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; )     

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; )     

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)     

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)     

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)     

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; )     

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)     

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)     

Improvement of the indolo-{alpha}-pyrone fluorescence method for quantitative determination of endogenous indole-3-acetic acid in lettuce seedlings

The indolo--pyrone fluorescence method was modified to measurethe endogenous amount of indole-3-acetic acid (IAA) in lettuceseedlings, because impurities in lettuce extracts made the indolo--pyronesolution turbid, if the reaction of the extracts with aceticanhydride to give indolo--pyrone was disrupted with 3 ml distilledwater. This turbid solution caused light scattering, resultingin overestimating the IAA amount in lettuce seedlings. We could,however, obtain a clear indolo--pyrone solution by disruptingthe reaction with 0.2 ml distilled water and 2.8 ml acetic acidin place of 3 ml distilled water. We examined the reliabilityof the new method and found that the use of acetic acid substantiallyslowed down the rate of indolo--pyrone breakdown and did notdecrease the sensitivity of the method. The effect of gibberellicacid on the endogenous amount of IAA in lettuce seedlings wasalso examined. ¹ One of the authors (S. K.) received for this work a grantfrom Osaka City University to study abroad. (Received January 11, 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; )     

Induction of Sexual Agglutinability by the Absorbed a Peptidyl Factor in a Mating Type Cells of Saccharomyces cerevisiae

The sexual agglutinability of inducible cells was induced byabsorbed pheromone in the absence of external a pheromone.The absorption needed no metabolic activity, but the inductiondid. This induction of sexual agglutinability by absorbed pheromonewas not accompanied or preceded by G1 arrest. The role of thebinding substance for pheromone in this induction is discussed. (Received November 13, 1980; Accepted December 19, 1980)     

A Mutation Leading to Constitutive Expression of High Sexual Activities in the Yeast Saccharomyces cerevisiae

An a mating type mutant of the yeast Saccharomyces cerevisiaewhich expressed high sexual activities during vegetative growthwas isolated and characterized. Its constitutive sexual agglutinabilitywas higher than the sexual agglutinability of its parental straininduced by pheromone. It produced a pheromone and -pheromone-inactivatingsubstances in larger amounts than its parental strain. It alsoproduced large pear-shaped cells (shmooed cells) without pheromone,was more sensitive to pheromone, and grew vegetatively moreslowly than its parental strain. When the mutant was crossedto a wild type strain isogenic with the parental strain, amating type segregants with high constitutive sexual agglutinabilityshowed self-shmooing. However, in a mating type segregants self-shmooingwas not observed regardless of the degree of their sexual agglutinability.The cross between a and segregants, both of which carried themutation, had higher frequency of zygote formation than thecrosses between a and cells one of which or both of which wereof wild type. (Received September 9, 1985; Accepted February 8, 1986)     

{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; )     

In Vitro Translation of {alpha}-Amylase mRNA from Dry Wheat Embryos

The poly(A)⁺RNA fraction was separated from ungerminated wheatembryos and translated in both wheat germ extract and rabbitreticulocyte lysate systems. The polypeptides synthesized invitro were immunoprecipitated using rabbit anti-wheat -amylaseantibody. Fluorography after SDS-polyacrylamide gel electrophoresisshowed a band of molecular weight of 43,000, which was the sameas that of the -amylase precursor polypeptide. The -amylaseprecursor synthesized from the mRNA of dry embryos belongedto the group II isozyme subset, characterized by an acidic isoelectricpoint. ²Present address: Matsumae High SchoolMatsumae-cho, Hokkaido049-15, Japan. (Received February 1, 1988; Accepted June 1, 1988)     

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; )     

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)     

Enzymatic Degradation of Chlorophyll in Chenopodium album

The breakdown of chlorophyll (Chi) in crude extracts of Chenopodiumalbum (white goose foot) in the dark was examined. Derivativesof pheophorbide were formed when Chi or chlorophyllide wasincubated with depigmented crude extracts. The formation ofpheophorbide was completely prevented by heat treatment of extracts,indicating that the reaction was enzymatic, and the presenceof a Mg-releasing enzyme, the so called Mg-dechelatase, waspostulated. This hypothesis was strongly supported by the observationthat the formation of pheophorbide was inhibited by 51% by 10mM MgCl₂. Analysis by high-performance thin-layer chromatography(HPTLC) and liquid chromatography (HPLC) showed that the appearanceof chlorophyllide , pheophorbide 13²-hydroxychlorophyllide and pyropheophorbide was accompanied by a concomitant decreasein levels of Chi The formation of 13²-hydroxychloro-phyllide was not clearly an enzymatic reaction and requires furtherexamination. It appears that Chl is degraded in a crude extractof C. album via the following enzymatically catalyzed reactions (Received September 10, 1990; Accepted November 15, 1990)     

Phosphorylation and subcellular location of {alpha}-L-fucosidase in Iymphoid cells from patients with I-cell disease and pseudo-Hurler polydystrophy

Mannose 6-phosphate is a recognition marker used by many newlymade acid hydrolases for their transport to lyso-somes. Previously,we investigated the incorporation of ³²Pi into -L-fucosidaseof lymphoid cell lines from a healthy individual (control) andan I-cell disease patient [DiCioccio and Miller, Glycobiology,1, 595–604 (1991)]. Phosphoserine was found in immunoprecipitable-L-fucosidase of both control and I-cell lymphoid cells, butmannose 6-phosphate was identified only in enzyme of controlcells. Extension of this investigation to lymphoid culturesof a pseudo-Hurler polydystrophy patient also identified onlyphosphoserine in -L-fucosidase. Using [³H] mannose instead of³²Pi, the precise identification of mannose 6-phosphate in -L-fucosidaseof control cells, and its absence in -L-fucosidase of I-celland pseudo-Hurler cells, was established. The stoichiometryof phosphorylation of -L-fucosidase in I-cell, pseudo-Hurlerand control lymphoid cells was 3, 4 and 10 mol Pi/mol enzyme,respectively. -L-Fucosidase was located in lysosomes isolatedfrom control, I-cell and pseudo-Hurler lymphoid cells by subcelluarfractionation on Percoll density gradients. Both I-cell andpseudo-Hurler lymphoid cells displayed normal intralysosomalactivity of -L-fucosidase despite lack of the mannose 6-phosphatemarker. Thus, I-cell and pseudo-Hurler lymphoid cells must possessa mannose 6-phosphate-independent mechanism for directing -L-fucosidaseto lysosomes. Phosphorylation of -L-fucosidase in pseudo-Hurlerand I-cell lymphoid cultures was found almost exclusively inintracellular and not in extracellular enzyme, suggesting thatphosphoserine may participate in the localization of -L-fucosidasein lysosomes of these cells. -L-fucosidase I-cell disease lysosome phosphorylation pseudo-Hurler polydystrophy     

Changes in {alpha} and {beta}-Amylase Activities during Germination of Seeds of Alfalfa (Medicago sativa L.)

We examined the methods available for the assay of -amylasein alfalfa (Medicago sativa) and found the Phadebas test mostsuitable. The Phadebas assay and activity staining on ampholinegels after isoelectrofocusing revealed that an amylase is presentin the dry seeds of alfalfa and that its activity decreasesrapidly after the second day of seed germination. An amylasewas purified by affinity chromatography and gel filtration.The kinds of sugar generated from soluble starch by the purifiedamylase resembled those generated by other -amylases from plants,in particular those from mung bean (Vigna radiata). These resultsindicate that the amylase in alfalfa seeds belongs to the familyof -amylases. The molecular weight and isoelectric point ofthe -amylase were determined to be 43 kDa and 4.92, respectively. The Pantrac assay and activity staining on immobiline gels afterisoelectrofocusing revealed that the activities of ß-amylasesincreased during the initial 4 to 5 days of germination. Furthermore,treatment of whole seedlings with cycloheximide or actinomycinD inhibited the increase in activity of ß-amylasesbut did not affect the reduction in activity of -amylase. During germination of alfalfa seeds, -amylase activity decreaseswhile, in contrast, ß-amylase activity increases (inthe cotyledons of germinating seeds), changes that are specificto the germinating seeds of alfalfa. (Received September 8, 1990; Accepted February 20, 1991)