不同芸豆品种种子发育过程中贮藏蛋白积累研究

以半蔓生型奶花芸豆(Y09)和直立型奶花芸豆(Y06)为材料,通过SDS-PAGE分析研究了开花后种子形成过程中贮藏蛋白(SP)的积累规律.结果表明:2个芸豆品种开花后种子形成过程中,子叶、胚蛋白积累变化趋势基本一致,但其变化幅度存在显著差异,其中,在开花后20d内,半蔓生型芸豆Y09的籽粒蛋白积累量大于直立型芸豆Y06,但在开花后25~40d,Y06籽粒蛋白积累量显著高于Y09;两芸豆品种的子叶和胚均含有丰富的贮藏蛋白(SP)(14.4~97.4kD),其中子叶的41.0、43.9、39.4kD3种蛋白含量较高,约占子叶总蛋白含量(光密度)50%以上,而胚蛋白亚基分布相对均匀;两品种的子叶、胚中蛋白亚基差异较小,只有个别亚基存在差异.     

Development of Vacuolar Membranes during Elongation of Cells in Mung Bean Hypocotyls

The development of vacuolar membrane in the elongating hypocotylsof the mung bean was investigated. The hypocotyls from 3-day-oldseedlings were dissected into the dividing, elongating and matureregions. The diameter of protoplasts prepared from the matureregions was about 3-fold greater than the diameter of thosefrom the dividing region. The activity of inorganic pyrophosphatase,an enzyme associated with the vacuolar membrane, was detectableeven in the dividing region. The level of pyrophosphatase wasquantified by slot-blot analysis with the pyrophosphatase-specificantibody. The relative amount of pyrophosphatase per cell, calculatedon the basis of DNA content, increased about 4-fold during cellmaturation. When the densities of vacuolar membranes were comparedby sucrose density gradient centrifugation, there was no markeddifference among the preparations from three regions. Furthermore,most of the major proteins were common to the three purifiedpreparations of vacuolar membranes. From the results, it appearsthat most components of vacuolar membrane may be synthesizedde novo and added to the existing membrane during cell elongation.Furthermore, it is proposed that the H⁺-pyrophosphatase mayactively hydrolyze its substrate to maintain the internal acidityof expanding vacuoles, because pyrophosphate was present ata concentration of more than 70 µM in the dividing andelongating regions. (Received August 7, 1989; Accepted January 11, 1990)     

Partial Purification of a Soluble Gibberellin-Binding Protein from Mung Bean Hypocotyls

A soluble binding protein specific for GA₄, GA₇ and GA₉ waspartially purified from mung bean hypocotyls, and its characteristicswere examined. Affinity chromatography using immobilized GA₃coupled to Sepharose 4B via the C-7 carboxyl group was veryeffective for purification of the protein. The molecular weightof the protein in its native state was estimated to be 150–200kDa by gel-permeation chromatography. This protein may be aheterooligomer consisting of two subunits (23 kDa and 35 kDa).The optimum pH for binding of GA₄ to the protein was around6.0 and the apparent dissociation constant (K_d) was 310^-7 M. (Received April 24, 1992; Accepted December 16, 1992)     

Properties of Two N-Linked Glycoproteins Associated with the Nuclear Envelope in Mung Bean Hypocotyls

Nuclei from mung bean (Vigna radiata) hypocotyls contained twoglycoproteins of 50 and 49 kDa, respectively, that reacted withconcanavalin A. The glycoproteins were released from the nuclearenvelope by treatment with 2 M KCl but not with nucleases. Theglycoproteins, tentatively named gp50 and gp49, were isolatedand characterized. Gel-permeation chromatography suggested thatgp50 and gp49 seem to exist as a complex with other components.The glycoproteins could be detected only in the nuclear fractionby immunoblot analysis with specific antibodies, and they werenot detected in endoplasmic reticulum, plasma membrane, vacuolarmembrane or mitochondria. Agglutinin I from Ulex europeaus,peanut agglutinin, soybean agglutinin and wheat germ agglutininall failed to bind to the glycoproteins. Treatment with glycopeptidaseF removed all oligosaccharides from the glycoproteins and decreasedtheir molecular masses by about one thousand daltons each. Theseresults suggest that the glycoproteins contained N-linked, high-mannose-typeoligosaccharides with six or seven hexose residues. gp50 andgp49 seemed to be isoforms of a single glycoprotein becausethe two proteins had some common properties. Nuclear fractionsfrom azuki bean (Phaseolus angularis) and soybean (Glycine max)contained proteins that were immunologically similar to gp50and gp49. (Received March 18, 1995; Accepted May 24, 1995)     

Mechanism of the Decline in Vacuolar H -ATPase Activity in Mung Bean Hypocotyls during Chilling

The mechanism responsible for the decrease in the activity of vacuolar H⁺ -ATPase during chilling was investigated in seedlings of mung bean (Vigna radiata). After chilling at 0°C for 3 d, the activity of vacuolar H⁺ -ATPase, calculated on the basis of membrane protein, decreased to 47% of the original value. Of the nine subunits of the ATPase, the specific contents of at least six subunits, of 68, 57, 44, 38, 37, and 32 kD, decreased in vacuolar membranes after chilling, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These subunits were released by treatment with chaotropic anions such as thiocyanate. The level of the 16-kD subunit did not change. Immunoblot analyses showed the decrease in the levels of the subunits of 68, 57, and 32 kD. Furthermore, the specific activity of the ATPase purified from chilled hypocotyls was two-thirds of that of the enzyme from nonchilled seedlings, and the enzyme from chilled tissue retained only a small amount of the 32-kD subunit. These results suggest that a selective release of the peripheral subunits of the ATPase from the membrane and a partial degradation of the ATPase complex may occur in vivo during chilling.     

Chill-Induced Changes in the Activity and Abundance of the Vacuolar Proton-Pumping Pyrophosphatase from Mung Bean Hypocotyls

Changes in the properties of extractable vacuolar H+-pumping pyrophosphatase (V-PPase) and vacuolar ATPase activities in chilling-sensitive seedlings of mung bean (Vigna radiata) were investigated. Following chilling at 4[deg]C for 48 h, both hydrolytic and proton-pumping activities of the V-PPase increased 1.5- to 2-fold over controls and remained elevated even after 72 h at low temperatures. Vacuolar ATPase levels did not change significantly throughout the chilling regime. However a large increase in alcohol dehydrogenase activity during chilling suggests a shift toward fermentative metabolism, which can be expected to decrease ATPase activity in situ. Western blotting of vacuolar membrane-enriched fractions from control and treated plants has confirmed that the changes in V-PPase activity are mirrored by increases in the amount of pump protein. Results suggest a specific role for the V-PPase in protecting chill-sensitive plants from the injurious effects of low temperatures via the maintenance of the proton gradient across the vacuolar membrane.     

Control of Seed Protein Accumulation in Field Bean (Vicia faba L.)

A study was made of the changes during development in the totalamino acid and 3, 4 dihydroxyphenylalanine (DOPA) content ofbean pod phloem sap, employing EDTA to aid phloem exudation.Two field bean lines, Dacre B and D, selected for their lowand high seed protein content respectively, were compared. Throughoutdevelopment, the sap samples of Dacre D had a greater aminoacid concentration than those from Dacre B. The sap of DacreB contained a higher proportion of DOPA than that of Dacre D.These two lines of Dacre were also studied with respect to accumulationof protein and uncombined amino acid in cotyledons grown bothin vitro and in vivo. Dacre D accumulated more total proteinthan Dacre B but contained a similar amount of uncombined aminoacids when grown in vivo. However, the amount of total proteinaccumulated was similar when the cotyledons were grown in vitro.The data suggest that the supply of nutrients to the pod maybe the basis of the different protein concentrations in themature seed of these lines. Vicia faba L., field bean, phloem sap, cotyledon culture, amino acids, DOPA, protein     

Hydrolysis of Crystalloid Storage Protein in Castor Bean Seed Endosperm During and Following Germination

Hydrolysis of the insoluble crystalloid storage proteins ofcastor bean endosperm during germination released buffer-solublepolypeptides with molecular weights in the presence of sodiumdodecyl sulphate of 30000–40000. These polypeptides appearto be dimers since the addition of 2-mercaptoethanol decreasestheir molecular weights to 15000–22000. Hydrolysis ofthe crystalloid proteins was detected 12–18 h after seedimbibition (HAI), which is before the completion of germination;maximum rates were attained at 30 HAI. During this period, parallelincreases in free amino acids were observed. Hydrolysis of thecrystalloid proteins during early germination was insensitiveto cycloheximide treatment and therefore did not require newlysynthesized proteases. Hydrolysis was effected by proteaseswhich were made in an inactive form during seed developmentand activated upon seed imbibition. Key words: Castor bean, crystalloid storage protein hydrolysis, seed germination, endosperm     

Generation of Amylosucrase Variants That Terminate Catalysis of Acceptor Elongation at the Di- or Trisaccharide Stage

An amylosucrase gene was subjected to high-rate segmental random mutagenesis, which was directed toward a segment encoding amino acids that influence the interaction with substrate molecules in subsites −1 to +3. A screen was used to identify enzyme variants with compromised glucan chain elongation. With an average mutation rate of about one mutation per targeted codon, a considerable fraction (82%) of the clones that retained catalytic activity were deficient in this trait. A detailed characterization of selected variants revealed that elongation terminated when chains reached lengths of only two or three glucose moieties. Sequencing showed that the amylosucrase derivatives had an average of no more than two amino acid substitutions and suggested that predominantly exchanges of Asp394 or Gly396 were crucial for the novel properties. Structural models of the variants indicated that steric interference between the amino acids introduced at these sites and the growing oligosaccharide chain are mainly responsible for the limitation of glucosyl transfers. The variants generated may serve as biocatalysts for limited addition of glucose moieties to acceptor molecules, using sucrose as a readily available donor substrate.Amylosucrase (AS) is a glucosyltransferase (EC 2.4.1.4) that was first isolated from bacteria belonging to the genus Neisseria and that transfers a d-glucose (Glc) residue, typically obtained from sucrose (Suc) as the donor, to acceptor molecules such as Glc itself, d-fructose (Fru), or α-1,4-glycosidically linked Glc oligomers and polymers, particularly glycogen (9, 14, 19, 20, 21, 25, 36). While use of Fru as the acceptor gives rise to Suc isomers (or reformation of Suc), use of Glc as the acceptor leads to further elongation to form α-1,4-linked malto-oligosaccharides (designated G2, G3, etc.), which are extended to amylose-like glucans. The enzyme consists of a single polypeptide chain consisting of about 640 amino acid residues (22). It has been categorized as a member of family 13 of the glycoside hydrolases (10). Catalysis by AS involves a two-step mechanism (13, 30). The first step is a nucleophilic attack by the Asp286 side chain at Suc C-1 to displace Fru and form an AS-glucosyl intermediate. Subsequently, this activated ester is typically attacked by a hydroxy group at the nonreducing end of a growing glucan chain, resulting in chain elongation. It can also be attacked slowly by water. The latter reaction, yielding Glc, is an essential step for glucan formation in the presence of Suc as the sole substrate, as neither Suc itself nor Fru serves as a chain initiator (2).Three-dimensional structures have been determined for AS, the AS-Glc intermediate, and various complexes consisting of AS or the inactive Glu328Gln variant and Glc, Suc, or G7 (13, 16, 32, 33). In combination with generation and characterization of AS variants, this has yielded a wealth of information about the reaction mechanism and the residues involved in catalysis and substrate binding (1, 30). These investigations also identified some residues that influence glycogen binding or chain elongation. Thus, replacement by Ala of Asp394, Arg446, or Arg415, which contact an active-site-bound maltoheptaose molecule at subsite +1 or +4 (33), increased Suc hydrolysis and the percentage of G2 and/or G3 formed at the expense of polymer synthesis (2). Furthermore, replacement by Ala of Arg226, which contacts G7 at subsite +2 (33), led to a larger fraction of insoluble glucans instead of short products (2). A twofold reduction in activation of the enzyme by glycogen was a consequence of the Phe417Ala change, an amino acid residue found to be located at the AS surface where binding of a second maltoheptaose molecule has been observed (4).As glucansucrases like AS use Suc as an inexpensive donor substrate and have fairly broad acceptor ranges (3, 17, 24, 31), they are biotechnologically interesting as catalysts for the glucosylation of carbohydrate molecules, as well as noncarbohydrate molecules. Thus, suppression of the undesired formation of glucans and of the multiple addition of sugar moieties to acceptor molecules is of considerable interest. In order to obtain enzyme variants, we used a segmental random mutagenesis method and a screen to identify AS variants with deficiencies in polymer synthesis. For selection of the positive variants obtained, chain elongation properties were characterized, amino acid changes were identified, and structural modeling was used to interpret the findings.     

Characterization of 2,4-D Binding to the Auxin-binding Protein Purified from Etiolated Mung Bean Seedlings

New acetylenic nematicidal compound, penipratynolene (1), methy (2′R)-4-(2′-hydroxy-3′-butynoxy)benzoate, together with two known compounds, 6-methoxycarbonylpicolinic acid (2) and 2,6-pyridinedicarboxylic acid (3), were isolated from the culture filtrate of Penicillium bilaiae Chalabuda. The structures of 1–3 were established by spectroscopic methods. The absolute configuration of 1 was confirmed by using a modified version of Mosher’s method. Compounds 1–3 showed nematicidal activity of 77%, 52%, and 98%, respectively, by a bioassay at 300 mg/l with the root-lesion nematode Pratylenchus penetrans.     

Variation in the Accumulation of Seed Storage Protein Among Genotypes of Phaseolus vulgaris (L.)

Differences in the accumulation of total seed protein and globulin-1 (G1) protein were detected among three inbred lines of common bean. Total protein accumulation ranged from 2.3 to 3.7 milligrams per cotyledon pair per day among lines. In all lines the dry weight and protein accumulation ceased and a loss of chlorophyll in the cotyledons occurred when the moisture content had fallen to 50% of fresh weight. G1 was first detected and rapid accumulation began 14 days after flowering in two lines, whereas in the cultivar Endogava zurundi namame, rapid accumulation was delayed until 20 days after flowering. Rates of G1 accumulation ranged from 1.0 to 1.8 milligrams G1 per cotyledon pair per day among lines. G1 accumulation ceased 6 days before the end of total protein accumulation in Sanilac. A steady rate of protein accumulation was observed in Sanilac, but pauses in the accumulation of G1 and of total protein were documented in Endogava zurundi namame. The rate of G1 accumulation preceding and following the pause in Endogava zurundi namame was 2.7 milligrams G1 per cotyledon pair per day, nearly double that of the other lines.     

Quantification of the In Vitro Radiosensitivity of Mung Bean Sprout Elongation to 6MV X-Ray: A Revised Target Model Study

In this study, a revised target model for quantifying the in vitro radiosensitivity of mung bean sprout elongation to 6-MV X-rays was developed. The revised target model, which incorporated the Poisson prediction for a low probability of success, provided theoretical estimates that were highly consistent with the actual data measured in this study. The revised target model correlated different in vitro radiosensitivities to various effective target volumes and was successfully confirmed by exposing mung beans in various elongation states to various doses of 6-MV X-rays. For the experiment, 5,000 fresh mung beans were randomly distributed into 100 petri dishes, which were randomly divided into ten groups. Each group received an initial watering at a different time point prior to X-ray exposure, resulting in different effective target volumes. The bean sprouts were measured 70 hr after X-ray exposure, and the average length of the bean sprouts in each group was recorded as an index of the mung bean in vitro radiosensitivity. Mung beans that received an initial watering either six or sixteen hours before X-ray exposure had the shortest sprout length, indicating that the maximum effective target volume was formed within that specific time period. The revised target model could be also expanded to interpret the “two-hit” model of target theory, although the experimental data supported the “one-hit” model. If the “two-hit” model was sustained, theoretically, the target size would be 2.14 times larger than its original size to produce the same results.     

Effect of Different Cytokinins on Ethylene Production by Mung Bean Hypocotyls in the Presence of Indole-3-Acetic Acid or Calcium Ion

Kinetin has been shown to act synergistically with indole-3-acetic acid (IAA) or calcium ion (Ca²⁺) to stimulate ethylene production. Several commercially available cytokinins (kinetin, kinetin-riboside, benzyladenine, benzyladenine-riboside, isopentenyladenine, isopentenyladenine-riboside, and zeatin) as well as noncytokinin bases (adenine and xanthine) were administered to mung bean (Phaseolus aureus Roxb.) hypocotyls to study their effects, alone or in combination with IAA or Ca²⁺, on ethylene production. In the presence of IAA or Ca²⁺, all cytokinins tested synergistically stimulated ethylene production and were as effective or nearly as effective as kinetin. Noncytokinin bases (adenine and xanthine) were, however, inactive in this system.     

RASCAL Is a New Human Cytomegalovirus-Encoded Protein That Localizes to the Nuclear Lamina and in Cytoplasmic Vesicles at Late Times Postinfection

The products of numerous open reading frames (ORFs) present in the genome of human cytomegalovirus (CMV) have not been characterized. Here, we describe the identification of a new CMV protein localizing to the nuclear envelope and in cytoplasmic vesicles at late times postinfection. Based on this distinctive localization pattern, we called this new protein nuclear rim-associated cytomegaloviral protein, or RASCAL. Two RASCAL isoforms exist, a short version of 97 amino acids encoded by the majority of CMV strains and a longer version of 176 amino acids encoded by the Towne, Toledo, HAN20, and HAN38 strains. Both isoforms colocalize with lamin B in deep intranuclear invaginations of the inner nuclear membrane (INM) and in novel cytoplasmic vesicular structures possibly derived from the nuclear envelope. INM infoldings have been previously described as sites of nucleocapsid egress, which is mediated by the localized disruption of the nuclear lamina, promoted by the activities of viral and cellular kinases recruited by the lamina-associated proteins UL50 and UL53. RASCAL accumulation at the nuclear membrane required the presence of UL50 but not of UL53. RASCAL and UL50 also appeared to specifically interact, suggesting that RASCAL is a new component of the nuclear egress complex (NEC) and possibly involved in mediating nucleocapsid egress from the nucleus. Finally, the presence of RASCAL within cytoplasmic vesicles raises the intriguing possibility that this protein might participate in additional steps of virion maturation occurring after capsid release from the nucleus.Cytomegalovirus (CMV) is a highly prevalent betaherpesvirus that can cause severe multiorgan disease in immunocompromised individuals (45). The ability of this virus to infect an exceptionally wide variety of different cell types substantially contributes to pathogenesis (5, 68). CMV tropism is largely determined by a finely tuned interplay between cellular and viral factors, many of which act at the earliest stages of infection (30, 68). We recently showed that the cellular protein vimentin is required for efficient onset of infection in primary human foreskin fibroblasts (HF). Interestingly, the degree of reliance on the presence and integrity of vimentin intermediate filaments is dependent on the virus strain, with the broadly tropic strain TB40/E being more negatively affected than the HF-adapted, attenuated strain AD169 (44).Serial passage of clinical isolates in HF or in endothelial cells (EC) has produced strains with different tropisms. The attenuated strains AD169 and Towne were developed as vaccine candidates by propagation in HF for more than 50 (AD169) and 125 (Towne) serial passages (19, 53, 61). During this process, both strains, compared to clinical isolates, accumulated multiple mutations and genomic deletions, resulting in the loss of more than 19 open reading frames (ORFs) (8). The number of serial passages in HF of another commonly used strain, Toledo, has been more moderate (19, 54, 58). This, however, did not prevent the emergence of numerous genomic mutations, including the inversion of an ∼15-kb fragment (8, 16, 56). As a consequence of these changes, productive infections by AD169, Towne, and Toledo are largely restricted to HF. In contrast, propagation of clinical isolates in EC has yielded a series of strains with more-intact genomes and broader tropisms, such as TB40/E, VHL/E, and FIX (VR1814) (25, 60, 71). These strains retain the ability to grow in a wider variety of cell types, including EC, epithelial cells, and dendritic cells (DC), in addition to HF (23, 28, 59, 60, 68).The UL128, UL130, and UL131A gene products were recently identified as essential mediators of CMV infection of EC and epithelial cells (26, 72, 73) and of virus transfer from infected EC to monocyte-derived DC (23). Each of these proteins is independently required for the broader tropisms of EC-propagated CMV isolates (63, 64), and the presence of mutations affecting their functionality has been directly linked to the inability of AD169, Towne, and Toledo to initiate productive infections in EC and epithelial cells (26, 72, 73).We have shown that mature Langerhans-type DC differentiated in vitro from CD34⁺ hematopoietic progenitor cells are highly permissive to direct infection with TB40/E or VHL/E, with 48 to 72% of cells in culture expressing the viral immediate-early genes IE1 and IE2 at 48 h postinfection (hpi) (28). In contrast, only 2 to 5% and 0% of mature Langerhans cells were IE1/IE2 positive after exposure to Towne and Toledo, respectively. However, productive infection was detected in 12 to 17% of cells infected with AD169, despite the fact that this strain lacks expression of the UL131A gene as a consequence of a frameshift mutation (26). These results suggested the existence of additional viral genes with products involved in mediating tropisms for specific cell types, such as DC. To identify possible candidates, we compared the amino acid sequence of each ORF found in the genome of TB40-BAC4, a sequenced clone of the TB40/E strain in a bacterial artificial chromosome (BAC) (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"EF999921","term_id":"157779983","term_text":"EF999921"}}EF999921) (69), to the sequence of each ORF found in AD169 and AD169-BAC (accession numbers {"type":"entrez-nucleotide","attrs":{"text":"X17403","term_id":"59591","term_text":"X17403"}}X17403 and {"type":"entrez-nucleotide","attrs":{"text":"AC146999","term_id":"38093733","term_text":"AC146999"}}AC146999) (10, 49), Towne and Towne-BAC (accession numbers {"type":"entrez-nucleotide","attrs":{"text":"FJ616285","term_id":"222354438","term_text":"FJ616285"}}FJ616285, {"type":"entrez-nucleotide","attrs":{"text":"AC146851","term_id":"37654163","term_text":"AC146851"}}AC146851, and {"type":"entrez-nucleotide","attrs":{"text":"AY315197","term_id":"124303521","term_text":"AY315197"}}AY315197) (17, 18, 49), and Toledo-BAC (accession number {"type":"entrez-nucleotide","attrs":{"text":"AC146905","term_id":"37777314","term_text":"AC146905"}}AC146905) (49). The product of a putative ORF, originally identified by Murphy et al. and named conserved ORF 29 (c-ORF29) (49), was considered of particular interest because the amino acid sequence of the putative protein encoded by Toledo and Towne was extended by 79 residues compared to the putative protein encoded by TB40/E and AD169. This led to our speculation that that the extended version might result in a nonfunctional version of the c-ORF29-encoded protein. We thus focused our studies on the products of this ORF.Here, we show for the first time that CMV c-ORF29 encodes a protein expressed at early to late times postinfection (p.i.) and localizes to the nuclear rim in peculiar invaginations of the nuclear lamina and in cytoplasmic vesicular structures at late times p.i. Based on this localization pattern, we named this gene product nuclear rim-associated cytomegaloviral protein, or RASCAL. Surprisingly, no difference was observed in the distributions of RASCAL during infection of HF with TB40/E or Towne, suggesting that the intracellular trafficking of this protein is not affected by the presence of the additional residues at the C terminus of RASCAL from strain Towne (RASCAL_Towne). Ectopic expression of RASCAL in human embryo kidney 293T (HEK293T) cells further revealed that this protein requires the presence of the nuclear egress complex (NEC) member UL50 to reach the nuclear rim, while coimmunoprecipitation (co-IP) assays provided evidence for the existence of an interaction between RASCAL and UL50. These findings suggest that RASCAL may be a new component of the NEC with possible roles in remodeling the nuclear lamina during nucleocapsid egress from the nucleus.     

Effects of the Inhibitory Protein of Ethylene Synthesis on ATP Levels in Mung Bean Hypocotyl Segments

The inhibitory protein of ethylene synthesis purified from mungbean seeds reduced ATP levels in mung bean hypocotyl segments.When the segments were incubated with 0.5mM IAA for 6 hr toinduce ethylene-producing activity, the presence of the inhibitoryprotein suppressed the ethylene production and ATP content inthe tissue about 82 and 60%, respectively. Similar suppressiveeffects were also observed for endogenous ethylene productionand ATP contents in tissue not treated with IAA. (Received June 20, 1981; Accepted October 24, 1981)     

Evidence That a Precursor Glycoprotein Is Cleaved to Yield the Major Glycoprotein of Avian Tumor Virus

A glycoprotein designated pr90, which is recognized by anti-gp85 serum, is present in lysates of pulse-labeled transformed cells. Under chase conditions, a reduction in the level of labeled pr90 is observed concomitant with the appearance of labeled, cell-associated viral glycoprotein.     

UDP-GlcNAc:Glycoprotein GlcNAc-Transferase is Located in the Golgi Apparatus of Developing Bean Cotyledons

The transport and accumulation of phytohemagglutinin in developing bean (Phaseolus vulgaris L.) cotyledons is accompanied by the transient presence of N-acetylglucosamine (GlcNAc) residues on the oligosaccharide sidechains of this glycoprotein. These peripheral GlcNAc residues can be distinguished from those in the chitobiose portion of the oligosaccharide sidechains by their sensitivity to removal by the exoglycosidase β-N-acetylglucosaminidase. GlcNAc residues sensitive to removal by β-N-acetylglucosaminidase are present not only on phytohemagglutinin, but also on other newly synthesized proteins. The enzyme UDPGlcNAc:glycoprotein GlcNAc-transferase which transfers GlcNAc residues to glycoproteins was first described by Davies and Delmer (Plant Physiol 1981 68: 284-291). The data presented here show that this enzyme is associated with the Golgi complex of developing cotyledons.     

The Lack of Correlation between ATP Accumulation in Seeds at the Early Stage of Germination and Seed Quality

Seeds of various species were treated with polyethyleneglycolin the absence or presence of AMP and phosphoenolpyruvate, redriedand examined for ATP accumulation at the early stage of germinationand for the rate of germination under suboptimal temperatures.The pretreatments resulted in seeds with various levels of accumulatedATP but in most comparative experiments no correlation was foundbetween the ATP accumulation and the rate of germination. Similarpretreatments with naturally aged seeds led to the same conclusion.The fact that ATP accumulation is a result of ATP synthesisas well as ATP utilization was demonstrated by a protein inhibitionexperiment which showed a possibility to produce seeds withrelatively high ATP accumulation levels at the early stage ofgermination, but with very low germination ability. Key words: AMP, PEP, Polyethyleneglycol, Pretreatment     

Peptide Mapping Reveals Considerable Sequence Homology among the Three Polypeptide Subunits of G1 Storage Protein from French Bean Seed

The major storage protein, G1 globulin, of bean (cv. Tendergreen) seeds was subjected to limited proteolysis with trypsin, chymotrypsin, papain, proteinase K, and protease V8 and to cleavage with cyanogen bromide and 2-(2-nitrophenylsulfanyl)-3-methyl-3′bromoindolenine. Mapping of peptides separated from each of the three G1 subunits by polyacrylamide gel electrophoresis revealed that many proteolytic cleavage sites were present at similar positions on the subunits. Evidence was adduced that the G1 subunits are homologous in amino acid sequence for about 61% of their length. The remaining region (possibly COOH-terminal) of the subunits appears to be heterologous, with the α subunit bearing an additional methionine residue.