Purification and Properties of 1-AminocycIopropane-l-carboxylate Synthase of Mesocarp of Cucurbita maxima Duch. Fruits

1-Aminocyclopropane-1-carboxylate (ACC) synthase, which formsAGC from S-adenosylmethionine (SAM), was purified to homogeneityfrom sliced and aged mesocarp tissue of Cucurbita maxima Duch.cv Ebisu fruits, and its enzymatic properties were determined.The specific activity of the purified enzyme was 220 mU/mg proteinat 30°C at 50 µM SAM. Native ACC synthase has a relativemolecular mass of 160 ± 10 kDa and consisted of two subunitsof about 84±3 kDa. S-adenosylhomocysteine (SAH), S-methylmethionine(SMM) and L-methionine did not serve as substrate. The enzymereaction was competitively inhibited by aminoethoxyvinylglycine(AVG) (Ki, 2.5 µM), aminooxyacetic acid (Ki, 40 µM)and SAH (Ki, 30 µM). The reaction was also strongly inhibitedby semicarbazide, and less effectively by homocysteine. Theenzyme was rapidly inactivated by its substrate, SAM in thepresence of pyridoxalphosphate (PLP), but in the absence ofPLP, SAM-induced inactivation was much slower. Inactivationdid not occur by SAH and SMM, SAM analogs without substrateactivity. Pyridoxal phosphate was an essential cofactor to beadded to a reaction mixture for maximum activity, but an enzymepreparation from which pyridoxal phosphate was removed by SephadexG-25 gel filtration exhibited one-eighth activity which wasinhibited by semicarbazide, this indicating that a small amountof pyridoxal phosphate is firmly bound to the enzyme. (Received May 6, 1986; Accepted May 20, 1986)     

Inactivation of 1-aminocyclopropane-1-carboxylate (ACC) oxidase

The enzyme 1-aminocyclopropane-1-carboxylate (ACC) oxidase,which catalyses the final step in the biosynthesis of ethylene,showed a non-linear time-course in vitro and activity decayedwith a half-life of around 14 min. This loss of activity wasstudied using tomato ACC oxidase purified from Escherichia coiltransformed with the cDNA clone pTOM13. Inactivation was notdue to end-product inhibition by dehydroascorbic acid or cyanide.Preincubatlon of enzyme in the combined presence of Fe²⁺ ascorbateand ACC, which together allowed catalytic turnover, resultedin almost total loss of ACC oxidase activity. Enzyme Inactivatedby catalysis could not be reactivated by passage through SephadexG-25 or by treating with combina tions of DTT and CO₂ A non-lineartime-course and inactivation in the presence of all substratesand cofactors was also shown for the enzyme assayed in vivowith melon fruit discs. Using the purified tomato enzyme a distinctascorbate-dependent inactivation was also observed, which occurredIn the absence of catalysis and was prevented, although notreversed, by catalase. This ascorbate-dependent inactivationmay thus be due to H₂O₂ attack on ACC oxidase. Key words: 1-aminocyclopropane-1-carboxylate (ACC) oxidase, catalase, catalytic inactivation, ethylene     

Biochemical properties of 1-aminocyclopropane-1-carboxylate N-malonyltransferase activity from early growing embryonic axes of chick-pea (Cicer arietinum L.) seeds

In the present work, certain biochemical characteristics ofthe enzyme 1-aminocyclopropane-1-carboxylate N-malonyltransferase(ACC N-MTase) which is responsible for the malonylation of 1-aminocyclopropane-1-carboxylate(ACC) in chickpea (Cicer arietinum) are described. Phosphatebuffer was the most appropriate buffer with regard to enzymestability and, therefore, ACC N-MTase was extracted, assayedand purified in the presence of this buffer. ACC N-MTase waspartially purified approximately 900-fold from embryonic axesof chick-pea seeds using ammonium sulphate precipitation, hydrophobicinteraction and molecular filtration chromatography. By gelfiltration chromatography on Superose-12, the molecular massof the enzyme was estimated to be 54 4 kDa. ACC N-MTase hadan optimal pH and temperature of 7.5 and 40C, respectively,as well as a K_m for ACC and malonyl-CoA of 400 M and 90 M,respectively. D-Phenylalanine was a competitive inhibitor ofACC N-MTase with respect to ACC (K_i of 720 M), whereas co-enzymeA was a competitive product inhibitor with respect to malonyl-CoA(K_i of 300 M) and a non-competitive inhibitor with respectto ACC (K_i of 600 M). Under optimal assay conditions, ACC N-MTasewas strongly inhibited by (a)divalent [Zn²⁺>Mg²⁺>>Co²⁺>Co²⁺>(NH₄)²⁺>Fe²⁺]and monovalent metal cations (Li⁺>Na⁺>K⁺), without activitybeing detected in the presence of Hg²⁺, and (b) PCMB or mersalicacid, suggesting that sulphydryl group(s) are involved at theactive site of the enzyme. Key words: ACC-N-malonyltransferase, Cicer arietinum, embryonic axes, ethylene, germination, seeds     

Inhibition by 1-Aminocyclobutane-l-Carboxylate of the Activity of 1-Aminocyclopropane-l-Carboxylate Oxidase Obtained from Senescing Petals of Carnation {Dianthus caryophyllus L.) Flowers

We partially purified 1-aminocyclopropane-l-carboxy-late (ACC)oxidase from senescing petals of carnation {Dianthus caryophyllusL. cv. Nora) flowers and investigated its general characteristics,and, in particular, the inhibition of its activity by ACC analogs.The enzyme had an optimum pH at 7-7.5 and required Fe²⁺, ascorbateand NaHCO₃ for its maximal activity. The K_m for ACC was calculatedas 111-125 µM in the presence of NaHCO₃. Its M_r was estimatedto be 35 and 36 kDa by gel-filtration chromatography on HPLCand SDS-PAGE, respectively, indicating that the enzyme existsin a monomeric form. These properties were in agreement withthose reported previously with ACC oxidases from different planttissues including senescing carnation petals. Among six ACCanalogs tested, l-aminocyclobutane-l-carboxylate (ACBC) inhibitedmost severely the activity of ACC oxidase from carnation petals.ACBC acted as a competitive inhibitor with the K_i of 20-31 µM.The comparison between the K_m for ACC and the K_i for ACBC indicatedthat ACBC had an affinity which was ca. 5-fold higher than thatof ACC. Whereas ACC inactivated carnation ACC oxidase in a time-dependentmanner during incubation, ACBC did not cause the inactiva-tionof the enzyme. Preliminary experiments showed that ACBC andits N-substituted derivatives delayed the onset of senescencein cut carnation flowers. (Received August 19, 1996; Accepted November 26, 1996)     

Tomato vesicles as a model system for studying in situ activity of 1-aminocyclopropane-1-carboxylic acid oxidase

A model system is described which could be used for the studyof ACC oxidase in vivo. The enzyme is localized within sedimentablevesicles isolated from the locular tissue of ripening tomatofruit. These vesicles display linear ACC oxidase activity overa period of at least 3 h and this activity is not dependenton the essential cofactors (Fe²⁺ and ascorbate) needed for theenzyme in vitro. This system has been used to demonstrate thepresence of an inhibitors) of ACC oxidase activity in the locularjuice and also to study the effects of ionophores and uncouplerson the in vivo enzyme activity. Key words: ACC oxidase, tomato, Lycopersicon esculentum     

Characterization and Induction of the Activity of 1-Aminocyclopropane-1-Carboxylate Oxidase in the Wounded Mesocarp Tissue of Cucurbita maxima

1-Aminocyclopropane-1-carboxylate (ACC) oxidase (ethylene-formingenzyme) was isolated from wounded mesocarp tissue of Cucurbitamaxima (winter squash) fruit, and its enzymatic properties wereinvestigated. The enzyme required Fe²⁺ and ascorbate for itsactivity as well as ACC and O₂ as substrates. The in vitro enzymeactivity was enhanced by CO₂. The apparent K_m value for ACCwas 175 µM under atmospheric conditions. The enzyme activitywas inhibited by sulfhydryl inhibitors and divalent cationssuch as Co²⁺, Cu²⁺, and Zn²⁺. ACC oxidase activity was induced at a rapid rate by woundingin parallel with an increase in the rate of ethylene production.The exposure of excised discs of mesocarp to 2,5-norbornadiene(NBD),an inhibitor of ethylene action, strongly suppressed inductionof the enzyme, and the application of ethylene significantlyaccelerated the induction of the activity of ACC oxidase inthe wounded mesocarp tissue. These results suggests that endogenousethylene produced in response to wounding may function in promotingthe induction of ACC oxidase. (Received January 13, 1993; Accepted April 15, 1993)     

Monoclonal Antibodies against Apple 1-Aminocyclopropane-l-Carboxylate Synthase

1-Aminocyclopropane-l-carboxylate (ACC) synthase from applefruits was purified over 5,000-fold by conventional column chromatography.By immunizing mice with this partially purified enzyme preparation,8 hybridoma lines producing monoclonal antibodies against appleACC synthase were isolated. While all 8 clones immunoprecipitatednative ACC synthase, only two clones recognized the putative(48 kDa) ACC synthase on Western blots. When a partially purifiedACC synthase preparation was incubated with S-adenosyl-L-[carboxyl-¹⁴C]methionine(AdoMet), only one radioactive protein of 48 kDa was detectedon sodium dodecyl sulfate-poly-acrylamide gel electrophoresis.This radioactive protein was specifically immunoprecipitatedby the monoclonal antibodies, indicating that apple ACC synthaseis specifically radiolabeled by its substrate AdoMet, as istomato ACC synthase. Thus, the monoclonal antibodies recognizedboth native and AdoMet-inactivated forms of ACC synthase. Whilethese antibodies failed to im-munoprecipitate ACC synthase isolatedfrom ripe tomato fruits, ripe avocado fruits or auxin-treatedmungbean hypocotyls, they were effective in immunoprecipitatingthe enzyme isolated from ripe pear fruits. (Received August 11, 1990; Accepted October 17, 1990)     

Purification,properties and partial amino-acid sequence of 1-aminocyclopropane-1-carboxylic acid oxidase from apple fruits

The enzyme which converts 1-aminocyclo-propane-1-carboxylic acid (ACC) into ethylene, ACC oxidase, has been isolated from apple fruits (Malus x domestica Borkh. cv. Golden Delicious), and for the first time stabilized in vitro by 1,10-phenanthroline and purified 170-fold to homogeneity in a five-step procedure. The sodium dodecyl sulfate-denatured and native proteins have similar molecular weights (approx. 40 kDa) indicating that the enzyme is active in its monomeric form. Antibodies raised against a recombinant ACC oxidase over-produced in Escherichia coli from a tomato cDNA recognise the apple-fruit enzyme with high specificity in both crude extracts and purified form. Glycosylation appears to be absent because of (i) the lack of reactivity towards a mixture of seven different biotinylated lectins and (ii) the absence of N-linked substitution at a potential glycosylation site, in a sequenced peptide. Phenylhydrazine and 2-methyl-1-2-dipyridyl propane do not inhibit activity, indicating that ACC oxidase is not a prosthetic-heme iron protein. The partial amino-acid sequence of the native protein has strong homology to the predicted protein of a tomato fruit cDNA demonstrated to encode ACC oxidase.     

Purification of Xyloglucan Hydrolase/Endotransferase from Cell Walls of Azuki Bean Epicotyls

An enzyme involved in the breakdown of xyloglucans was purifiedfrom an extract of cell walls of azuki bean epicotyls obtainedwith 1 M NaCl and purified by column chromatography on severaldifferent resins. The purified enzyme gave a single band ofa protein with a molecular mass of about 32 kDa after SDS-PAGE.The enzyme hydrolyzed the xyloglucans of high molecular massfrom azuki cell walls to yield fragments of about 50 kDa withoutproduction of any oligo- or monosaccharides. Moreover, the enzymehad hardly any effect on xyloglucans of less than 60 kDa. Theenzyme also hydrolyzed xyloglucans from tamarind, but it didnot react with cellulose derivatives. In the presence of pyridylamino-labeledxyloglucan oligosac-charides as acceptor substrates, the enzymecatalyzed the transfer of 50-kDa products to the oligosaccharides.The K_m value of the enzyme for xyloglucans of 540 kDa was similarin the presence and in the absence of xyloglucan oligosaccharidesas acceptors: 1.0 mg ml^–1. These results suggest thatthe enzyme was an endotransferase but had unusual acceptor specificity,preferring smaller acceptors such as water. (Received September 9, 1996; Accepted March 16, 1997)     

Inhibition of Auxin-Induced Ethylene Production by Lycoricidinol

Lycoricidinol, a natural growth inhibitor isolated from bulbsof Lycoris radiata Herb. strongly suppressed auxin-induced ethyleneproduction from the hypocotyl segments of etiolated mung bean(Vigna radiata Wilczek) seedlings. The inhibitor did not significantlyinhibit ethylene formation from its immediate precursor, 1-aminocyclopropane-1-carboxylicacid (ACG), during short-term (up to 4 h) incubation. The ACCcontent in tissue treated with IAA was reduced by lycoricidinolin close parallel with the inhibition of ethylene production.Examination of radioactive metabolites in tissues labeled with3,4-¹⁴C-methionine indicated that reduction of the ACC contentwas not due to any possible promotive effect of lycoricidinolon conjugation of ACC with malonate. Lycoricidinol showed noinhibitory effect on the activity of ACC synthase if appliedin vitro, but it almost completely abolished the increase inthe enzyme activity when applied in vivo during incubation ofthe tissue with IAA. Lycoricidinol also strongly inhibited incorporationof ¹⁴C-leucine into protein in the tissue. The suppression ofthe enzyme induction and, in turn, that, of ethylene productionby lycoricidinol were interpreted as being due to the inhibitionof protein synthesis. (Received September 30, 1983; Accepted December 8, 1983)     

S-adenosylmethionine-dependent inactivation and radiolabeling of 1-aminocyclopropane-1-carboxylate synthase isolated from tomato fruits

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase was partially purified from the homogenate of wounded tomato (Lycoperiscon esculentum Mill.) pericarp tissue by (NH₄)₂SO₄ fractionation followed by conventional column chromatography with diethylaminoethyl-Sepharose, Sephadex G-150, Affi-Gel blue and hydroxylapatite. The partially purified ACC synthase preparation attained a specific activity of about 12,000 nmoles per hour per milligram protein. Employing this enzyme preparation, we confirmed that the ACC synthase was inactivated by its substrate, S-adenosyl-l-methionine (SAM), during its catalytic action. When the partially purified enzyme preparation was incubated with [3,4-¹⁴C]SAM and the resulting proteins were analyzed by sodium dodecyl sulfate-gel electrophoresis, only one radioactive protein band was observed. This protein was thought to be ACC synthase based on its molecular mass of 50 kD and on the fact that it was specifically bound to a monoclonal antibody against ACC synthase (AB Bleecker et al. 1986 Proc Natl Acad Sci USA 83, 7755-7759). These results suggest that the substrate SAM acts as an enzyme-activated inactivator of ACC synthase by covalently linking a fragment of SAM molecule to the active site of ACC synthase, resulting in the inactivation of the enzyme.     

Immunochemical Difference of Wound-Induced 1-Aminocyclopropane-1-carboxylate Synthase from the Auxin-Induced Enzyme

Immunochemical cross-reactivity of wound- and auxin-induced1-aminocyclopropane-1-carboxylate (ACC) synthase was examinedwith the antibody against wound-induced ACC synthase purifiedfrom mesocarp of winter squash (Cucurbita maxima Duch.). Theantibody recognized ACC synthase from wounded hypocotyls ofwinter squash and from wounded pericarp of tomato fruits, butnot the enzyme from IAA-treated hypocotyls of winter squash,tomato and mung bean. These results indicate that the primarystructure of the wound-induced enzyme is different from thatof the auxin-induced enzyme in the same species, and impliesthat there are two different genes for ACC synthase, one forwound induction and the other for auxin induction. (Received June 14, 1988; Accepted July 20, 1988)     

Preliminary characterization of 1-aminocyclopropane-1-carboxylate oxidase properties from embryonic axes of chick-pea (Cicer arietinum L.) seeds

For a deeper understanding of the germination of chick–pea(Cicer arietinum) seeds, which is dependent upon ethylene synthesis,a crude extract containing authentic ACC oxidase (ACCO) activitywas isolated in soluble form from the embryonic axes of seedsgerminated for 24 h. Under our optimal assay conditions (200mM HEPES at pH 7.0, 4µM FeS0₄, 6 mM Na–ascorbate,1 mM ACC, 20% 0₂, 3% CO₂ , and 10%glycerol) this enzyme was5–fold more active than under the conditions we used initiallyin the present work. The enzyme has the following K_m: 28 µMfor ACC (approximately 4–fold less than in vivo), 1.2%for O₂ (in the presence of an optimal CO₂ concentration of 3%),and 1% for CO₂ in the presence of O₂ (20%). The enzyme is inhibitedby phenanthroline (PNT) (specific chelating agent of ferrousion), and competitively inhibited (K₁, =0.5 mM) by 2–aminoisobutyricacid (AIB), and the enzymatic activity was not detectable inthe absence of CO₂. Under optimal assay conditions, the enzymehas two optimum temperatures (28 C and 35 C) and is inhibitedby divalent metal cations (Zn²⁺> CO²⁺>Ni²⁺>Cu²⁺>Mn²⁺>Mg²⁺) and by salicylic acid, propylgallate, carbonyl cyanidem–chlorophenyl hydrazone (CCCP), dinitrophenol (DNP),and Na–benzoate. The in vitro ACCO activity which we recoveredin soluble form is equivalent to approximately 80–85%of the apparent activity evaluated in vivo. Key words: ACC oxidase, Cicer arietinum, ethylene, germination, seeds     

Turnover of 1-aminocyclopropane-1-carboxylic Acid synthase protein in wounded tomato fruit tissue

Ethylene production in plant tissues declines rapidly following induction, and this decline is due to a rapid decrease in the activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, a key enzyme in ethylene biosynthesis. To study the nature of the rapid turnover of ACC synthase in vivo, proteins in wounded ripening tomato (Lycopersicon esculentum) fruit discs were radiolabeled with [³⁵S]methionine, followed by a chase with nonradioactive methionine. Periodically, the radioactive ACC synthase was isolated with an immunoaffinity gel and analyzed. ACC synthase protein decayed rapidly in vivo with an apparent half-life of about 58 min. This value for protein turnover in vivo is similar to that previously reported for activity half-life in vivo and substrate-dependent enzyme inactivation in vitro. Carbonylcyanide-m-chlorophenylhydrazone and 2,4-dinitrophenol, potent uncouplers of oxidative phosphorylation, strongly inhibited the rapid decay of ACC synthase protein in the tissue. Degradation of this enzyme protein was moderately inhibited by the administration of aminooxyacetic acid, a competitive inhibitor of ACC synthase with respect to its substrate S-adenosyl-l-methionine, α,α′-dipyridyl, and phenylmethanesulfonyl fluoride or leupeptin, serine protease inhibitors. These results support the notion that the substrate S-adenosyl-l-methionine participates in the rapid inactivation of the enzyme in vivo and suggest that some ATP-dependent processes, such as the ubiquitin-requiring pathway, are involved in the degradation of ACC synthase proteins.     

Purification and Characterization of a Vegetative Lytic Enzyme Responsible for Liberation of Daughter Cells during the Proliferation ofChlamydomonas reinhardtii

A vegetative lytic enzyme (VLE) of Chlamydomonas reinhardtiimediates digestion of the cell walls of mother cells (sporangia)to allow release of daughter cells after mitotic cell divisionin the vegetative cell cycle. This enzyme is secreted into theculture medium concurrently with the appearance of daughtercells in synchronized cultures. Using an assay that monitorsdigestion of the mother cell wall, we purified VLE by ion-exchangeand gel-filtration chromatography from the medium of synchronizedcultures. The purified enzyme was a basic glycoprotein withan apparent molecular mass of 120 kDa on gel filtration and130 kDa on SDS-PAGE. Thus, VLE appeared to behave as a monomer.The enzyme acted specifically on the mother cell wall and wasunable to digest the cell walls derived from single vegetativecells. The enzymatic activity was inhibited by PMSF, p-APMSF,TLCK, HgCl₂, iodoacetate, EGTA, EDTA and 1,10-phenanthroline.VLE cleaved several synthetic model peptides on the carboxylside of a Lys or Arg residue, indicating that it is a proteasethat acts on protein in the mother cell wall in vivo to releasethe daughter cells. (Received November 30, 1994; Accepted March 22, 1995)     

Inhibition of 1-Aminocyclopropane-l-carboxylic Acid Synthase Activity by Polyamines, Their Related Compounds and Metabolites of S-adenosylmethionine

Basic amino acids, monoamines, diamines and polyamines inhibitedthe activity of 1-aminocyclopropane-1-carboxylic acid (ACC)synthase extracted from wounded mesocarp tissue of winter squashfruit (Cucurbita maxima Duch.). Among the amines tested, polyamineswere highly effective, while the synthetic triamine, 1,8-diamino-4-aminomethyloctane,was an even stronger inhibitor than the polyamine spermine.Polyamines inhibited ACC synthase activity in a non-competitivemanner, while metabolic inhibitors such as aminoethoxyvinylglycineand aminooxyacetic acid inhibited ACC synthase activity competitively,showing much lower Ki values than those of polyamines. ACC synthaseactivity was also inhibited by intermediates of the methionine-recyclingpathway, 5'-methylthioadenosine and -keto--methylthiobutyricacid and by S-adenosylhomocysteine, a product of transmethylationof S-adenosylmethionine. It appears that polyamines not only inhibit ACC synthase activitybut also suppress the induction of the enzyme. However, unlikeprevious reports, polyamines did not inhibit in vivo ethyleneforming enzyme activity in the wounded mesocarp tissue. (Received October 24, 1985; Accepted January 10, 1986)     

Monomeric and Dimeric Forms and the Mechanism-Based Inactivation of 1-Aminocyclopropane-1-Carboxylate Synthase

The molecular mass of 1-aminocyclopropane-1-carboxylate (ACC)synthase from a variety of sources was examined by both high-performancegel-filtration chromatography and polyacryl-amide gel electrophoresisin the presence of sodium dodecylsulfate. Enzymes used wereprepared from wounded or non-wounded pericarp of ripe tomatofruits and wounded mesocarp of winter squash fruits, as wellas from cells of E. coli that had been transformed with cDNAsfor the wound-induced or ripening-induced ACC synthases of tomatoand the wound-induced or auxininduced enzymes from winter squash.The enzymes from tomato fruit tissues were isolated in a monomericform, whereas the enzymes synthesized in E. coli from cDNAsfor tomato ACC synthase were isolated in a dimeric form. ACCsynthases of winter squash obtained either from fruit tissuesor from transformed E. coli cells were isolated in dimeric forms.ACC synthase in the monomeric form was less sensitive to theinactivation that is associated with the catalytic reaction(the mechanism-based inactivation) than the enzyme in the dimericform. A plausible mechanism relating the difference in molecularform to sensitivity to the mechanism-based inactivation of tomatoACC synthase is discussed. (Received February 1, 1993; Accepted May 17, 1993)     

Induction of 1-Aminocyclopropane-1-carboxylic Acid Synthase in Wounded Mesocarp Tissue of Winter Squash Fruit and the Effects of Ethylene

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase activityincreased rapidly after wounding of mesocarp tissue of wintersquash fruit (Cucurbita maxima Duch.) and reached a peak at16 h after excision and then declined sharply. The rise in ACCsynthase activity was followed by increases in the endogenousACC content and the rate of ethylene production. The activityof ethylene forming enzyme (EFE) also increased rapidly in theexcised discs of mesocarp of winter squash fruit. ACC synthase activity was strongly inhibited by aminoethoxyvinylglycinewith a Ki value of 2.1 µM. Michaelis-Menten constant ofACC synthase for S-adenosylmethionine was 13.3 µM. Ethylene suppressed the induction of ACC synthase in the woundedmesocarp tissue. The suppression by ethylene increased withthe increasing concentrations of applied ethylene and the maximumeffect was obtained at about 100 µl 1^–1 ethylene,at which point the induction was suppressed by 54%. Ethylenedid not inhibit ACC synthase activity, nor did it suppress theinduction of EFE, but rather it slightly enhanced the latter. (Received August 24, 1984; Accepted October 29, 1984)