The effect of abscisic acid on the differential expression of α-amylase isozymes in barley aleurone layers

Summary The treatment of barley aleurone layers with gibberellic acid (GA₃) results in the synthesis of two groups of -amylase isozymes. Addition of abscisic acid (ABA) at the same time as GA₃ inhibited the synthesis of both groups of isozymes. However, midcourse ABA addition (12 h or later after GA₃) had a more inhibitory effect on the high pI -amylase group than on the low pI -amylase group. This midcourse inhibition was detectable within 2 h of ABA addition. Northern analysis results using cDNA probes for the high pI and low pI -amylase groups paralleled the protein synthesis results for both isozyme groups. High pI -amylase mRNA levels began to decrease within 2 h of midcourse ABA treatment and were less than 10% of the original level by 4 h. The levels of low pI -amylase mRNA were decreased less by midcourse ABA addition than were high pI mRNA levels. Cordycepin and cycloheximide blocked the effects of midcourse ABA addition on -amylase mRNA. These observations indicate that ABA inhibits -amylase expression at the pretranslational level and that protein and RNA synthesis are required for midcourse ABA action to occur. Our results also show that -amylase mRNA, which has been thought to be very stable, is degraded after midcourse ABA treatment.     

Hormonal regulation of gene expression in the “slender” mutant of barley (Hordeum vulgare L.)

The slender mutant of barley resembles a normal barley plant treated with high doses of gibberellic acid (GA₃). Expression of GA₃-regulated and abscisic acid (ABA)-regulated mRNAs was studied in the endosperm and roots of mutant and wild-type (WT) plants.Production of -amylase (EC 3.2.1.1) by WT embryoless half-grains was dependent on the presence of GA₃, and was prevented by ABA. In contrast, -amylase was produced by half-grains of the slender mutant in the absence of added GA₃, although it was still reduced by ABA. The spectrum of -amylase mRNAs in slender embryoless half-grains incubated in the absence of added GA₃ was the same as in WT endosperm half-grains incubated in the presence of GA₃. These results indicate that the endosperm of the slender mutant exhibits similar properties to WT endosperm treated with GA₃.In roots the expression of an ABA-inducible mRNA was similar in slender and WT seedlings either treated with exogenous ABA or exposed to dehydration. This result, and the effect of ABA on -amylase production by the endosperm, indicate that the slender plants retain sensitivity to ABA.Abbreviations ABA abscisic acid - AMV avian myeloblastosis virus - GA gibberellin - GA₁ gibberellin A₁ - GA₃ gibberellic acid - WT wild-type     

Variation of seed α-amylase inhibitors in the common bean

Variation of seed -amylase inhibitors was investigated in 1 154 cultivated and 726 non-cultivated (wild and weedy) accessions of the common bean, Phaseolus vulgaris L. Four -amylase inhibitor types were recognized based on the inhibtion by seed extracts of the activities of porcine pancreatic -amylase and larval -amylase and larval -amylase of the Mexican bean weevil, Zabrotes subfasciatus Boheman. Of the 1 880 accessions examined most (1 734) were able to inhibit porcine pancreatic -amylase activity, but were inactive against the Z. subfasciatus larval -amylase; 41 inhibited only the larval -amylase activity, 52 inhibited the activities of the two -amylases, and 53 did not inhibit the activity of either of the -amylases. The four different inhibitor types were designated as AI-1, AI2, AI-3, and AI-0, respectively. These four inhibitor types were identified by the banding patterns of seed glycoproteins in the range of 14–20 kDa by using SDSpolyacrylamide gel electrophoresis. Additionally, four different banding patterns were recognized in accessions with AI-1, and were designated as AI-1a, 1b, 1c, and 1d. Two different patterns of the accessions lacking an -amylase inhibitory activity were identified and designated as AI-0a and AI-0b. The largest diversity for seed -amylase inhibitors was observed in non-cultivated accessions collected from Mexico where all eight inhibitor types were detected. The possible relationships between the variation of seed -amylase inhibitors and bruchid resistance are discussed.     

The role of the endoplasmic reticulum in the synthesis and transport of α-amylase in barley aleurone layers

The subcellular site of -amylase (EC 1.6.2.1) synthesis and transport was studied in barley aleurone layers incubated in the presence or absence of gibberellic acid (GA₃). Using [³⁵S]methionine as a marker, the site of amino-acid incorporation into organelles isolated from aleurone layers incubated with and without GA₃ was determined following purification by isopycnic sucrose-density-gradient centrifugation. Incorporation of radioactivity into trichloroacetic-acid-insoluble proteins was greatest in those fractions exhibiting activity of an endoplasmic reticulum (ER) marker enzyme. Further fractionation of densitygradient fractions by sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis showed that a major portion of the radioactivity in the ER fractions was present in a protein co-migrating with marker -amylase. This protein was identified as authentic -amylase by immunoadsorbent chromatography and affinity chromatography. The newly synthesized -amylase associated with the ER was shown to be sequenstered within the lumen of the ER by experiments which showed that the enzyme was resistant to proteolytic degradation. The labelled -amylase sequestered in the ER can be chased from this organelle when tissue is incubated in unlabelled methionine following a 1-h pulse of labelled methionine. The isoenzymic forms of -amylase found in tissue homogenates and incubation media of aleurone layers incubated with and without GA₃ were characterized after chromatography on diethylaminoethyl cellulose. In homogenates of GA₃-treated aleurone layers, five peaks of -amylase activity were detected, while in homogenates of aleurone layers incubated with-out GA₃ only three peaks of activity were found. In incubation media, four isoenzymes were found after GA₃ treatment and two were found after incubation without GA₃. We conclude that at least five -amylase isoenzymes are synthesized by the ER of barley aleurone layers and that this membrane system is involved in the sequestration and transport of four of these isoenzymes.Abbreviations CHA cyclohepataamylose - DEAE-cellulose diethylaminoethyl-cellulose - ER endoplasmic reticulum - GA₃ gibberellic acid - SDS-PAGE sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis     

Gibberellic acid and abscisic acid modulate protein synthesis and mRNA levels in barley aleurone layers

Using in vivo pulse labeling, changes in the pattern of protein synthesis were detected in isolated barley aleurone layers treated with fibberellic acid (GA₃). GA₃ greatly altered the relative rates of synthesis of many polypeptides, increasing some, notably -amylase, and decreasing others. -Amylase synthesis increased until it was the major product (over 60%) of protein synthesis after 24h. The pulse-labeled pattern of secreted polypeptides was also changed by GA₃. There was the expected increase in -amylase together with a number of other polypeptides but there was reduced secretion of several polypeptides also.Cell-free translation of RNA isolated from control and hormone-treated tissues was used to measure changes in mRNA levels. GA₃ caused many changes, particularly in the level of mRNA for -amylase. In vitro synthesized -amylase, identified by immunoaffinity chromatography, had an Mr of 46 000. This polypeptide was partially processed to a polypeptide with Mr 44 000 by the addition of dog pancreas membranes to the in vivo translation mixture. The level of mRNA for -amylase began to increase 2–4 h after GA₃ was added and reached a maximum level of about 20% of total mRNA after 16 h. Thus after 16 h, the synthesis of -amylase as a proportion of total protein synthesis, continued to increase while the level of its mRNA as a proportion of total mRNA remained constant. These results indicate that protein synthesis was modified more extensively than we can account for by changes in mRNA.Abscisic acid (ABA) reversed all of the effects of GA₃ on protein synthesis and mRNA levels. It also promoted synthesis of a small number of new polypeptides and increased the level of some mRNAs. GA₃ reversed the accumulation of ABA-promoted mRNAs. Although, ABA strongly suppressed the increase in the level of translatable mRNA for -amylase, there was an even stronger inhibition of enzyme synthesis and accumulation.We conclude that both GA₃ and ABA regulate protein synthesis both positively and negatively in aleurone cells largely by regulating levels of mRNA and in the case of -amylase, possibly also by changing the efficiency of translation of its mRNA.     

Expression of α-amylase and other gibberellin-regulated genes in aleurone tissue of developing wheat grains

Aleurone tissue from freshly harvested immature wheat grains (Triticum aestivum L. cv. Sappo) which is normally unresponsive to gibberellic acid can be made responsive by subjecting the tissue to a pre-incubation treatment in a simple buffered medium prior to the addition of the growth substance. The effectiveness of this treatment is dependent on grain age, with grains less than 15–20 days post anthesis failing to become converted to a responsive state whilst tissue from grains older than this become increasingly susceptible. Tissue from grains of a certain age (approx. 25–28 days post anthesis) produce small amounts of -amylase following this treatment even in the absence of exogenously applied growth substance. Using different ³²-labelled complementary-DNA probes for -amylase in wheat it was demonstrated that the failure of freshly harvested tissue to produce -amylase was correlated with the absence of the appropriate mRNA species. Inability to accumulate -amylase mRNA in response to gibberellic acid was removed by the pre-iccubation treatment and also by enforced drying. The gibberellin-regulated expression of other unidentified genes also responds to pre-incubation or drying. Induction of gibberellin-responsiveness in immature aleurone cells did not extend to the secretion of acid phosphatase, protease and ribonuclease.Abbreviations cDNA complementary DNA - dpa days post anthesis - GA gibberellin - GA₃ gibberellic acid     

Endogenous gibberellin A1 level and α-amylase activity in germinating rice seeds

The role of endogenous gibberellin A₁ (GA₁) in the induction of -amylase activity was investigated during germination of rice (Oryza sativa L.) seeds. The level of endogenous GA₁ and the -amylase activity in the seeds of normal rice, cv. Nipponbare, increased simultaneously from 3 days after the imbibition of water. The -amylase activities in the dwarf rice, cv. Waito-C and Tan-ginbozu, were less than that in the normal rice. The level of endogenous GA₁ and -amylase activity were decreased in proportion to the concentration of a growth retardant, uniconazole. The retardation in -amylase activity caused by the treatment of uniconazole was recovered by the application of exogenous GA₁. These results indicate that the endogenous GA₁ biosynthesized de novo regulates -amylase production in germinating rice seeds.Abbreviations GA(s) gibberellin(s) - ABA abscisic acid - AE fraction acidic ethyl acetate-soluble fraction - HPLC high performance liquid chromatography - R _t retention time - GC-SIM gas chromatography-selected ion monitoring     

The effects of gibberellic acid and abscisic acid on α-amylase mRNA levels in barley aleurone layers studies using an α-amylase cDNA clone

Summary Two cDNA clones were characterized which correspond to different RNA species whose level is increased by gibberellic acid (GA₃) in barley (Hordeum vulgare L.) aleurone layers. On the criteria of amino terminal sequencing, amino acid composition and DNA sequencing it is likely that one of these clones (pHV19) corresponds to the mRNA for -amylase (1,4--D-glucan glucanohydrolase, EC 3.2.1.1.), in particular for the B family of -amylase isozymes (Jacobsen JV, Higgins TJV: Plant Physiol 70:1647–1653, 1982). Sequence analysis of PHV19 revealed a probable 23 amino acid signal peptide. Southern hybridization of this clone to barley DNA digested with restriction endonucleases indicated approximately eight gene-equivalents per haploid genome.The identity of the other clone (pHV14) is unknown, but from hybridization studies and sequence analysis it is apparently unrelated to the -amylase clone.Both clones hybridize to RNAs that are similar in size (1500b), but which accumulate to different extents following GA₃ treatment: -amylase mRNA increases approximately 50-fold in abundance over control levels, whereas the RNA hybridizing to pHV14 increases approximately 10-fold. In the presence of abscisic acid (ABA) the response to GA₃ is largely, but not entirely, abolished. These results suggest that GA₃ and ABA regulate synthesis of -amylase in barley aleurone layers primarily through the accumulation of -amylase mRNA.Abbreviations ABA abscisic acid - CHA cyclohepta-amylose - CMC carboxymethyl cellulose - GA₃ gibberellic acid     

Expression of α-amylase in response to wounding in mung bean

The activity of -amylase (EC 3.2.1.1) in mung bean (Vigna radiata (L.) Wilczek) cotyledons increased markedly in response to wounding. The changes in enzyme activity were in parallel with those in enzyme content. The level of -amylase mRNA also notably increased in wounded cotyledons and attained its maximum level during the period between 1 and 2 d after wounding. The level of mRNA for phenylalanine ammonia-lyase, which is one of the well-characterized stress-inducible proteins, also increased after wounding, but the increase in mRNA level was faster than that of -amylase mRNA. On the other hand, the content of mRNA for actin, a housekeeping protein, was almost the same in wounded and unwounded cotyledons. The increase in -amylase mRNA level in wounded cotyledons was severely inhibited by -amanitin and cordycepin. -Amylase expression in the first leaves of mung-bean seedlings was also induced by wounding.Abbreviations PAL phenylalanine ammonia-lyase - SSC standard saline citrate We greatly acknowledge Prof. Richard Meagher, Department of Genetics, University of Georgia, Athens, USA for the gift of soybean actin gene clone. We also thank Mr. Kaoru Ishiwata for technical assistance.     

The responsiveness of Avena fatua aleurone protoplasts to gibberellic acid

The sensitivity to gibberellic acid (GA₃) of aleurone protoplasts isolated from a single harvest of an inbred line of Avena fatua seed that had been after-ripened over anhydrous CaCl₂ at 25±2°C and 4±2°C for three years was assessed. Protoplasts isolated from aleurones of seed stored at 25°C produced substantially more -amylase in response to 10^–7 M GA₃ than those isolated from aleurones of seed stored at 4°C. The apparent difference in responsiveness does not appear to be due to a change in the duration of the lag phase between addition of GA₃ and the production of -amylase. The dose response of aleurone protoplasts to GA₃, measured as -amylase production, is complex and appears to have three phases. Protoplasts from seed stored at both temperatures respond appreciably to 10^–14 M GA₃. With increasing concentrations of GA₃, up to 10^–9 M, -amylase production increases similarly in protoplasts from both lots of seed, reaching a level approximately 2.7–3.8 times greater than when no GA₃ is applied. GA₃-induced -amylase production increases markedly as the concentration is raised from 10^–9 M to 10^–6 M, and the response then appears to be saturated. Over this part of the response curve protoplasts from the two seed lots differ markedly in their responsiveness to GA₃. Those from seed stored at 25°C produce considerably more -amylase, >130-fold higher than the minus GA₃ control, than those from seed stored at 4°C, <35-fold higher than the minus GA₃ control. This apparent difference in the responsiveness of aleurone protoplasts to GA₃ could be correlated with the loss of embryo dormancy in seed stored at 25°C. Seed stored at 4°C retained the dormancy characteristics present immediately after harvesting.     

Properties and synthesis de novo of auxin-induced α-amylase in pea cotyledons

Analysis of starch-degrading enzymes in a crude extract of detached cotyledons of Pisum sativum L. by polyacrylamide gel electrophoresis (PAGE) demonstrated the presence of one band of -amylase (EC 3.2.1.1) activity. The activity of only this amylase was promoted in cotyledons incubated with 2,4-dichlorophenoxyacetic acid (2,4-D). The auxin-induced -amylase from pea cotyledons was purified to homogeneity, as judged by the criterion of a single band after PAGE. The relative molecular mass (M_r), estimated by gel filtration, was approx. 42 000 and the enzyme contained no carbohydrate moiety. Sodium dodecylsulfate-PAGE yielded a single band that corresponded to an M_r of 41 000. The isoelectric point was 5.85 and the aminoacid composition was similar to that of -amylase from other plants. When [³H]leucine was fed to detached dry cotyledons prior to incubation, the radioactivity in -amylase from cotyledons incubated in the presence of 2,4-D was found to be approx. 10-fold higher than that from cotyledons incubated in distilled water. When -amylase from cotyledons incubated with ²H₂O that contained 2,4-D and the tritiated amylase were centrifuged together in a CsCl density gradient, the peak of enzymatic activity of deuterated -amylase was shifted to a denser fraction than the peak of radioactivity of the tritiated enzyme. These results show that auxin-induced -amylase in pea cotyledons is synthesized de novo.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PAS periodic acid-Schiff - pI isoelectric point - SDS sodium dodecyl sulfate We are very grateful to Mr. Kazuo Itoh and Mrs. Matsumi Doe for carrying out the analysis of amino-acid composition.     

Genetics of α-amylases from rye endosperm

Summary Fifteen inbred lines of rye, F₁ and F₂ progenies from crosses between lines were studied using polyacrylamide gel electrophoresis. Conventional genetic analysis of -amylase zymograms showed that the 19 bands detected in the endosperm of germinating caryopses were controlled by three linked structural loci and one independent modifying locus, which influenced the electrophoretic mobility of isozymes. Two codominant alleles were found at the -Amy1, -Amy2 structural loci and the M--Amy modifying locus while the -Amy3 locus had three alleles. Double-banded expression of the -amylase alleles was probably due to the simultaneous presence of modified and unmodified forms of isozymes on the zymogram.This work was supported by Polish Academy of Sciences under project MR-II/7 and was also a part of the author's PhD Thesis     

Amylase activity and growth in internodes of deepwater rice

Isoelectrofocusing, product analysis, thermal denaturation studies and affinity chromatography on cycloheptaamylose-Sephadex were used to identify the amylolytic enzymes in internodes of deepwater rice (Oryza sativa L.). Amylolytic activity in internodes of deepwater rice consists of -amylase (sometimes separated into two isoforms) and of -amylase. During submergence of whole plants, -amylase activity increases in young, growing internodes, but -amylase activity declines. Although non-growing, mature internodes contain higher levels of -amylase than do the elongating younger internodes, the effect of submergence on amylase activities in both tissues follows the same trend. Submergence, gibberellic acid (GA₃) and ethylene all promote -amylase activity in growing and non-growing internodes of excised deepwater-rice stem sections. Inhibitor studies showed that submergence and ethylene promote -amylase activity in the absence of endogenous gibberellin (GA), and GA₃ enhances -amylase activity when ethylene action is inhibited. Therefore, ethylene and GA appear to increase -amylase activity independently of each other. Enhanced -amylase activities are probably responsible for the mobilization of carbohydrates which are needed to support internode elongation during submergence of deepwater rice.Abbreviations CHA cycloheptaamylose - GA₃ gibberellic acid - NBD 2,5-norbornadiene - TCY tetcyclacis     

Gibberellin perception at the plasma membrane of Avena fatua aleurone protoplasts

A functional assay for gibberellin (GA) receptors is described based on the induction of -amylase gene expression in isolated aleurone protoplasts of Avena fatua L. by GA₄ immobilised to Sepharose beads. A 17-thiol derivative of GA₄, shown to be biologically active with aleurone protoplasts, has been coupled to epoxy-activated Sepharose 6B. This GA₄-17-Sepharose induces high levels of -amylase when incubated with isolated aleurone protoplasts, while cells of the intact aleurone layer do not respond appreciably to the immobilised GA₄. In order to eliminate the possibility that GA₄ may be released from the Sepharose when incubated with protoplasts, aleurone layers and isolated aleurone protoplasts have been co-incubated, and their responses to GA₄, GA₄-17-Sepharose and control Sepharose estimated by determining the relative amounts of -amylase mRNA induced in each tissue. Evidence from these experiments is consistent with the view that GA₄17-Sepharose induces -amylase gene expression in aleurone protoplasts by interacting with the protoplast surface. This indicates that GA receptors may be located at, or near, the external face of the aleurone plasma membrane.Abbreviation GA(n) gibberellin A(n) We thank Professor Jake MacMillan and Drs. Peter M. Chandler (CSIRO, Division of Plant Industry, Canberra, Australia), Peter Hedden and Johnathan Weir (Unilever, Port Sunlight, UK) for helpful discussions and suggestions. Computer graphics were performed by the University of Bristol Molecular Recognition Centre.     

Extracellular and mycelial amylases of the thermophilic fungus Malbranchea sulfurea

The thermophilic fungus Malbranchea sulfurea produces extracellular -amylase whereas -glucosidase is mainly cell bound. Extraction of the cell bound enzyme was maximum with one molar NaCl, followed by Triton ×100 and Urea-Na₂SO₃ extractants. Supplementation of Triton ×100 in growth medium significantly affected the presence of enzymes at various locations. A role for cell bound -amylase and -glucosidase has been suggested in rapid starch utilization by the fungus during early growth phase.     

The mode of secretion of α-amylase in barley aleurone layers

The involvement of the endomenbrane system of barley (Hordeum vulgare L.) aleurone cells in the secretion of gibberellin-induced hydrolases has been investigated at the biochemical level. Our results show that at least 40–60% of the -amylase activity in homogenates of aleurone layers occurs in a membrane-bound, latent form. The latent -amylase can be assayed quantitatively following disruption of membranes by treatment with Triton X-100, ethanol, sonication, or osmotic shock and shear. The association of -amylase with the membrane is not an artifact arising from homogenization of the tissue, and acid protease is also enriched in the same subcellular fraction as the -amylase. The membrane fraction with which the -amylase is associated has many properties of the endoplasmic reticulum (ER). When membranebound -amylase is prepared in buffers containing 3 mM MgCl₂ two fractions from a sucrose step gradient contain most of the -amylase activity. These fractions are enriched in the ER marker enzyme, NADH-dependent cytochrome-c reductase, and show densities characteristic of smooth and rough ER during subsequent purification on continuous gradients. In step gradients prepared with ethylenediaminete-traacetic-acid-treated membranes, -amylase activity is contained primarily in one fraction having the density of smooth ER. Electron microscopy of the purified fractions is consistent with -amylase being associated with smooth and rough ER. However, it has not been ruled out that the enzyme is also associated with plasma membrane, Golgi membranes, or tonoplast. Examination of the isoenzyme patterns of secreted, of total-homogenate and of membrane-associated -amylases, as well as the results from pulsechase experiments using L-[³H]leucine for labeling of -amylase, are all consistent with the hypothesis that membrane-associated -amylase is an intermediate in the secretory process.Abbreviations CNTPE N-carbobenzoxy-L-tyrosine p-nitrophenylester - Cyt oxidase Cytochrome oxidase - ER endoplasmic reticulum - EDTA ethylenediaminetetraacetic acid - GA₃ gibberellic acid - IDPase inosine diphosphatase - K⁺-ATPase pH 6.5 K⁺-stimulated adenosine triphosphatase - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - NADH: Cyt c reductase cyanide-insensitive NADH-linked cytochrome-c reductase - RER rough endoplasmic reticulum - Tris tris-(hydroxymethyl)-aminomethane     

Involvement of the Golgi apparatus in the secretion of α-amylase from gibberellin-treated barley aleurone cells

Localisation of -amylase (EC 3.2.1.1) in barley aleurone cells treated with gibberellic acid has been achieved using protein A-gold-labelled polyclonal antibodies. Gold particles were located almost exclusively over the lumen of the rough endoplasmic reticulum and cisternae of the Golgi apparatus. The label was most concentrated over the Golgi apparatus. This indicates that the Golgi is involved in the secretion of -amylase protein from aleurone cells.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - PBS phosphate-buffered saline     

Regulation of α-amylase activity in Amaranthus caudatus seeds by methyl jasmonate, gibberellin A3, benzyladenine and ethylene

Methyl jasmonate (JA-Me) at 10^–3 Minhibited Amaranthus caudatus seed germination anddecreased -amylase activity. Exogenous gibberellin A₃(GA₃) and ethylene, but not benzyladenine (BA), increased activity ofthe enzyme in the presence of JA-Me, with ethylene being the most effective. Theinhibitor of ethylene action, 2,5-norbornadiene (NBD) inhibited seed germinationand decreased -amylase activity. The inhibitory action of JA-Me onAmaranthus caudatus seed germination is associated with theinhibition of -amylase activity. Exogenous GA₃ and ethylenecontrol both -amylase activity and seed germination in the presence of JA-Me.     

Evidence for disaggregation of oligomeric G(o)alpha induced by guanosine-5;-3-O-(thio)triphosphate activation

Myristoylated G_o was expressed in and highly purified from Escherichia coli strain JM109 cotransformed with pQE60 (G_o) and pBB131 (N-myristoyltransferase, NMT). Non-denaturing gel electrophoresis and gel filtration analysis revealed that the G_o, in its GDP-bound form, could form oligomers involving dimer, trimer, tetramer, pentamer, or hexamer and guanosine 5"-3-O-(thio)triphosphate (GTPS) activation induced disaggregation of the G_o oligomers to monomers. The G_o was crosslinked by a cross-linker, N,N"-1,4-phenylenedimaleimide (p-PDM), yielding multiple crosslinked products. In contrast, no obvious cross-linking occurred when G_o was pretreated with GTPS. Immunoblot analysis also demonstrated oligomerization of the purified G_o proteins and its disaggregation triggered by GTPS. These results provided direct evidence for the disaggregation–coupling theory and the disaggregation action of GTPS may further elucidate the regulatory role of GDP/GTP exchange in G protein-coupled signal transduction pathways.     

Inheritance of seed α-amylase inhibitor in the common bean and genetic relationship to arcelin

The inheritance of seed -amylase inhibitor in the common bean and the genetic relationships among the variants and six arcelin variants in the common bean were investigated by crossing between accessions containing different AI and arcelin variants. All seed proteins in parental, F₁ and F₂ seeds from the crosses were examined by Western-blot analysis. All F₁ seeds gave combined AI banding patterns from parents on the blotting membranes. The segregation of F₂ seeds for AI variants indicated that the polypeptides of AI variants were inherited as single co-dominant units. Moreover, AI and arcelin behaved as a single block in crosses, indicating a close linkage relationship between the genes controlling these proteins.