Sugar-Induced Increase of Calcium-Dependent Protein Kinases Associated with the Plasma Membrane in Leaf Tissues of Tobacco

The sugar-inducible expression of genes for sporamin and [beta]-amylase in leaf explants of sweet potato (Ipomoea batatas) and that of a [beta]-glucuronidase-fusion gene, with the promoter of the gene for [beta]-amylase in leaves of tobacco (Nicotiana tabacum), requires Ca2+ signaling (M. Ohto, K. Hayashi, M. Isobe, K. Nakamura [1995] Plant J 7: 297-307), and it was inhibited by staurosporin and K252a, inhibitors of protein kinases. Autophosphorylation activities of several potential protein kinases in leaves of tobacco were significantly higher in younger leaves than in mature leaves. However, the autophosphorylation activities of these proteins in mature leaves, especially those of the major autophosphorylatable proteins with apparent molecular masses of 56 and 54 kD, increased upon treatment of leaf discs with a 0.3 M solution of sucrose, glucose, or fructose, did not increase with sorbitol or mannitol treatments, and the increase by sucrose was inhibited by cycloheximide. Autophosphorylation of the 56- and 54-kD protein in vitro was dependent on Ca2+ and inhibited by staurosporine, K-252a, and by W-7. These results suggest that they belong to the family of calcium-dependent protein kinases. They were concentrated in the plasma membrane fraction and were released from membrane vesicles by high salt or with sodium carbonate. The possible functions of these sugar-inducible calcium-dependent protein kinases associated with the plasma membrane are discussed.     

Calcium/Calmodulin-Dependent Protein Kinase II in Squid Synaptosomes

The Ca2+/calmodulin (CaM)-dependent protein kinase II system in squid nervous tissue was investigated. The Ca2+/CaM-dependent protein kinase II was found to be very active in the synaptosome preparation from optic lobe, where it was associated with the high-speed particulate fraction. Incubation of the synaptosomal homogenate with calcium, calmodulin, magnesium, and ATP resulted in partial and reversible conversion of the Ca2+/CaM-dependent protein kinase II from its calcium-dependent form to a calcium-independent species. The magnitude of this conversion reaction could be increased by inclusion of the protein phosphatase inhibitor NaF or by substitution of adenosine 5'-O-(3-thiotriphosphate) for ATP. When [gamma-32P]ATP was used, proteins of 54 and 58 kilodaltons (kDa) as well as proteins greater than 100 kDa were rapidly 32P-labeled in a calcium-dependent manner. Major 125I-CaM binding proteins in the synaptosome membrane fraction were 38 and 54 kDa. The Ca2+/CaM-dependent protein kinase II was purified from the squid synaptosome and was shown to consist of 54- and 58-60-kDa subunits. The purified kinase, like Ca2+/CaM-dependent protein kinase II from rat brain, catalyzed autophosphorylation associated with formation of the calcium-independent form. These studies, characterizing the Ca2+/CaM-dependent protein kinase II in squid neural tissue, are supportive of the putative role of this kinase in regulating calcium-dependent synaptic functions.     

Localization of Sorbitol Oxidase in Vacuoles and Other Subcellular Organelles in Apple Cotyledons

To investigate the function and subcellular localization ofsorbitol oxidase, free cells, protoplasts and isolated vacuolesof apple cotyledons (Malus pumila Mill. var. domestica Schneid.)were examined by differential and sucrose density gradient centrifugation.Twenty percent of the activity of sorbitol oxidase in the wholetissue was contained in the subcellular fraction (d=1.06) whichcorresponded closely to the main peaks of activity and proteinafter the recentrifugation of the 150,000?g pellet of rupturedvacuoles with a linear sucrose density gradient. The enzymethus appears to be derived from the tonoplast membrane. Thistonoplast membrane-bound sorbitol oxidase may play an importantrole in the transport of vacuolar sorbitol into the cytoplasm,rather than in the transport of sorbitol into the vacuole. About10% of the enzyme activity also occurred in the subcellularfraction having a density of 1.12–1.16, which coincidedwith the peaks of acid phosphatase and ATPase activities. Thereforesorbitol oxidase may also be associated with the plasma membrane.Furthermore, 30–40% of its activity was located in theinterspace between the cell wall and the plasma membrane, orperhaps attached weakly to them. These results suggest thatsorbitol is transported into the cytoplasm by being convertedto glucose by sorbitol oxidase. ¹ This paper is contribution A-138 of the Fruit Tree ResearchStation. (Received January 20, 1982; Accepted May 18, 1982)     

Immunological Studies on ATPases in Mung Bean Hypocotyl Plasma Membrane: Proposal of the Presence of Two Molecular Species of ATPase

ATPase in a highly purified plasma membrane fraction from mungbean hypocotyls was solubilized by lysolecithin and fractionatedby glycerol density gradient centrifugation. Lysolecithin activatedATPase activity in the lower but not in the upper half of theactivity peak after glycerol density gradient centrifugation.Antibody against maize root plasma membrane ATPase [Nagao etal. (1987) Plant Cell Physiol. 28: 1181] reacted to a 100-kDapolypeptide which was localized only at the lower half of theactivity peak. Antibody against a 67-kDa polypeptide, whichwas proposed to be a subunit of a new type of ATPase in mungbean hypocotyl plasma membrane (Mito et al. the preceding paper),reacted only to its own antigen which was present mainly inthe upper half of the activity peak. The activity peak fractioncontained a low-molecular-mass polypeptide binding N.N'-dicyclohexylcarbodiimide.We propose the presence in mung bean hypocotyl plasma membraneof two distinct ATPases which differ from each other in polypeptideconstitution and in their response to lysolecithin. (Received September 2, 1987; Accepted May 20, 1988)     

Partial Purification and Characterization of An ATPase in Mung Bean Hypocotyl Plasma Membrane: Suggestion for A New Type of Higher Plant Plasma Membrane ATPase

A vanadate-sensitive and nitrate-resistant ATPase was solubilizedwith Zwittergent 3–14 from a highly purified plasma membranefraction of mung bean hypocotyls and partially purified by glyceroldensity gradient centrifugation and phenyl-Sepharose columnchromatography. Either phosphatidylcholine or phosphatidylserinein addition to Mg^{2 +} was required for the enzyme activity, whereasK⁺, phosphatidylethanolamine and lysophosphatidylcholine hadno effect on the activity. The purified enzyme preparation containedtwo major polypeptides with molecular masses of 67 and 55 kDaas analyzed by SDS-polyacrylamide gel electrophoresis. Whenthe plasma membrane fraction was incubated with [-³²P]ATP, a45-70-kDa polypeptide(s) was labeled, and the label could berapidly chased with cold ATP. When the fraction was incubatedwith [¹⁴C]N,N'-dicyclohexylcarbodiimide, an inhibitor for theATPase, a 15-20-kDa polypeptide was labeled. We propose thatthe enzyme is a new type of higher plant plasma membrane ATP-aseand is composed of 67- and 55-kDa subunits and probably alsoa 15-20-kDa subunit. ¹Present address: Takarazuka Institute, Sumitomo Chemical IndustriesLtd., Takatsukasa, Takarazuka, Hyogo 665, Japan (Received September 2, 1987; Accepted May 20, 1988)     

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.     

Purification and Some Properties of Pheophorbidase in Chenopodium album

A novel enzyme, pheophorbidase, which catalyzes the conversionof pheophorbide a to C-13²-carboxylpyropheophorbide a, was purifiedfrom Chenopodium album leaves. The purified enzyme showed twobands of 28 kDa and 29 kDa on SDS-PAGE. The molecular mass ofthe native pheophorbidase was 105 kDa. The N-terminal aminoacid sequence for the 28-kDa protein could be determined, whereasthe N-terminus of the 29-kDa protein was blocked. Immunochemicaland enzyme activity analyses revealed that pheophorbidase islocated in an extra-plastidic part of the cell. (Received September 7, 1998; Accepted October 26, 1998)     

Tryptophan Oxidizing Enzyme and Basic Peroxidase Isoenzymes in Arabidopsis thaliana (L.) Heynh.: Are They Identical?

The in vitro conversion of [³H]tryptophan by a plasma membraneenriched fraction from Arabidopsis thaliana (L.) Heynh. seedlings,grown in liquid culture, revealed indole-3-acetaldoxime (IAOX)as the only detectable reaction product. The pH optimum of thereaction was at pH 8, the K_m value for tryptophan 12 µM.The formation of IAOX was stimulated about 10-fold by H₂O₂ Incubationexperiments with solubilized proteins and membrane vesiclesshowed that the investigated enzyme(s) were bound covalent tothe plasma membrane. Tryptophan oxidizing enzyme (TrpOxE) andperoxidase activity were not only found in the plasma membrane,but also in the culture medium. Specific IAOX forming activitywas 74-fold and 6-fold higher compared to the crude extractand the plasma membrane fraction, respectively. After isoelectricfocusing of solubilized plasma membrane and precipitated mediumproteins, TrpOxE activity co-migrated with two prominent highpI peroxidase bands stained with benzidine-guaiacol. The zonesof the IEF gel with peroxidase and TrpOxE activity were analyzedby SDS PAGE and revealed in all fractions a main protein bandof ca. 55 kDa. TrpOxE activity and peroxidase activity wereboth inhibited by antisera directed against tobacco and horseradishperoxidase. TrpOxE activity and peroxidase activity were determinedduring plant development. TrpOxE activity peaked after 8 and42 days, whereas peroxidase activity was consistently presentduring the whole life cycle. The inhibitory effects of indolederivatives, especially indole-3-glyoxylic acid, on (i) seedlingdevelopment and (ii) on TrpOxE and peroxidase activity werealso compared. (Received November 1, 1991; Accepted September 2, 1992)     

Hormone Effects on the Membrane Potential and on Sucrose-Induced Depolarization of Young Citrus Leaves

The effect of IAA, GA₃ and ABA on transmembrane potential difference(Em) and on sucrose-induced depolarization has been studiedin young Citrus leaves. The addition of any of these hormonesto the perfusion solution (short-term experiments) did not affectEm or sucrose-induced depolarization. Hormonal treatments ofyoung leaves on the tree resulted, after 4 to 16 days (long-termexperiments), in an increase of Em for GA₃- and ABA-treatedleaves, while in IAA-treated ones no hyperpolarization was found.Only in ABA treated leaves this membrane hyperpolarization couldbe related to an enhancement of sucrose uptake. (Received April 28, 1992; Accepted September 21, 1992)     

Biochemical, Immunochemical and Immunohistochemical Identification of Myosin Heavy Chains in Cultured Cells of Catharanthus roseus

In the pollen tubes of the lily Lilium longiflorum, myosin,composed of 170-kDa heavy chains is responsible for the intracellulartransport of organelles [Yokota and Shimmen (1994) Protoplasma177: 153]. Polypeptides of 170 kDa with similar antigenicityto this pollen-tube myosin have also been found in other angiospermcells [Yokota et al. (1995) Protoplasma 185: 178]. To clarifythe role of this type of myosin in cytoplasmic streaming, weprepared partially purified myosin fraction from cultured cellsof Catharanthus roseus by co-precipitation with F-actin. Ina motility assay in vitro with this fraction, rhodamine-phalloidin-labeledF-actin moved with an average velocity of 10.7 µm s^-1.This sliding velocity was similar to that of the cytoplasmicstreaming observed in intact cultured cells. Antibodies raisedagainst the 170-kDa heavy chain of pollen-tube myosin recognizedonly a single polypeptide of 170 kDa in this partially purifiedfraction. The same polypeptide was also identified by theseantibodies in a crude extract of proteins from cultured cells.The myosin-specific fluorescence was concentrated around thenuclei and was associated with particles of various sizes. Duallocalization using antibodies against myosin and against actinrevealed that these particles were preferentially co-localizedwith actin filaments. On the other hand, no component of thecrude extract or of the partially purified myosin fraction cross-reactedwith antibodies against heavy chains of myosin II from animalcells. These results suggest that the 170-kDa polypeptide is the myosinheavy chain and that this myosin generates the motive forcefor cytoplasmic streaming in cultured cells of Catharanthusroseus. (Received March 28, 1995; Accepted September 14, 1995)     

NAD (P)H-Duroquinone Reductase in the Plant Plasma Membrane

The intracellular distribution of NADPH- and NADH-dependentduroquinone reductase (NAD (P)H-DQR) from etiolated zucchinihypocotyls (Cucurbita pepo L.) was investigated. About 80% ofthis enzyme is in the supernatant fraction and is probably cytosolic.Particulate NAD (P)H-DQR was largely (42%) found in associationwith the plasma membrane and was strongly stimulated by TX100.Another 33% of NAD (P)H-DQR was associated with mitochondria,and minor fractions with the endoplasmic reticulum (8%) andother particles. All these fractions were little or not stimulatedby TX100. The distribution of detergent-activated NAD (P)H-DQRis thus distinct from microsomal NADH- and NADPH-CCR. The plasma membrane was purified from microsomal fractions bymetrizamide plus sucrose density gradient centrifugation orby PEG/dextran phase partitioning. Both types of particle preparationspeaked at a density (d) of 1.165 g cm^–3 in sucrose gradientsand contained substantial TX100-sensitive NADH-DQR, TX100-stimulatedNAD (P)H-DQR, together with traces of NADH-CCR and trapped ‘soluble’enzyme (MDH, NADP-malic enzyme) activities. In isopycnic gradientsof unfractionated microsomes, however, trapped enzymes peakedat d 1.155 whereas NAD (P)H-DQR peaked at d 1.165 and GSII atd 1.170, probably revealing plasma membrane heterogeneity. Furtherevidence of heterogeneity was provided by fractionation of plasmamembrane vesicles on dextran step-gradients. Most of the trapped MDH was released to the supernatant by sonicationor treatment with 0.0125% TX100. Under these conditions mostof the NAD (P)H-DQR sedimented with the membranes. It is concludedthat NAD (P)H-DQR is bound to the inside of plasma membranevesicles, but a fraction (7 to 31%) may be ‘soluble’and sequestered within the vesicle lumen. Part of the detergent-sensitiveNADH-DQR may be externally bound and accessible to non-permeatingsubstrates. Key words: Cucurbita, NAD (P)H-quinone reductase, plasma membrane     

Relationship between the Stability of Antenna Chl {alpha}-Binding Proteins CP43/47 and the Accumulation of the Extrinsic 30-kDa Protein in PS II in Euglena

The accumulation of the extrinsic 30-kDa protein of PS II inthylakoid membranes was studied in relation to the assemblyof PS II antenna Chl-proteins CP43/47 and light-harvesting Chia/b-protein (LHC) in greening cells of Euglena. We showed previouslythat the 30-kDa protein accumulates in the membrane later thanthe reaction center-binding Dl protein (Mizobuchi and Yamamoto1989). In the present study, the rate of accumulation of theapoproteins of LHC and CP43/47 was shown to be similar to thatof the Dl protein. By contrast, we found by non-denaturing lithiumdodecyl sulfate-polyacrylamide gel electrophoresis that, atthe early stages of greening, newly assembled antenna Chi a-bindingproteins CP43 and CP47 were more susceptible to detergent thanwere the other Chl-protein complexes. Prolonged illuminationwas required for the stabilization of CP43 and CP47, and thetime course of the light-dependent formation of stable CP43/47paralleled that of the accumulation of the extrinsic 30-kDaprotein. These results are discussed in terms of the relationshipbetween the stabilization of CP43/47 and the accumulation ofthe 30-kDa protein in the thylakoid membrane and the roles ofthese two processes in the assembly of the oxygen-evolving systemin Euglena are examined. (Received February 18, 1991; Accepted June 17, 1991)     

Purification and Characterization of Sucrose Synthase from Peach (Prunus persica) Fruit

Sucrose synthase (EC 2.4.1.13 [EC] ) was purified from peach fruit(Prunus persica) to a single band of protein on SDS-PAGE byammonium sulfate fractionation, DEAE-cellulose (DE-52) chromatography,Sepharose CL-6B gel filtration, PBA-60 affinity chromatographyand Sephadex G-200 gel filtration. The molecular weight wasestimated to be 360,000 by gel filtration. The enzyme was foundto be a tetramer of identical 87-kDa subunits. The maximum activityfor the synthesis and cleavage of sucrose was observed at pH8.5 and pH 7.0, respectively. The enzymatic reaction followedtypical Michaelis-Menten kinetics in both directions, with thefollowing parameters: K_m(fructose), 4.8 mmM; K_m(UDPglucose),0.033 mM; K_m(sucrose), 62.5 mM; K_m(UDP), 0.080 mM. Other properties,such as substrate specificity and the effects of divalent cations,were also investigated. The relationship between the enzymeand the accumulation of sucrose in peach fruit is discussed. Present address: Laboratory of Horticulture, Faculty of Agriculture,Nagoya University, Chikusa, Nagoya 464, Japan. (Received May 2, 1988; Accepted September 14, 1988)     

Assembly and activation of the NADPH:O2 oxidoreductase in human neutrophils after stimulation with phorbol myristate acetate

Phagocytic leukocytes contain an activatable NADPH:O2 oxidoreductase. Components of this enzyme system include cytochrome b558, and three soluble oxidase components (SOC I, SOC II, and SOC III) found in the cytosol of resting cells. Previously, we found that SOC II copurifies with, and is probably identical to, a 47-kDa substrate of protein kinase C. In the present study we investigated the change in location of several of these oxidase components after activation of intact neutrophils with phorbol myristate acetate (PMA) and separation of subcellular fraction on sucrose density gradients. On Western blots with fractions of resting cells, the alpha subunit of cytochrome b558 was detected with a monoclonal antibody as a doublet of Mr 22,000 and 24,000 in the specific granules and as a single band of Mr 24,000 in the plasma membrane. PMA induced an increase of cytochrome b558 in the plasma membrane, including the Mr 22,000 band. PMA also induced translocation of the 47-kDa protein from the cytosol to the membrane fraction, as revealed by in vitro phosphorylation experiments. When NADPH oxidase activity was determined in a cell-free system in the presence of sodium dodecyl sulfate and GTP with plasma membranes from resting cells, cytosol from PMA-treated cells was deficient compared with cytosol from resting cells. This deficiency could be partially restored by the addition of SOC I. Concomitantly, SOC I activity appeared in the plasma membranes of PMA-treated cells. These studies support the hypothesis that PMA stimulation of neutrophils results in assembly of oxidase components from the cytosol and the specific granules in the plasma membrane with subsequent expression of NADPH oxidase activity.     

Apoptosis induced in neuronal cells by C-terminal amyloid ß-fragments is correlated with their aggregation properties in phospholipid membranes

Rat brain proteins presenting high-affinity binding of S-adenosyl-L-homocysteine were solubilized and purified. Extraction of binding protein was carried out in the presence of Triton X-100 and 1 M NaCl; this solubilized fraction exhibits similar kinetic properties than the membrane proteins. Purification was performed using affinity chromatography on S-adenosyl-L-homocysteine carboxyhexyl Sepharose 48 conjugate. The analysis of the affinity gel eluate by SDS-PAGE showed high purification ratios for two proteins exhibiting 54 and 68 kDa. Three activity peaks were separated when solubilized membrane proteins were submitted to isoelectric focusing; the activity peaks corresponded to proteins of pH, 6.0, 6.5, and 7.2. SDS-PAGE separation of proteins contained in each peak showed protein aggregation; a 54-kDa subunit was present in each aggregate. Solubilized membrane proteins were labeled by photoaffinity labeling with tritiated S-adenosyl-L-homocysteine; the 54-and 68-kDa proteins were found among the specifically labeled proteins. Finally, according to the previous data from the literature, the purified S-adenosyl-L-homocysteine binding proteins do not seem to be the same as adenosine receptors or phosphatidylethanolamine-N-methyltransferase.     

Distribution of Vacuolar H+-Pyrophosphatase and a Membrane Integral Protein in a Variety of Green Plants

Vacuolar membranes isolated from several species including fernand moss exhibited pyro-phosphate-dependent H⁺ transport activity.On immunoblot analysis, H⁺ -pyrophosphatase was detected inthe vacuolar membranes. A membrane integral protein of 23,000daltons was not found in the membranes of Chara, Conocephalum,or Kalanchoë. Thus, H⁺-pyrophosphatase may be a universalenzyme among green plants, but the 23-kDa protein is not a commonprotein of central vacuoles. (Received September 10, 1993; Accepted November 29, 1993)     

The 57-Kilodalton Phosphoprotein of Spiroplasma melliferum Is Autophosphorylated

The partially purified 57-kDa protein of Spiroplasma melliferum was autophosphorylated when incubated with ATP in the presence of ZnCl₂. Autophosphorylation was also apparent by showing the in situ phosphorylation of the 57-kDa protein band separated by polyacrylamide gel electrophoresis under nondenaturing conditions. The autophosphorylation was affected neither by the pH of the reaction mixture nor by the presence of NaF. The steady state level of the phosphorylated 57-kDa protein remained constant for up to 15 min, suggesting the absence of a phosphoprotein phosphatase activity in the preparation. As the initial phosphorylation rate did not decrease upon a 100-fold dilution of the 57-kDa protein under constant substrate concentration, it is suggested that the autophosphorylation is an intramolecular process. Received: 7 June 1996 / Accepted: 6 July 1996     

Proteolytic Cleavage of a Spectrin-Related Protein by Calcium-Dependent Protease in Neurospora crassa

To investigate the functional significance of a cytoskeletal spectrin-like protein, we studied its localization pattern in Neurospora crassa and sought the answer to whether it is a substrate for another apically localized protein, the calcium-dependent protease (CDP II). Immunoblots of crude extracts from exponentially growing mycelia, separated by one- and two-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis using antichicken α/β-spectrin antibodies, revealed a single band of approximately relative mass (Mr) 100 kDa with an isoeletric point (pI) in the range of 6.5 to 7.0. Despite rigorous efforts, we could not confirm the presence of an Mr 240- to 220-kDa spectrin-like protein in N. crassa. The immunofluorescence- and immunogold-labeling Mr 100-kDa protein showed its predominance along the plasma membrane of the conidia during the swelling phase of germination. In contrast, in the germ tubes and the growing hyphae, the localization was polarized and concentrated mainly in the apical region. The in vitro proteolysis experiments showed that indeed this protein is a preferred substrate of CDP II which is, as mentioned previously, also localized in the apical regions of the hyphae. These results indicate a putative functional relationship between these two proteins (spectrin-like protein and CDP II) in the dynamics of tip growth.