3-Methyleneoxindole and Plant Growth Regulation

The campound 3-methyleneoxindole was synthesized and tested in three “biological systems for auxin activity: Root initarion in pea stem currings, extension growth of etiolated pea stem segments and Avena first internodes. In none of the systems was found any effect — positive or negative of methyleneoxindole. It thus seems improbable that the compound participates in the growth regulation of these plants.     

Nitrate Reductase Activity: Phytochrome Mediation of Induction in Etiolated Peas

THE extractable activity of nitrate reductase from higher plant leaves is inducible by light and shows, under natural growth conditions, a pattern of diurnal variation¹. Studies on the nature of light involvement have generally used the green leaf as experimental material, implying that photosynthesis supports the induction process^1,2. We have examined the role of light for the induction of nitrate reductase activity in the etiolated terminal buds of field peas (Pisum arvense cv. Century). Treatments consisted of brief exposure of intact plants to broad bands of light, followed by a period in darkness before extraction for enzyme assay. These light treatments exclude the possibility of photosynthesis as a process contributing to induction. Under these conditions, induction is shown to be reversibly controlled by red and far red light, an effect ascribable to the pigment phytochrome.     

The Binding of Indole-3-acetic Acid and 3-Methyleneoxindole to Plant Macromolecules

Homogenates of pea (Pisum sativum L., var. Alaska) seedlings exposed to ¹⁴C-indole-3-acetic acid or ¹⁴C-3-methyleneoxindole, an oxidation product of indole-3-acetic acid, were extracted with phenol. In both cases 90% of the bound radioactivity was found associated with the protein fraction and 10% with the water-soluble, ethanol-insoluble fraction. The binding of radioactivity from ¹⁴C-indole-3-acetic acid is greatly reduced by the addition of unlabeled 3-methyleneoxindole as well as by chlorogenic acid, an inhibitor of the oxidation of indole-3-acetic acid to 3-methyleneoxindole. Chlorogenic acid does not inhibit the binding of ¹⁴C-3-methyleneoxindole. The labeled protein and water-soluble, ethanol-insoluble fractions of the phenol extract were treated with an excess of 2-mercaptoethanol. Independently of whether the seedlings had been exposed to ¹⁴C-indole-3-acetic acid or ¹⁴C-3-methyleneoxindole, the radioactivity was recovered from both fractions in the form of a 2-mercaptoethanol-3-methyleneoxindole adduct. These findings indicate that 3-methyleneoxindole is an intermediate in the binding of indole-3-acetic acid to macromolecules.     

3-Methyleneoxindole: an affinity label of glutathione S-transferase pi which targets tryptophan 38.

The compound 3-methyleneoxindole (MOI), a photooxidation product of the plant auxin indole-3-acetic acid, functions as an affinity label of the dimeric pi class glutathione S-transferase (GST) isolated from pig lung. MOI inactivates the enzyme to a limit of 14% activity. The k for inactivation by MOI is decreased 20-fold by S-hexylglutathione but only 2-fold by S-methylglutathione, suggesting that MOI does not react entirely within the glutathione site. The striking protection against inactivation provided by S-(hydroxyethyl)ethacrynic acid indicates that MOI reacts in the active site region involving both the glutathione and the xenobiotic substrate sites. Incorporation of [(3)H]MOI up to approximately 1 mol/mol of enzyme dimer concomitant with maximum inactivation suggests that there are interactions between subunits. Fractionation of the proteolytic digest of [(3)H]MOI-modified GST pi yielded Trp38 as the only labeled amino acid. The crystal structure of the human GST pi-ethacrynic acid complex (2GSS) shows that the indole of Trp38 is less than 4 A from ethacrynic acid. Similarly, MOI may bind in this substrate site. In contrast to its effect on the pi class GST, MOI inactivates much less rapidly and extensively alpha and mu class GSTs isolated from the rat. These results show that MOI reacts preferentially with GST pi. Such a compound may be useful in novel combination chemotherapy to enhance the efficacy of alkylating cancer drugs while minimizing toxic side effects.     

高等植物的3-羟基-3-甲基戊二酰辅酶A还原酶

介绍了植物3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)的结构和调控,并简略讨论了HMGR调控与植物类异戊二烯途径的关系.     

Bacterial Spoilage of Thawed Frozen Peas

The main groups of bacteria developing in peas were isolated on differential media. Leuconostoc mesenteroides and Streptococcus lactis predominated and smaller numbers of catalase positive 'coryneform'bacteria were also regularly present. Strep. cremoris , streptococci (group D), catalase positive cocci and Gram negative rods were less regularly isolated.
Bacterial spoilage of untreated peas was usually accompanied by the development of a yellow colour and a reduction in pH. In pure culture in peas leuconostocs and group N streptococci produced an acid pH while the catalase positive organisms produced alkali, sometimes with ammoniacal odours. In mixed cultures in peas the products of a leuconostoc and a 'coryneform'tended to neutralize each other. A leuconostoc and a 'coryneform'did not interfere with each others'growth when grown from similar inocula. The behaviour of streptococci is still being investigated. The type and rate of spoilage probably depends on the balance between ammonia production and acid production in the system.     

Occurrence of 3-Hydroxy-3-methyl-glutaryl Coenzyme A Reductase in Sweet Potato

Illumination of the dark-grown Euglena gracilis, both the wild-green type and a permanently bleached mutant, for 4 hr at 2,000 lux caused about 6-fold increase of the cellular content of total l-ascorbic acid. The increase was mainly due to an increase of reduced-form l-ascorbic acid. From the action spectrum only blue light was found to be effective for the increase. Darkening stopped the increase and reillumination started a renewed increase. The activity of l-gulono-γ-lactone dehydrogenase, catalyzing the last step of l-ascorbic acid biosynthesis, was also increased two fold by illumination for 2 hr, and was changed in parallel to that of the cellular content of l-ascorbic acid depending on the presence or absence of illumination. The augmentation of l-ascorbic acid formation was markedly inhibited by various inhibitors and uncouplers, but not by dichlorophenyldimethylurea. The results in sum suggest that the light-dependent increase of l-ascorbic acid formation in E. gracilis is not primarily associated with photosynthesis, but is apparently related to the adaptation of the dark-grown cells to the illuminated state.     

Subcellular Localization of IAA Oxidase in Peas

Indoleacetic acid (IAA) oxidase has been reported to be involved in plant growth because of its alleged role in the control of endogenous IAA levels. This purported role was reevaluated in terms of the properties and subcellular location of the enzyme in etiolated pea (Pisum sativum L. var. Alaska) epicotyls.     

Controlling Cholesterol Synthesis beyond 3-Hydroxy-3-methylglutaryl-CoA Reductase (HMGCR)

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGCR) is the target of the statins, important drugs that lower blood cholesterol levels and treat cardiovascular disease. Consequently, the regulation of HMGCR has been investigated in detail. However, this enzyme acts very early in the cholesterol synthesis pathway, with ∼20 subsequent enzymes needed to produce cholesterol. How they are regulated is largely unexplored territory, but there is growing evidence that enzymes beyond HMGCR serve as flux-controlling points. Here, we introduce some of the known regulatory mechanisms affecting enzymes beyond HMGCR and highlight the need to further investigate their control.     

Mechanism of Gibberellin-Dependent Stem Elongation in Peas

Stem elongation in peas (Pisum sativum L.) is under partial control by gibberellins, yet the mechanism of such control is uncertain. In this study, we examined the cellular and physical properties that govern stem elongation, to determine how gibberellins influence pea stem growth. Stem elongation of etiolated seedlings was retarded with uniconozol, a gibberellin synthesis inhibitor, and the growth retardation was reversed by exogenous gibberellin. Using the pressure probe and vapor pressure osmometry, we found little effect of uniconozol and gibberellin on cell turgor pressure or osmotic pressure. In contrast, these treatments had major effects on in vivo stress relaxation, measured by turgor relaxation and pressure-block techniques. Uniconozol-treated plants exhibited reduced wall relaxation (both initial rate and total amount). The results show that growth retardation is effected via a reduction in the wall yield coefficient and an increase in the yield threshold. These effects were largely reversed by exogenous gibberellin. When we measured the mechanical characteristics of the wall by stress/strain (Instron) analysis, we found only minor effects of uniconozol and gibberellin on the plastic compliance. This observation indicates that these agents did not alter wall expansion through effects on the mechanical (viscoelastic) properties of the wall. Our results suggest that wall expansion in peas is better viewed as a chemorheological, rather than a viscoelastic, process.     

Co-sedimentation of Actin, Tubulin andMembranes in the Cytoskeleton Fractions from Peas and Mouse 3T3 Cells

Pea stem tissue (Pisum sativum L. var. Alaska) was homogenizedin a recently-developed cytoskeleton-stabilizing buffer, CSB,(Abeand Da vies, 1991) and homogenates electrophoresed and blottedon to membranes. Blots probed individually withantibodies toactin, alpha-tubulin, and beta-tubulin, revealed bands withapparent molecular weights of 42, 46, and 48–50 kDa,respectively.Blots probed with all three antibodies simultaneously revealedall three bands which could be distinguished in thesame lane.Homogenates of mouse 3T3 cells yielded an actin band at about42 kDa, but both alpha- and beta-tubulin appeared atabout 50kDa and thus could not be distinguished on blots probed simultaneously.This ‘triple-blotting technique’ was, therefore,suitablefor pea tissue, but not for mouse tissue. In pea tissue, sedimentabletubulin and actin were found maximally in the 4000 xg pelletand less in successive 15000 and l00000xg pellets. Both EGTAand Mg²⁺ which had been found earlier to beessential for stabilityof the actin cytoskeleton as revealed by fluorescence microscopy,were essential for co-sedimentation of actinand tubulin. Incontrast to the results with pea stems, only the actin componentof the cytoskeleton could be isolated from mouse 3T3 cells usingCSB. Pea tissue was homogenized in CSB without PTE and the resultingcytoskeletal pellets resuspended in actin- or tubulin-solubilizingbuffers with and without PTE. In the absence of PTE, the bufferssolubilized their appropriate cytoskeletal protein, but littleof the other protein, while in the presence of PTE both proteinswere quite effectively solubilized by both buffers. Incontrast,in CSB with or without PTE, both proteins remained in the sedimentablefraction. These results, taken together withother evidence,indicate that microtubules, as well as microfilaments are importantcomponents of the sedimentable cytoskeletonfraction of peasand that the membrane system is intimately involved in organizationof the cytoskeleton in peas. Key words: Actin, tubulin, membranes, detergent, Ca²⁺, Mg²⁺, cytoskeleton     

The Induction of Fibre Differentiation in Peas

The problem studied in this work was that of the internal controlof the formation of strands of fibres in Pisum sativum. It isshown that fibre differentiation is dependent on stimuli originatingin young leaf primordia. Removing these primordia early enoughprevents fibre differentiation; changing the position of theleaves experimentally changes the position of the fibres aswell. It was demonstrated that some stimuli for fibre differentiationmust flow through the strands at the time they differentiate.The evidence for this flow is in experiments concerning theability of very young fibre strands to regenerate after cutsas well as in experiments concerning their pattern of joining.The stimuli which originate in the leaves and control the differentiationof fibres and xylem are shown to differ in at least one component:auxin does not cause fibre differentiation and no surgical treatments,carried out on very young tissues, caused the replacement ofpart of a strand of fibres byor xylem or vice versa.     

Rat Brain 3-Hydroxy-3-Methylglutaryl-CoA Reductase: Subcellular Distribution and Cold Lability

Abstract: Data are provided indicating that the rat brain 3-hydroxy-3-methyl-glutaryl-CoA reductase is similar to the enzyme from other tissues as far as diurnal rythmicity, cold lability and half-life measurements at 0°C are concerned. The enzyme activity in the brain decreased with age of the animals. Subcellular fractionation studies demonstrate that while 77% of the activity was associated with the microsomal fraction, 19% of the enzyme activity was recovered in the mitochondrial fraction. The possible function of such a mitochondrially located 3-hydroxy-3-methylglutaryl-CoA reductase in rat brain is discussed.     

Identification of Pig Brain Aldehyde Reductases with the High-Km Aldehyde Reductase, the Low-Km Aldehyde Reductase and Aldose Reductase, Carbonyl Reductase, and Succinic Semialdehyde Reductase

Four NADPH-dependent aldehyde reductases (ALRs) isolated from pig brain have been characterized with respect to substrate specificity, inhibition by drugs, and immunological criteria. The major enzyme, ALR1, is identical in these respects with the high-Km aldehyde reductase, glucuronate reductase, and tissue-specific, e.g., pig kidney aldehyde reductase. A second enzyme, ALR2, is identical with the low-Km aldehyde reductase and aldose reductase. The third enzyme, ALR3, is carbonyl reductase and has several features in common with prostaglandin-9-ketoreductase and xenobiotic ketoreductase. The fourth enzyme, unlike the other three which are monomeric, is a dimeric succinic semialdehyde reductase. All four of these enzymes are capable of reducing aldehydes derived from the biogenic amines. However, from a consideration of their substrate specificities and the relevant Km and Vmax values, it is likely that it is ALR2 which plays a primary role in biogenic aldehyde metabolism. Both ALR1 and ALR2 may be involved in the reduction of isocorticosteroids. Despite its capacity to reduce ketones, ALR3 is primarily an aldehyde reductase, but clues as to its physiological role in brain cannot be discerned from its substrate specificity. The capacity of succinic semialdehyde reductase to reduce succinic semialdehyde better than any other substrate shows that this reductase is aptly named and suggests that its primary role is the maintenance in brain of physiological levels of gamma-hydroxybutyrate.     

Malonic Semialdehyde Reductase,Succinic Semialdehyde Reductase,and Succinyl-Coenzyme A Reductase from Metallosphaera sedula: Enzymes of the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in Sulfolobales

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates during autotrophic CO₂ fixation in various members of the Crenarchaea. In this cycle, as determined using Metallosphaera sedula, malonyl-coenzyme A (malonyl-CoA) and succinyl-CoA are reductively converted via their semialdehydes to the corresponding alcohols 3-hydroxypropionate and 4-hydroxybutyrate. Here three missing oxidoreductases of this cycle were purified from M. sedula and studied. Malonic semialdehyde reductase, a member of the 3-hydroxyacyl-CoA dehydrogenase family, reduces malonic semialdehyde with NADPH to 3-hydroxypropionate. The latter compound is converted via propionyl-CoA to succinyl-CoA. Succinyl-CoA reduction to succinic semialdehyde is catalyzed by malonyl-CoA/succinyl-CoA reductase, a promiscuous NADPH-dependent enzyme that is a paralogue of aspartate semialdehyde dehydrogenase. Succinic semialdehyde is then reduced with NADPH to 4-hydroxybutyrate by succinic semialdehyde reductase, an enzyme belonging to the Zn-dependent alcohol dehydrogenase family. Genes highly similar to the Metallosphaera genes were found in other members of the Sulfolobales. Only distantly related genes were found in the genomes of autotrophic marine Crenarchaeota that may use a similar cycle in autotrophic carbon fixation.The thermoacidophilic autotrophic crenarchaeum Metallosphaera sedula uses a 3-hydroxypropionate/4-hydroxybutyrate cycle for CO₂ fixation (9, 28, 29, 35) (Fig. ​(Fig.1).1). A similar cycle may operate in other autotrophic members of the Sulfolobales (31) and in mesophilic marine group I Crenarchaea (Cenarchaeum sp., Nitrosopumilus sp.). This cycle uses elements of the 3-hydroxypropionate cycle that was originally discovered in the phototrophic bacterium Chloroflexus aurantiacus (15, 22-25, 41, 42). It involves the carboxylation of acetyl coenzyme A (acetyl-CoA) to malonyl-CoA by a biotin-dependent acetyl-CoA carboxylase (12, 29). The carboxylation product is reduced to malonic semialdehyde by malonyl-CoA reductase (1). Malonic semialdehyde is further reduced to 3-hydroxypropionate, the characteristic intermediate of the pathway (9, 31, 35). 3-Hydroxypropionate is further reductively converted to propionyl-CoA (3), which is carboxylated to (S)-methylmalonyl-CoA by propionyl-CoA carboxylase. Only one copy of the genes encoding the acetyl-CoA/propionyl-CoA carboxylase subunits is present in most Archaea, indicating that this enzyme is a promiscuous enzyme that acts on both acetyl-CoA and propionyl-CoA (12, 29). (S)-Methylmalonyl-CoA is isomerized to (R)-methylmalonyl-CoA, which is followed by carbon rearrangement to succinyl-CoA catalyzed by coenzyme B₁₂-dependent methylmalonyl-CoA mutase.Open in a separate windowFIG. 1.Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle in M. sedula and other autotrophic Sulfolobales. Enzymes: 1, acetyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonate semialdehyde reductase (NADPH); 4, 3-hydroxypropionate-CoA ligase (AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, propionyl-CoA carboxylase, identical to acetyl-CoA carboxylase; 8, (S)-methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH), identical to malonyl-CoA reductase; 11, succinic semialdehyde reductase (NADPH); 12, 4-hydroxybutyrate-CoA ligase (AMP forming); 13, 4-hydroxybutyryl-CoA dehydratase; 14, crotonyl-CoA hydratase; 15, (S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺); 16, acetoacetyl-CoA β-ketothiolase. The highlighted steps are catalyzed by the enzymes studied here.Succinyl-CoA is converted via succinic semialdehyde and 4-hydroxybutyrate to two molecules of acetyl-CoA (9), thus regenerating the starting CO₂ acceptor molecule and releasing another acetyl-CoA molecule for biosynthesis. Hence, the 3-hydroxypropionate/4-hydroxybutyrate cycle (Fig. ​(Fig.1)1) can be divided into two parts. The first part transforms one acetyl-CoA molecule and two bicarbonate molecules into succinyl-CoA (Fig. ​(Fig.1,1, steps 1 to 9), and the second part converts succinyl-CoA to two acetyl-CoA molecules (Fig. ​(Fig.1,1, steps 10 to 16).The second part of the autotrophic cycle also occurs in the dicarboxylate/4-hydroxybutyrate cycle, which operates in autotrophic CO₂ fixation in Desulfurococcales and Thermoproteales (Crenarchaea) (27, 37), raising the question of whether the enzymes in these two lineages have common roots (37). The first part of the cycle also occurs in the 3-hydroxypropionate cycle for autotrophic CO₂ fixation in Chloroflexus aurantiacus and a few related green nonsulfur phototrophic bacteria (19, 22, 23, 32, 49).The two-step reduction of malonyl-CoA to 3-hydroxpropionate in Chloroflexus is catalyzed by a single bifunctional 300-kDa enzyme (30). The M. sedula malonyl-CoA reductase is completely unrelated and forms only malonic semialdehyde (1), and the enzyme catalyzing the second malonic semialdehyde reduction step that forms 3-hydroxypropionate is unknown. In the second part of the 3-hydroxypropionate/4-hydroxybutyrate cycle a similar reduction of succinyl-CoA via succinic semialdehyde to 4-hydroxybutyrate takes place. The enzymes responsible for these reactions also have not been characterized.In this work we purified the enzymes malonic semialdehyde reductase, succinyl-CoA reductase, and succinic semialdehyde reductase from M. sedula. The genes coding for these enzymes were identified in the genome, and recombinant proteins were studied in some detail. Interestingly, succinyl-CoA reductase turned out to be identical to malonyl-CoA reductase. We also show here that enzymes that are highly similar to succinyl-CoA reductase in Thermoproteus neutrophilus do not function as succinyl-CoA reductases in M. sedula.     

Isolation of Neutral Growth Inhibitors in Dwarf Peas

From seedlings of dwarf pea (Pisum sativum L., var. Progress No. 9) grown under red light, three neutral growth inhibitors were isolated which interfered with the responses of these plants to GA₃. The compounds were identified as β-sitosterol, α-stearoyl glycerol and pisatin, of which the glyceride produced remarkable inhibition when applied to terminal buds. Though its anti-gibberellin activity was not very strong, pisatin produced inhibition of the straight-growth of Avena coleoptile segments caused by IAA.     

Starch Gel Electrophoresis of Plant NADH-Nitrate Reductase and Nitrite Reductase

Isozymes of both nitrate reductase (NR) and nitrite reductase(NiR) have been found in plant tissues, mainly after partialpurification. We have used starch gel electrophoresis to examineboth NR and NiR in crude extracts. Only one NR and one NiR enzymewere found in wheat tissues and no difference in mobilitiescould be detected between root and leaf enzymes. It was confirmedthat some tissues of corn have two NiR isozymes.