Acid invertase in germinating Lactuca sativa seeds: Evidence for de novo synthesis

Acid invertase activity in germinating lettuce seeds is first observed after 15 hr germination, from when it rises steadily at least till 30 hr of germination. The enzyme was purified about 500-fold using ammonium sulphate fractionation followed by isoelectric focussing. Labelling the enzyme with ³⁵SO₄ or leucine-¹⁴C during development of its activity, followed by purification suggests that acid invertase is synthesized de novo during germination. The possible significance of acid invertase in the metabolism of the seed is discussed.     

Increased conformational stability of mitochondrial soluble ATPase (F1) by substitution of H2O for D2O.

Between 20 and 40 °C D₂O inhibits the hydrolytic activity of soluble mitochondrial ATPase F₁. The effect of D₂O is proportional to its concentration in the incubation mixture and at nearly 100% D₂O in the incubation mixture the ATPase activity is inhibited by 50–60%. The effect of D₂O is mainly on the V of the reaction. At temperatures above 45 °C, D₂O does not inhibit the activity. D₂O protects against the denaturation of the enzyme that is observed at relatively high temperatures and against the cold-induced inactivation of F₁. The intensity of fluorescence of 8-anilino-1-naphthalene sulfonate incubated with F₁ increases as the enzyme becomes inactivated by low temperatures; in D₂O the changes of fluorescence are almost nil. These observations indicate that H (or D) bonding between the solvent and the protein as well as the strength of the hydrophobic interactions within the enzyme as determined by the solvent are of central importance in determining the overall activity of F₁ and the stability of the enzyme to denaturing conditions. Moreover, the data indicate that the enzyme may exist in two different conformations, each with a characteristic activation energy. It is also proposed that D₂O may be employed with success in the isolation and purification of labile enzymes.     

Regulation of Alcohol Dehydrogenases in Maize Scutellum during Germination

The pattern of change in the activity of alcohol dehydrogenase in maize (Zea mays L.) scutellum during seed germination is not altered by 10 μg/ml cycloheximide or 50 μg/ml actinomycin D. The enzyme does not become density labeled when maize seeds are germinated in the presence of D₂O and ¹⁵NH₄Cl, indicating that no new alcohol dehydrogenase molecules are synthesized after the onset of germination. However, the activity of an endogenous inhibitor for alcohol dehydrogenase is increased after germination. The increase of this inhibitor is concomitant with the decline of alcohol dehydrogenase activity, indicating that the activity of alcohol dehydrogenase during seed germination is controlled by the level of the inhibitor.     

The biosynthesis of ribonuclease and its accumulation in protein bodies in the cotyledons of mung bean seedlings

Germination and seedling growth of mung bean are accompanied by a 7- to 10-fold increase in the ribonuclease content of the cotyledons. The increase occurs during the first 4 days of seedling growth and precedes the senescence of the cotyledons. Separation of the RNases in the cotyledons by polyacrylamide gel electrophoresis indicates the presence of several minor bands in seeds imbibed for 24 hr. On the second day of seedling growth a new major band with an R_f of 0.76 is present. In 4- to 5-day old seedlings this major band accounts for nearly all the RNase activity in the tissue. The characteristics of this RNase show that it is a plant ribonuclease I (pH optimum of 5.0; MW 16,000; activity preferentially inhibited by purine nucleotides; no activity toward DNA; no phosphodiesterase activity). When the seedlings are grown in 66% D₂O the RNase activity undergoes a density shift of 0.61% indicating that the increase in enzyme activity is due to the de novo synthesis of the enzyme molecules. A method is described for the isolation of protein bodies from protoplasts of storage parenchyma cells. Fractionation of protoplast lysates on Ficoll gradients results in the recovery of a high proportion (75%) of intact protein bodies. On these gradients RNase activity comigrates with α-mannosidase, a protein body marker enzyme indicating that the newly synthesized RNase accumulates in the protein bodies. We suggest that the synthesis of RNase in the cotyledons and its accumulation in the protein bodies indicates that protein bodies may function in the degradation of cellular macromolecules other than the reserves stored within them.     

Density-labelling studies on the photocontrol of phenylalanine ammonia lyase levels in Cucumis sativus

The de novo synthesis of PAL is demonstrated to occur sometime between imbibition and the end of a 4-hr white light treatment. H₂OD₂O transfer experiments indicate that PAL synthesis may occur during the light period whilst D₂O-H₂O transfer experiments indicate that synthesis of inactive PAL may occur during dark growth followed by activation by light. Neither of these observations is conclusive. De novo synthesis of PAL occurs in excised hypocotyls of gherkin and tuber discs of potato either in darkness or in light. It is concluded that there is as yet no evidence which definitively shows that light controls PAL levels by regulating the rate of de novo synthesis.     

Hydrogen-Deuterium Exchange Effects on β-Endorphin Release from AtT20 Murine Pituitary Tumor Cells

Abundant evidences demonstrate that deuterium oxide (D₂O) modulates various secretory activities, but specific mechanisms remain unclear. Using AtT20 cells, we examined effects of D₂O on physiological processes underlying β-endorphin release. Immunofluorescent confocal microscopy demonstrated that 90% D₂O buffer increased the amount of actin filament in cell somas and decreased it in cell processes, whereas β-tubulin was not affected. Ca²⁺ imaging demonstrated that high-K⁺-induced Ca²⁺ influx was not affected during D₂O treatment, but was completely inhibited upon D₂O washout. The H₂O/D₂O replacement in internal solutions of patch electrodes reduced Ca²⁺ currents evoked by depolarizing voltage steps, whereas additional extracellular H₂O/D₂O replacement recovered the currents, suggesting that D₂O gradient across plasma membrane is critical for Ca²⁺ channel kinetics. Radioimmunoassay of high-K⁺-induced β-endorphin release demonstrated an increase during D₂O treatment and a decrease upon D₂O washout. These results demonstrate that the H₂O-to-D₂O-induced increase in β-endorphin release corresponded with the redistribution of actin, and the D₂O-to-H₂O-induced decrease in β-endorphin release corresponded with the inhibition of voltage-sensitive Ca²⁺ channels. The computer modeling suggests that the differences in the zero-point vibrational energy between protonated and deuterated amino acids produce an asymmetric distribution of these amino acids upon D₂O washout and this causes the dysfunction of Ca²⁺ channels.     

Changes in the activity of phosphoenolpyruvate carboxylating enzymes in germinating yellow lupin seeds

The rate of phosphoenolpyruvate carboxylation by extracts from germinating lupin seeds was measured through the H¹⁴CO₃ fixation. PEP carboxylation in seed axes increased during their imbibition, mainly as a result of the increase in the activity of PEP carboxylase [EC 4.1.1.31]. However, the activity of PEP carboxykinase [EC 4.1.1.38], present during the first 3 hours of imbibition, as well as the activity of PEP-carboxykinase [EC 4.1.1.49], after 24 hours of imbibition, have also been shown. Possible physiological role of the changes in the activity of PEP carboxylases during lupin seeds germination is discussed.     

Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis)

Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.     

Evidence for de novo synthesis of isocitratase and malate synthesis in germinating peanut cotyledons

Evidence for de novo synthesis of isocitratase and malate synthetase in cotyledons of germinating peanut (Arachis hypogaea L.) was obtained by the density labeling method. When dry peanut cotyledons were cultured in H₂¹⁸O, a 2.4% increase in the buoyant density of malate synthetase in a cesium chloride gradient was observed. In 100% D₂O the buoyant density shift was 5.5% for isocitratase and 3.5% for malate synthetase in comparison to the water controls. These data suggest that isocitratase and malate synthetase do not pre-exist in some inactive form in the cotyledons, but are completely synthesized after onset of germination from a pool of amino acids which do not derive directly from hydrolysis of storage proteins.     

The development of ribonuclease and acid phosphatase during germination of Pisum arvense

1. Development of ribonuclease activity in the cotyledons of germinating peas is biphasic, the time of appearance of the two phases depending on the conditions of growth. 2. Acid phosphatase exhibits a single phase of development. 3. Cycloheximide inhibits development of ribonuclease activity in phase II but not in phase I. 4. (14)C-labelled amino acids are not incorporated into ribonuclease isolated during phase I. 5. The buoyant density of ribonuclease isolated during phase I is not affected by imbibition of the seed in 80% deuterium oxide. 6. Acid phosphatase was isolated from the supernatant fraction of the cotyledons of germinating peas and partially purified. 7. Development of acid phosphatase activity during germination is inhibited by treatment of the seed with cycloheximide or actinomycin D. 8. Partial purification of acid phosphatase from peas germinated in the presence of (14)C-labelled amino acids suggests that the enzyme is radioactively labelled. 9. Germination of peas in the presence of 80% deuterium oxide results in an increase in the buoyant density of acid phosphatase. 10. The results suggest that increase in ribonuclease activity during the first 4 days of germination does not result from synthesis of protein de novo, but that the corresponding increase in acid phosphatase activity does result from synthesis de novo.     

Phosphoenol-pyruvate-carboxylase activity in cotton and Sorghum seeds and its relation to seedling development

Cotton (Gossypium hirsutum) seeds and Sorghum vulgare caryopses are able to incorporate CO₂ through a PEP-carboxylating enzyme (EC 4.1.1.38). The enzyme activity is optimal at pH 8.2 and is unaffected by ATP, GDP or acetyl CoA. The partially purified cotton enzyme is stimulated by inorganic phosphate with an apparent Km of 0.3 mM. The enzymes from both cultivars are inhibited by pyrophosphate, malate, and aspartate but not by succinate. Kinetic studies for Sorghum and cotton seed enzymes show apparent Km values for carbonate of 5 mM and 1.2 mM and for PEP of 36 M and 5 mM, respectively. The V_max values are 90 and 3.3 nmol min^-1 mg protein^-1, respectively.A two-fold increase in the enzyme activity from cotton seeds occurs after 2 h under laboratory germination conditions after which the activity drops sharply to 1/3 of the original activity after 5 h imbibition. No such change was observed in Sorghum caryopses enzyme. A correlation between PEP-carboxylase activity and seed vigor in both cultivars was demonstrated.Abbreviations GOT glutamicoxaloacetic-transaminase - MDH malic dehydrogenase-NADH₂ - RH relative humidity     

Properties and Partial Purification of 1-Aminocyclopropane-1-carboxylate Synthase

We studied the regulation of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity in tomato (Lycopersicon esculentum Mill.) fruit tissue and attempted the purification of this enzyme. The increase of ACC synthase activity in wounded tomato pericarp was inhibited by cordycepin and cycloheximide. Density labeling studies showed a 0.75% increase in the buoyant density of ACC synthase isolated from tomato pericarp tissue that had been incubated on ²H₂O as compared to ACC synthase from H₂O-treated tissue. These data are consistent with the hypothesis that ACC synthase is synthesized de novo following wounding of tomato pericarp tissue. SDS-gel electrophoresis and fluorography showed that the pattern of incorporation of l-[³⁵S]methionine into protein changed with time after wounding of the tissue. Radioactive protein bands that were not detected 1 hour after wounding, became apparent 2 to 3 hours after wounding.     

Purification and characterisation of rape seed phospholipase D

Phospholipase D (PLD, phosphatidylcholine:phosphatidohydrolase, EC 3.1.4.4) has been isolated from matured dry winter rape seed (Brassica napus L.), variety Lirajet). Final purification of the soluble enzyme was achieved by two-step ammonium sulphate precipitation, hydrophobic interaction chromatography and native PAGE followed by electroelution. The specific activity of the final electrophoretically homogeneous preparation was increased about 700 times during the purification process with an overall yield of 4.6%. The activity of purified soluble PLD depends strictly on the presence of Ca²⁺ (120 mM). The pH optimum of rape seed PLD was in the range 5.5–6. The K_m value for phosphatidylcholine depends on the ratio between SDS and substrate concentration. No polymorphism of PLD was detected by SDS-PAGE and size exclusion chromatography of the purified enzyme. The purified enzyme was a monomer with a molecular mass of 105 000 Da determined by SDS-PAGE and of 90 000–100 000 Da assessed by size exclusion chromatography. The amino acid composition of PLD was also determined. Similar intensities of immunochemical cross reactions were demonstrated between PLD extracted from rape seed, soybean, castor bean and sunflower using immunoabsorption technique with the immune serum previously prepared against partially purified rape seed PLD. Data obtained in this study and those gathered from the literature indicate close similarities in molecular, enzymatic and antigenic characteristics between PLDs of oil seeds of different species.     

Protein kinase in Cicer embryonic axes

A dramatic increase in the cAMP-independent protein kinase activity (10-fold) was observed in the first 10 hr of germination of excised embryonic axes of Cicer. The lag phase of enzyme induction was very short since a 6-fold stimulation of protein kinase activity was witnessed after two hr of imbibition of the axes. The increase in protein kinase activity is ascribed to de novo biosynthesis of the enzyme. Conclusive proof for the de novo biosynthesis of protein kinase was obtained by labelling the proteins in vivo with [³⁵S]-sulphate and subsequently recovering the label predominantly in the methionine residues of the purified enzyme. The purification of the enzyme to electrophoretic homogeneity (453-fold) was achieved by ion exchange chromatography followed by affinity chromatography on a Casein-Sepharose column. Polyacrylamide gel electrophoresis of the [³⁵S]-labelled enzyme revealed a single radioactive band by autoradiography, that co-electrophoresed with the Coomassie Blue stained band of protein kinase. The M, of the purified protein kinase is 94 000 as determined by molecular sieving on Sepharose CL-6B. SDS-PAGE data indicated that the enzyme is composed of two subunits of M,s 49 000 and 62 000. Chemical characterization of the reaction product of protein kinase revealed that phosphorylation occurs at serine and threonine residues of the substrate, casein.     

Enzyme Profiles in Seedling Development and the Effect of Itaconate, an Isocitrate Lyase-directed Reagent

Changes in levels of isocitrate lyase, malate synthase, and catalase have been investigated during germination of flax (Linum usitatissimum L.) in the presence and absence of itaconate. Germination was accompanied by a rapid increase in these enzymes during the first 3 days. The presence of 38 millimolar itaconate inhibited the incidence of seed germination and the growth of embryo axes as well as the appearance of isocitrate lyase but did not alter the levels of malate synthase, catalase, or NADP⁺-isocitrate dehydrogenase. The specific activity for the latter enzyme was constant throughout germination. Oxalate or succinate, each at 38 millimolar, had no effect upon germination of flax seeds. Itaconate did not inhibit the activities of malate synthase, catalase, or NADP⁺-isocitrate dehydrogenase in vitro but was a potent noncompetitive inhibitor of isocitrate lyase (K_i:17 micromolar at 30 C, pH 7.6). Itaconate (at 38 millimolar) did not alter the appearance of malate synthase but reduced the incidence of germination, onset of germination, and growth of the embryo axis as well as the specific activity of isocitrate lyase in seedlings of Zea mays, Vigna glabra, Glycine hispida, Vigna sinensis, Trigonella foenumgraecum, Lens culinaris, and Medicago sativa. The incidence and onset of germination of wheat seeds were unaltered by the same concentration of itaconate but seedlings did not contain isocitrate lyase or malate synthase. The data suggest that itaconate may be isocitrate lyase-directed in inhibiting the germination of fatty seeds.     

Role of the testa in preventing cellular rupture during imbibition of legume seeds

Studies with the seeds of soybean, navy bean, pea, and peanut were made to determine the extent of leakage of intracellular enzymes during imbition. Embryos with intact testae from all four species were found to leak detectable activities of either intracellular enzymes of the cytosol (glucose-6-phosphate dehydrogenase) or enzymes found in both the cytosol and organelles (malate dehydrogenase, glutamate dehydrogenase, glutamate oxaloacetate transaminase, and NADP-isocitrate dehydrogenase) after 6 hours imbition at 25 C. Pea and peanut embryos with testae leaked considerably lower levels of activity for these enzymes than did those of soybean and bean. Leakage of mitochondrial marker enzymes (fumarase, cytochrome c oxidase, and adenylate kinase) was not detected from embryos with testae, suggesting that a differential diffusion of intracellular components out of cells occurred. Soybean and bean embryos without testae leaked high, and proportionally (per cent dry seed basis) similar, levels of all cytosol, cytosol-organelle, and mitochondrial marker enzymes and protein during imbibition, indicating that cell membranes were not differential to leakage and that they had ruptured. Pea and peanut embryos without testae leaked detectable activities of all cytosol and cytosol-organelle enzymes, although fumarase was the only detectable mitochondrial marker enzyme leaked, suggesting that some degree of differential leakage may have occurred in these species. The outermost layers of embryo cells of seeds without testae of all four species absorbed and sequestered the nonpermeating pigment Evan's blue after 5 to 15 minutes imbibition, indicating that membranes had ruptured. This occurred to a much lesser extent in seeds with intact testae. Both soybean and bean embryos without testae were observed to disintegrate during imbibition, whereas those of pea and peanut did not. These data indicate that seeds of certain legumes are susceptible to cellular rupture during imbibition when seed coats are damaged or missing.     

S-adenosylmethionine decarboxylase of corn seedlings

S-Adenosylmethionine decarboxylase (EC 4.1.1.50) has been purified 500-fold in 30% yield from the extract of etiolated corn seedlings (cv. Golden Crossbantam Bell). This preparation had a molecular weight of approximately 25,000. The K_m value was 5 micromolar for S-adenosylmethionine. Methylglyoxal bis(guanylhydrazone), hydroxylamine, and sulfhydryl reagents (such as p-hydroxymercuriphenylsulfonate and N-ethylmaleimide) were effective inhibitors of this enzyme. Germination of corn seed was accompanied by a rapid increase in enzyme activity and maximum activity occurred in 5-day-old seedlings.     

Characterization of the increased lysophospholipase activity in gibberellic Acid-treated barley aleurone layers

A lysophospholipase (LPL) activity appears in the aleurone of barley (Hordeum vulgare L. cv Himalaya) half seeds during imbibition on moist agar. Secretion of LPL by half seeds is promoted by GA₃; the increase in secretory rate is almost linear from 10⁻¹⁰ to 10⁻⁶ molar GA₃. LPL activity is likewise promoted in isolated aleurone layers by GA₃. Its secretion into the incubation medium requires the continued presence of GA₃ and commences after a 10 to 14 hour lag period when 10 millimolar Ca²⁺ is present. In the absence of Ca²⁺, the lag period remains unchanged but attainment of the maximum secretory rate is delayed. Ca²⁺ alone has very little effect either on LPL activity accumulated in the aleurone layer or in the surrounding medium. However, 50 millimolar Ca²⁺ together with GA₃ dramatically increase the level of secreted activity and of total (accumulated and secreted) activity.

The metabolic inhibitors cycloheximide and actinomycin D inhibit the accumulation of LPL activity in the aleurone and also the secreted activity. Actinomycin D added after the lag period results in a much lower inhibition. The increase in LPL activity in response to GA₃ occurs as a result of de novo synthesis; LPL activity from barley half seeds incubated in 80% D₂O in the presence of GA₃ undergoes a shift to higher density compared with the activity from similar controls incubated in H₂O. The characteristics of the GA₃ enhancement of LPL activity are compared specifically with α-amylase and generally with other GA₃-controlled hydrolases.

     

Thiamin Phosphorylation by Thiamin Pyrophosphotransferase during Seed Germination

Thiamin pyrophosphotransferase activity was present in seedling extracts from several monocot and dicot species of agronomic as well as noncultivated plants. Changes in thiamin pyrophosphotransferase activity and thiamin pyrophosphate content were followed for 6 days in soybean (Merr.) seedlings. Maximum enzyme activity occurred 48 to 96 hours from imbibition. Thiamin pyrophosphate content peaked sharply at 36 hours and was preceded by increased thiamin pyrophosphotransferase activity. Addition of pyrithiamin, an inhibitor of in vitro thiamin pyrophosphotransferase activity, to the imbibition medium at various times inhibited subsequent fresh weight gains of soybean seedlings. These results indicated that, although not among the earliest phosphorylation events after initiation of water imbibition by soybean seeds, a substantial increase in thiamin pyrophosphate content did precede the onset of rapid seedling growth and development. Since both enzyme activity and thiamin appear to be available in unimbibed soybean seeds, ATP or other nucleoside triphosphate concentration may represent an important factor in modulating thiamin phosphorylation during early seedling development.     

Agmatine deiminase in rice seedlings

Agmatine deiminase activity in rice embryos increased gradually upto 24 hr during germination and then decreased. Gibberellic acid and kinetin inhibited the activity when added to the germination medium. The enzyme was purified 717 fold with specific activity 788.5 nkat/mg protein and yield 8.8%. The M_r of the native enzyme was 18.3 x 10⁴ and the enzyme was a dimer of two identical subunits. The pH and temperature optimum of the enzyme were 6.0 and 28° respectively. The enzyme followed typical Michaelis-Menten kinetics with a K_m value of 1.5 x 10^?2 M. The enzyme activity was inhibited by various divalent cations and spermidine and spermine, but putrescine showed no effect.