Different Intracellular Localization of Two Cytochrome P-450 Systems, Ipomeamarone 15-Hydroxylase and Cinnamic Acid 4-Hydroxylase, in Sweet Potato Root Tissue Infected with Ceratocystis fimbriata

Ipomeamarone 15-hydroxylase activity was mainly recovered inthe pellet fraction between centrifugations at 10,000 and 100,000?gfrom a crude extract of Ceratocystis fimbriata-infected sweetpotato root tissue, whereas cinnamic acid 4-hydroxylase activitywas found between centrifugations at 300 and 10,000?g. Whenparticles in the crude extract were fractionated by sucrosedensity gradient centrifugation, the rough-surfaced microsomeswere distributed over a wide density range from 1.09 to 1.14g cm^–3, judging from the distributions of protein, RNAand NADPH-cytochrome c reductase activity. Phosphorylcholine-glyceridetransferase activity was only in the lighter half of the microsomalfraction (density: 1.09–1.11 g cm^–3). Ipomeamarone15-hydroxylase activity was found in heavier half of the microsomalfraction (density: 1.10–1.14 g cm^–3). We proposethat this tissue has two rough-surfaced endoplasmic reticulumspecies, only one of which carries phosphorylcholine-glyceridetransferase, and that the cytochrome P-450 system is localizedon the species lacking the enzyme. Cinnamic acid 4-hydroxylaseactivity was mainly found in a fraction that had densities of1.17–1.19 g cm^–3 and contained vesicular particlesof various sizes. ¹ Present address: Laboratory of Food Hygienics, Faculty ofAgriculture, Kagawa University, Miki-cho, Kida-gun, Kagawa 761-07,Japan. (Received September 6, 1984; Accepted December 27, 1984)     

Purification of Sulphite-Cytochrome c Reductase of Thiobacillus novellus and Reconstitution of Its Sulphite Oxidase System with the Purified Constituents

Sulphite-cytochrome c reductase (sulphite: ferricytochrome coxidoreductase, EC 1.8.2.1 [EC] ) derived from Thiobacillus novelluswas purified by chromatography on a DEAE-cellulose column andby gel filtration with a Sephadex G-100 column. Although thereductase thus purified moved as a single band both in gel filtrationand in isoelectric focusing it was always split into two bandsby polyacrylamide gel electrophoresis; the one had the enzymaticactivity and showed absorption spectrum of cytochrome, whilethe other had no activity and was colourless, in contrast withthe results reported by Charles and Suzuki [(1966) Biochim.Biophys. Acta 128: 522]. The enzymatic properties of the purifiedreductase were almost the same as those of the enzyme obtainedby Charles and Suzuki. Cytochrome c-551 free of the reductase activity was obtained.Its molecular weight was determined to be 23,000 by polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate.The cytochrome seemed to exist in the organism as a complexwith the reductase or a subunit of the enzyme. In the stateof the complex with the enzyme, the cytochrome was reduced veryquickly on addition of sulphite, while the cytochrome free ofthe reductase activity was hardly reduced by the enzyme withsulphite. A sulphite oxidase system was reconstituted with the reductase,cytochrome c-550 and cytochrome oxidase highly purified fromthe bacterium. ¹ Present address: Water Research Institute, Nagoya University,Nagoya 464, Japan ² Present address: Institute for Biological Science, SumitomoChemical Co., Ltd., Takarazuka, Hyogo 665, Japan (Received January 23, 1981; Accepted March 9, 1981)     

Induction of Cytosolic Acetyl-Coenzyme A Carboxylase in Pea Leaves by Ultraviolet-B Irradiation

Levels of subunits of two acetyl-coenzyme A carboxylases werehigh in small leaves of Pisum sativum, decreased with growth,and remained constant in fully expanded leaves. Irradiationof fully expanded leaves induced the cytosolic isozyme only.This result suggests a key role for the cytosolic enzyme inprotection against UV-B. ¹Present address: Laboratory of Molecular Genetics, BiotechnologyInstitute, Akita Prefectural College of Agriculture, 2-2 Minami,Ohgata, Akita, 010-04 Japan ²Present address: Laboratory of Plant Molecular Biology, Schoolof Agricultural Sciences Nagoya University, Nagoya, 464-01 Japan     

Studies on anaerobic biphasic growth of a denitrifying bacterium, Pseudomonas stutzeri

Growth of Pseudomonas stutzeri(VAN NIEL strain) in the presenceof a limiting amount of nitrate under anaerobic conditions ischaracterized by 2 logarithmic phases separated distinctly byan intermediate phase where the growth rate is very low. Inthe first logarithmic phase nitrate is reduced stoichiometricallyto nitrite stage, and in the second phase nitrite is reducedto nitrogen gas. The nitrite reducing activity of cells in the second growthphase is 3–4 times higher than that of cells in the firstphase. The rise in nitrite reducing activity is correlated witha remarkable increase in the content of cytochromes a₂ and c-552. ¹Present address: Department of Biochemistry, Hiroshima UniversitySchool of Dentistry, Hiroshima, Japan. ²Present address: Institute of Molecular Biology, Faculty ofScience, Nagoya University, Nagoya, Japan. (Received June 16, 1969; )     

Glycogen biosynthesis in Chromatium strain D II. Purification and properties of glycogen synthetase

Glycogen synthetase, ADP-glucose-a (l4) glucan transglucosylase[E.C. 2.4.1.11 [EC] ] from a purple sulfur bacterium, Chromatium,was purified to a homogeneous state and its enzymic propertieswere studied. The molecular weight of the enzyme was 8.6?10⁴dalton as determined by analytical gel filtration on a columnof Sephadex G-100. Since sodium dodecyl sulfate-polyacrylamidegel electrophoresis gave the molecular weight value of 8.4?10⁴to the monomeric form of the enzyme, we concluded that Chromatiumglycogen synthetase is comprised of a single polypeptide chain.The optimal pH of teh transglucosylation reaction was between8.0–8.5. The enzyme molecule utilized only ADP-glucoseas the glucose donor. The k_m value was determined as 3.8?10_-4M by the radioisotopic method of measuring the incorporationof ¹⁴C-glucose into the acceptor glycogen, and 6.1?10^-5M bythe enzyme coupling method. The most effective glucose acceptor(primer) was proved to be a long-chain a (16) branched a (14)polyglucan, e.g. Chromatium and cow glycogen, whereas short-chainmalto-oligosaccharides were much less efficient in the chain-elongationreaction. ¹ Part I of this series is Ref. (9). (Received February 13, 1974; )     

Metabolism of p-Hydroxybenzoate via Hydroxyquinol by Trichosporon cutaneum WY2-2: Characterization of the Pathway using Superoxide Dismutase as a Stabilizer of Hydroxyquinol

Trichosporon cutaneum WY2-2 was shown to metabolize p-hydroxybenzoatevia protocatechuate and hydroxyquinol. Using superoxide dismutaseas a stabilizer of hydroxyquinol, the conversion of protocatechuateto hydroxyquinol and the ring fission process of hydroxyquinolwere confirmed. Hydroxyquinol was chemically identified as theproduct of protocatechuate hydroxylase reaction. Partially purifiedprotocatechuate hydroxylase was highly specific for protocatechuate;its K_m values for protocatechuate and NADH were 17.6 and 12.4µM, respectively. It catalyzed equimolar CO₂ formation,NADH oxidation and O₂ consumption from protocatechuate. Hydroxyquinoldioxygenase was highly specific for hydroxyquinol, with a K_mof 2.9 µM. ¹A preliminary account of this work was presented at the 81stMeeting of the Chubu-branch of Agricultural Chemical Societyof Japan, Gifu, October, 1980. ²Present address: Biological Institute, Faculty of Science,Nagoya University, Nagoya 464, Japan. ³Present address: Shin Nihon Chemical Co. Ltd... 19-10, Showa-cho,Anjoh, Aichi 446, Japan. (Received November 15, 1985; Accepted August 27, 1986)     

Stimulation by Fungal Elicitor of Inositol Phospholipid Turnover in Tobacco Suspension Culture Cells

The effects of fungal elicitor on inositol phospholipid turnoverand induction of phenylalanine ammonia-lyase (PAL) activityin tobacco suspension culture cells were investigated. Tobaccocells labeled by [³H]inositol in vivo were treated with Phytophthoranicotianae elicitor and [³H]metabolites of inositol phospholipidturnover were examined. Stimulation of inositol phospholipidturnover was observed preceding the induction of PAL activity;inositol 1,4-bisphosphate increased 15 times over the control10 min after the elicitor treatment. Increase of inositol 1,4,5-trisphosphatewas only 38% of the control. Phosphatidylinositol and phosphatidylinositol4-phosphate transiently decreased by 21 and 35%, respectively.Phosphatidylinositol 4,5-bisphosphate was not affected significantlyby the elicitor. Inositol 1,4-bisphosphate was preferentiallyelevated by elicitation than 1,4,5-trisphosphate suggestingthat the regulatory mechanism of inositol phospholipid turnoverin tobacco cells is different from that in animal cells. Phosphatidylinositolkinase but not phospholipase C was activated by the elicitorin vitro. Elicitor-dose dependency curves in the induction ofPAL activity and in the stimulation of inositol phospholipidturnover showed a similar feature suggesting that inositol phospholipidturnover is involved in the elicitor-signal transduction intobacco cells. ¹Present address: The Johns Hopkins University School of Hygieneand Public Health, 615 N. Wolfe St., Baltimore, Maryland 21205,U.S.A. ²Present address: Nagoya University BioScience Center and GraduateSchool of Agricultural Sciences, Nagoya University, Chikusa-ku,Nagoya, 464-01 Japan.     

Inhibition of Hydroxyquinol Auto-Oxidation by a Cell Extract of Trichosporon cutaneum

A cell extract from acetate-grown Trichosporon cutaneum WY2-2inhibited auto-oxidation of phenolics, especially that of hydroxyquinol.It prevented auto-oxidation of hydroxyquinol without directinteraction with hydroxyquinol. Bovine erythrocyte superoxidedismutase had similar characteristics as the cell extract, andthe elution patterns of superoxide dismutase activity and ofthe inhibitory activity to hydroxyquinol auto-oxidation froma Sephadex G-150 column coincided. These results indicate thatthe inhibitory activity in the cell extract is mainly due tosuperoxide dismutase. High activity of superoxide dismutase(20–30 unit/mg protein) and its isozyme profiles suggestan intimate relation between the regulation of superoxide dismutaseand catabolism of phenolics via hydroxyquinol. ¹Present address: Biological Institute, Faculty of Science,Nagoya University, Nagoya 464, Japan. ²Present address: Shin Nihon Chemical Co. Ltd., 19-10, Showa-cho,Anjoh, Aichi 446, Japan. (Received November 15, 1985; Accepted July 3, 1986)     

Mating reaction in Saccharomyces cerevisiae VI. Effect of 2-deoxyglucose on conjugation

The effect of 2-deoxyglucose (2-dG) on the mating reaction ofSaccharomyces cerevisiae was investigated and the followingresults were obtained. 1) The cell fusion process of the mating reaction was completelyinhibited by 0.05% 2-dG added to a culture medium containing2% D-glucose. This inhibition was partially reversed by raisingthe glucose concentration in the medium. 2) Sexual cell agglutination was hardly affected by 2-dG. 3) 2-dG at concentrations inhibiting cell fusion considerablysuppressed the incorporation of ¹⁴C-glucose into the cell wallpolysaccharides, glucan and mannan. 4) Glucose uptake and protein synthesis were only slightly inhibitedby 2-dG. 5) No enhancement of bulk polysaccharide synthesis was detectedduring mating. ¹Present address: Biological Institute, Faculty of Science,Nagoya University, Chikusa-ku, Nagoya 464, Japan. (Received April 20, 1974; )     

Isolation of a new phytoalexin-like compound, ipomeamaronol, from black-rot fungus infected sweet potato root tissue, and its structural elucidation

A new phytoalexin-like compound was isolated from sweet potatoroot tissue infected by the black-rot fungus, Ceratocystis fimbriata.Its chemical structure was similar to ipomeamarone, and thecompound was identified as 14-hydroxy-ipomeamarone and calledipomeamaronol. ¹This paper constitutes Part 105 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. ²Present address: Institute for Biochemical Regulation, Facultyof Agriculture, Nagoya University, Nagoya. (Received November 7, 1972; )     

Protein Kinase Inhibitors Inhibit Stimulation of Inositol Phospholipid Turnover and Induction of Phenylalanine Ammonia-Lyase in Fungal Elicitor-Treated Tobacco Suspension Culture Cells

Elicitor prepared from Phytophthora nicotianae stimulated inositolphospholipid turnover and induced phenylalanine ammonia-lyaseactivity in tobacco suspension culture cells [Kamada and Muto(1994) Plant Cell Physiol. 35: 397]. Protein kinase inhibitors,K252a and staurosporine inhibited both responses. These resultssuggest that inositol phospholipid turnover plays an importantrole in PAL induction through protein kinases. In addition,their mode of inhibition were different, proposing that severaltypes of protein kinases are involved in these elicitor-inducedresponses. ¹Present address: The Johns Hopkins University School of Hygieneand Public Health, 615 N. Wolfe St., Baltimore, Maryland 21205,U.S.A. ²Present address: Nagoya University BioScience Center and GraduateSchool of Agricultural Sciences, Nagoya University, Chikusa-ku,Nagoya, 464-01 Japan.     

The occurrence and properties of methenyltetrahydrofolate cyclohydrolase in plants

Methenyltetrahydrofolate cyclohydrolase (E.C. 3.5.4.9 [EC] ), whichis responsible for the enzymatic conversion of 5,10-methenyl-H₄FAto 10-formyl-H₄FA, has been found in various plant tissues.The enzyme was partially purified from pea seedlings and someof its properties were investigated. It was unstable, but wasstabilized by the addition of 25% glycerol. The enzyme was purifiedabout 60-fold by fractionation with ammonium sulfate and columnchromatography on DEAE-cellulose in the presence of 25% glycerol.Optimum pH for the reaction was 7.7. Michaelis constants for5,10-methenyl-H₄FA in the forward reaction, and for 10-formyl-H₄FAin the reverse reaction were 4x10^–5M and 2x10^–4M,respectively. The apparent equilibrium constant for the reactionwas calculated as 50. Enzyme activity was greatly inhibitedby the reduced forms of folate derivatives. The probable participationof this enzyme in the regulation of folate coenzyme levels inplant tissues has been suggested. ¹ Studies on the enzymatic synthesis and metabolism of folatecoenzymes in plants, VI. (For Part V, see Reference (5) ). Partof this paper was presented at the 22nd annual meeting of theJapan Vitamin Society held at Hiroshima on October 14, 1970. ² Present address: Sizuoka Eiwa Junior College, Ikeda, Shizuoka. (Received September 9, 1972; )     

Some properties of shikimate: NADP oxidoreductase produced in sweet potato root tissue after slicing

The activity of shikimate: NADP oxidoreductase [EC 1. 1. 1.25] in sweet potato root tissue increased soon after slicing.Enzyme preparations obtained from both sliced tissue and fromfresh tissue probably contained a single enzyme component, andthey showed identical chromatographical behaviour. K_m values of the enzyme for NADP and shikimate were 1.0x10^–4Mand 1.3 x 10^–3M, respectively. Enzyme activity was potentlyinhibited by SH-inhibitors such as p-chloromercuribenzoate andoxidized glutathione. Enzyme activity was affected neither by mononucleotides suchas ATP, ADP and AMP, divalent cations, Mg⁺⁺, Ca⁺⁺ and Mn⁺⁺,nor by metabolites such as tryptophan, phenylalanine, tyrosineand t-cinnamic acid which are involved in aromatic compoundsyntheses. The enzyme rapidly lost its activity. This inactivation reactionshowed a time course consisting of two steps of the first-orderreaction. The inactivated enzyme preparation was not reactivatedby thiol compounds such as cysteine, 2-mercaptoethanol and glutathione,although these reagents, to a certain extent, protected theenzyme from inactivation. The results suggest that denaturationof the enzyme protein was involved in inactivation of the enzyme. ¹Part 74 of the phytopathological chemistry of sweet potatowith black rot and injury. ²Present address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Setagaya-ku, Tokyo. (Received August 5, 1968; )     

Enzymes involved in the last steps of the biosynthesis of mannitol in brown algae

Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17 [EC] ) and mannitol-1-phosphatase(EC number yet unassigned) were detected in the brown algae,Spatoglossum pacificum and Dictyota dichotoma. The enzymes wereextracted from algal fronds and their properties were investigatedusing partially purified preparations. Mannitol-1-phosphatase shows maximum activity at pH 7. The enzymehad a narrow substrate specificity. The Km value for mannitol-1-phosphateis 8.3x10^–4 M (30°C, pH 7.0). The enzyme is activatedby Mg⁺⁺ and Mn⁺⁺and is strongly inhibited by PCMB, Hg⁺⁺and NaF. Mannitol-1-phosphate dehydrogenase showed maximum activitiesat pH values 6.5 and 10.2 in reductive and oxidative reactions,respectively. The dehydrogenase also showed narrow substratespecificity; mannitol-1-phosphate and NAD or fructose-6-phosphateand NADH₂ are utilized, respectively, in oxidative and reductivereactions by the enzyme. Km values for these substrates andthe coenzymes are 2.5x10^–4 M and 7.1x10^–5 M forthe first pair and 2.8x10^–4 M and 1.3x10^–5 M forthe latter pair. This enzyme was strongly inhibited by PCMBand Hg⁺⁺, but was only slightly affected by adenosine phosphates. Possible roles of these enzymes in the biosynthesis of mannitolin brown algae are discussed. ¹ Contributions from the Shimoda Marine Biological Station ofTokyo Kyoiku University, No. 233. This work was supported inpart by a Grant-in-Aid for Co-operative Research from the Ministryof Education, Japan and in part by a grant to one of us (T.Ikawa) from the Matsunaga Science Foundation. ² Present address: Chemical and Physical Laboratory, HoechstJapan Research Laboratory, Minamidai, Kawagoe, Japan. (Received February 22, 1972; )     

Distribution of Fallover in the Carboxylase Reaction and Fallover-Inducible Sites among Ribulose 1,5-Bisphosphate Carboxylase/Oxygenases of Photosynthetic Organisms

The biphasic reaction course, fallover, of carboxyla-tion catalysedby ribulose 1,5-bisphosphate carboxylase/ox-ygenase (RuBisCO)has been known as a characteristic of the enzyme from higherland plants. Fallover consists of hysteresis in the reactionseen during the initial several minutes and a very slow suicideinhibition by inhibitors formed from the substrate ribulose-l,5-bisphosphate(RuBP). This study examined the relationship between occurrenceof fallover and non-catalytic RuBP-binding sites, and the putativehysteresis-inducible sites (Lys-21 and Lys-30S of the largesubunit in spinach RuBisCO) amongst RuBisCOs of a wide varietyof photosynthetic organisms. Fallover could be detected by followingthe course of the carboxylase reaction at 1 mM RuBP and thenon-catalytic binding sites by alleviation of fallover at 5mM RuBP. RuBisCO from Euglena gracilis showed the same linearreaction course at both RuBP concentrations, indicating an associationbetween an absence of fallover and an absence of the non-catalyticbinding sites. This was supported by the results of an equilibriumbinding assay for this enzyme with a transition state analogue.Green macroalgae and non-green algae contained the plant-type,fallover enzyme. RuBisCOs from Conjugatae, Closterium ehrenbergii,Gona-tozygon monotaenium and Netrium digitus, showed a muchsmaller decrease in activity at 1 mM RuBP than the spinach enzymeand the reaction courses of these enzymes at 5 mM RuBP werealmost linear. RuBisCO of a primitive type Conjugatae, Mesotaeniumcaldariorum, showed the same linear course at both RuBP concentrations.Sequencing of rbcL of these organisms indicated that Lys-305was changed into arginine with Lys-21 conserved. ⁷ On leave from Research and Development Center, Unitika Ltd.,23 Kozakura, Uji, Kyoto, 611 Japan. ⁸ Present address: Department of Applied Biological Chemistry,Faculty of Agriculture, Tohoku University, Tsutsumidori-Ama-miyamachi, Sendai, 981 Japan. ⁹ Present address: National Institute for Basic Biology, Myodaiji,Okazaki, 444 Japan. ¹⁰ Present address: Department of Environmental Biology, TokyoPharmaceutical University, Hachioji, Tokyo, 192-03 Japan.     

Orthovanadate Suppresses Accumulation of Phenylalanine Ammonia-Lyase mRNA and Chalcone Synthase mRNA in Pea Epicotyls Induced by Elicitor from Mycosphaerella pinodes

Orthovanadate delayed accumulation of mRNAs encoding phenylalanineammonia-lyase and chalcone synthase in pea epicotyls inducedby an elicitor from Mycosphaerella pinodes. However, accumulationof mRNA for a putative P-type ATPase was not affected. The relationshipbetweenthe ATPase and defense responses is discussed. ³Present address: Plant Pathology Laboratory, School of Agriculture,Nagoya University, Chikusa, Nagoya, 464-01 Japan.     

Kinetics of Phenylalanine Ammonia-Lyase and the Effect of L-{alpha}-aminooxy-{beta}-phenylpropionic Acid on Enzyme Activity and Radicle Growth in Germinating Lettuce Seeds

The effects of different concentrations of L--aminooxy-ß-phenyIpropionicacid (AOPP), an analog of L-phenylalanine, on the activity ofphenylalanine ammonia-lyase (PAL, EC 4.3.1.5 [EC] ) and the growthof radicles in 24 h old germinating lettuce (Lactuca salivaL.) seeds were investigated. AOPP causes a significant inhibitionof PAL activity in the seeds (85% inhibition at 10⁴ M). It alsocauses a stimulation of radicle growth at that concentration.The results show that the inhibition of PAL by AOPP may be dueto an irreversible binding of the inhibitor to the enzyme leadingto its inactivation. AOPP also inhibits ethylene biosynthesisin germinating lettuce seeds which could probably explain thestimulation of radicle growth in these seeds. The enzyme shows typical Michaelis-Menten kinetics. The K_m forL-phenylalanine is 4.2 x 10⁵ M. The enzyme does not show anytyrosine ammonia-lyase activity. Various substrate analogs suchas D-phenylalanine, p-fluorophenylalanine, ß-phenyllacticacid, tryptophan and the product of the enzyme reaction, trans-cinnamicacid, inhibit the enzyme competitively. A number of intermediatesand endproducts of the phenylpropanpid pathway, except chlorogenicacid, do not show any inhibition. ¹Scientific contribution number 1423 from the New HampshireAgricultural Experiment Station. (Received May 9, 1986; Accepted September 8, 1986)     

Purification and properties of a nitrite reductase from Porphyra yezoensis Ueda

Nitrite reductase was extracted from the red alga Porphyra yezoensisUeda and purified through precipitation with ammonium sulfate,column chromatographies, and polyacrylamide gel disk electrophoresis.The enzyme preparation thus obtained showed a single band ondisk electrophoresis. The absorption spectrum had three maxima at 385 nm (Soret band),580 nm (-band), and 278 nm; the ratio of absorbance of the Soretband to the -band was 4.3. The molecular weight and the numberof amino acid residues were estimated to be 63,000 and 601,respectively. The enzyme activity was optimal at around pH 7.5, and its activitywas heat labile as indicated by reduction of activity by about70% when heated at 37°C for 10 min. The enzyme used ferredoxin and methyl viologen, but not NADP⁺or NAD⁺, as the electron carriers. Moreover, reduced forms ofthe latter two showed no effect on its activity. Km values ofthis enzyme for NO₂^–, Fd, and MV were 8.1 x 10^–4M, 4.3 x 10^–8 M, and 3.7 x 10^–4 M, respectively.Almost half of its activity was lost when potassium cyanidewas added at a concentration as low as 10^–5 M, and theKi value was 1.8 x 10^–5 M. Thus, the nitrite reductaseof Porphyra must be systematically grouped in EC 1.7.7.1 [EC] . Itresembled closely that of Chlorella, except for the amountsof some amino acids. ¹ Present address: Department of Biological Sciences, Universityof Tsukuba, Sakura-Mura, Ibaraki, 300-31 Japan. ² Present address: Department of Fisheries, College of Agricultureand Veterinary Medicine, Nihon University, Shimouma, Setagaya-ku,Tokyo, 154 Japan. (Received June 10, 1975; )     

Some Properties of the Arginine Decarboxylase in Vicia faba Leaves

Growth of Vicia faba seedlings is accompanied by a rapid increasein arginine decarboxylase (EC 4.1.1.19 [EC] ) in the leaves and epicotyl.Increased enzyme activity was observed under saline conditionsin the presence of NaCl and with osmotic stress by mannitol.The partially purified enzyme (about 86-fold) readily decarboxylatedL-arginine, while D-arginine, L-homoarginine, L-ornithine andL-lysine were decarboxylated very slowly, and L-citrulline andL-glutamic acid were not decarboxylated. The K_m value was 5.8?10^–4M for L-arginine. The optimal pH and temperature for activitywere 8.5 and 45?C, respectively. p-Chloromercuribenzoate andN-ethylmaleimide were effective inhibitors of the enzyme. Inhibitionby spermidine, putrescine and agmatine suggested a possiblefeed-back mechanism in the pathway of polyamine biosynthesis. (Received October 11, 1983; Accepted February 24, 1984)     

Properties of glutamate dehydrogenase purified from green tobacco callus tissue

Glutamate dehydrogenase [L-glutamate : NAD(P) oxidoreductase(deaminating) EC 1.4.1.3 [EC] .] has been purified from the mitochondrialfraction of green tobacco callus tissue. The enzyme was stableat –20?C for several months. The pH optimum for the aminationreaction was 7.8. But the optimum for the deamination reactionwas indistinct because it was in an extremely alkaline domain.Relative activities of the enzyme for amination were 50 withNADH and 10 with NADPH, and those for deamination were 5 withNAD and 1 with NADP at pH 7.9. The enzyme was inactivated by EDTA, but its activity partiallyrestored by the addition of divalent cations such as Ca²⁺, Mn²⁺,Zn²⁺, Cu²⁺ and Mg²⁺. Ca²⁺, Mn²⁺ and Zn²⁺ activated the reductiveamination 141, 122 and 39% respectively, but these divalentcations scarcely affected the oxidative deamination. Citrate and fumarate acted as inhibitors for reductive amination,and oxaloacetate for oxidative deamination of the enzyme reaction.These inhibitions were counteracted by the addition of Ca²⁺.ATP and ADP exerted an inhibitory effect on both directionsof the enzyme reaction. The inhibitory effect was hardly preventedby the addition of AMP. Ca²⁺ caused considerable recovery fromthe inhibition of ATP and ADP. Amino acids scarcely affectedthe enzyme activity. Michaelis constants were 0.28 mM for NAD, 0.065 mM for NADH,2.19 mM for a-ketoglutarate, 43.6 mM for ammonium chloride and4.24 mM for L-glutamate. ¹To whom requests for reprints should be addressed. (Received June 25, 1980; )