Synthesis and assembly of ribulosebisphosphate carboxylase enzyme during greening of barley plants

The synthesis and activation of ribulosebisphosphate carboxylase was studied in etiolated barley leaves during increasing periods of light irradiation. Comparisons were made among enzymatic activity, ¹⁴C-amino acid incorporation into anti-ribulosebisphosphate carboxylase precipitable and 16S protein, and total mass of enzyme. A major portion of newly synthesized anti-ribulosebisphosphate carboxylase specific protein preceded light-induced increase in enzyme activity by a significant period of time. These findings are consistent with a model in which both subunits of ribulosebisphosphate carboxylase are synthesized in response to an early event in greening and subsequently become associated to active oligomeric carboxylase species.     

Partial purification of the NADPH-dependent aldehyde reductase from bovine cardiac muscle.

An aldehyde reductase catalyzing the NADPH-dependent reduction of long-chain aldehydes has been purified 690-fold from bovine cardiac muscle. Based on the results obtained during gel filtration, this enzyme has an apparent molecular weight of 34,000. The pI of the aldehyde reductase was 6.1 and the enzymatic activity had a sharp pH optimum at 6.4. The enzyme catalyzed the reduction of aromatic aldehydes and aliphatic aldehydes having eight or more carbon atoms. Short-chain aldehydes, aldoses, or ketoses or long-chain methyl ketones were not utilized as substrates by this enzyme. However, the methyl ketone, pentadecan-2-one, was a competitive inhibitor of this enzyme with an apparent K_i = 10 μm when tetradecanal was the variable substrate. The reaction was not reversible when ethanol or hexadecanol was employed as substrate, utilizing either NAD⁺, or NADP⁺ as a cofactor. The addition of 10 mm pyrazole to the incubation medium had no effect on the enzymatic activity.     

Enhancement of UDP-galactose:mucin galactosyltransferase activity by spermine

A microsomal preparation prepared from the mucosal lining of canine trachea catalyzed the transfer of galactose from its uridine diphosphate derivative to sialidase-treated ovine submaxillary mucin. Maximal incorporation occurred at 30 mm mn²⁺. When the concentration of mn²⁺ in the reaction mixture was reduced to 2.5 mm, approximately two-thirds of the enzymatic activity was lost, but full activity could be restored by the addition of 1 mm spermine. Under these conditions spermine did not affect the K_m for UDP-galactose, but lowered the K_m for sialidase-treated ovine submaxillary mucin and Mn²⁺ by a factor of 10. The effect of spermine was abolished with increasing concentrations of Mn²⁺, and in the absence of the metal, enzymatic activity was lost and could not be restored by the addition of spermine. Spermidine also stimulated activity at low levels of Mn²⁺, but to a lesser degree than spermine. A slight stimulatory effect was consistently derived from putrescine as well, while cadaverine, putreanine, and monoamines were ineffectual. Spermine had a similar effect on the enzymatic transfer of GalNAc to a protein core acceptor but had little or no effect on the enzymatic transfer of sialic acid to sialidase-treated ovine submaxillary mucin, galactose to N-acetylglucosamine, or fucose to sialidase-galactosidase-treated fetuin. Similar results were obtained with enzyme preparations prepared from canine submaxillary glands. Other polycationic compounds such as protamine, histone, and polylysine also stimulated enzymatic activity at suboptimal concentrations of mn²⁺.     

Alternative Routes for the Synthesis of 5-Aminolevulinic Acid in Maize Leaves : II. Formation from Glutamate

Intact plastids from greening maize (Zea mays L.) leaves converted [¹⁴C]glutamate and [¹⁴C]2-ketoglutarate (KG) to [¹⁴C]5-aminolevulinic acid (ALA). Glutamate appeared to be the immediate precursor of ALA, while KG was first converted to glutamate, as shown by the effect of various inhibitors of amino acid metabolism. Plastids from greening leaves contained markedly higher activity as compared with etioplasts or chloroplasts. The synthesis of ALA by intact plastids was light dependent. The enzyme system resides in the stroma of plastids or may be lightly bound to membranes. The solubilized system showed maximal activity around pH 7.9 and required Mg²⁺, ATP, and NADPH although dependence on the latter was not clear-cut. A relatively high level of activity could be extracted from etioplasts. Maximal activity was obtained from plastids of leaves which had been illuminated for 90 minutes, after which activity declined sharply. The enzyme system solubilized from plastids also catalyzed the conversion of putative glutamate 1-semialdehyde to ALA in a reaction which was not dependent on the addition of an amino donor.

The system in maize greatly resembled the one which had been reported from barley. It is suggested that this system is the one responsible for the biosynthesis of ALA destined for chlorophyll formation.

     

Light induction of phosphoenolpyruvate carboxylase in etiolated maize leaf tissue

An antibody for phosphoenolpyruvate carboxylase was used to isolate and to quantitate the enzyme from greening maize (cv. KOU 6) leaves. The increase in enzyme activity during greening was due to de novo synthesis, which was paralleled by increases in enzyme protein and incorporation of leucine. The light-induced activity was due to one specific isoenzyme. The action spectrum for enzyme synthesis had red and blue peaks.     

Modulation of δ-aminolevulinic acid dehydratase activity by the sorbitol-induced osmotic stress in maize leaf segments

Osmotic stress induced with 1 M sorbitol inhibited δ-aminolevulinic acid dehydratase (ALAD) and aminolevulinic acid (ALA) synthesizing activities in etiolated maize leaf segments during greening; the ALAD activity was inhibited to a greater extent than the ALA synthesis. When the leaves were exposed to light, the ALAD activity increased for the first 8 h, followed by a decrease observed at 16 and 24 h in both sorbitol-treated and untreated leaf tissues. The maximum inhibition of the enzyme activity was observed in the leaf segments incubated with sorbitol for 4 to 8 h. Glutamate increased the ALAD activity in the in vitro enzymatic preparations obtained from the sorbitol-treated leaf segments; sorbitol inhibited the ALAD activity in the preparations from both sorbitol-treated and untreated leaves. It was suggested that sorbitol-induced osmotic stress inhibits the enzyme activity by affecting the ALAD induction during greening and regulating the ALAD steady-state level of ALAD in leaf cells. The protective effect of glutamate on ALAD in the preparations from the sorbitol-treated leaves might be due to its stimulatory effect on the enzyme.     

Isolation and characterization of the hatching enzyme from the amphibian,Xenopus laevis

The proteolytic activity released at the time of Xenopus laevis embryo hatching, termed the hatching enzyme, was purified and characterized in terms of its physical and enzymatic properties. Using predominantly isoelectric focusing and preparative ultracentrifugation, the enzyme was purified 2200-fold over the starting crude hatching media. From disc gel electrophoretic experiments, the most highly purified form of the enzyme had two enzymatically active charge isomers present with molecular weights of 62,500. With time, the purified enzyme gave rise to a family of enzymatically active charge isomeric proteins. The enzymatic activity of hatching enzyme toward its ¹²⁵I-labeled natural substrate, the fertilization envelope, was optimal at pH 7.7 and was ionic strength dependent. The enzyme was inhibited by Zn²⁺ and by EDTA. From inhibition by the site-specific reagents diisopropylfluorophosphate and phenylmethylsulfonylfluoride, we concluded that the enzyme was of the serine protease type, although its inhibition by Zn²⁺ and EDTA prevents a clear and unequivocal classification of the protease. This enzyme is different from the hatching enzymes reported in fish and echinoderms, on the basis of size, but it is similar to that described in Rana chensinensis on the basis of size and specificity.     

Glycollate oxidase inhibition and its effect on photosynthesis and pigment formation in Hordeum vulgare

Investigations of the effect of 2-hydroxy-3-butynoic acid and its methyl ester on photosynthesis in Hordeum vulgare are reported. In the presence of either of these compounds the assimilation of ¹⁴CO₂ was greatly decreased. The labelling patterns showed massive accumulation of glycollate and greatly reduced incorporation into sucrose and other products of photosynthesis. The inhibition was specific for the S(+) enantiomers. In greening barely the S(+) enantiomers inhibited formation of chloroplast pigments, and this was paralleled by inhibition of glycollate oxidase. This was the only enzyme of the glycollate pathway whose activity was significantly decreased after inhibitor treatments. Of a range of metabolises tested, only supplementations with glycine and glutamate or glycine, serine and succinate fully restored greening.     

Effects of Light Intensity on Photosynthetic Carboxylative Phase Enzymes and Chlorophyll Synthesis in Greening Leaves of Hordeum vulgare L

The effects of various light intensities on in vivo increases in activities of phosphoriboisomerase, phosphoribulokinase and ribulose-1, 5-diP carboxylase and on synthesis of chlorophyll were studied in greening leaves of Hordeum vulgare L.

Each enzyme was already present in dark-grown plants, but further increases in activities required both a light treatment of the intact plant and a favorable temperature. The amount of enzymatic activity and chlorophyll developed was governed by light intensity.

Measured activities of phosphoriboisomerase and ribulose 1,5-diP carboxylase were highly correlated with synthesis of chlorophyll at all intensities studied. Measured activity of phosphoribulokinase was correlated with synthesis of chlorophyll only at saturating or near saturating light intensities. At decreasing light intensities the response curves of this enzyme differed from those of chlorophyll and of phosphoriboisomerase and ribulose-1, 5-diP carboxylase. A lag period of phosphoribulokinase increased with decreasing light intensity. After the lag period a rapid rate of increase occurred which did not level off during 48 hours of illumination. Thus, a different control mechanism may be operative in inducing increased activity of this enzyme.

     

Occurrence and some properties of carbonic anhydrases from legume root nodules

Carbonic anhydrase activity (hydration of CO₂ was found in homogenates of leaves (116–500 units.mg^?1 protein) and root nodules (27–255 units.mg^?1 protein) from 8 legume genera inoculated in each case with a host specific Rhizobium. No enzyme, or only trace amounts (2–7 units.mg^?1 protein), were detected in root extracts, The enzymatic activity was inhibited in all cases by azide and acetazolamide. The sizes of nodule and leaf carbonic anhydrases, estimated by gel filtration of partially purified preparations from Phaseolus vulgaris, were around 45 000 and 205 000 respectively. These enzymes also differed in sensitivity to inhibitors. More than 99% of the activity present in Vicia faba nodules was recovered as a soluble enzyme and only a trace was located in the isolated bacteroids.     

Involvement of xylem sap zeatin-O-glucoside in cucumber shoot greening

A fraction of xylem sap collected from squash (Cucurbita maxima) root and separated with gel filtration showed strong promoting activity in a greening bioassay using etiolated cucumber cotyledons. This activity decreased with waterlogging of the root. The promoting factor was purified with sequential gel-permeation, and reverse-phase and normal-phase column chromatographies. Zeatin-O-glucoside (ZOG) was identified as the promoting factor by monitoring liquid chromatography tandem mass spectrometry. Although the glycosyl conjugate of zeatin was thought to be inactive, ZOG had a promoting activity that was 100 times higher than those of zeatin and zeatin riboside between concentrations of 10^–9 to 10^–4 M in the greening bioassay. These results suggest that in some developmental stages, the conjugation of cytokinins with a sugar moiety, such as glucose, might be a key factor in the control of shoot greening by roots.     

Synthesis of Isocitrate Lyase in Sunflower Cotyledons during the Transition in Cotyledonary Microbody Function

Density-labeling with 10 millimolar K¹⁵NO₃/70% ²H₂O has been used to investigate isocitrate lyase synthesis during greening of sunflower (Helianthus annuus L.) cotyledons when the glyoxysomal enzyme activities sharply decline and the transition in cotyledonary microbody function occurs. A density shift of 0.0054 (kilograms per liter) was obtained for the profile of isocitrate lyase activity in the CsCl gradient with respect to the ¹H₂O control. Quantitative evaluation of the density-labeling data indicates that about 50% of the isocitrate lyase activity present towards the end of the transition stage in microbody function is due to enzyme molecules newly synthesized during this stage.     

Some Characteristics of Practical Relevance of the β -Galactosidase from Potential Probiotic Strains ofPropionibacterium acidipropionici

In the present study, factors influencing the synthesis and activity of β-galactosidase of two strains of Propionibacterium acidipropionici with some probiotic properties are described for the first time. The enzyme 6-phospho-β-D-galactosidase of the PEP-PTS system was not detected, suggesting that P. acidipropionici metabolize lactose only by using β-galactosidase. The highest enzymatic activities were obtained from cultures developed in a basal broth medium containing 1.0% sodium lactate or 0.25% lactose. Maximum β-galactosidase activity from cell-free extracts of the strains was obtained at pH 7.0 and 50°C, but a high activity was even detected at 37°C. The enzyme was competitively inhibited by lactose and activated by glucose and sodium lactate. The remaining activities after heating cell-free extracts up to 20 min at 60°C were 70% and 25% of untreated control activities for P. acidipropionici Q4 and CRL 1198, respectively. Cations like Mg²⁺, Mn²⁺, Li⁺, Na⁺, and K⁺ acted as stimulators of the β-galactosidase activity whereas Ca²⁺, Co²⁺, Ni²⁺, Hg²⁺ and Cu²⁺ showed inhibitory effect in different extent. These results suggest that the environmental conditions commonly present in the human's intestine may be adequate for the synthesis and activity of β-galactosidase from these strains of Propionibacterium. The enzyme resist the cooking temperature of Swiss-type cheeses in different extent depending on the strain tested and most of the cations present in milk stimulate the enzymatic activity. Our results suggest that a cheese would be an appropriate vehicle for delivery of β-galactosidase from propionibacteria to the host and efforts to develop a Swiss-type probiotic cheese for lactose intolerant persons should be done.     

THYMIDINE TRIPHOSPHATE SYNTHESIS IN TETRAHYMENA : I. Studies on Thymidine Kinase

The amount of thymidine-H³ converted to thymidine-H³ monophosphate in 30 min formed the basis for assays of thymidine kinase in cell extracts from Tetrahymena pyriformis. The optimal concentration of adenosine triphosphate is lower than that required by other cell types. Thymidine triphosphate does not exercise any feedback control of the enzyme. Other deoxyprimidine nucleotides were tested, but these also failed to exhibit any feedback inhibition. At suboptimal adenosine triphosphate levels, thymidine triphosphate and other deoxypyrimidine nucleotides stimulate the reaction, suggesting that these nucleotides may act either directly or indirectly as phosphate donors in the crude enzyme preparations. This possibility was affirmed when thymidine triphosphate and deoxycytidine triphosphate were shown to be capable of limited phosphorylation of thymidine. Comparison of enzymatic activities in logarithmically growing culture and stationary phase culture, in which nuclear DNA synthesis has virtually ceased, reveals no change in enzymatic activity. The results suggest that thymidine kinase is a constitutive enzyme in Tetrahymena.     

Environmental regulation of enzymes in the microbodies and mitochondria of dark-grown, greening, and light-grown Euglena graclis

Catalase activity is demonstrated histochemically in the microbodies of aerated cultures of Euglena gracilis strain Z grown on inorganic media supplemented with acetate or glucose. Although this enzyme can also be assayed photometrically in cell-free extracts of acetate-supplemented cells, it is below the level of detectability in extracts of glucose-supplemented cells, there being an order of magnitude fewer microbodies in the latter than the former. Even acetate-supplemented cultures (dark-grown, greening, or continuously light-grown) fail to exhibit detectable catalase activity when CO₂ is removed from the air by Ascarite.Negative results were obtained with histochemical techniques considered optimal for the demonstration of cytochrome oxidase; under other conditions, however, a KCN-sensitive enzyme was revealed in the mitochondrial matrix. This (unidentified) enzyme is first observed in mitochondria after 20–24 hr of greening, reaches a maximum intensity at about 48 hr, and becomes undetectable by 72 hr of greening. Poisoning of photosynthesis by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) results in loss of activity of this mitochondrial enzyme.     

Purification,properties and regulation of the level of bovine S-adenosylmethionine decarboxylase during lymphocyte mitogenesis

S-Adenosylmethionine decarboxylase was purified from the livers of calves treated with methylglyoxal bis (guanylhydrazone) to elevate the level of the enzyme. Purified bovine S-adenosylmethionine decarboxylase was similar in specific activity and subunit molecular weight (32 000) to the enzymes previously isolated from rat and mouse. The bovine liver enzyme immunologically crossreacted with S-adenosylmethionine decarboxylase from resting and mitogenically activated bovine lymphocytes. The rate of enzyme synthesis in activated lymphocytes was determined by labeling the cells with [³H]leucine and isolating the radioactive decarboxylase by affinity chromatography and sodium dodecyl sulfate gel electrophoresis. The rate of enzyme syntheis was increased 10-fold by 9 h after mitogen treatment, which accounts for the initial increase in cellular enzymatic. There was no further incraese in the rate of S-adenosylmethionine decarboxylase synthesis that correlated with a second elevation of activity occuring at approx. 24 h after mitogenic activation. It was concluded that the second increase in enzyme activity was due to lengthening the intracellular half-life of the enzyme by 2-fold.     

Apparent Catalase Synthesis in Sunflower Cotyledons during the Change in Microbody Function: A Mathematical Approach for the Quantitative Evaluation of Density-labeling Data

Density-labeling with 10 mm K¹⁵NO₃/70% ²H₂O has been used to investigate catalase synthesis in different developmental stages of sunflower (Helianthus annuus L.) cotyledons. A mathematical approach is introduced for the quantitative evaluation of the density-labeling data. The method allows, in the presence of preexisting enzyme activity, calculation of this synthesized activity (apparent enzyme synthesis) which results from the balance between actual enzyme synthesis and the degradation of newly synthesized enzyme at a given time. During greening of the cotyledons, when the catalase activity declines and the population of leaf peroxisomes is formed, the apparent catalase synthesis is lower than, or at best equal to, that occurring during a developmental stage when the leaf peroxisome population is established and catalase synthesis and degradation of total catalase are in equilibrium. This result suggests a formation, in fatty cotyledons, of the leaf peroxisomes by transformation of the glyoxysomes rather than by de novo synthesis.     

Partial purification and characterization of prostaglandin A isomerase from rabbit serum.

Prostaglandin A isomerase has been purified 120-fold from rabbit serum by the use of ammonium sulfate fractionation, isoelectric focusing, and Sephadex G-200 chromatography. The molecular weight of the enzyme was estimated to be 110,000 from the elution volume on Sephadex G-200. Prostaglandin A isomerase is a heterogeneous protein with respect to charge. This has been concluded from the spread of enzymatic activity over 1 pH unit after isoelectric focusing. The enzymatic activity is inhibited by N-ethylmaleimide but not by other sulfhydryl blocking agents. The K_m was determined to be 5 × 10^?5m.     

Structural and functional characterization of polyethylene terephthalate hydrolase from Ideonella sakaiensis

Polyethylene terephthalate (PET) hydrolase from Ideonella sakaiensis (IsPETase) can be used to degrade PET. In order to use IsPETase in industry, we studied the enzymatic activity of IsPETase in different conditions containing environmental and physicochemical factors commonly found in nature. We observed that salts and glycerol enhanced the enzymatic activity, while detergents and organic solvents reduced the enzymatic activity. IsPETase hydrolyzed p-nitrophenyl (p-NP) esters instead of naphthyl esters. To make IsPETase an enzyme capable of hydrolyzing naphthyl esters, site-directed mutagenesis was carried out based on the structural information provided by the crystal structure. We found that the IsPETase^S93M, IsPETase^W159F, and IsPETase^N241F mutants can hydrolyze naphthyl esters. IsPETase engineering can direct researchers to use this α/β-hydrolase protein scaffold to design enzymes that can hydrolyze a variety of polyesters.     

Purification and Properties of a Maltotetraose- and Maltotriose-Producing Amylase from Chloroflexus aurantiacus

A maltotetraose- and maltotriose-producing amylase which is stable at alkaline pHs and high temperatures was detected in the culture filtrate of a strain of Chloroflexus aurantiacus J-10-F1, a thermophilic, green, photosynthetic bacterium. The enzyme was purified to homogeneity, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by means of ultrafiltration, ammonium sulfate fractionation, and DEAE-cellulose, hydroxyapatite, and high-performance liquid chromatographies. The molecular mass of the purified enzyme was estimated to be about 210,000 Da. The isoelectric point of the enzyme was estimated to be 6.24 by polyacrylamide gel electrofocusing. The amylase was stable up to 55°C and at alkaline pHs of up to 12.0. The optimum pH and temperature of the enzyme activity were 7.5 and 71°C, respectively. Metal ions such as Hg²⁺, Zn²⁺, Cu²⁺, Mn²⁺, and Ni²⁺ strongly inhibited the enzyme activity. The enzyme activity was reactivated specifically by Ca²⁺ after the enzyme was treated with 1 mM EDTA. This enzyme could digest various kinds of raw-starch granules from corn, cassava, and potato. Both maltotetraose and maltotriose were formed as the main enzymatic products from soluble starch.