosoglutathione but not glutathione

In cultured rat cerebellar granule cells, glutamate or N-methyl-D-aspartate (NMDA) activation of the NMDA receptor caused a sustained increase in cytosolic Ca²⁺ levels ([Ca²⁺]_i), reactive oxygen species (ROS) generation, and cell death (respective EC₅₀ values for glutamate were 12, 30, and 38 µM) but no increase in caspase-3 activity. Removal of extracellular Ca²⁺ blocked all three glutamate-induced effects, whereas pretreatment with an ROS scavenger inhibited glutamate-induced cell death but had no effect on the [Ca²⁺]_i increase. This indicates that glutamate-induced cell death is attributable to [Ca²⁺]_i increase and ROS generation, and the [Ca²⁺]_i increase precedes ROS generation. Apoptotic cell death was not seen until 24 h after exposure of cells to glutamate. S-nitrosoglutathione abolished glutamate-induced ROS generation and cell death, and only a transient [Ca²⁺]_i increase was seen; similar results were observed with another nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine, but not with glutathione, which suggests that the effects were caused by NO. The transient [Ca²⁺]_i increase and the abolishment of ROS generation induced by glutamate and S-nitrosoglutathione were still seen in the presence of an ROS scavenger. Glial cells, which were present in the cultures used, showed no [Ca²⁺]_i increase in the presence of glutamate, and glutamate-induced granule cell death was independent of the percentage of glial cells. In conclusion, NO donors protect cultured cerebellar granule cells from glutamate-induced cell death, which is mediated by ROS generated by a sustained [Ca²⁺]_i increase, and glial cells provide negligible protection against glutamate-induced excitotoxicity. cytosolic calcium concentration; N-methyl-D-aspartate; reactive oxygen species