Effect of GA3, kinetin and indole acetic acid on carbohydrate metabolism in chickpea seedlings germinating under water stress

Enhanced amylase activity was observed during a 7-day-growth period in the cotyledons of PEG imposed water stressed chickpea seedlings grown in the presence of GA₃ and kinetin, when compared with stressed seedlings. During the first 5 days of seedling growth, the seedlings growing under water deficit conditions as well as those growing in the presence of PGRs had a higher amylase activity in shoots than that of control seedlings. Neither GA₃ nor kinetin increased the amylase activity of roots whereas IAA reduced root amylase activity. Activity of acid and alkaline invertases was maximum in shoots and at a minimum in cotyledons. Compared with alkaline invertase, acid invertase activity was higher in all the tissues. The reduced acid and alkaline invertase activities in shoots of stressed seedlings were enhanced by GA₃ and kinetin. Roots of stressed seedlings had higher alkaline invertase activity and GA₃ and IAA helped in bringing the level near to those in the controls. GA₃ and kinetin increased the sucrose synthase (SS) and sucrose phosphate synthase (SPS) activities in cotyledons of stressed seedlings, whereas they brought the elevated level of SPS of stressed roots to near normal level. The higher level of reducing sugars in the shoots of GA₃ and kinetin treated stressed seedlings could be due to the high acid invertase activity observed in the shoots, and the high level of bound fructose in the cotyledons of stressed seedlings could be due to the high activity of SPS in this tissue.     

Substrate inhibition of maize endosperm sucrose synthase by fructose and its interaction with glucose inhibition

Sucrose synthase (EC 2.4.1.13) was purified to homogeneity from developing maize (Zea mays L.) endosperm. Substrate saturation and inhibitor kinetics were examined for the sucrose synthase reaction. The K_m-values for fructose and uridine diphosphate glucose (UDPGlc) were estimated to be 7.8 mM and 76 μM, respectively. Fructose concentrations over 20 mM inhibited sucrose synthase in an uncompetitive manner with respect to UDPGlc. Glucose was also found to be an uncompetitive inhibitor with respect to both fructose and UDPGlc. At inhibitory concentrations of fructose, the apparent K_i for glucose increased linearly with increasing fructose concentration. The results suggest an ordered kinetic mechanism for sucrose synthase where UDPGlc binds first and UDP dissociates last. Fructose and glucose both inhibit by binding to the enzyme-UDP complex. Fructose and glucose, which are present in maize endosperm as the products of invertase, could inhibit sucrose synthase, especially in basal regions of the kernel where hexosesmay accumulate.     

The resting metabolism of dodder seeds

Extracts of mature seeds of Cuscuta reflexa were examined for any deficiency in key enzymes. The activities of malate dehydrogenase, β-amylase and fructose 1,6-diphosphate aldolase exceeded 5.0 μmol substrate/min/g, while those of starch phosphorylase, α-amylase, acid phosphatase, phosphogluconate dehydrogenase (decarboxylating), aspartate aminotransferase, glucose 6-phosphate dehydrogenase, fructose 1,6-diphosphatase and alanine aminotransferase fell within the range 1 to 5 μmol/min/g and hexokinase, isocitrate dehydrogenase and alkaline phosphatase were below 1 μmol substrate/min/g seed powder. No activity of the following were found: acid invertase, alkaline invertase, phytase and glutamate dehydrogenase. Some of these observations were made also for seeds of Cuscuta campestris and Cuscuta indicora.     

Control of the hexose content of potato tubers

The amounts of glucose and fructose in a range of harvested tubers of Solanum tuberosum were compared with the labelling of these hexoses by [U-¹⁴C]sucrose supplied to the tubers. Hexose content varied. Fructose was more heavily labelled than glucose. There was no correlation between the amounts of glucose and fructose in the tuber and their labelling. The maximum catalytic activities of α-glucan phosphorylase, acid invertase, alkaline invertase, sucrose synthase, α-amylase and β-amylase in tubers stored for 17 weeks at 5° and at 10° were estimated. The values showed no clear correlation with hexose content, but provided sound evidence that starch breakdown was phosphorolytic. It is suggested that the amounts of glucose and fructose in mature harvested tubers may be determined more by the partitioning of the translocated sucrose during the development of the tubers than by the metabolism of the harvested tuber.     

Alkaline β-fructofuranosidases of tuberous roots: Possible physiological function

Summary Alkaline invertase of roots of carrot (Daucus carota L.) did not hydrolyze raffinose while the acid invertase from the same tissue showed with this sugar ca. 60% of the activity found with sucrose. The activity of the two invertases was inhibited by fructose to a different extent, the K _i value being ca. 4×10^–2 M and 3×10^–1M, respectively, for the alkaline and the acid invertases from the roots of both carrot and turnip (Brassica rapa L.). It is proposed that fructose inhibition of acid invertase is of no physiological significance but that, in contrast, hexoses might regulate the activity of alkaline invertase.Comparing several species and cultivars, it was found that the content of reducing sugars and the activity of alkaline invertase of mature tuberous roots showed a positive correlation. This indicates that alkaline invertase may participate in the regulation of the hexose level of the cell, as was previously suggested for sugar-cane. A scheme is presented which proposes a way of participation of alkaline invertase in such a regulation, assuming that this enzyme is located in the cytoplasm and acid invertase is membrane-bound and mainly located at the cell surface.     

Reciprocal regulation of fructose 1,6-bisphosphatase and phosphofructokinase by fructose 2,6-bisphosphate in swine kidney

The effect of fructose 2,6-P₂, AMP and substrates on the coordinate inhibition of FBPase and activation of PFK in swine kidney has been examined. Fructose 2,6-P₂ inhibits the activity of FBPase and stimulates the activity of PFK in the presence of inhibitory concentrations of ATP. Under similar conditions 2.2 μM fructose 2,6-P₂ was required for 50% inhibition of FBPase and 0.04 μM fructose 2,6-P₂ restored 50% of the activity of PFK. Fructose 2,6-P₂ also enhanced the allosteric activation of PFK by AMP and it increased the extent of inhibition of FBPase by AMP. Fructose 2,6-P₂, AMP and fructose 6-P act cooperatively to stimulate the activity of PFK whereas the same latter two effectors and fructose 1,6-P₂ inhibit the activity of FBPase. Taken collectively, these results suggest that an increase in the intracellular level of fructose 2,6-P₂ during gluconeogenesis could effectively overcome the inhibition of PFK by ATP and simulataneously inactivate FBPase. When the level of fructose 2,6-P₂ is low, a glycolytic state would be restored, since under these conditions PFK would be inhibited by ATP and FBPase would be active.     

Effect of kinetin (6-furfuryl aminopurine) on changes in activities of some enzymes of lipid-carbohydrate metabolism in relation to growth of isolated cotyledons of vegetable marrow (Cucurbita pepo L.)

Of the 0–35 ppm concentration employed, kinetin (6-furfurylaminopurine) at 25 ppm resulted in the maximum increase in size and fresh weight of excised cotyledons of vegetable marrow (Cucurbita pepo L.). The lipolysis of total lipids and triglycerides increased on kinetin treatment. The content of reducing sugars rose markedly and that of sucrose reduced drastically with kinetin treatment. No lipase activity could be detected in the control. The activity of this enzyme was always higher with kinetin treatment alone as compared to the combined action of kinetin and detergent. The activities of isocitrate lyase and invertase were maximum at the 3rd day of incubation and even more pronounced with kinetin treatment. The activities of sucrose phosphate synthase paralleled with the amount of sucrose.     

番茄叶片糖与转化酶的日变化研究

为探讨转化酶在叶片糖分含量和光合作用日变化中的作用,测定了番茄(Lycopersicon esculentumL.)叶片光合速率、转化酶活性、淀粉、蔗糖、葡萄糖和果糖含量,并分别分析了12:00前后它们间的相关关系.结果表明:番茄日间叶片净光合速率在10:00和16:00出现一大一小两个高峰;6:00~18:00叶片中淀粉和果糖呈现持续升高,而蔗糖和葡萄糖为先升后降趋势;光合速率同叶片蔗糖含量和胞质转化酶活性存在高度正相关,淀粉和果糖含量同光合速率未表现出显著相关性.由此可知,胞质转化酶在蔗糖代谢方面有明显的作用;果糖可能是通过抑制胞壁转化酶活性,促进了蔗糖外运.     

Expression of fructokinase isoforms in the sugarcane culm

Two isoforms of fructokinase (FRK), FRK1 and FRK2, are present in sugarcane (Saccharum spp. var N19) internodal tissue. Both isoforms are highly specific for fructose as the hexose substrate. FRK2 is inhibited by fructose concentrations exceeding 0.1 mM and 50% inhibition is attained at 230 μM (K_i (Fru) = 160 μM), while FRK1 activity is not negatively affected even at 1.0 mM fructose. The ratio of FRK2 to FRK1 activity is dependent on the developmental stage of the tissue. FRK1 appears to be the isoform that is preferentially expressed in mature tissue. Total FRK activity decreases during tissue maturation. This is the result of changes in expression of the isoforms and not inactivation of existing protein. A mathematical method that allows accurate estimation of the activities of the two isoforms of FRK in crude sugarcane extracts is presented.     

Proteinaceous Inhibitor Versus Fructose as Modulators of Pteris deflexa Invertase Activity

An acid invertase from the fern Pteris deflexa Link was purified and the effect of reaction products on enzyme activity was studied. Fructose and glucose were competitive and non-competitive inhibitors of the enzyme, respectively. Since proteins suppressed glucose and fructose inhibition of the enzyme, an invertase modulation by reaction products is unlikely; nevertheless, an invertase proteinaceous inhibitor previously reported could have a role in this respect. The purified enzyme was an heterodimer M r 90,000 Daltons composed of subunits of 66,000 and 30,000 Daltons. The enzyme had β -fructofuranosidase activity and hydrolyzed mainly sucrose but also raffinose and stachyose, with K m of 3.22, 10.80 and 38.50 mM, respectively. Invertase activity with an optimum pH at 5.0 was present in almost every leaf fern tissue. Pinnas (sporophyll leaflets) had the higher enzyme levels. Invertase histochemical and immunochemical localization studies showed the enzyme mainly in phloem cells. Epidermis, collenchyma and parenchyma cells also showed invertase protein.     

Gibberellin A3 reverses the effect of salt stress in chickpea (Cicer arietinum L.) seedlings by enhancing amylase activity and mobilization of starch in cotyledons

The percentage germination of chickpea seeds (Cicer arietinum L.cv. PBG-1) gradually decreased with increasing concentration of NaCl in the growth medium and was completely inhibited with 200 mM NaCl. In the presence of 75 mM NaCl, only 51% of the seeds germinated. Gibberellic acid (GA3) and kinetin at 6 µM concentration induced the maximum increase in % germination and seedling growth under salt stress. However, IAA further inhibited both the germination and growth of stressed seedlings. The reduction in amylase activity in cotyledons of stressed seedlings was partially reversed with GA3 and kinetin whereas IAA did not show any positive effect. GA3 was more effective than kinetin in enhancing the reduced germination and seedling growth of chickpea seeds along with amylase activity in cotyledons under NaCl induced saline conditions. The reduced uptake of radiolabelled 14C sucrose by cotyledons and its reduced distribution in the shoots and roots of stressed seedlings was increased with addition of GA3 in the medium. Cotyledonary amylase was separated into amylase 1 and amylase 2 by sephadex G 150 column chromatography. The reduced activities of both amylase 1 and amylase 2 in cotyledons under salt stress was returned to near normal levels with GA3 and there was also an increase in starch utilization, resulting in its lower concentration in cotyledons of GA3-supplemented stressed cotyledons.     

Purification and properties of a neutral invertase from the roots of Cichorium intybus

Multiple activity peaks of neutral invertase (EC 3.2.1.26) were found in chicory roots ( Cichorium intybus L. var. foliosum cv. Flash). The main activity peak was purified by a combination of anion-exchange chromatography, hydrophobic interaction chromatography, chromatofocusing and gel filtration. This protocol produced a 77-fold purification and a specific activity of 1.6 μmol (mg protein)⁻¹ min⁻¹. The mass of the enzyme was 260 kDa as estimated by gel filtration and 65 kDa on SDS-PAGE. Optimal activity was found between pH 7 and 7.5. The purified enzyme exhibited hyperbolic saturation kinetics with a K_m between 10 and 20 mM for sucrose. No other products than glucose and fructose could be detected. Raffinose was hydrolyzed at a rate of 2.4% relative to sucrose whereas the enzyme did not hydrolyze maltose, cellobiose, trehalose, 1-kestose, 1.1-nystose or inulin. Neutral invertase activity was completely inhibited by HgCl₂ and AgNO₃ and partially inhibited by CoCl₂, and ZnSO₄ (1 mM). Pyridoxal phosphate (K_i≅ 500 μ M ), Tris (K_i≅ 1.2 m M ), glucose and fructose (K_i≅ 16 m M ) were strong inhibitors of the enzyme. Fructose and Tris behaved as competitive inhibitors. A possible role for the enzyme's activity in vivo is discussed.     

Changes in enzyme activity during elongation and tuberization of stolons of Solanum tuberosum L. cultured in vitro

Isolated stolons of Solanum tuberosam L. were cultured in vitroin the presence of kinetin, which induced tuber initiation orgibberellic acid which inhibited initiation. Progressive changes in enzyme activity, at the locus of tuberinitiation, were monitored at specified intervals. In the presenceof kinetin soluble invertase activity was decreased with timewhereas gibberellic acid (GA) evoked substantial increases inactivity. Acid phosphatase activity was enhanced by GA but changedonly slightly under tuber inducing conditions. Enzymic hydrolysisof glucose-6-phosphate and fructose-6-phosphate decreased duringthe course of tuber induction but increased in the presenceof GA. In contrast hydrolysis of 3'AMP was stimulated undertuber inducing conditions. GA evoked substantial increases in peroxidase activity duringthe initial stages of incubation while under tuber inducingconditions increased activity was only observed after 5 days.Substantially higher levels of IAA oxidase activity were associatedwith tuber initiation. RNase activity decreased with time undertuber inducing conditions but showed an initial stimulationin the presence of GA. These results are discussed with referenceto the role of these enzymes in carbohydrate metabolism andthe regulation of hormone levels. (Received February 29, 1972; )     

Synthesis, biological evaluation and structure-activity relationships of glycyrrhetinic acid derivatives as novel anti-hepatitis B virus agents

Fifty-seven derivatives of glycyrrhetinic acid (GA) were synthesized, and their anti-hepatitis B virus (HBV) activity was evaluated in HepG 2.2.15 cells. Among them, sixteen compounds showed greater anti-HBV activity than GA, especially, compounds 29, 32, 35, 41 exhibited significantly inhibitory activities against HBV DNA replication with IC(50) values of 5.71, 5.36, 8.90 and 9.08 μM, respectively. The structure-activity relationships (SARs) of GA derivatives were discussed for exploring novel anti-HBV agents.     

Glycyrrhetinic acid and its analogs: A new class of antifilarial agents

Although a number of chemicals have been isolated from Glycyrrhiza glabra, only a few have been evaluated for their biological significance. As part of our drug discovery program for antifilarial agents from Indian medicinal plants, the roots of G. glabra were chemically investigated, which resulted in the isolation and characterization of an antifilarial agent, glycyrrhetinic acid (GA, 1a) effective against microfilariae (mf) in vitro (LC100: 12.5 μM; IC₅₀: 1.20 μM), but was inactive against adult worms. Further, GA (1a) was converted into six analogs (2a–7a) and their antifilarial potential was evaluated by studying in vitro motility and MTT reduction assays employing mf and adult worms of Brugia malayi. The results showed that out of six GA analogs, the benzyl amide analog (6a) killed adults and mf at 25 and 50 μM concentration, respectively, and inhibited 49% MTT reduction potential of the adult parasites. The IC₅₀ values were found to be 8.8 and 2.2 μM for adults and mf, respectively. The SI of the compound was >60. On the other hand the octylamide analog (7a) required much higher concentration to adversely affect the parasites. Finally, both active amide analogs (6a and 7a) were in vivo evaluated using B. malayi-jird model, which showed that analog 6a possesses promising macrofilaricidal activity at 100 mg/kg, s.c. ×5 days and around 40% of the treated animals showed calcified masses of worm fragments in peritoneal cavity of the animals. To the best of our knowledge this is the first ever report on the antifilarial potential of GA analogs. Further work on optimization of the antifilarial lead is under progress.     

宁夏枸杞果实糖积累和蔗糖代谢相关酶活性的关系

通过对枸杞果实发育过程中果实生长模式、蔗糖、果糖、葡萄糖和淀粉含量及糖代谢相关酶活性的测定,研究了宁夏枸杞果实生长发育过程中糖的代谢积累与相关酶活性的关系.结果表明:(1)宁夏枸杞果实发育呈双S"曲线,果实主要以积累己糖为主.(2)蔗糖磷酸合成酶(SPS)活性在果实发育初期处于下降的趋势,在花后19d开始上升,果实转色后又逐渐下降;蔗糖合成酶(SS)活性总体表现为SS分解方向的活性大于SS合成方向的活性,说明枸杞果实发育过程中,SS的活性主要以分解方向的为主;酸性转化酶(AI)和中性转化酶(NI)的活性随果实发育呈上升趋势,但在果实成熟后期有所下降,且AI和NI活性高于合成酶类的活性,较高的转化酶类活性促进了果实内部己糖的积累.(3)在枸杞果实生长发育中,葡萄糖和果糖含量与AI和NI均呈极显著正相关,而与其它酶不具有相关性.说明AI和NI在宁夏枸杞果实的糖代谢中起着主要的调控作用.     

Ovarian monosaccharides (glucose and fructose): hormonal effects and their role in final oocyte maturation and egg quality in catfish Heteropneustes fossilis, Bloch

In the catfish H. fossilis, ovarian glucose and fructose levels showed significant annual variations that could be correlated with the gonado-somatic index. The concentration of the sugars, which was low in resting phase of gonad increased with the onset of oogenesis, reaching the peak in prespawning (June) phase. Glucose is the predominant monosaccharide in the late oogenesis, while fructose in early oogenesis and resting phase. Administration of a single injection of HCG (100 IU/fish) decreased glucose at 4 hr but increased fructose (6 hr) in the resting phase. In the preparatory phase (active oogenesis), fructose decreased significantly at 4 hr and glucose at 6 and 12 hr. In the prespawning phase, HCG administration induced a significant decrease in glucose level at 2 hr but increased it significantly at 16 and 18 hr. Fructose concentration, on the other hand, decreased > or = 4 hr. The involvement of different energy substrates (glucose, fructose, 2-deoxyglucose and pyruvate) on final oocyte maturation and viability of eggs were demonstrated in an in vitro study, employing incubation of mature oocytes in medium containing one or the other energy substrates. Glucose at 4 and 8 mM concentrations increased the viability of eggs without affecting germinal vesicle break down (GVBD) and at high concentrations (16 and 32 mM) marginally increased GVBD but decreased viability. Pyruvate at high concentrations (> or = 8 mM) produced a positive effect on both GVBD and viability. Fructose and 2-deoxyglucose did not elicit any effect on these parameters. The results suggest that glucose has an active role in catfish oogenesis and maintaining egg quality and HCG influences monosaccharide metabolism in a season-dependent manner.     

In vitro propagation of Caralluma tuberculata and evaluation of antioxidant potential

Present study describes rapid in vitro propagation of Caralluma tuberculata, a traditional medicinal plant, and antioxidant potential of calli and plants extracts. The highest callus induction rate (93.3%) with maximum weight of calli 5.2 g was achieved from shoot tip explants on MS medium supplemented with 9.04 μM 2,4-D and 4.44 μM BA. The maximum shoot induction rate (71.1%) with mean number of shoots 3.66 ± 1.53 and 4.6 cm average shoot length was observed on 13.32 μM BA, 4.52 μM 2,4-D and 2.89 μM GA3 appended in MS medium. The developed shoots were best rooted in the presence of 5.07 μM IAA with 3.0 ± 0.15 roots per plantlet. The plants were successfully acclimatized under in vivo conditions. The plants and calli extracts exhibited good antioxidant activities, however, plant extract activities were more pronounced. The phenolic compounds in plant and calli extracts were 0.16% and 0.057%, respectively. While the flavonoids were 0.092% in plant and 0.039% in calli extract. Total Phenolics, flavonoids; DPPH radical scavenging activity and reducing power potential distributed among different fractions depending upon polarity of the solvent. The highest DPPH scavenging activity and reducing power was exhibited by water fractions; 4.95 mg/mL and 0.729 OD at 10 mg/mL, respectively. The micropropagation protocol can be successfully used for large-scale multiplication and conservation of germplasm of this threatened plant. Furthermore, antioxidant value describes importance of this valuable plant as food and medicine.     

Antioxidant responses in roots of two contrasting <Emphasis Type="Italic">Sedum alfredii</Emphasis> Hance ecotypes under elevated zinc concentrations

Effects of different zinc concentrations on antioxidant responses in the roots of the hyperaccumulating ecotype (HE) and nonhyperaccumulating ecotype (NHE) of Sedum alfredii Hance were investigated under hydroponic conditions. Growth of NHE was inhibited significantly when Zn concentration was >-50 μM, whereas high Zn concentrations were beneficial for HE growth, and 500 μM Zn induced a significant increase in the root biomass and reducing activity. Malondialdehyde content and electrical conductivity of the NHE roots increased significantly; however, no changes were observed in HE when the Zn concentration was >10 μM, suggesting a severe damage to the membrane of the NHE roots. Proline content in NHE roots increased rapidly, whereas it was low in HE roots even at high Zn concentrations, suggesting that proline may not play an important role in Zn hyperaccumulation. The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (GPX) in NHE roots increased significantly when the Zn concentration was >10 μM and decreased sharply when the Zn concentration was >-500 μM. For roots of HE, in contrast, no significant changes were observed in SOD, CAT, APX, and GPX activities at low Zn concentrations, whereas a high Zn concentration (≥500 μM) led to a marked enzyme activation, which was in accordance with Zn accumulation in shoots. The results suggest that antioxidant enzymes were important for Zn detoxification in NHE at low Zn concentrations (10–250 μM) and were more critical for Zn detoxification and hyperaccumulation in HE under elevated Zn concentrations (500–1000 μM).