Energy-driven Protein Release from Germinating Pollen of Petunia hybrida

The gradual release of proteins from Petunia hybrida pollenduring 5 h of germination in vitro is severely inhibited bythe uncouplers 2,4-dinitrophenol (DNP) and carbonylcyanide-m-chlorophenylhydrazonc(CCCP) as well as by the ATPase inhibitors N,N'-dicyclohexylcarbodiimde(DCCD) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl).In the presence of these so-called energy poisons, a small amountof protein is given up to the culture medium in the first hourand no further release is seen. Cyclohexiinide, an inhibitorof protein synthesis, also inhibits much of the later gradualprotein export, and gel electrophoresis (SDS-PAGE) coupled withautoradiography suggests that a small fraction of the proteinsreleased is newly synthesized but they are different from mostof the major proteins exported to the medium. It is concludedthat in addition to the diffusible proteins passively exportedin the first hour, a major proportion of protein is releasedfrom the pollen by an energy-driven process, inhibited by thethiol reagent N-ethylmaleimide, during the 5 h of germination.A small number of these proteins is synthesized during pollengermination. Petunia hybrida L, pollen protein release, energy-dependent, germination, protein synthesis     

The Female-specific Protein (Vitellogenic Protein) in Crustacea with Particular Reference to Orchestic gammarella (Amphipoda)

Comparative electrophoretic studies of male and female hemolymphin Crustacea have led to the discovery of a lipoglycoproteinfraction present in females and absent from males. The female-specificprotein fraction also contains a pigment which has been identifiedin a small number of species and appears to be a carotenoid.Further observations indicated that the presence of this fractionis coincident with the presence of maturing ovocytes in theovary. The major protein component of the vitellus is also alipoglycocarotenoprotein complex. Comparative analyses haveshown that the female-specific protein fraction present in hemolymphand the major protein component of the vitellus are electrophoreticallyand immunochemically identical. Moreover, in the Amphipod Orchestiagammarella both components appear to have the same molecularweight, estimated as approximately 3 x 10⁵ by Sephadex G 200gel filtration. Although there is ample evidence to supportthe idea that the female-specific protein is synthesized externallyto the ovary, the site of synthesis still remains unknown. Experimentallyinduced sex reversal in O. gammarella indicates that the synthesisof the female specific protein is under ovarian control.     

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     

Characterization of Seed Protein Fractions from Castanea spp

Seed proteins of Castanea sativa and C. crenata were extractedby sequential procedures. Albumins accounted for over 20% ofthe total protein. Globulins were the main storage proteinsand represented close to half of the total protein. Additionof 2-mercaptoethanol was essential for maximum extraction ofthe globulins, a fraction with an amino acid composition similarto that of the 1 IS storage protein from legumes. Negligibleamounts of prolamins (<3%) were present in the seeds. Glutelinsrepresented just under 30% of the total protein. The proteinfractions were characterized by electrophoresis in polyacrylamidegels. The glutelin fraction contained unextracted globulinseven when 2-mercaptoethanol was used. Key words: Castanea spp, seeds, storage protein     

A Possible Role for Urease as a Storage Protein in Aspergillus tamarii

A marked decrease in mycelial urease activity during the endogenousphase of undifferentiated Aspergillus tamariicultures was foundto be independent of preparative procedures but related to thedepletion of external nutrients. The enzyme, which was synthesizedduring the active growth stage, was produced in similar quantitieswith ammonium or urea as sole nitrogen source and at its peakrepresented c. 8·5 per cent of the total soluble proteinpool of the mycelium. It was found to show maximum activityat pH 8·20–8·65 when measured in cell-free,phosphate-buffered extracts. Isolation of urease from differentstages of the endogenous phase by affinity chromatography hasshown that the observed decrease in activity was due to breakdownof the enzyme protein in mature cultures, followed by the progressivedeactivation of residual enzyme during the autolytic stage.Since selective inhibition of 80–90 per cent of activityby acetohydroxamic acid in media containing urea as the onlynitrogen source or total repression of urease synthesis by L-histidinein ammonium-grown cultures did not interfere with normal growth,it was concluded that in A. tamarii urease fulfils the functionof a storage protein with a measure of catalytic activity. Aspergillus tamarii, urease, storage protein, nitrogen metabolism     

Cloning and Characterization of the nrtA Gene That Encodes a 45-kDa Protein Involved in Nitrate Transport in the Cyanobacterium Synechococcus PCC 7942

A 45-kDa protein in the cytoplasmic membrane of the cyanobacteriumSynechococcus PCC 7942 is involved in the active transport ofnitrate [Omata et al. (1989) Proc. Natl. Acad. Sci. USA 86:6612]. The gene coding for this protein (designated herein asnrtA) has been cloned and sequenced. The nrtA gene encodes aprotein of 443 amino acids with a calculated molecular weightof 48424. The deduced amino acid sequence of the protein is46.5% homologous to that of a 42-kDa cytoplasmic membrane proteinthat is synthesized under carbon-limited conditions in SynechococcusPCC 7942. (Received July 16, 1990; Accepted December 5, 1990)     

The Effect of Infection by Erysiphe graminis f.sp. hordei on Protein Synthesis in Vivo in Leaves of Barley

The effect of infection by Erysiphe graminis f.sp. hordei onprotein synthesis in a susceptible cultivar of Hordeum vulgarehas been studied using in vivo labelling. Results have indicateda decline in protein synthesis in the chloroplasts at 1, 3 and5 days after inoculation and in the cytoplasm at 3 and 5 daysafter inoculation. The most extensive effect of the pathogenwas on the synthesis of a 32 Kd protein synthesised in the chloroplasts. (Received May 16, 1984; Accepted July 10, 1984)     

Cloning of a Novel Rat Gene, DB83, That Encodes a Putative Membrane Protein

Using a partial cDNA sequence and a 5'-RACE technique, we isolateda novel cDNA from rat liver referred to as DB83. DB83 had fourhydrophobic trans-membrane domains and one N-myristoylationsite as well as multiple possible phosphorylation sites. Thedb83 gene was highly expressed in the liver and significantlyin brain, lungs and kidneys. We suggest that DB83 is a tissue-specificputative membrane protein.     

Isolation and Mapping of a Family of Putative Zinc-finger Protein cDNAs from Rice

To understand the functions of rice homologues of the Arabidopsisflowering-time gene CONSTANS (CO) and salt-tolerance gene STO,we performed a similarity search of the single-run sequencedata of cDNA clones accumulated by the Rice Genome ResearchProgram, and isolated seven rice cDNA clones (S3574, C60910,S12569, R2931, R1479, R1577, and E10707) coding for proteinscontaining one or two zinc-finger-like motifs. Comparison ofthe deduced amino acid sequences between these cDNAs and theCO gene revealed significant similarities (46%-;61%) in theregion of zinc-finger motifs. A domain having a high contentof basic amino acids at the C-terminus of the CO protein wasfound in the corresponding region of proteins predicted fromcDNAs S3574, C60910, and S12569. Two amino acid sequences, "CCADEAAL"and "FCV(L)EDRA," which were present inside each zinc-fingerin the Arabidopsis regulatory protein STO, were also found ineach of the two zinc-finger regions of proteins predicted fromcDNAs R2931, R1479, R1577, and E10707. Using restriction fragmentlength polymorphism (RFLP) linkage analysis, we determined thechromosomal location of the seven cDNA clones. The positionof R2931 on the RFLP linkage map was closely linked to Hd-3,one of the putative quantitative trait loci (QTL) controllingheading date in rice.     

A Novel Nuclear-Encoded Protein, NDH-Dependent Cyclic Electron Flow 5, is Essential for the Accumulation of Chloroplast NAD(P)H Dehydrogenase Complexes

The chloroplast NAD(P)H dehydrogenase (NDH) complex, which reducesplastoquinones in thylakoid membranes, is involved in PSI cyclicelectron flow and chlororespiration. In addition to land plants,the NDH complex is conserved in cyanobacteria. In this study,we identified a novel NDH-related gene of Arabidopsis, NDH-dependentcyclic electron flow 5 (NDF5, At1g55370). Post-illuminationincreases in chlorophyll fluorescence were absent in ndf5 mutantplants, which indicated that NDF5 is essential for NDH activity.Sequence analysis did not reveal any known functional motifsin NDF5, but there was some homology in amino acid sequencebetween NDF5 and NDF2, a known NDH subunit. NDF5 and NDF2 homologswere present in higher plants, but not cyanobacteria. A singlehomolog, which had similarity to both NDF5 and NDF2, was identifiedin the moss Physcomitrella patens. Immunoblot analysis showedthat NDF5 localizes to membrane fractions of chloroplasts. Thestability of NdhH, a subunit of the NDH complex, as well asNDF5 and NDF2, was decreased in ndf5, ndf2 and double ndf2/ndf5mutants, resulting in a loss of NDH activity in these mutants.These results indicated that both NDF5 and NDF2 have essentialfunctions in the stabilization of the NDH complex. We proposethat NDF5 and NDF2 were acquired by land plants during evolution,and that in higher plants both NDF5 and NDF2 are critical toregulate NDH activity and each other's protein stability, aswell as the stability of additional NDH subunits.     

Cloning and Characterization of the Ribosomal Protein Genes in the spc Operon of a Prokaryotic Endosymbiont of the Pea Aphid, Acyrthosiphon kondoi

To correlate a prokaryotic endosymbiont in the pea aphid, Acyrthosiphonkondoi, with the endosymbionts in related aphid species as wellas with free-living bacteria and subcellular organelles, andto study the mode of its gene expression within aphid cells,we have cloned and characterized the genes encoding ribosomalproteins S3, L16, L29, S17, L14, L24, L5, S14, S8, L6, L18,S5, L30, L15 and secretion protein Y (Sec Y) from the S10 andspc ribosomal protein gene operons of this endosymbiont. Theorganization of these genes is identical to that in Escherichiacoli, and their nucleotide sequences are highly similar (87%identity) to the corresponding E. coli genes. They are muchless similar to the corresponding chloroplast and mitochondrialgenes. The guanine plus cytosine G+C content of the genes ofthe A. kondoi endosymbiont is much higher than those of theendosymbionts in related aphid species reported so far. It appearseither that the A. kondoi endosymbiont is derived from an ancestralbacterium different from those in other aphids or that its G+Ccontent increased in a relatively short time after the evolutionarydivergence of its host.     

Transient Increase in the Level of mRNA for a Germin-Like Protein in Leaves of the Short-Day Plant Pharbitis nil during the Photoperiodic Induction of Flowering

A cDNA corresponding to an in vivo labeled protein whose levelincreased during flower-inductive darkness in the cotyledonof the short-day plant Pharbitis nil Choisy cv. Violet was isolatedand characterized. The deduced amino-acid sequence of the protein(designated PnGLP; P. nil germin-like protein) showed homologyto that of a GLP of Sinapis alba, a leaf protein, the mRNA accumulationof which showing circadian oscillations. PnGLP mRNA was detectedspecifically in the cotyledon and leaf, in particular, in theyoung expanded cotyledon and leaf. Accumulation of PnGLP mRNAincreased transiently during flower-inductive darkness and peakedat a time that corresponded approximately to the critical nightlength. This mRNA peak was reduced by a brief exposure to redlight at the 8th hour of darkness. The level of PnGLP mRNA peakedabout 10 h from the beginning of the dark period, whereas itwas reported that the level of mRNA for GLP of a long-day plantS. alba increased about 14 h from the beginning of the lightperiod. Thus, the different time courses of accumulation ofthe mRNAs for leaf-specific GLPs appear to reflect the differencesin photoperiodic responses of each plant. (Received May 16, 1996; Accepted July 5, 1996)     

Some Characteristics of Protein Kinases in Lemna paucicostata

The three protein kinases of Lemna paucicostata that are separableby DEAE-Sephacel chromatography have been designated PI, PIIand PIII [Kato et al. (1983) Plant & Cell Physiol. 24: 841].The optimum pH for the PI and PII enzymes was 7.5 and for thePHI enzyme 7.0. The activities of these enzymes were stimulatedby divalent cations, the maximum stimulation being producedby 5 nw Mg^{2 $} for PI, by 3 mM Co^{2 $} for PII and by 1 mM Mn^2$ for PIII. The cytokinins; benzyladenine, kinetin and zeatin,inhibited the activity of the PIII enzyme. The molecular weightsof the PI and PII enzymes did not change after incubation withcAMP even though their activities were regulated by this compound. (Received October 17, 1983; )     

Identification of the Product of ndhA Gene as a Thylakoid Protein Synthesized in Response to Photooxidative Treatment

A 76 amino acid sequence of NDH-A (the protein encoded by plastidndhA gene) from barley (Hordeum vulgare L.) was expressed asa fusion protein with rß-galactosidase in E. coli.The corresponding antibody generated in rabbits was used toinvestigate localization, expression and synthesis in vitroof NDH-A. NDH-A was identified as a 35 kDa polypeptide localizedin thylakoid membrane. Western blots shows a large increasein NDH-A levels when barley leaves were incubated under photooxidativeconditions, which was more pronounced in mature-senescent leavesthan in young leaves. Immunoprecipitation of the [³⁵S]methioninelabelled proteins, synthesized in vitro by isolated chloroplasts,demonstrated the synthesis in chloroplasts of the NDH-A 35 kDapolypeptide when barley leaves had been incubated under photooxidativeconditions. The results indicate that ndh genes may be involvedin the protection of chloroplasts against photooxidative stress,particularly in mature-senescent leaves. (Received November 13, 1995; Accepted February 5, 1996)     

The Relation Between Ion Absorption and Protein Synthesis in Beet Disks

Disks of red beet storage tissue were incubated under asepticconditions permitting the development of various metabolic processescommonly associated with aged disks, and the effects of chloramphenicoland puromycin on protein synthesis, on the development of invertaseactivity and ion absorption capacity, and on ion absorptionper se were determined. Low concentrations of chloramphenicoland puromycin inhibit the development of ion absorption capacitybut stimulate invertase development and protein synthesis, whilehigher concentrations inhibit all three processes. In contrastion absorption itself is unaffected by puromycin, but is sensitiveto quite low concentrations of chloramphenicol The D-threo andL-threo isomers of chloramphenicol have sharply contrasted effectson the development, as distinct from the utilization, of ionabsorption capacity. The D isomer inhibits the development ofion absorption capacity more effectively than the L isomer whichin turn inhibits absorption more effectively than the D isomer. A reappraisal is made of the hypothesis that ion absorptionis directly linked with protein turnover and to account forthe results a model is proposed in which D-threo-chloramphenicolis active both as an uncoupler of oxidative phosphoryalationand as an inhibitor of protein synthesis, while L-threo-chloramphenicolacts only in the former capacity and puromycin only in the latter.It is concluded that the inhibition of ion uptake by chloramphenicolcannot be attributed to a contemporaneous effect on proteinsynthesis. However, the results are consistent with the involvementof ATPase proteins in ion uptake.     

Accumulation of a High Molecular Weight Protein during Cold Hardening of Wheat (Triticum aestivum L.)

Electrophoretic patterns of soluble protein fractions from cold-tolerantwinter wheats (Triticum aestivum L. cv. Frederick and cv. Norstar)and cold-sensitive spring wheat (T. aestiaum L. cv. Glenlea)were analysed in hardened and unhardened plants. One and two-dimensionalgel electrophoresis analysis reveals that cold hardening conditionsinduce changes in the soluble protein patterns. The most importantis the accumulation of a high molecular weight protein in therange of 200 kDa. This protein accumulated at higher concentrationin cold-tolerant cultivars compared to the coldsensitive onesuggesting a correlation between the degree of freezing toleranceand the accumulation of this specific protein. In addition,the intensity of three protein bands (mol wt 48, 47 and 42 kDa)increased while that of five others (mol wt 93, 89, 80, 67 and63 kDa) decreased during hardening. These changes occured inthe three cultivars suggesting that they are part of the metabolicadjustments in response to low temperature rather than a specificchange associated with the development of cold hardiness. (Received April 23, 1987; Accepted June 5, 1987)     

Degradation of Protein Bodies in Germinating Seeds of Brassica campestris L. var. sarson Prain

Histochemical investigations were carried out on dry and germinatingseeds of Brassica campestris var. sarson, to study the degradationof protein bodies with globoidal inclusions. During germination,a wave of protein body degradation sets in from the radicularend of the embryo, passing through the hypocotyl and shoot apex,and ending in the cotyledons. The digestion of protein bodiesis of the internal type. The various isolated parts of the embryoshowed a similar pattern of protein body digestion to that ofthe whole embryo, except that in some cells the globoids persistedeven after complete digestion of protein bodies; the rate wasfaster in the comparatively more expanded part of the isolatedorgans. No specific factor controlling the initiation of thewave of protein body digestion could be ascertained. Brassica campestris var. sarson, yellow sarson, seed, germination, protein bodies, degradation     

Studies on the Inactivation of Protein Synthesis in Mesophilic Bacteria Induced by Chilling

The mechanism of inactivation of in vivo protein synthesis (incorporationof phenylalanine into protein at 20C), by chilling at 0C,in Escherichia coli Q13 and Pseudomonas aeruginosa was studied.In vitro protein synthesis with poly(U) or R17 phage RNA asmRNA showed that the protein-synthesizing system itself wasnot damaged by the chilling. In contrast, the ability of E.coli Q13 and P. aeruginosa to take up phenylalanine decreasedby 100% and 90%, respectively, after the 8-day chilling period.A significant part of the phenylalanine pool leaked out of thecells during the chilling period. Intracellular ATP levels andthe energy balance did not alter greatly in E. coli Q13, butchanged considerably in P. aeruginosa due to chilling. These results strongly suggest that the inactivation of thein vivo protein synthesis is due to damage of membrane functions,probably those related to the amino acid transport system, andnot to the inactivation of the protein-synthesizing system itself. (Received January 28, 1985; Accepted August 6, 1985)