Sylva Leblová

A Comparative Study of the Effect of Heavy Metal Ions on Ribulose-1,5-bisphosphate Carboxylase and Phosphoenolpyruvate Carboxylase

Summary The Cd 2+ , Cu 2+ , Zn 2+ and Pb 2+ ions inhibited ribulose-1,5-bisphosphate carboxylase (RuBPC) from barley and phosphoenolpyruvate carboxylase (PEPC) from maize in vivo and in vitro . PEPC was more sensitive to inhibition by the heavy metal ions tested than RuBPC was. The Cd 2+ ions at concentrations 5μM caused a time-dependent loss of activity of both enzymes. CU 2+ , Zn 2+ and Pb 2+ at concentrations 5μM inhibited only PEPC. A time-dependent activation of RuBPC by 5 μM Cu 2+ or Zn 2+ was observed. PEP, glycine and glucose-6-phosphate protected PEPC against Cd 2+ -induced inactivation while Mg 2+ ions were without this effect. Na 2 C0 3 and Mg 2+ ions protected RuBPC against inhibition by Cd 2+ ions, but rib ulose-1,5-bisphosphate (RuBP) was ineffective. The mechanism of a heavy metal toxicity on photosynthesis is discussed.

Uptake of Lead and Cadmium by Maize Seedlings and the Effect of Heavy Metals on the Activity of Phosphoenolpyruvate Carboxylase Isolated from Maize

Maize seeds and five-day-old maize seedlings were incubated in media containing Pb2+ at concentrations of 50, 100, and 200 mg 1-1 and Cd2+ at concentrations of 1, 5, 10 and 50 mg 1-1. After five days of incubation, both heavy metals were determined by means of AAS following wet mineralisation of roots and shoots. The results obtained indicate that Pb2+ were transported to shoots from roots at a lower rate than Cd2+.Phosphoenolpyruvate carboxylase (PEPC) isolated from germinating maize seeds was inhibited to a comparable degree by solutions containing 0.001 mmol 1-1 Pb2+, 0.01 mmol 1-1 Cd2+, and 0.005 mmol 1-1 Cu2+. The enzyme was protected against this inhibition by the addition of mercaptoethanol, the substrate (PEP), or the cofactor (Mg2+). The inhibition increased during a 20 min incubation of the enzyme with salts of the metals. Mn2+, Ni2+, and Co2+ ions could partially substitute for the metal cofactor Mg2+. Km values for these metal ions were as follows: for Mg2+ 0.07 mmol 1-1 in the range from 0 to 0.30 mmol 1-1 Mg2+; 0.71 mmol 1-1 for 0.30 to 2.50 mmol 1-1 Mg2+; for Mn2+ 0.36 mmol 1-1; for Ni2+ 0.34 mmol 1{-1}; and for Co2+ 0.20 mmol 1-1. The activity of the enzyme reached with Mn2+ 85 %, with Ni2+ 65 %, and with Co2+ 55 % of the activity recorded with Mg2+.

Pyruvate metabolism in germinating seeds during natural anaerobiosis

Lactate as well as ethanol is formed in seeds of soybean, maize, pea, bean, lentil and broad-bean in the course of germination during the so-called natural anaerobiosis. After 0 to 30 h of germination a concentration peak of lactate appears. Maximum in ethanol content is found after 40 h. The amount of ethanol is higher big more than one order of magnitude as compared to the amount of lactate. Both products of anaerobiosis occur in germinating seeds irrespective of the type of reserve substances.In contrast to alcohol dehydrogenase lactate dehydrogenase (EC 1. 1. 1. 27) is present in the dry seeds too. Its activity decreases during the first 12 h of germination. It is in this stage that its substrate, lactate, is usually present at a maximal concentration. During the later stages of germination the amount of lactate decreases and enzyme activity rises. There exists a reciprocal relationship between enzyme activity and substrate concentration. In the case of alcohol dehydrogenases (EC 1. 1. 1. 1) the maximum concentration of ethanol precedes the peak of enzyme activity.AbstractZjistily jsme, že u soji, kukuřice, hrachu, fazolu, čočky a bobu se tvoří při klíčení během tzv. přirozené anaerobiosy jak laktát, tak také ethanol. Nejprve dosahuje maxima koncentrace laktát v rozmezí prvých 0–36 hodin klíčení a poté přibližně po 40 hodinách klíčení ethanol. Množství ethanolu je o více než řád vyšší než množství laktátu. Oba produkty anaerobiosy se vyskytují v klíčících semenech bez ohledu na typ reservních látek.Laktátdehydrogenasa (EC 1.1.1.27) je přítomna na rozdíl od alkoholdehydrogenasy i v suchém semeni. Její aktivita během prvých 12 h klíčení klesá. Právě v té době je obvykle maximální koncentrace jejího substrátu, tzn. laktátu. V dalších hodinách klíčení se množství laktátu snižuje a aktivita enzymu stoupá. Mezi aktivitou enzymu a množstvím substrátu existuje reciproký stav. Pokud jde o aktivitu alkoholdehydrogenasy (EC 1.1.1.1), maximum koncentrace ethanolu předchází maximu aktivity enzymu.

Stucture of phosphoenolpyruvate carboxylase from maize leaves

Phosphoenolpyruvate carboxylase (PEPC) from maize leaves has an M r of 400000. The native enzyme molecule is a homotetramer. The amino acid composition of PEPC is determined. The enzyme contains 8 half‐cystine residues per subunit. The role of half‐cystine residues and the steric arrangement of the enzyme protein molecule are discussed.

Occurrence of ethanol in pea plants in the course of growth under normal and anaerobic conditions

Abstract1)At a so-called natural anaerobiosis during the first 48 hours of germination the concentration of ethanol in pea tissues increases (according to the cultivation conditions) up to 40 μmol per gram fresh weight.2)In a nitrogen atmosphere the content of ethanol in pea seedlings increases as well, and after a 90 hour incubation in N2 it can reach even 100 μmol ethanol per gram fresh weight. In older plants the content increases the most markedly in cotyledons, but considerable amounts were revealed also in stems and roots. Its increase in vegetative organs of plants cultivated both in light and darkness is more or less identical. Ethylalcohol can be formed by the vegetative organs themselves, as proved by the increase of this metabolite in plants deprived of reserve organs; in addition, however, it is evidently transported into them from reserve parts. Ethanol formed under anaerobiosis is catabolyzed after transferring plants to the air.Abstract1.Za tzv. přirozené anaerobiosy během prvých 48 hodin klíčení stoupne koncentrace ethanolu (podle podmínek, za kterých jsou rostliny pěstovány) v pletivech hrachu až na 40 μmolů na gram čerstvé váhy.2.Rovněž v atmosféře dusíku stoupá obsah ethanolu v klíčících semenech hrachu a může např. po 90hodinové inkubaci v N2 dosáhnout dokonce 100 μmolů ethanolu na gram čerstvé váhy. U rostlin starších stoupá obsah nejvýrazněji v dělohách, ale značně také v osách a kořenech. Vzestup ve vegetativních orgánech rostlin pěstovaných na světle i ve tmě je víceméně shodný. Vegetativní orgány mohou ethanol samy tvořit, jak dokazuje vzestup tohoto metabolitu u rostlin zbavených reservních orgánů, vedle toho však je do nich zřejmě také transpotován z reservních částí. Ethanol vytvořený za anerobiosy se po přenesení rostlin na vzduch odbourává.

On plant alcohol dehydrogenases

We have found in a number of plants (lentil, lupine, bean, barley, oats, rye, wheat, cucumber, melon, flax, sunflower and rape) that varying amounts of ethanol are formed under natural anaerobiosis and, that in later growth periods these plants continue to react to anaerobiosis by formation of ethanol. When the testa has opened in germinating plants or, when plants are transferred from the anaerobic atmosphere to air, ethanol disappears.Plants contain alcohol dehydrogenases, the activity of which depends on the alcohol concentration in their tissue; the maximum concentration is reached during natural anaerobiosis, rising in the course of further growth when the plants are kept in a nitrogen atmosphere.Alcohol dehydrogenases of the plants studied are localised in the soluble cell fraction notsedimenting at 120 000 g, their pH optimum is in the weakly alkaline region and their Michaelis constants are equal in order of magnitude (10−5m). They are all inhibited in the same way by Zn2+, Cu2+, Hg2+, B4O72− ions, p-chloromercuric benzoate, iodoacetate, EDTA and phenantroline, which may be considered as evidence of the presence of −SH groups. The specific activity of alcohol dehydrogenase preparations is higher in plants grown in light than in plants grown in the dark.The specific activity of plant alcohol dehydrogenases can be increased by precipitation with ammonium sulphate by at most one order of magnitude, while all the activity is lost by this purification process in the case of cereals.The following isoenzyme composition of ADH was found by means of electrophoresis on polyacrylamide: the enzyme from poas and sunflower, for example, is composed of three, that from wheat and oats six, the enzyme from maize and barley of five isoenzymes.AbstractZjistily jsme, že u celé řady rostlin (čočky, lupiny, fazolu, pelušky, ječmene, ovsa, žita, pšenice, okurky, melounu, lnu, slunečnice, řepky olejky) se za přirzené anaerobiosy tvoří větší nebo menší množství ethanolu a že všechny tyto rostliny reagují i v dalším růstovém, údobí na anaerobiosu tvorbou ethanolu. Ethanol po prasknutí testy u klíčících rostlin nebo přenesení rostlin z anaerobní atmosféry na vzduch mizí.Rostliny obsahují alkoholdehydrogenasy, jejichž aktivity je zévislá na koncentraci ethanolu vpletivech: dosahuje maxima během přirozené anaerobiosy a v dalším růstu se zvýší při přechovávání rostlin v atmosféře dusíku.Alkoholdehydrogenasy studovaných rostlin jsou lokalisované v rozpustné buněčné frakei nesedimentující při 120 000 g, mají pH optimum v mírně alkalické oblasti a Michaelisovy konstanty jsou řádově stejné (10−5m). Inhibice ionty Zn2+, Cu2+, Hg2+, B4O72−, p-chlormerkutibenzoátem, jodacetátem, EDTA a fenantrolinem jsou obdobné a svědčí o přítomnosti −SH skupin. Specifická aktivita preparátu alkoholdehydrogenasy je vyšší u rostlin pěstovaných na světle než ve tmě.Srážení síranem amonným dovolí zvýšit specifickou aktivitu rostlinných alkoholdehydrogenas maximálně o jeden řád, u obilovin se u tohoto stupně čištění aktivita ztrácí.Elektroforesou na polyakrylamidu jsme zjistily isoenzymové složení ADH: Např. enzym z hrachu a slunečnice obsahuje 3, pšenice a oves 6 a kukuřice a ječmen 5 isoenzymů.

Soybean alcohol dehydrogenase

Abstract Alcohol dehydrogenase was prepared from germinating soybean seeds. Specific activity was increased from 511 to 31316 units. The coenzyme is NAD with a K m of 10 −4 M. Allyl alcohol is oxidized faster than ethanol; with the latter substrate, the K m is 1.3 × 10 −2 M, and the pH optimum 8.7. The enzyme catalyses acetaldehyde reduction, with a K m of 10 −2 M and a pH opt of 7.1. The MW is 53(±5) × 10 −3 .

Kinetics of the reaction catalysed by rape alcohol dehydrogenase

Abstract The kinetics of the enzyme reaction of ethanol oxidation and acetaldehyde reduction catalysed by alcohol dehydrogenase (ADH) (EC 1.1.1.1) isolated from germinating rape seeds obeys the bi-bi ordered mechanism of Theorell and Chance. The enzyme reaction depends on the pH and temperature. The K m values for the basic substrates have the lowest values around the pH optimum of the reaction. The enzyme is most stable at pH 6.5–7. The K m values for ethanol and NAD increase with increasing temperature. The maximum rate of the ethanol oxidation satisfies the Arrhenius equation. The activation energy for the given temperature range is 40.11 kJ/mol. The rape ADH is denatured by heating above 60° but the enzyme-NAD complex is thermally more stable than the enzyme alone.

The regulation of lactate dehydrogenase activity in soy-bean seedlings

An electrophoretically homogeneous lactate dehydrogenase was isolated from soybean seedlings, the specific activity of which was approximately 1800 times higher than the crude extract. From the dependence of the rate of reaction catalyzed by lactate dehydrogenase on substrate concentration, Michaelis constants and Hill coefficients were determined for four natural substrates,i.e. lactate, pyruvate, NAD and NADH. The enzyme from soy-bean plants is non-competitively inhibited by oxalate and mesoxalate,i.e. by the compounds analogous to the substrate. At pyruvate concentrations above 0.8 mM, the rate of reaction catalyzed by lactate dehydrogenase from soy-bean plants decreases, fructose diphosphate and ATP function as inhibitors as well. The inhibition by ATP is pH dependent, which seems to be of importance for the regulation of enzyme activityin vivo.

Comparative study of plant alcohol dehydrogenases

Alcohol dehydrogenase was isolated both from monocotyledons and dicotyledons, some of them with proteins (bean, pea), others with lipids (rape, sunflower) and still others with sugars (rice) as reserve substances. Molecular weights of the isolated dehydrogenases ranged from 53 000 to 80 000. Plant alcohol dehydrogenases (ADH) catalyze the oxidation of ethanol as well as the reduction of acetaldehyde. pH optimum for the oxidation is in the alkaline region, for the reduction it is near neutrality. The Michaelis constants for ethanol oxidation are, with the exception of rice, higher than those for reduction of acetaldehyde. The specificity of plant ADH toward alcohols is relatively broad and only quantitatively different in the individual plants. Inhibitors of the ADH’s studied are oximes, amides and intermediates of sugar metabolism, such as malate, acetate or succinate. The degree of inhibition brought about by the inhibitors studied differs from plant to plant but the inhibition type is the same.