Gibberellin-like Substances in the Sporophores of Agaricus bisporus (Lange) Imbach

Sporophores of cultivated Agaricus bisporus (Lange) Imbach,were shown to contain a gibberellin-like substance active inthe dwarf maize (d-5), -amylase and other bioassays. Ethyl-acetateextraction followed by paper, column, and thin-layer chromatographyrevealed the presence of one major active substance. Ficin hydrolysisof dried sporophore powder, after the complete removal of freesubstances, released more gibberellin-like substances, one ofwhich appeared identical to the free compound. The free substance was predominantly in the lamellae and residualpileus tissue. The major active substance released by ficinoccurred mostly in the lamellae but also in substantial equalamounts in both stipes and pilei. No activity was found in extractsof dikaryotic vegetative mycelium on malt agar. The level ofactivity in extracts from sporophores stored at – 20 °Cfell sharply after 7 d, and then remained constant over a periodof 6 weeks. The content of gibberellin-like substances in youngand old whole sporophores showed wide variation between experiments.In most cases young 2-d tissue had higher levels than old, 11-dtissue on a fresh-weight basis. Purified sporophore extractsand authentic gibberellins had no stimulating effect on growthof sporophores or of cultured vegetative mycelium. The inhibitorsof diterpene biosynthesis, CCC, and AMO-1618 induced a smallincrease in mycelial growth rate. Ethyl-acetate extraction ofhorse-straw compost prior to inoculation with Agaricus bisporusshowed the presence of gibberellin-like activity in significantamounts.     

Gibberellin-like Substances from Norway Spruce (Picea abies)

By use of methanol extraction, two different consecutive partition procedures, repeated polyvinylpyrrolidone column chromatography, silicic acid partition column chromatography and the dwarf rice, lettuce, and barley half-seed bioassays, several gibberellin-like substances were detected in elongating shoots of Picea abies (L.) Karst. No significant differences in the content of gibberellin-like substances could be detected between juvenile and mature trees. The shoot axes were found to contain fewer gibberellin-like substances than the needles.     

CHANGES IN THE CONTENT OF GIBBERELLIN-LIKE SUBSTANCES IN RIPENING SEED AND POD OF LUPINUS LUTEUS

The amount of gibberellin-like substances in seed, pod, embryoand "endosperm" of Lupinus luteus in relation to their developmentwas studied by means of the rice seedling test. The amount of gibberellin-like substances per seed increasedremarkably at the first stage of development, when the growthof seed and pod was very slow, and attained its maximum at suchan early stage as the dry weight of seed reached a value ofonly 10 per cent and that of pod 28 per cent of their respectivemaximum levels. Changes in amount of gibberellin-like substancespresent in the pod were very closely related to the changesin growth of pod. Gibberellin-like substances equivalent to0.3 µg gibberellin A₃ per seed and 0.028 µg perpod were found at the 20th day after anthesis, and no activityin mature seed and 0.09 µg per pod at the 50th day. Noactivity was found in the embryo itself at all stages, indicatingthat the gibberellin-like substances in seed occur only in the"endosperm," i.e. in the tissues that will eventually form testain fully ripened stage. (Received December 18, 1962; )     

{alpha} Glucosidase activity of excised roots of Lycopersicon esculentum

Homogenates of excised roots of Lycopersicon esculentum possessboth invertase and glucosidase activity. These were separatedby Sephadex G-100 filtration and their ability to hydrolysea number of different sugars was determined. (Received May 29, 1974; )     

GIBBERELLIN-LIKE SUBSTANCES OCCURRING IN THE SEED OF PHARBITIS NIL CHOIS. AND THEIR CHANGE IN CONTENTS DURING THE SEED DEVELOPMENT

The amount of gibberellin-like substances in the seed of Pharbitisnil increased in parallel with the growth of the seed, and attained20 days after anthesis to a maximum of 0.115 µg gibberellinA₃ eqivalent per seed, when the seed reached its maximum freshweight or four-fifths of its final dry weight. At this maximumlevel, 0.03 µg and 0.1 µg gibberellin A₃ equivalentswere localized in the embryo and in the "endosperm", respectively.Three gibberellin-like substances (Factors I, II and III) wereseparated upon paper chromatography. In view of the changesin amount of the factors with respect to the seed maturation,these factors, especially Factor II, in the "endosperm" andembryo were assumed to participate in the initial or the mainpart of growth of the embryo. Dwarf rice seedling and maizedwarfs 1, 3 and 5 responded to the three factors nearly in thesame way. Pharbitis dwarf, however, responded only to FactorI, but not to Factors II and III. (Received February 21, 1963; )     

Intracellular Localization and Properties of NADH-Glutamate Dehydrogenase from Vitis vinifera L.: Purification and Characterization of the Major Leaf Isoenzyme

Glutamate dehydrogenase was partially purified from grapevine(Vitis vinifera L. cv. Soultanina) tissues and its activityand isoenzymic pattern were studied. Seven anodal migratingisoenzymes were revealed after PAGE. Leaf protoplasts were isolatedfrom in vitro-grown axenic shoot cultures and used to studythe intracellular localization of GDH. Results revealed thatthe enzyme was associated with the mitochondrial fraction. Theisoenzyme with the lowest electrophoretic mobility, which accountedfor 35 to 40% of total activity, was purified 2050-fold to homogeneityfrom leaves. The purification method included ammonium sulphatefractionation, DEAE-cellulose chromatography, Sephadex G-200gel filtration and NAD-sepharose affinity chromatography. Themolecular weight of the native enzyme was estimated to be 252kDa and it consisted of identical 42.5 kDa subunits. pH optimumfor the aminating reaction was 8.0 and for the deaminating reaction9.3. At optimum pH conditions the apparent K_m values for ammonium,as ammonium chloride and ammonium sulphate, -ketoglutarate,NADH, glutamate, and NAD⁺ were 45.0, 13.0, 2.1, 0.069, 18.0,and 0.195 mM, respectively. The amination reaction of GDH wasfully activated with about 100 µM Ca²⁺ while the deaminationreaction was not affected by the addition of Ca²⁺. The isoenzymesof GDH showed different magnitude of their activating responseto calcium ions. Key words: Vitis vinifera L., glutamate dehydrogenase     

Simultaneous microdetermination of homovanillic acid and 5-hydroxyindoleacetic acid in brain tissue using Sephadex G-10 and QAE-Sephadex A-25

Homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in animal brains were simultaneously purified by two steps of column chromatography on Sephadex G-10 and QAE-Sephadex A-25. Perchloric acid extracts of brain tissue were directly passed through a column of Sephadex G-10. The gel retained both HVA and 5-HIAA, thereby separating them from Cl0^?₄ which interferes with subsequent purification process and from endogenous substances which give blank fluorescence. HVA was loosely adsorbed on the gel and was easily desorbed with dilute acetic acid. This effluent was successively passed onto a column of QAE-Sephadex A-25 placed beneath the G-10 column and the adsorbed HVA was eluted with 0.1 M Na₂HPO₄. The 5-HIAA remaining on the Sephadex G-10 without being desorbed by acetic acid was eluted with dilute ammonia. The recovery of both acid metabolites by this column procedure was more than 90%. Thus, it is possible to determine the levels of HVA and 5-HIAA in single brains of small rodents.     

Chromactivating hormones of Pandalus borealis; Isolation and purification of a light-adapting hormone

1.

1. Light-adapting hormone activity (distal-retinal-pigment hormone) from eyestalks of the prawn Pandalus borealis was purified by partition between an organic and an aqueous phase in two different systems and subsequent chromatography on Sephadex G-25. It was then separated into 4 chemically different components by chromatography on CM-Sephadex.     

Purification and Properties of Alkaline Proteinase from Aspergillus oryzae

Alkaline proteinase was purified from culture extract of a strain of Aspergillus oryzae. The process consists of the Amberlite IRC-50 adsorption, column chromatography on DEAE-cellulose and CM-cellulose and Sephadex G-100 gel filtration. The molecular weight of the enzyme was estimated to be about 23,000 by a gel filtration method. Alkaline proteinase showed neither carboxypeptidase activity nor aminopeptidase activity, but degraded 10101010 poly-l,α-glutamic acid, poly-l-lysine, 10101010 and 10101010. The enzyme was completely inhibited by diisopropylphos-phorofluoridate (10^?2 m) or potato inhibitor (250 μg/ml).     

Changes in Levels of Naturally Occurring Gibberellin-like Substances during Germination of Seed of Lycopersicon esculentum Mill.

Naturally occurring gibberellin-like substances possessing acidic,basic, and neutral properties were detected, by paper partitionchromatography, in ethanolic extracts of tomato seed and ofetiolated seedlings after 72 and 116 hours' growth. Dwarf maizemutants of the d-1 and d-5 types, ‘Meteor’ pea seedlingsand young ‘Potentate’ tomato plants were used asbioassay material. Hydrolysis of seed and seedling proteinsby ficin in phosphate buffer, pH 6.2, after removal of ethanol-solublesubstances, liberated more and different ‘bound’gibberellin-like substances. It is suggested that protein hydrolysisduring germination is an important means of liberating thesesubstances at different stages of seedling development. Acidic substances were present in all the extracts prepared,but in general two with Rfs 0.25 and 0.55 in iso-propanol: ammonia:water : : 10:1:1 v/v were differentiated on d-2 and d-5 maizerespectively. Neutral substances in dry seed extracts chromatographedin the same solvent, had Rfs of 0.05, 0.35, and 0.95 and thesewere found only in the ethanolic (‘free’) extracts.They were active on d-1 and d-2 maize and ‘Meteor’pea. Basic gibberellin-like substances with Rfs of 0.05 and0.35 were found in ‘free’ extracts of both dry seedand etiolated seedlings after 116 hours' growth which were activeon d-2 maize only. Two others with Rfs 0.45 and 0.95 were extractedfrom seedlings after 72 hours' growth and these were activeon young ‘Potentate’ tomato plants. It is suggested that certain gibberellin-like substances, capableof reversing dwarfism in test plants, may be responsible formorphogenetic or other responses not involving stem extensionin the parent species. Changes were found in the levels of gibberellin-likesubstances but there was no evidence of changes in levels ofseed inhibitors relative to seed growth substances.     

Purification and Properties of Wall-bound α-Glucosidase from Suspension-cultured Rice Cells

Wall-bound α-glucosidase (EC 3.2.1.20) has been solubilized from suspension-cultured rice cells with Sumyzyme C and Pectolyase Y-23 and isolated by a procedure including fractionation with ammonium sulfate, Sephadex G-100 column chromatography, CM-cellulose column chroma-tography, Sephadex G-200 column chromatography, and preparative disc gel electrophoresis. The molecular weight of the enzyme was 64,000. The enzyme readily hydrolyzed maltose, maltotriose, and amylose, but hydrolyzed isomaltose and soluble starch more slowly. The Michaelis constant for maltose of the enzyme was estimated to be 0.272 mm. The enzyme produced panose as the main α- glucosyltransferred product from maltose.     

Isolation of a polysaccharide from the inner cell wall, intine, of pollen of Cryptomeria japonica

Isolation and solubilization of the intine of pollen grainsof Cryptomeria japonica were attempted. The intine was separatedeffectively from other cell fragments by passing it througha column of glass beads and dissolving it in an EDTA solution.The extract was fractionated by column chromatography on DEAE-celluloseand gelfiltration through Sephadex G-200, and a polysaccharidecomponent was obtained. This was found to consist of galactose,rhamnose, arabinose, xylose and uronic acid. (Received October 13, 1976; )     

A New Method for Separation of Gibberellins and Growth Inhibitors in Plant Extracts using Sephadex Columns

A method is described for estimating gibberellin levels in plantmaterial, using a separation method avoiding organic solvents.High molecular weight inhibitors were separated from growth-promotingfractions by column chromatography on Sephadex. Of the severalgrades of Sephadex tried, G15 was found to give the sharpestseparation. Ammonium chloride was included in the sample appliedto the column to increase the retention of anionic substances.The inhibitory effect of other substances, probably abscisicacid, which are eluted from the gel together with the gibberellins,was reversed by addition of kinetin to test solutions in thelettuce hypocotyl bioassay. Using this method an increase ingibberellin level in cotyledons of Phaseolus multiflorus duringgermination was measured in terms of GA₃ activity, with referenceto the lettuce hypocotyl bioassay.     

Penicillin Induction of Gibberellin and {alpha}-Amylase Biosynthesis in Rice Endosperm

Penicillin induces the synthesis of -amylase in embryoless riceendosperm and enhances the gibberellin-induced response. Penicillininduction of -amylase can be prevented by inhibitors of nucleicacid and protein synthesis, CCC and 2,4-DNP. A characteristic gibberellin-like activity in the extracts frompenicillin-treated endosperms becomes detectable after 12 hfrom the addition of penicillin. This gibberellin-like activityis located on paper chromatograms at the R_F typical for GA₃and its formation is blocked by CCC, an inhibitor of GA biogenesis.Glucose has no effect on the biosynthesis of either gibberellinor -amylase induced by penicillin. The time-course study of the levels of different constituentsshows that penicillin probably induces RNA and DNA synthesisin the first place, which results in gibberellin biosynthesis,which in turn stimulates the synthesis of -amylase. The possiblemode of action of penicillin in higher plants is discussed.     

An Extension of the Logistic Model of Plant Growth

The kinetics of growth of Dactylis glomerata L. were studiedunder controlled temperature and nutritional conditions at threelevels of irradiance (35·55 and 85 W m^–s). Thedry weights of the root and shoot parts of the plants were measuredeach week between the fourth and eleventh weeks after sowing. The growth kinetics were found to be dependent on the levelof irradiance, but no significant differences in the root: totaldry weight ratio were observed. To characterize the effect of illuminance, the experimentalgrowth curves were analysed initially using the logistic model,the adequacy of which is discussed. An extension of the logisticmodel is proposed, represented by the kinetic equation dM/dt= kM, with < 1 and where M is the dry weight of the plant.It is shown that this relationship allows a distinction to bemade between two kinds of plant material according to theirfunctions in the growth process. Dactylis glomerata L., illuminance, growth curves, kinetic analyses, logistic model, shoot:root ratio, partition of assimilates     

Lectin-like activity of lupin seed conglutin {gamma}, a glycoprotein previously referred to as a storage protein

The lupin seed glycoprotein conglutin was capable of bindingvarious exogenous and endogenous N-glycosylated proteins andalso specifically bound to a Sephadex G-75 column. The bindingactivity of conglutin was abolished by either addition of 10mM EDTA or by enzymic deglycosylation and acid treatment. Inthe latter case activity could be restored by the addition ofMn and Ca ions. Immunological homology at polypeptide level of conglutin withother legume lectins was found. Key words: Conglutin , glycoprotein, legume lectin, Lupinus albus     

Approach to elaborating immunochemical methods for removing peptides from the blood of kidney failure patients

A homogenic peptide (m. w. 2100 dalton) was isolated from blood serum of patients suffering from renal failure by gel chromatography on a Sephadex G-25 column and by partition chromatography on a column with cellulose MN-300. The peptide was not detectable in the blood of healthy donors. The amino acid composition of the peptide was studied. To obtain an immunosorbent, the peptide was conjugated by the bifunctional reagent, glutaric aldehyde. An optimal conjugation variant promoting the formation of conjugates with a molecular weight of 60000 dalton was developed. The molecular weight of the conjugates was measured by gel chromatography on a Sephadex column G-50 and with the aid of disc electrophoresis in polyacrylamide gel.     

Root, Callus, and Cell Suspension Cultures, from Atropa belladonna, L. and Atropa belladonna, Cultivar lutea Doll

Root, callus, and cell suspension cultures have been establishedfrom seedlings of Atropa belladonna, L. and Atropa belladonna,cultivar lutea Döll. The growth of these cultures is described.Callus cultures transferred to auxin (-naphthaleneacetic acid)-freemedium initiated roots and shoots. Excised root cultures havebeen established from such roots and plants from such shoots.Extracts of the cultures have been submitted to the Vitali—Morinreaction and following chromatography, to the Dragendorff reaction.Cultured excised roots and plants raised from shoots initiatedon cultured callus were shown to contain atropine (hyoscyamine)and reactive substances corresponding in Rf to hyoscine andcuscohygrine. These alkaloids were absent from cultured callusand cultured cell suspensions and from leaves when initiatedwithout roots on callus. The cultured calluses and cell suspensionscontained choline (0.022–0.027 g per 100 g dry weightof root callus). The growth of cell suspension cultures wasnot inhibited by incorporating atropine sulphate, L-hyoscyamine,L-hyoscine hydrobromide, or DL-scopoline nitrate in the culturemedium at 250 mg/I. These alkaloids were absorbed by the cells,a high proportion of the added alkaloid could be recovered fromthe cultures even after 4 weeks' growth and no evidence wasobtained of the presence of degradation products of the alkaloids.The suppression of alkaloid formation in actively growing callusand cell suspension cultures is discussed.     

Organization of enzymes in the shikimate pathway of Phaseolus mungo seedlings

DEAE-cellulose and Sephadex G-100 column chromatography showedthe separability of 3-dehydroquinate synthase, 3-dehydroquinatehydro-lyase-shikimate: NADP oxidoreductase complex, shikimatekinase and 5-enolpyruvylshikimate-3-phosphate synthase of Phaseolusmungo seedlings. The approximate molecular weights of theseenzymes were estimated using a gel filtration column. (Received October 30, 1978; )