Control of Glutamate Dehydrogenase from Green Tobacco Callus Mitochondria by Ca2$ and pH

Glutamate dehydrogenase (GDH) (EC 1.4.1.3 [EC] .) purified from greentobacco callus mitochondria was activated markedly by Ca^2$ inthe amination reaction. This activation was detectable evenat concentrations below 5 µM Ca^2$. Saturation curves for the three substrates of the aminationreaction showed normal Michaelis-Menten kinetics in the presenceof 1 mM of Ca^2$, but pronounced substrate inhibition occurredwithout Ca^2$. The effect of Ca^2$ was chiefly on the maximalvelocity. The saturation curve for NH₄Cl in the presence of Ca^2$ was modulatedby a change in pH. The apparent K_m value for NH₄Cl markedlydecreased whereas that for -ketoglutarate increased slightlywhen the pH was raised from 7.3 to 9.0. In contrast, the K_mfor NADH was little affected by raising the pH. The characteristicof GDH which increases its affinity for NH₄Cl when the pH israised may be compatible with the detoxification of ammonia. ¹ Present address: Mochida Pharmaceutical Co., Ltd. (Received August 24, 1981; Accepted November 28, 1981)     

The isozymic nature and kinetic properties of glutamate dehydrogenase from safflower seedlings

Levels of glutamate dehydrogenase (GDH) [L-glutamate: NAD oxidoreductase(deaminating), EC 1.4.1.2 [EC] ] from safflower roots and cotyledonsincreased (?2.7) and decreased ( ?5.7), respectively, as a functionof seedling age. No significant changes in enzyme levels weredetected during hypocotyl development. GDH preparations of thedifferent organs were resolved by polyacrylamide gel electrophoresisinto 2 to 4 isozymes. The isozymic pattern was influenced byseedling age and organ tested. The slowest moving isozyme (No.1) appears to be responsible for the changes in GDH levels observedin cotyledons and roots. We isolated isozyme 1 and GDH fractionchiefly containingisozyme 2, by DEAE-cellulose chromatography. GDH was purified approximately 53-fold from the particulatefraction of cotyledons. The pH optima for NADH and NAD activitieswere 8.2 and 8.9, respectively. Michaelis constants were foundto be: -ketoglutarate, 8mM; glutamate, 4 mM; ammonium, 35.4mM; NAD, 0.26 mM; NADH, 0.065 mM. Km values of isozymes 1 and2 were similar. The binding order of substrates in die reductiveamination reaction was NADH, -ketoglutarate and NH₄⁺. (Received July 17, 1972; )     

Regulation of NADH-Glutamate Dehydrogenase Activity by Phytochrome, Calcium and Calmodulin in Zea mays

Regulation by the active form of phytochrome (P_FR) and the effectof Ca²⁺ and calmodulin was examined with glutamate dehydrogenase(GDH) of Zea mays. A brief irradiation (5 min) to 5 day oldplants with red light resulted in 5-6 fold increase in GDH activity.This effect was nullified when red light was followed immediatelyby far-red light. The photoreversibility showed that P_FR regulatesGDH activity in vivo. To the enzyme extract obtained after EGTAtreatment, when Ca²⁺ was added in vitro, GDH was activated by6 fold. The maximum response by Ca²⁺ was obtained at 80 µM.Both P_AR and Ca²⁺ effects were found to be age dependent. Theenzyme activity was inhibited by compound 48/80 in partiallypurified extracts and the effect was reversed by calmodulin.The purified GDH, however, was not activated directly by calmodulin;it required the presence of another protein factor which wasseparated by gel permeation column by HPLC. Neither anticalmodulindrugs nor addition of calmodulin had any effect on nitrate re-ductaseactivity. (Received July 13, 1988; Accepted October 31, 1988)     

An NADP-Glutamate Dehydrogenase from the Green Alga Bryopsis maxima. Purification and Properties

NADP-glutamate dehydrogenase (EC 1.4.1.4 [EC] ; NADP-GDH) was purifiedto electrophoretic homogeneity from the multinuclear-unicellulargreen marine alga in Sipho-nales, Bryopsis maxima, and its propertieswere examined. M_r of the undenatured enzyme was 280 kDa, andthe enzyme is thought to be a hexamer of 46 kDa subunit protein.Optimum pHs for the reductive amination and oxidative deaminationwere 7.5 and 8.2-9.0 respectively. The enzyme displayed NADPH/NADH-specificactivities with a ratio of 18 :1. Apparent K_m values for 2-oxoglutarate,ammonia, NADPH, glutamate and NADP⁺ were 3.0, 2.2, 0.03, 3.2and 0.01 mM respectively. The enzymochemical characteristicsof the GDH were studied and compared to those of other species.The B. maxima GDH was insensitive to 5 mM Ca²⁺ and to 1 mM EDTAin contrast to higher plant NAD-GDHs. Chemical modificationswith DTNB and pCMBS suggested that cysteine residues are essentialfor the enzymatic activity as in other species GDHs. The GDHwas not affected by 1 mM purine nucleotides, suggesting thatthe enzyme is not allosteric, in contrast to animal NAD(P)-GDHsand fungal NAD-GDHs. (Received August 12, 1996; Accepted January 7, 1997)     

Glutamate transport and cellular glutamine metabolism: regulation in LLC-PK1 vs. LLC-PK1-F+ cell lines

The glutamate (Glu) transporter may modulate cellular glutamine(Gln) metabolism by regulating both the rates of hydrolysis andsubsequent conversion of Glu to -ketoglutarate andNH⁺₄. By delivering Glu, a competitiveinhibitor of Gln for the phosphate-dependent glutaminase (PDG) as wellas an acid-load activator of glutamate dehydrogenase (GDH) flux, thetransporter may effectively substitute extracellularly generated Glufrom the -glutamyltransferase for that derived intracellularly fromGln. We tested this hypothesis in two closely related porcine kidneycell lines, LLC-PK₁ and LLC-PK₁-F⁺,the latter selected to grow in the absence of glucose, relying on Glnas their sole energy source. Both cell lines exhibited PDG suppressionas the result of Glu uptake while disrupting the extracellularL-Glu uptake, withD-aspartate-acceleratedintracellular Glu formation coupled primarily to the ammoniagenicpathway (GDH). Conversely, enhancing the extracellular Glu formationwith p-aminohippurate and Glu uptakesuppressed intracellular Gln hydrolysis whileNH⁺₄ formation from Glu increased. Thus theseresults are consistent with the transporter's dual role in modulatingboth PDG and GDH flux. Interestingly, PDG flux was actually higher inthe Gln-adapted LLC-PK₁-F⁺cell line because of a two- to threefold enhancement in Gln uptake despite greater Glu uptake than in the parentalLLC-PK₁ cells, revealing theimportance of both Glu and Gln transport in the modulation of PDG flux.Nevertheless, when studied at physiological Gln concentration, PDG fluxfalls under tight Glu transporter control as Gln uptake decreases,suggesting that cellular Gln metabolism may indeed be under Glutransporter control in vivo.

     

Purification and properties of DNA topoisomerase II from Daucus carota cells

Topoisomerase II was partially purified from Daucus carota cellsby a procedure including ammonium sulphate fractionation, ion-exchange,and affinity chromatography steps. The type II enzyme, identifiedfor its ability to unknot knotted P4 DNA and decatenate Trypanosomacruzi kDNA, requires ATP and Mg²⁺ for activity. The unknottingactivity was sensitive to an inhibitor of the mammalian typeII enzyme, the drug VP16 (IC₅₀ 32 mmol m^–3), whereas inhibitorsof DNA gyrase showed a limited effect on activity. The SDS-PAGEanalysis of the dsDNA cellulose fraction revealed the presenceof four polypeptides of apparent molecular masses of 72, 71,34, and 33 kDa among which only a polypeptide of about 70 kDacrossreacted with antibodies against yeast topoisomerase II.Immunoprecipitation experiments with monoclonal antibodies tothe and ß isoforms of the human enzyme confirmedthe recognition of a polypeptide of 70 kDa. The sedimentationcoefficient (S) of the topoisomerase II in the phosphocellulosefraction, calculated by analytical glycerol gradient, was 6.1corresponding to a molecular mass of about 123 kDa. Resultssuggest the presence in carrot of a protein of molecular massof 70 kDa having the typical properties of an eukaryotic topoisomeraseII and carrying epitopes recognized by MoAbs to both human and ß enzymes. The 70 kDa polypeptide might then representthe monomer of a homodimer enzyme of 123 kDa. Key words: Daucus carota, topoisomerase II, immunoprecipitation     

Glycosidases from Cotyledons of Pisum sativum L.

-Mannosidase and ß-N-acetylglucosaminidase were purifiedfrom extracts of cotyledons of germinating Pisum sativum L.A 13-fold purification of a-mannosidase free from ß-N-acetylglucosaminidaseactivity was achieved by precipitation in ammonium sulphate,column chromatography on DEAE-cellulose, and treatment with2 M pyridine. ß-N-Acetylglucosaminidase was purified200-fold by the use of (NH₄)₂SO₄, and chromatography on ConcanavalinA¹-Sepharose and Sephacryl-200. This preparation showed no measurablecontamination by -mannosidase activity. Both glycosidases appearto be glycoproteins and demonstrate optimal activity at pH valuesof 4.0–4.5. Both glycosidases appear to have very similarmolecular weights, with -mannosidase being slightly larger thanß-N-acetylglucosaminidase. An extensive search forthe activity of aspartylglycosylamine amido hydrolase in peacotyledons proved unsuccessful.     

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)     

Comparative Studies of NAD-Malic Enzyme from Leaves of Various C4 Plants

Using butyl-TSK-gel chromatography, we purified NAD-malic enzyme(ME) (EC 1.1.1.39 [EC] ), which is involved in C₄ photosynthesis,to electrophoretic homogeneity, from leaves of Amaran-thus tricolor.Molecular weights of the native and SDS-denatured enzyme fromA. tricolor were 490 kDa and 61 kDa, respectively. During assayof the enzyme there was a slow reaction transient in the formof a lag before a steady-state rate was reached. The durationof this lag was inversely proportional to the concentrationof each substrate and the activator, fructose- 1,6-bis-phosphate(FBP). The optimal pH of the reaction fell with decreasing concentrationsof either malate or FBP. High pH prolonged the lag in reaction. Double reciprocal plots of the enzymatic activity as a functionof the concentration of malate yielded straight lines and didnot show any cooperativity for binding of malate. The enzymefrom A. tricolor was not inhibited by either HCO^–₃ orCO₂. At different concentrations of malate, the nature of theactivating effect of FBP was compared among the purified enzymesfrom A. tricolor and the C₄ monocots Eleusine coracana and Panicumdichotomiflorum. At low levels of malate, FBP markedly stimulatedthe enzyme from each species. In contrast, at saturating levelsof malate, the response of enzymes to increasing concentrationsof FBP was different and depended on the source of enzyme. The immunochemical properties of the enzymes from the threespecies were compared using an enzyme-linked immunoadsorbentassay with antisera raised against the purified enzymes fromthe three species. Different cross-reactivities were observedamong the enzymes from different sources. The N-terminal aminoacid sequences of NAD-MEs from the three species were determinedand some differences were found among the three enzymes. ²Permanent address; Tohoku National Agricultural ExperimentStation, Morioka, 020-01 Japan. ³Permanent address; National Grassland Research Institute, Nishinasuno,Tochigi, 329-27 Japan. (Received December 12, 1988; Accepted February 17, 1989)     

Rat cerebellar granule cells are protected from glutamate-induced excitotoxicity by S-nitrosoglutathione but not glutathione

In cultured rat cerebellar granule cells, glutamate or N-methyl-D-aspartate (NMDA) activation of the NMDA receptor caused a sustained increase in cytosolic Ca²⁺ levels ([Ca²⁺]_i), reactive oxygen species (ROS) generation, and cell death (respective EC₅₀ values for glutamate were 12, 30, and 38 µM) but no increase in caspase-3 activity. Removal of extracellular Ca²⁺ blocked all three glutamate-induced effects, whereas pretreatment with an ROS scavenger inhibited glutamate-induced cell death but had no effect on the [Ca²⁺]_i increase. This indicates that glutamate-induced cell death is attributable to [Ca²⁺]_i increase and ROS generation, and the [Ca²⁺]_i increase precedes ROS generation. Apoptotic cell death was not seen until 24 h after exposure of cells to glutamate. S-nitrosoglutathione abolished glutamate-induced ROS generation and cell death, and only a transient [Ca²⁺]_i increase was seen; similar results were observed with another nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine, but not with glutathione, which suggests that the effects were caused by NO. The transient [Ca²⁺]_i increase and the abolishment of ROS generation induced by glutamate and S-nitrosoglutathione were still seen in the presence of an ROS scavenger. Glial cells, which were present in the cultures used, showed no [Ca²⁺]_i increase in the presence of glutamate, and glutamate-induced granule cell death was independent of the percentage of glial cells. In conclusion, NO donors protect cultured cerebellar granule cells from glutamate-induced cell death, which is mediated by ROS generated by a sustained [Ca²⁺]_i increase, and glial cells provide negligible protection against glutamate-induced excitotoxicity. cytosolic calcium concentration; N-methyl-D-aspartate; reactive oxygen species     

Phosphoproteins and Protein Kinase Activities Intrinsic to Inner Membranes of Potato Tuber Mitochondria

Inside-out submitochondrial particles (IO-SMP) were isolatedand purified from potato (Solanum tuberosum L. cv.) tubers.When these IO-SMP were incubated with [ ³²P]ATP more then 20proteins became labelled as a result of phosphorylation. The³²P incorporation was stimulated by the oxidising reagent ferricyanide.Except for a 17 kDa protein which was phosphorylated only inthe absence of divalent cations, the protein phosphorylationrequired Mg²⁺. The time for half-maximum ³²P incorporation was4 mm for the 22 kDa phospho-F₁ -subunit and 2 min for the 28kDa phospho-F₀ b-subunit of the proton-ATPase. The K_m for ATPfor the detected phosphoproteins was between 65 µM and110 µM. The pH optimum for protein phosphorylation ininner membranes was between pH 6 and 8, and for the F₁ -subunitand the F₀ b-subunit the pH optima were 6.5–8 and pH 8,respectively. A 37 kDa phosphoprotein was phosphorylated ona histidine residue while the remainder of the inner membraneproteins were phosphorylated on serine or threonine residues.Two autophosphorylated putative kinases were identified: oneat 16.5 kDa required divalent cations for autophosphorylation,while another at 30 kDa did not. A 110 kDa protein was labelledonly with [-³²P] suggesting adenylylation. ³ Present address; Novartis Seeds AB, Box 302, S-261 23 Landskrona,Sweden.     

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     

Purification by Affinity Chromatography of {alpha}-Amylase--A Main Amylase in Cotyledons of Germinating Vigna mungo Seeds

In Vigna mungo cotyledons, the -amylase activity increased markedlyduring germination at 27°C in the dark, while the activityof other amylases was very low. The -amylase was purified from4-day-old cotyledons by affinity chromatography on epoxyactivatedSepharose 6B substituted with rß-cyclodextrin andby column chromatography on Bio-Gel P-200. Gel filtration andpolyacrylamide gel electrophoresis showed that the enzyme existsmostly as a monomer (43,000 daltons), but partially aggregatesto form dimer, trimer and further multimers. Ca²⁺ protectedthe -amylase against heat inactivation. Incubation of the enzymewith 5 mM EDTA or dialysis against 10 mM EDTA resulted in a50–90% loss of activity. The inactivation was partiallyreversed by the addition of Ca²⁺. Other properties, such asthe amino acid composition, K_m value, pH optimum and activationenergy were similar to those of other plant -amylases. (Received May 6, 1981; Accepted June 22, 1981)     

Effect of Applied Nitrogen on N2 Fixation, Assimilation of Nitrate and Ammonia in Nodules of Field-grown Moong (Vigna radiata)

A field experiment was conducted to study the effect of nitrogenapplication at 15, 30 and 45 kg ha^–1 of urea at pre-flowering(PF) and pod initiation (PI) stages on the activity of nitrogenase(N₂ase), nitrate reductase (NR) and other related parametersin the nodules of moong (Vigna radiata). Nitrogen applied atPF or PI stage was found to be inhibitory to N₂ase and glutaminesynthetase (GS) activities except at 15 kg N ha^–1 whenapplied at PF in the case of N₂ase. At both the stages therewas increase in NR and glutamate dehydrogenase (GDH) activitieswith the application of nitrogen. Seed yield increased by 18per cent with the application of 15 kg N ha^–1 at PI stagewhereas nitrogen application at PF stage only increased strawyield significantly. Nitrate reductase, nitrogenase, nitrogen application, ammonia assimilation, Vigna radiata     

Ammonium excretion and glutamate dehydrogenase activity of zooplankton in Great South Bay, New York

In Great South Bay, nanoplankton, (<20 sµm) accountedfor the largest fraction (56%) of zooplankton glutamate dehydrogenase(GDH) activity over a one year period. Microzooplankton (20–200µm) and macrozooplankton (>200 µm) accountedfor 20% and 24%, respectively. Total zooplankton ammonium regenerationin Great South Bay could account for 74% of the ammonium requirementby phytoplankton in winter, but in summer when phytoplanktondemand was greater, and zooplankton population was low, it suppliedless than 5%. This study suggests that the smallest zooplanktonfraction, less than 20 µm, can be the most important asregards nitrogen regeneration in estuarine environments. MacrozooplanktonGDH activity in Great South Bay ranged from 0.18 mg atoms NH⁺₄-Nm^–3 d^–1 in winter to 3.34 mg atoms NH⁺₄-N m^–3d^–1 in spring. Over an annual period, the averaged GDH/excretionratio was 20.4 3.5 (n = 10), and this ratio agrees well withobservations by other investigators. Observed macrozooplanktonexcretion rates showed a strong correlation with the excretionrates indirectly estimated from GDH activities. The GDH/excretionratio seems to vary depending on the internal physiologicalstates of zooplankton as well as food availability.     

Purification and Characterization of Two Isozymes of Chlorophyllase from Mature Leaves of Chenopodium album

Chlorophyllase (Chlase) was purified from mature leaves of Chenopodiumalbum, and its enzymatic properties were investigated. Chlasewas extracted from acetone powder of C. album and purified bythe following chroma-tographic procedures: hydrophobic chromatography,Con A Sepharose, Heparin affinity chromatography, Mono Q ion-exchangechromatography, and gel-filtration. Con A Sepharose affinitychromatography and gel-filtration were the most effective stepson the purification. On Mono Q chromatography, the Chlase preparationseparated into two major and one minor fractions that exhibitedChlase activity. The two major Chlases were purified to homogeneity.Their molecular masses were estimated as 41.3 kDa and 40.2 kDaby SDS-PAGE. The optimum pH and K_m values of these two Chlaseswere similar. Their N-terminal amino acid sequences were almostidentical except for a deletion in the tenth amino acid residuein one of the Chlase; there was no homologous protein detectedby database search. ³Present address: Department of Biology and Geoscience, Facultyof Science, Shizuoka University, 836 Ohya, Shizuoka, 422 Japan.     

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

Previous studies have shown that inhibition of L-type Ca²⁺ current (I_Ca) by cytosolic free Mg²⁺ concentration ([Mg²⁺]_i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I_Ca, rat cardiac myocytes and tsA201 cells expressing L-type Ca²⁺ channels were whole cell voltage-clamped with patch pipettes in which [Mg²⁺] ([Mg²⁺]_p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca²⁺ channels (_1C/_2A/₂), increasing [Mg²⁺]_p from 0.2 mM to 1.8 mM decreased peak I_Ca by 76 ± 4.5% (n = 7). Mg²⁺-dependent modulation of I_Ca was also observed in cells loaded with ATP--S. With 0.2 mM [Mg²⁺]_p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP_2A) produced large changes in I_Ca amplitude; however, with 1.8 mM [Mg²⁺]_p, these same manipulations had no significant effect on I_Ca. With mutant channels lacking principal PKA phosphorylation sites (_1C/S1928A/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p had only small effects on I_Ca. However, when channel open probability was increased by _1C-subunit truncation (_1C1905/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p greatly reduced peak I_Ca. Correspondingly, in myocytes voltage-clamped with pipette PP_2A to minimize channel phosphorylation, increasing [Mg²⁺]_p produced a much larger reduction in I_Ca when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg²⁺ modulates the extent to which channel phosphorylation regulates I_Ca. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca²⁺ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A     

A Protein from Immature Raspberry Fruits which Inhibits Endopolygalacturonases from Botrytis cinerea and other Micro-organisms

A polygalacturonase-inhibiting protein (PGIP) was purified fromimmature raspberry fruits using ion exchange chromatography.The protein was composed of a single polypeptide chain withM_r of 38·5 kDa and a pI residing above pH 10. Kineticstudies suggested that the inhibition was of a non-competitivenature. The PGIP inhibited two endopolygalacturonases (endo-PG)purified from Botrytis cinerea and an endo-PG produced by Aspergillusniger to varying degrees but did not inhibit two exo-PGs purifiedfrom B. cinerea, bacterial endopectate lyases and bacterialendo-PGs. The concentration of PGIP at various stages of flowerand fruit development was determined. The inhibitor was notdetected in the flower, but reached a maximum of 69 units g^–1in the immature green fruit decreasing to 9 units g^–1as fruits matured. The N-terminal amino-acid sequence was determined. Key words: Polygalacturonase-inhibiting protein, Rubus idaeus, red raspberry, Botrytis cinerea, pectinases     

Regulation of the Cytoplasmic pH of Chara corallina in the Absence of External Ca2+: Its Significance in Relation to the Activity and Control of the H+ Pump

Effects of removal of external Ca²⁺ on the cytoplasmic pH (pH_c)of Chara corallina have been measured with the weak acid 5,5-dimethyl-oxazolidine-2,4-dione(DMO) as a function of external pH (pH₀) and of the externalconcentration of K⁺. Removal of Ca²⁺ always decreased pH_c whenpH₀ was below about 6.0; the decrease was about 0.2–0.4units at pH₀ 5.0, increasing to about 0.5 units at pH₀ 4.3.When pH₀ was 6.0 or higher the removal of Ca²⁺ had little orno effect on pH_c. This situation was not altered by changingthe concentration of K⁺, though in some experiments at pH₀ 5.0–5.2there was a slight decrease in pH₀ (about 0.2 units) when K⁺was increased from 0.2 to 2.0 mol m^–3, an effect apparentlyreversed when K⁺ was higher (5.0 or 10.0 mol m^–3). Theresults suggest that H⁺ transport continues in the absence ofexternal Ca²⁺, despite previous suggestions to the contrary,and that the H⁺ pump does not necessarily run near thermodynamicequilibrium with its chemical driving reaction. They indicate,rather, that the H⁺ pump is under kinetic control and providefurther evidence for the inadequacy of present models for theoperation of the H⁺ pump in charophyte cells, especially inrelation to its proposed role in regulating pH_c. Key words: Chara corallina, Cytoplasmic pH, Calcium     

Structural and Kinetic Properties of NADP-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L.

NADP-malic enzyme (EC 1.1.1.40 [EC] ), which is involved in Crassulaceanacid metabolism (CAM), was purified to electrophoretic homogeneityfrom the leaves of the inducible CAM plant Mesembryanthemumcrystallinum. The NADP-malic enzyme, which was purified 1,146-fold,has a specific activity of 68.8 µmol (mg protein)^–1min^–1. The molecular weight of the subunits of the enzymewas 64 kDa. The native molecular weight of the enzyme was determinedby gel-filtration to be 390 kDa, indicating that the purifiedNADP-malic enzyme is a hexamer of identical subunits. The optimalpH for activity of the enzyme was around 7.2. Double-reciprocalplots of the enzymatic activity as a function of the concentrationof L-malate yielded straight lines both at pH 7.2 and at pH7.8 and did not reveal any evidence for cooperativity of bindingof L-malate. The K_m value for L-malate was 0.35 mM. Hill plotsof the activity as a function of the concentration of NADP⁺indicated positive cooperativity in the binding of NADP⁺ tothe enzyme with a Hill coefficient (nH) of 2.0. An S_0.5 value(the concentration giving half-maximal activity) of 9.9 µMfor NADP⁺ was obtained. Oxaloacetate inhibited the activityof the NADP-malic enzyme. Effects of succinate and NaHCO₃ onthe activity of NADP-malic enzyme were small. (Received October 30, 1991; Accepted May 1, 1992)