{alpha}-Amylase synthesis by a cell free system and plant growth substances

A microsomal preparation from the aleurone layer pre-treatedwith GA or H-ol had the ability to synthesize -amylase whenit was incubated with an appropriate medium. -Amylase synthesiswas inhibited by the addition of p-fluorophenylalanine or bypre-treatment with RNase. The synthesized -amylase was separatedinto three isozymes by disc-electrophoresis (Received July 25, 1970; )     

Regulation of sexual agglutinability in Saccharomyces cerevisiae of {alpha} and {alpha} types by sex-specific factors produced by their respective opposite mating types

The sexual agglutinability of haploid cells of heterothallicSaccharomyces cerevisiae was repressed when they were culturedin the absence of easily fermentable sugars, such as glucoseand mannose. The repression was reversed by the action of hormone-likesubstances of the opposite mating types. The substance producedby mating type cells was identical to subtsance-I which isknown to induce sexual agglutinability of inducible matingtype cells. The mating type cells produce a new hormone-likesubstance which induces or enhances sexual agglutinability of mating type cells. A crude fraction of the mating type-specific substance ( substance-I)was obtained by passing the culture filtrate of mating typecells through Amberlite CG-50 (H⁺ form), followed by elutionwith 1.5 M ammonia. ² On leave from Osaka City University. (Received December 25, 1975; )     

ON THE EFFECTS OF PLANT EXTRACTS ON THE {alpha}-AMYLASE ACTIVITY IN RICE ENDOSPERM

Effects of ethanol extracts obtained from several plant materialson -amylase activity in rice endosperm were examined. Promotingeffect on the -amylase activity was estimated in terms of gibberellinA₃ equivalence using the embryoless endosperm. Inhibiting effectwas examined using intact seed. On chromatography of the extractfrom Prunus, the zones showing promotion of the -amylase activityalso exhibited the activity for the elongation of the leaf sheathof rice seedling. But this was not the case with the extractsfrom Pharbitis, Allium and Lupinus. (Received April 13, 1966; )     

Part of the Lysyl Residues in Wheat {alpha}-Amylase is Methylated as N-{varepsilon}-Trimethyl Lysine

Amino acid analyses of wheat -amylase purified from germinatingseeds by affinity chromatography showed a high content of amodified lysyl residue. The modified residue was identifiedas N--trimethyl lysine. The presence of trimethyl lysine in-amylase is discussed in terms of isozymes. ¹ Present address: National Institutes of Health, Bldg. 10,Rm. 9B-15, Bethesda, MD 20205, U.S.A. (Received August 20, 1981; Accepted March 19, 1982)     

HORMONAL REGULATION ON THE INDUCTION OF {alpha}-AMYLASE ISOZYMES IN THE EMBRYO-LESS ENDOSPERM OF BARLEY

The -amylase induced by helminthosporol and gibberellic acidin the embryo-less endosperm of barley was separated into thethree fractions, 1, 2 and 3, by DEAE-cellulose column chromatography.A maximum amount of the 2. was induced by gibberellic acid andthat of the as by helminthosporol. After rechromatography, the2 induced by gibberellic acid and the as induced by helminthosporolshowed their respective single bands in an electrophoresis agargel zymogram. On the other hand, the ai induced by helminthosporoland gibberellic acid showed three bands. Dihydrohelminthosporic acid, a derivative of helminthosporol,induced the same isozymes as helminthosporol did. (Received May 8, 1967; )     

Purification by Affinity Chromatography of {alpha}-Amylase--A Main Amylase in Cotyledons of Germinating Vigna mungo Seeds

In Vigna mungo cotyledons, the -amylase activity increased markedlyduring germination at 27°C in the dark, while the activityof other amylases was very low. The -amylase was purified from4-day-old cotyledons by affinity chromatography on epoxyactivatedSepharose 6B substituted with rß-cyclodextrin andby column chromatography on Bio-Gel P-200. Gel filtration andpolyacrylamide gel electrophoresis showed that the enzyme existsmostly as a monomer (43,000 daltons), but partially aggregatesto form dimer, trimer and further multimers. Ca²⁺ protectedthe -amylase against heat inactivation. Incubation of the enzymewith 5 mM EDTA or dialysis against 10 mM EDTA resulted in a50–90% loss of activity. The inactivation was partiallyreversed by the addition of Ca²⁺. Other properties, such asthe amino acid composition, K_m value, pH optimum and activationenergy were similar to those of other plant -amylases. (Received May 6, 1981; Accepted June 22, 1981)     

An {alpha}-Glucan from Suspension-Cultured Rice Cells

An -glucan was isolated from 11-day-old suspension-culturedrice cells by extraction with hot Na-phosphate buffer (pH 6.8).The -glucan had []_D=+234? (C = 0.14, in water) and its averagemolecular weight was estimated to be about 1.4 ? 10⁴, basedon elution characteristics on acalibrated Sepharose CL-6B column.Upon partial acid hydrolysis, the -glucan gave mainly malto-oligosaccharides.The maximum absorption of the iodine complex of the -glucanin the presence of Na₂SO₄ was at 470 nm. The results of hydrolysisby , ß- and iso-amylases and methylation analysisindicated that the isolated -glucan is a highly branched polysaccharidewith an average chain length of 9. The exterior and interiorchain lengths of the -glucan were calculated to be 5 and 3,respectively. (Received July 23, 1986; Accepted February 7, 1987)     

Characterization of the mRNA for the {alpha}-Amylase from Wheat

Characterization of the mRNA for the -amylase from wheat wasmade by fractionation of the total membrane-bound polysomalRNA and by immunoprecipitation of in vitro synthesized -amylaseby its specific antibody. The content of the mRNA for the -amylasein the membrane-bound polysomal fraction from germinating wheatseeds was estimated as 5–10% of the total mRNA in thisfraction. RNA, separated in high resolution on acid-urea-agarosegels by electrophoresis, was recovered from the gels by a newmethod. Its translation products in rabbit reticulocyte lysateswere analyzed with a specific antibody against -amylase. Thesize of the mRNA for -amylase was estimated as 5.0?10⁵ daltons. ¹ Present address: National Institutes of Health, Bldg. 10,Rm. 9B-15, Bethesda, MD 20205, U.S.A. (Received March 6, 1982; Accepted August 4, 1982)     

SPLITTING OF {varepsilon}-CAPROLACTAM AND OTHER LACTAMS BY BACTERIA

-Caprolactam-utilizing bacteria split -caprolactam, -valerolactamand -butyrolactam, and produce the -amino acids correspondingto them. This activity is lost when cells are grown on 6-amino-caproicacid or ammonium adipate, and reappears when cells are incubatedwith either -caprolactam or -valerolactam as the sole majororganic nutrient. Chloramphenicol inhibits this adaptation.The enzyme splitting those lactams is one and the same. It maybe called "lactam-splitting enzyme". But attempts to demonstratethe enzymic activity in a cell-free system has not yet beensuccessful. (Received September 9, 1965; )     

Induction of sexual agglutinability of {alpha} mating-type cells as the primary action of the peptidyl sex factor from {alpha} mating-type cells in Saccharomyces cerevisiae

The effect of a pure preparation of substance-I_A (S-I_A) whoseamino acid sequence is identical to that of one of the factorpeptides (2), on sexual agglutinability and DNA synthesis wascomparatively studied. The optimum concentration of S-I_A forthe induction of sexual agglutinability of cells of an inducible strain was 1 ng/ml. The inducing action of S-I_A was detectedin 20 min and reached a maximum in 60 min. Only 8.7% inhibitionof DNA synthesis by S-I_A in the same strain was detected in1 hr and 40.4% inhibition in 2 hr at a concentration of 1 µg/ml.These results suggest that the primary action of the peptidyl sex fractor on a mating-type cells is the induction of sexualagglutinabiity. (Received October 25, 1977; )     

Promotion and inhibition of {alpha}-amylase production in barley endosperm by cyclic 3',5'-adenosine monophosphate and adenosine diphosphate

The possibility that gibberellin-induced -amylase synthesisin barley endosperm might be mediated by cyclic-3',5'-adenosinemonophosphate (3',5'-AMP) was examined. Promotion of -amylasesynthesis by 3',5'-AMP (5 mM) was observed in the absence ofgibberellic acid (GA₃) and in combination with GA₃ at concentrationsbelow 2 mµM. When combined with gibberellin at concentrationsabove 2 mµM, however, 3',5'-AMP reduced the amount of-amylase obtained. The cyclic nucleotide showed slight activityat concentrations as low as 0.05 mM. These promotions were shownto be due to increased synthesis of -amylase rather than toan increased secretion of the enzyme. Of a variety of adeninecompounds and nucleoside diphosphates tested only 3',5'-AMPand adenosine diphosphate (ADP) induced -amylase synthesis.Longer incubation times were necessary to obtain maximal -amylaseinduction with the nucleotides than with GA₃. ADP and 3',5'-AMPwere about one third and one fifth as active, respectively,as GA₃ in promoting -amylase synthesis, although GA₃ was morethan 10⁷ times more effective. AMO-1618 did not inhibit theaction of the nucleotides and methanolic extracts of the nucleotidesshowed no gibberellin-like activity. Both nucleotides were synergisticwith GA₃ in overcoming the inhibitory effects of acetate andcitrate buffers on -amylase synthesis. (Received February 24, 1969; )     

Temperature-Dependent Inhibitive Actions of {alpha},{alpha}'-Dipyridyl and Cycloheximide on the Senescence of Maize Leaves

Temperature-dependent inhibitive actions of ,'-dipyridyl andcycloheximide on the senescence of maize leaves were studied.,'-Dipyridyl effectively inhibited the loss of chlorophyll at25?C but not at 35?C. Gycloheximide was highly effective inpreserving chlorophyll at both of 25 and 35?C. Spectral analysisof senescent leaves at 35?C in ,'-dipyridyl showed simultaneousbleaching the carotenoid and chlorophyll. (Received February 20, 1984; Accepted June 14, 1984)     

Isolation of Subunits of Coupling Factor 1 from Maize and Spinach Chloroplasts and Properties of Combinations of Subunits with ATPase Activity

Subunits (, ß, ) and mixtures of subunits ( ß, , ß , ß ) were isolated without denaturationfrom a chloroform extract of chloroplast coupling factor 1 (CF₁)from maize (Zea mays var. Ushiku 5-4) and from spinach by fastprotein liquid chromatography (FPLC), on an anion-exchange columnof Mono-Q in the presence of n-octylglucoside (OG) and on achromatofocusing column of Mono-P. The ß -subunitcomplex (CF¹ ß ) was the minimum unit required forATPase activity, as was confirmed by the reconstituted complexof ß and subunits. An subunit isolated from maizeinhibited the ATPase activity of CF₁ ß from bothmaize and spinach. CF₁ ß was found to contain anOG-dependent Mg²⁺-ATPase. The ATPase activity of CF₁ ß required divalent cations, such as Mg²⁺ or Mn²⁺, for its expressionin the presence of OG; its optimum pH was 8.0 and it was markedlyinhibited by NaN₃. The enzyme hydrolyzed ATP in prefernece toGTP but not CTP, UTP, ADP, AMP or pNPP. Lineweaver-Burk plotsof its activity were curvilinear in the range of 0.6–0.7mM ATP.Mg²⁺. ¹Present address: Department of Biology, School of Education,Waseda University, Shinjuku-ku, Tokyo, 160 Japan. (Received February 15, 1989; Accepted April 20, 1989)     

BACTERIAL BREAKDOWN OF {varepsilon}-CAPROLACTAM AND ITS CYCLIC OLIGOMERS

Eleven different types of bacteria were isolated which werecapable of growing on -caprolactam, the monomeric material fornylon 6 polyamide, as the sole source of both carbon and nitrogen. The optimal concentration of -caprolactam for the bacterialgrowth was about 0.6% in a synthetic liquid medium enrichedwith a small amount of yeast extract. The bacterial strains grew also on -butyrolactam, -valerolactamand the -amino acids corresponding to these lactams and -caprolactam.Ammonium adipate was a good substrate for the growth of allthe strains. One strain of Corynebacterium aurantiacum was found to be capableof utilizing cyclic and linear oligomers of 6-aminocaproic acidwith an exception of cyclic dimer. The strains of corynebacteria required vitamin B₁ for growth. Metabolism of -caprolactam and related compounds is discussedbriefly. (Received September 9, 1965; )     

Physiological detection of a binding substance for the agglutinability-inducing pheromone, {alpha} substance-I in Saccharomyces cerevisiae

We found a substance which binds to substance-I to inactivatethe biological activity of the pheromone to induce sexual agglutinabilityof a mating type cells. Both living and boiled cells of thea mating type had the substance-I-absorbing action. The absorbingaction of living cells was detected almost equally at both 0and 28?C. Cell extract of the a mating type showed the substance-I-inactivatingaction. The biological activity of substance-I inactivatedby shaking with cell-free culture medium of the a mating typewas recovered by heating at 100?C, which destroyed the inactivatingaction of the culture medium with little effect on the substance-Iactivity, indicating that a substance in the culture mediuminactivated substance-I by binding to it. This is supportedby the fact that the inactivating action completely stoppedin 30 min, leaving a considerable amount of active substance-I,when the concentration of the inactivating substance was lowcompared with that of substance-I. The ability to produce thebinding substance as specific to the a mating type. The bindingsubstance was different from the a agglutination substance responsiblefor sexual agglutination. ² On leave from Osaka City University. (Received April 6, 1977; )     

Function of the {alpha} Subunit of Rice Heterotrimeric G Protein in Brassinosteroid Signaling

The subunit of plant heterotrimeric G proteins (G) plays pivotalroles in multiple aspects of development and responses to planthormones. Recently, several lines of evidence have shown thatG participates in brassinosteroid (BR) responses in Arabidopsisand rice plants. In this study, we conducted a comprehensiveanalysis of the roles of the rice G in the responses to BR usinga defective mutant of the G gene, T65d1. Decreased sensitivityto 24-epi-brassinolide (24-epiBL) in the T65d1 mutant was observedin many processes examined, e.g. in the inhibition of root growthand the promotion of coleoptile elongation. The T65d1 mutantalso showed similar phenotypes to those of BR-deficient mutants,such as the specifically shortened second internode and theconstitutive photomorphogenic growth phenotype under dark conditions.However, a negative feedback effect by 24-epiBL on the expressionof BR biosynthetic genes was observed in the T65d1 mutant, andthe levels of BR intermediates did not fluctuate in this mutant.To determine the epistatic relationship between the T65d1 mutantand d61-7, a weak allele of a rice BR receptor mutant, the twomutants were crossed. The T65d1/d61-7 double mutant showed noepistasis in the elongation inhibition of the internodes, theinternode elongation pattern, the leaf angle and the morphologicalabnormality of leaf, except for the vertical length of seedand the seed weight. Our results suggest that the rice G affectsthe BR signaling cascade but the G may not be a signaling moleculein BRI1-meditated perception/transduction.     

Effects of Gibberellins and Prohexadione on the Activities of Oryzain and {alpha}-Amylase in Rice Seeds

Oryzains, cysteine proteinases of rice seeds, are induced byGA₃ in germinating rice seeds [Abe et al. (1987) Agric. Biol.Chem. 51: 1509]. The effects of GA₁, GA₃, GA₄, GA₉, and GA₂₀on the production of oryzain and -amylase were investigatedin embryoless half- and whole-seeds of rice (cv. Nipponbare).When gibberellins (GAs) were incubated with embryoless half-seeds,GA₁, GA₃ and GA₄ induced oryzain and -amylase, but GA₉, andGA₂₀ did not. GA₉ and GAM induced oryzain and -amylase productionin whole seeds, but this production was inhibited by the simultaneousapplication of prohexadione, an inhibitor of 2ß- and3ß-hydroxylation of GAs. Prohexadione did not inhibitthe activities of oryzain and -amylase induced by GA₁. Theseresults suggest that GAs possessing the 3ß-hydroxylgroup induce activities of oryzain and -amylase in rice seedsand that GA₉ and GA₂₀ have activity only after they are convertedmetabolically to active GAs, probably GA₄ and GA₁, respectively.GA₁, was more active than GA₄ in both half seeds and wholeseeds incubation. Oryzain and -amylase activities induced byGA₄ were significantly inhibited in the presence of 10^–4M prohexadione. This suggests that the conversion of GA₁, toGA₄ (13-hydroxylation) might be inhibited at a high dose ofprohexadione in whole seeds. ⁴Present address: Institute of Food Development, Kyung Hee University,Suwon 449-701, Korea     

Effect of Gibberellic Acid on the Ultrastructure and {alpha}-Amylase Activity of Aleurone Cells of Avena sativa L.

The -amylase activity and ultrastructure of aleurone cells inseeds of Avena sativa L. were studied using seed halves withembryo (embryo seeds) which had imbibed water and seed halveswithout embryo (embryo-less seeds) which had imbibed water withor without GA₃. -Amylase activity was detected in the aleurone layers of embryoseeds that had imbibed water and embryo-less seeds that hadimbibed GA₃-water. The ultrastructure of aleurone cells withdetectable -amylase activity showed marked changes in the roughsurfaced endoplasmic reticulum (rER), in the flattened sacculesforming stacks and in the aleurone grains. The progressive changesin the rER were as follows: first, the number of slender rERincreased; then, the inner space became wider and showed roundor oval profile; and finally, the rER became slender again witha reduced number of adhering ribosomes. The flattened sacculesforming stacks were appressed to the surface of aleurone grains.With time, they decreased in number and finally disappeared.In parallel with the decrease of flattened saccules, digestionof proteinaceous material inside the aleurone grains proceeded. (Received February 24, 1987; Accepted September 3, 1987)     

Apoplastic {alpha}-Amylase in Pea is Enhanced by Heat Stress

The apoplastic -amylase (EC 3.2.1.1 [EC] ) in pea (Pisum sativum L.cv. Laxton's Progress No. 9) seedlings was enhanced by a 4-to 6-fold with heat stress. Increased levels of the enzyme persistedfor 48 h at least after the stress and subsequent heat treatmentsenhanced the activity further. These results indicate that thefunction of apoplastic -amylase in vegetative tissues may berelated to plant stress. (Received October 14, 1996; Accepted February 20, 1997)