Ethylene evolution in marine algae and a proteinaceous inhibitor of ethylene biosynthesis from red alga

Fronds of marine algae, especially green alga, Codium latum,and red alga, Porphyra tenera, evolved a quantity of ethylenewhen IAA was exogenously applied, while brown alga, Padina arborescens,evolved only a little. Propionic acid, when added together withIAA, noticeably enhanced IAA-induced ethylene evolution in P.tenera and P. arborescens. This evolution was also enhancedby added acrylic acid in P. arborescens but not in P. tenera.It was promoted by methionine, though only at a high concentration(0.1 M), in P. tenera but not in P. arborescens. The rate ofethylene evolution was highest at 12?C among the incubationtemperatures tested of 5, 12 and 15?C. The conversion of ¹⁴C-3-methionineto radioactive ethylene in P. tenera was remarkably inhibitedby a proteinaceous inhibitor from P. tenera. ¹Present address: Division of Environmental Biology, NationalInstitute for Environment, Yatabe, Ibaraki, Japan. (Received May 27, 1976; )     

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)     

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)     

CO2-Fixation, Glycolate Formation, and Enzyme Activities of Photorespiration and Photosynthesis during Greening and Senescence of Sunflower Cotyledons

Time-courses of ¹⁴CO₂-fixation and of enzyme activities involvedin photorespiration and photosynthesis were determined duringthe life span of cotyledons from sunflower seedlings (Helianthusannuus L.). Glycolate formation in vivo was estimated from theresults of combined labelling and inhibitor experiments. NADPH-glyceraldehyde-3-phosphatedehydrogenase, NADPH-glyoxylate reductase and chlorophyll werewell correlated with the time-course of ¹⁴CO₂-fixation (photosynthesis).There was, however, a considerable discrepancy between the developmentalsequence of photosynthesis and that of both ribulose-l,5-bisphosphatecarboxylase and glycolate oxidase. Furthermore, time-coursesof glycolate oxidase activity in vitro and of glycolate formationin vivo differed significantly. Therefore, the use of glycolateoxidase as a marker for the activity of photorespiration ingreening sunflower cotyledons may be questionable. Results from¹⁴CO₂-labelling experiments with cotyledons treated with theglycolate oxidase inhibitor 2-hydroxy butynoic acid suggestthat glycolate formation relative to CO₂-fixation is reducedin senescent cotyledons. Key words: Development, glycolate oxidase, photorespiration, ribulose-l,5-bisphosphate carboxylase, oxygenase     

Ethylene Production by the Kudzu Strains of Pseudomonas syringae pv. phaseolicola Causing Halo Blight in Pueraria lobata (Willd) Ohwi

Significant amounts of ethylene was produced by Pseudomonassolanacearum (all strains), P. syringae pv. phaseolicola (Kudzustrains isolated from Pueraria lobata) and Erwinia rhapontici(2 strains out of 22) out of 24 species, 3 subspecies and 38pathovars of plant pathogenic bacteria tested in yeast extract-peptonebroth. The bean strains of P. syringae pv. phaseolicola causinghalo blight in kindney bean plants did not produce ethylene.The Kudzu strains produced ethylene at a rate of 7 to 100?10^–9nl cell^–1 h^–1, which was 500 to 1,000 times higherthan that of P. solanacearum and several times higher than thatof Penicillium digitatum, the most potent ethylene producerknown among microorganisms. The presence of living cells was essential for ethylene productionby the Kudzu strains. The bacterium effectively produced ethylenefrom amino acids such as glutamate, aspartate and their amides.Although glucose and succinate were also good substrates forethylene biosynthesis, the rate of ethylene production was significantlysmaller than that with glutamate. Methionine, which is knownas the precursor of ethylene in plants, had no effect on ethyleneproduction by the bacterium. 1-Aminocyclopropane-1-carboxylicacid (ACC) also had no effect on ethylene production, and therewas not enough ACC in the bacterial cells to account for thehigh rate of ethylene production. Ethylene production from glutamatewas inhibited by n-propylgallate and EDTA, but not by aminoethoxyvinylglycine.These results indicate that ACC is not involved as an intermediatein the process of ethylene biosynthesis by the bacterium, suggestingthe presence of a pathway different from that of plant tissues. (Received September 4, 1984; Accepted October 27, 1984)     

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     

Effects of Free Radical Scavengers on Stress Ethylene in Soybean Leaves Hypersensitively Reacting to Tobacco Necrosis Virus

The hypersensitive reaction of soybean cuttings to tobacco necrosisvirus is characterized by a large stimulation of stress ethyleneinvolving a marked accumulation of free 1-aminocyclopropane-1-carboxylicacid (ACC) and a moderate increase in ethylene-forming enzyme(EFE) activity. The scavengers of hydroxyl radicals (OH^{dot})sodium benzoate, sodium formate, mannitol and dimethylsulphoxide,did not affect stress ethylene biosynthesis. Propyl gallate,an inhibitor of lipoxygenase enzymes, substantially reducedthe release of stress ethylene from hypersensitive leaves. Thisreduction was not attributable to an inhibitory effect on EFEactivity, but to a strong reduction of free ACC accumulationin leaf tissues. The results suggest that OH^{dot} and the lipoxygenasesystem are not involved in stress ethylene produced during thehypersensitive reaction of soybean to this virus. Glycine max Merr, soybean, ethylene, free radicals, hypersensitivity, tobacco necrosis virus     

黄荆中β-石竹烯对棉蚜的毒力和作用机理

为明确泰山野生黄荆Vitex negundo种子中的有效杀虫活性成分、杀虫作用及其毒理机制,本研究采用硅胶柱层析,GC-MS技术和生物活性追踪方法,测定了泰山黄荆种子中的杀虫活性成分;采用生物测定和生化分析法,研究了黄荆中的β-石竹烯和α-蒎烯对棉蚜Aphis gossypii的毒力及作用机制。结果表明：通过三级柱层析从黄荆中分离得到对棉蚜毒力高的馏分β-石竹烯和α-蒎烯,其含量分别达7.68%和5.45%。β-石竹烯和α-蒎烯对棉蚜的触杀毒力都较高,并以β-石竹烯的毒力最高,LD₅₀为0.65×10^-1 μg/头。β-石竹烯和α-蒎烯对棉蚜均具有强烈的忌避作用,处理棉蚜24 h的AFC₅₀分别为0.80×10³和0.89×10³ mg/L,其中也以β-石竹烯的忌避毒力最大。β-石竹烯和α-蒎烯以亚致死剂量处理棉蚜,对其繁殖力、排蜜频率和排蜜量均有显著不利影响。β-石竹烯和α-蒎烯处理棉蚜或离体酶,对乙酰胆碱酯酶、多酚氧化酶、羧酸酯酶和谷胱甘肽-S-转移酶都有明显抑制作用。结果显示β-石竹烯和α-蒎烯是黄荆种子提取物中的重要杀虫活性成分,并且其致毒机制存在多样性,开发应用价值大。     

Effects of lipophilic and water-soluble membrane probes on ethylene synthesis in apple and Penicillium digitatum

Several chemicals were used to probe the in situ ethylene formingenzyme systems in apple tissue and Penicillium digilatum. 2,4-Dinitrofluorobenzene,a membrane permeant probe, inhibited ethylene production effectivelyin apples but far less effectively in P. digitatum. In contrast,salicylaldehyde, another membrane permeant probe, effectivelyinhibited the P. digitatum system but, except at 0.1 mM concentration,little influenced the apple system. l,5-Difluoro-2,4-dinitrobenzene(DFDNB), a membrane permeant probe which cross-links proteinswith proteins and with phospholipids, strongly inhibited ethylenebiosynthesis in both apple and P. digitatum, whereas dimethylsuberimidate, the protein cross-linking reagent, inhibited slightlythe apple system but not P. digitatum system. Picrylsulfonate(TNBS), a non-permeant membrane probe, up to 0.1 mM, did notinhibit any of the two systems studied. However, in the presenceof exogenous methionine in the apple system and glutamate inP. digitatum, TNBS at 0.1 and 1 mM caused inhibition of ethylenesynthesis. These probes did not affect respiration of appleslices under similar incubating conditions, excepting for DFDNBwhich on longer incubation did inhibit respiration, but theeffect on ethylene synthesis was 15 times greater. Divalentcation ionophores, A23187 [GenBank] and X537 A, had no effect on ethylenesynthesis in both the systems. The water soluble iron chelatingagent, o-phenanthroline, was a more potent inhibitor of theapple system but minimally affected P. digitatum. In contrast,the lipophilic chelator, bathophenanthroline, was a more potentinhibitor of the P. digitatum system. Assay of the fatty acidcomposition of polar lipids from crude membrane fractions showedconsiderably greater linoleic to linolenic ratio in P. digitatumthan in apple. We suggest that the ethylene formations in appleand P. digitatum are sensitive to a modification of membranestructure and that specific chelator-sensitive metals (perhapsiron and copper) are involved in ethylene synthesis in boththese systems. ¹ On leave from the M.S. University of Baroda (India); presentaddress: Department of Plant Genetics, The Weizmann Instituteof Science, Rehovot, Israel. ²Present address: Agricultural Research Organization, The VolcaniCenter, Bet-Dagan, Israel. (Received February 23, 1979; )     

Induction of chitinase and {beta}-1,3-glucanase activity in sunflower suspension cells in response to an elicitor from Phytophthora megasperma f. sp. glycinea (Pmg). Evidence for regulation by ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC)

In heterotrophic cell suspensions of sunflower (Helianthus annuusL. cv. Spanners Allzweck) the effect of Pmg elicitor, a fungalelicitor preparation from Phytophthora megasperma f. sp. glycinea,on the induction of chitinase and ß-1,3-glucanaseactivity was studied in relation to changes in ethylene biosynthesis.Dose-response experiments with Pmg elicitor showed that theonset of the induction of intracellular chitinase and ß-1,3-glucanaseactivity coincided or followed a transient rise in ethyleneand particularly endogenous 1-aminocyclopropane-1-carboxylicacid (ACC) levels within 5 h of application. Treatment with5 µg ml^–1 elicitor stimulated ethylene and ACC levels1.6-fold and 4-fold, relative to control, respectively. Themolar ratio of ACC to ethylene changed from approximately 3:1in controls to 9:1 in treated cells. During further incubation,ethylene formation and, to a lesser degree, ACC levels declinedand the ACC/ethylene ratio increased to 56:1 in elicitor-treatedcells. On a protein basis, the activities of ß-1,3-glucanaseand chitinase increased approximately 5-fold and 8-fold, respectively,48 h after elicitor application. Additional treatment with theACC synthesis inhibitor aminoethoxyvinyiglycine (AVG) decreasedelicitor-induced enzyme activities and the levels of both ethyleneand ACC. Elicitor effects on chitinase and ß-1,3-glucanaseactivities could be fully restored when ACC was additionallyapplied. Concomitantly, the ACC/ ethylene ratio increased. Neithertreatments with ACC alone, which simultaneously increased internalACC and ethylene levels, nor treatments with AVG alone, whichsimultaneously reduced ACC and ethylene levels, could generallystimulate chitinase or ß-1,3-glucanase activitiesin the cells. It is suggested that ACC functions as a promotingfactor in the induction of chitinase and ß-1,3-glucanaseactivity triggered by Pmg elicitor and appears to reverse aninhibiting influence of ethylene. Key words: 1-Aminocyclopropane-1-carboxylic acid, chitinase, ß-1,3-glucanase, ethylene, Helianthus cellsuspension cultures, Phytophthora megasperma-elicitor     

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     

Alkaline phosphatase activity in Protogonyaulax tamarensis

A non-toxic strain of the marine dinoflagellate Protogonyaulaxtamarensis (= Gonyaulax tamarensis has been isolated from abloom in the Adriatic Sea, off the Emilia-Romagna coast. Culturesof the cells were grown in the laboratory in enriched seawaterat various initial ambient orthophosphate (P_i concentrations,ranging from 0.3 to 40.5 µM. The growth rate varied from0.3 to 0.8 divisions day^–1 depending on the P_i concentration.Alkaline phosphatase activity was inversely proportional toambient P levels. From measurements of kinetic parameters, thebinding of the artificial substrate p-nitrophenylphosphate tothe P.tamarensis alkaline phosphatase was quite strong (K_m=50µM). Maximal activity was observed at pH 8.4, althoughthe pH-activity curve was broad, in contrast to that of otheralkaline phosphatases. Protogonyaulax tamarensis alkaline phosphatase,measured over a 24h period, exhibited an apparent diurnal fluctuationin activity, in common with the enzyme from other dinoflagellates.     

Evidence for Involvement of the Superoxide Radical in the Conversion of 1-Aminocyclopropane-1-Carboxylic Acid to Ethylene by Pea Microsomal Membranes

Electron spin resonance (ESR) spectroscopy has provided evidencefor involvement of the superoxide anion (O₂^–) radicalin the conversion of l-aminocyclopropane-l carboxylic acid (ACC)to ethylene by microsomal membranes from etiolated pea seedlings.Formation of ethylene from ACC by the membrane system is oxygen-dependent,heat denaturable, inhibited by the radical scavenger n-propylgallate and sensitive to superoxide dismutase (SOD) and catalase.Addition of 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron)to the reaction mixture results in formation of the Tiron semiquinone(Tiron radical) ESR signal derived from O₂^–, and alsoinhibits ethylene production. The radical signal is oxygen-dependentand inhibited by SOD and catalase, but is formed both in thepresence and absence of ACC. Heat denaturation of the microsomalenzyme system completely blocks formation of the radical signal.The data collectively suggest that O₂^– generated by amembrane-bound enzyme facilitates the conversion of ACC to ethylene. (Received September 8, 1981; Accepted January 19, 1982)     

Photoconversion of protochlorophyll to chlorophyll a in a mutant of Chlorella regularis

Dark-grown cells of a mutant strain of Chlorella regularis containedchlorophyll a and protochlorophyll, phytyl ester of protochlorophyllide.Under illumination, protochlorophyll was quantitatively anddirectly converted into chlorophyll a. The photoconversion wasdependent on light intensity and temperature and proceeded ina cell-free preparation. The pathway of chlorophyll formation found in the mutant cellsis entirely different from that from protochlorophyllide byway of chlorophyllide a, which is generally observed in greenplants. ¹Present address: Division of Biology, Medical College of Miyazaki,Miyazaki 889-16, Japan. ²Present address: Division of Environmental Biology, The NationalInstitute for Environmental Studies, Ibaragi 300-21, Japan. (Received October 24, 1975; )     

Cyclic AMP in Rhizobia and Symbiotic Nodules

Cyclic adenosine 3',5'-monophosphate (cAMP) content of variouscultured rhizobia strains and tissues of legumes and non-leguminousplants was measured by enzyme immunoassays. Most rhizobia, culturedfor 44 to 165 h, contained cAMP ranging from 0.6 to 5 pmol mg^-1proteinexcept forAzorhizobium caulinodansORS571. The culture mediaalso contained varying amounts of cAMP depending on the strainof rhizobia.Azorhizobiumcells and their media contained no detectablecAMP. Nodules from most legumes and non-legumes had cAMP contentsranging from 2–70 pmol g^-1f.wt. However, nodules fromSesbaniarostrata,Crotalaria spectabilisandParasponia andersoniishowedundetectable cAMP levels, and those fromGlycine maxandVignaangularisoccasionally showed levels below the detection limit.The leaves of non-legumes mostly had cAMP levels below detectionlimit (approx. 1.0 pmol g^-1 f.wt), while the leaves ofa few legumes occasionally had detectable cAMP. The possiblerole of cAMP as a symbiotic signal is discussed. cAMP; legumes; modules; rhizobia; symbiosis     

Some Physiological Responses of Chlorella pyrenoidosa to 2,4-Dichlorophenoxyacetic Acid

An 18-h treatment of synchronously-grown Chlorella pyrenoidosawith 2,4-D did not significantly alter the size, dry weight,degree of synchrony, or pigment content of the cells, nor weredetectable quantities of ethylene produced. When Chlorella pyrenoidosawas treated with 5?10^–4 M 2,4-D, there was a statisticallysignificant stimulation of both net oxygen uptake and productionwhile 5?10 M 2,4-D inhibited both processes. When Chlorellapyrenoidosa was treated with 5?10^–4 M and 5?10^–3M 2,4-D, significantly greater amounts of glycollate were presentin the culture medium, even though an assay for glycollate dehydrogenaseshowed that the activity of this enzyme from 2,4-D-treated Chlorellapyrenoidosa was three times greater than in control cells. Looselybound 2,4-D was partitioned from a nonaqueously isolated chloroplastfraction, while other cell fractions failed to show detectablequantities of 2,4-D. It is postulated that in Chlorella pyrenoidosathe chloroplast is a target for 2,4-D action and that interferencein photorespiratory processes may underlie the observed responses.     

Regulation of ethylene production by phosphate in Penicillium digitatum

Inorganic phosphate regulated ethylene production in shake culturesof Penicillium digitatum. Decreasing the phosphate level ofthe medium from 100 to 0.01 mM markedly increased, about 100-fold,the rate of ethylene production, in 96 hr, which was confinedentirely to the fungal mycelium. Exogenous addition of between0.01 to 100 mM phosphate, to high ethylene producing, low-phosphatecultures strongly inhibited their ethylene production and increasedthe ATP content of the mycelium. Phosphate also inhibited ethyleneproduction in apple slices. Addition of calcium ions to theincubation medium stimulated the production of ethylene in appleslices, subhook epicotyl segments of pea and shake culturesof P. digitatum. We suggest that this stimulatory effect wascaused by the reduction of inhibitory levels of phosphate, whichcomplexed with calcium. Thus, phosphate in conjunction withcalcium may play an important role in regulating ethylene productionnot only in P. digitatum but also in higher plants. ¹ On leave from the Agricultural Research Organization, TheVolcani Center, Israel. ² On leave from the M.S. University of Baroda, India. (Received September 7, 1977; )     

Effects of Calcium on NAD(H)-Glutamate Dehydrogenase from Turnip (Brassica rapa L.) Mitochondria

The effect of Ca²⁺ and ammonia on mitochondrial NADH-glutamatedehydrogenase (GDH: EC 1.4.1.2 [EC] ) isolated from turnip root (Brassicarapa L.) activity was examined. Increasing the ammonia [(NH₄)₂SO₄]concentration led to significant substrate inhibition whichcould be reversed by micromolar levels of Ca²⁺. The sensitivityof the enzyme to ammonia inhibition and its reversal by Ca²⁺was affected by proteolysis. After treatment with various proteases,lower concentrations of Ca²⁺ were capable of fully activatingthe enzyme or overcoming the inhibitory effects of high ammonium,compared to non-treated enzyme. However, the protease-treatedenzyme was still sensitive to ethylene glycol-bis(ß-aminoethylether) N,N,N',N'-tetraacetate (EGTA). In contrast, NADH-GDHactivity was inhibited approx. 30% by organic mercurials (200µm), but the residual activity was not affected by thesubsequent additions of EGTA. NADH-GDH activity could also bestimulated by additions of high concentrations of NaCl (300mM) in the absence of added Ca²⁺. These results suggest thathydrophobic and -SH groups may be involved in the regulationof mitochondrial NADH-GDH activity by Ca²⁺. ² Present address: CSIRO Division of Horticulture, Urrbrae,S.A. 5064, Australia (Received April 18, 1990; Accepted July 23, 1990)