Characterization and localization of three soluble invertase forms from Lilium longiflorum flower buds

Three invertase forms (EC 3.2.1.26) were identified in soluble extracts from developing flower buds of Lilium longiflorum Thunb. cv. Nellie White. The enzymes were separable on a diethylaminoethyl (DEAE)-Sephacel column and designated invertase I. II or III according to the order of elution from Sephacel. To determine tissue specificity of these floral invertases, anthers were separated from tepal. pistil and filament tissue, and analyzed for invertase activity. Invertase I was localized primarily in anthers, with invertases II and III being present in much smaller amounts (less than 5% of the invertase I activity). Much higher levels of invertases II and III were found in the nonanther organs of the flower, where essentially no invertase 1 was detectable. Further purification of each form (using gel filtration. Con-A-Sepharose affinity chromatog-raphy and hydrophobic interaction chromatography on phenyl-agarose) resulted in 135- 189- and 202-fold purification of pooled fractions from DEAE-Sephacel. respectively, and established that each invertase form is a glycoprotein. Each was an acid invertase. with pH optima between 4.0 and 5.0 and an apparent molecular mass of 77 500 Da (as determined by Sephadex gel filtration). The invertases had sucrose K_m values of 1.0. 6.4 and 6.6 m M . and temperature optima of 40. 50 and 45°C. respectively. A temperature stability study revealed that invertase III was the most thermostable, followed by II and I. Invertases II and III had lower affinity to raffinose and stachyose than invertase I. All three enzymes were completely inhibited by Hg²⁺ or Ag⁺ ions at 1.7 m M . At this concentration. Cu^2- showed differential partial inhibition . Although fructan was shown to be present in both anther and nonanther tissues of Lilium flower buds, these invertases showed no sucrose:sucrose fructosyltransferase (EC 2.4.1.99) activity.     

FORMATION AND CHARACTERIZATION OF THREE TYPES OF YEAST INVERTASE

Three types of invertase (invertase I, II and III) are separatedfrom the soluble and insoluble fractions (4,500xg, 10 min supernatantand pellets of the homogenate, respectively) of baker's yeastby a DEAE cellulose column chromatography. The invertases Iand II are eluted with 0.1 M sodium acetate buffer (pH 3.9)and with 0.1 M sodium acetate buffer (pH 6.2) containing 0.1M NaCl from DEAE cellulose respectively, whereas the invertase-IIIremains adsorbed on the cellulose under these conditions. Theyare present in proportions of 2.5: 1 : 0.06 in the soluble fractionand 1.4: 1 : 0.12 in the insoluble fraction of the fresh baker'syeast cells. While in-vertase-II remains at a constant level,invertases I and III in the soluble fraction increase upon incubationof cells for the formation of invertase under the continuoussupply of sucrose. Invertases I and II differ from each other considerably in theoptimum pH and slightly in the response to (activation and inactivationby) crude papain and are identical with respect to the heatstability and probably to the affinity for sucrose. ¹Present address: Chemical Laboratory, Nippon Medical School,Konodai, Ichikawa-shi, Chiba-ken.     

Invertase activity associated with the walls of Solanum tuberosum tubers

Three fractions with invertase activity (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) were isolated from mature Solanum tuberosum tubers: acid soluble invertase, invertase I and invertase II. The first two invertases were purified until electrophoretic homogeneity. They are made by two subunits with an apparent M(r) value of 35,000 and their optimal pH is 4.5. Invertase I was eluted from cell walls with ionic strength while invertase II remained tightly bound to cell walls after this treatment. This invertase was solubilized by enzymatic cell wall degradation (solubilized invertase II). Their K(m)s are 28, 20, 133 and 128 mM for acid soluble invertase, invertase I, invertase II and solubilized invertase II, respectively. Glucose is a non-competitive inhibitor of invertase activities and fructose produces a two site competitive inhibition with interaction between the sites. Bovine serum albumin produces activation of the acid soluble invertase and invertase I while a similar inhibition by lectins and endogenous proteinaceous inhibitor from mature S. tuberosum tubers was found. Invertase II (tightly bound to the cell walls) shows a different inhibition pattern. The test for reassociation of the acid soluble invertase or invertase I on cell wall, free of invertase activity, caused the reappearance of all invertase forms with their respective solubilization characteristics and molecular and kinetic properties. The invertase elution pattern, the recovery of cell wall firmly bound invertase and the coincidence in the immunological recognition, suggest that all three invertases may be originated from the same enzyme. The difference in some properties of invertase II and solubilized invertase II from the other two enzymes would be a consequence of the enzyme microenvironment in the cell wall or the result of its wall binding.     

Invertases in Oat Seedlings: SEPARATION, PROPERTIES, AND CHANGES IN ACTIVITIES IN SEEDLING SEGMENTS

The soluble invertase activity in etiolated Avena seedlings was highest at the apex of the coleoptile and much lower in the primary leaf, mesocotyl, and root. The activity in all parts of the seedling consisted of two invertases (I and II) which were separated by chromatography on diethylaminoethylcellulose. Both enzymes appeared to be acid invertases, but they differed in molecular size, pH optimum, and the kinetic parameters K_m and V_max of their action on sucrose, raffinose, and stachyose. Invertase II had low stability at pH 3.5 and below, and exhibited high sensitivity to Hg²⁺, with complete inhibition by 2 micromolar HgCl₂. Segments of coleoptiles incubated in water lost about two-thirds of the total invertase activity after 16 hours. The loss of activity was due primarily to a decrease in the level of invertase II. The loss of invertase was decreased by indoleacetic acid, 2,4-dichlorophenoxyacetic acid, and α-naphthaleneacetic acid but not by β-naphthaleneacetic acid and p-chlorophenoxyisobutyric acid. Conditions that inhibited auxin-induced growth of the segments (20 millimolar CaCl₂ and 200 millimolar mannitol) also blocked the auxin effect on invertase loss.     

Purification and characterization of two soluble acid invertase isozymes from Japanese pear fruit

Two isozymes (AIV I and AIV II) of soluble acid invertase (EC 3.2.1.26) were purified from Japanese pear fruit through procedures including (NH(4))(2)SO(4) precipitating, DEAE-Sephacel column chromatography, Concanavalin A (ConA)-Sepharose affinity chromatography, hydroxyapatite column chromatography and Mono Q HR 5/5 column chromatography. The specific activities of purified AIV I and AIV II were 2670 and 2340 (nkat/mg protein), respectively. AIV I was a monomeric enzyme of 80 kDa, while AIV II may be also a monomeric enzyme, which is easy to be cleaved to 52 kDa and 34 kDa polypeptide during preparation by SDS-PAGE. The Km values for sucrose of AIV I and AIV II were 3.33 and 4.58 mM, respectively, and optimum pH of both enzyme activities was pH 4.5.     

A simple one-step procedure for the separation of calpain I, calpain II and calpastatin.

The soluble fraction from rabbit brain was adsorbed on a column of phenyl-Sepharose. By applying a linear gradient with decreasing salt concentration and increasing pH, it was possible to separate calpain I and calpain II from each other and from the endogenous inhibitor calpastatin. Both enzymes were capable of degrading endogenously labelled neuronal proteins, including slowly axonally transported soluble proteins and rapidly transported membrane-bound proteins, as well as casein.     

Acid and Neutral Invertases in the Mesocarp of Developing Muskmelon (Cucumis melo L. cv Prince) Fruit

Acid and neutral invertases were found in the mesocarp of developing muskmelon (Cucumis melo L. cv Prince) fruit and the activities of these enzymes declined with maturation of the fruit, concomitantly with the accumulation of sucrose. Neutral invertase was only present in the soluble fraction and acid invertase was present in both the soluble and cell-wall fractions. The cell-wall fraction contained three types of acid invertase: a NaCl-released invertase; an EDTA-released invertase, and a tightly bound invertase that still remained on the cell wall after treatment with NaCl and EDTA. The soluble acid and neutral invertases could be separated from one another by chromatography on DEAE-cellulose and they exhibited clear differences in their properties, namely, in their pH optima, substrate specificity, K_m values for sucrose, and inhibition by metal ions. The EDTA-released invertase and the soluble acid invertase were similar with regard to their chromatographic behavior on DEAE-cellulose, but the NaCl-released invertase was different because it was adsorbed to a column of CM-cellulose. The soluble acid invertase and two cell-wall bound invertases had very similar characteristics with regard to optimal pH and temperature, K_m value for sucrose, and substrate specificity.     

苜蓿悬浮细胞对盐胁迫的反应和适应

苜蓿悬浮细胞能够适应200mmol/L NaCl及其以下盐浓度的胁迫,适应细胞中游离脯氨酸、还原糖和Na~ 积累增加。400mmol/LNaCl对细胞生长明显抑制。细胞对盐胁迫的反应和适应中PM-ATPase和TM-ATPase起到重要作用,在适应细胞中两者的活力都明显增加。PM-ATPase活力的增加可受CHX的明显抑制。     

Rhythmic growth and carbon allocation in Quercus robur. Sucrose metabolizing enzymes in leaves

The high sucrose phosphate synthase (SPS) capacity and the low soluble acid invertase activity of mature leaves of the first flush of leaves remained stable during second flush development. Conversely, fluctuations of sucrose synthase (SS) activity were in parallel with the sucrose requirement of the second flush. Sucrose synthase activity (synthesis direction) in first flush leaves could increase in 'response' to sink demand constituted by the second flush growth. Only the ptotosynthates provided by flush mature leaves were translocated for a current flush, while the starch content of these leaves remained stable. After their emergence, second flush leaves showed an increase in SPS and SS (Synthetic direction) activities. The high sucrose synthesis in second flush leaves was used for leaf expansion. When young leaves were 30% fully expanded (stage II20), SPS activity showed little change whereas SS activity declined rapidly toward and after full leaf expansion. The starch accumulation in the young leaves occured simultaneously with their expansion. Developing leaves showed a high level of acid invertase activity until maximum leaf expansion (stage II1). In first and second flush leaves, changes in acid invertase activity correlated positively with changes in reducing sugar concentrations. Alkaline invertase and sucrose synthase (cleavage direction) activities showed similar changes with low values when compared with those of acid invertase activity, especially in second flush leaves. The present results suggest that soluble acid invertase was the primary enzyme responsible for sucrose catabolism in the expanding common oak leaf.     

Purification and characterization of membrane-bound inositolpolyphosphate 5-phosphatase

Membrane-bound inositolpolyphosphate 5-phosphatase was solubilized and highly purified from a microsomal fraction of rat liver. Its physiochemical and enzymological properties were compared with those of highly purified preparations of two types of soluble enzyme (soluble Type I and Type II) from rat brain. The molecular masses of the membrane-bound and soluble Type I enzymes were 32 kDa, while that of soluble Type II enzyme was 69 kDa, as determined by molecular sieve chromatography. The membrane-bound and soluble Type I enzymes showed similar broad peaks on isoelectric focusing (pI 5.8-6.4), while soluble Type II enzyme showed multiple peaks in the region between pI 4.0-5.8. All three enzymes required divalent cation for activity. Mg2+ was the most effective for both the membrane-bound and soluble Type I enzymes, while Co2+ enhanced soluble Type II enzyme activity about 1.5-fold relative to Mg2+ at 1 mM. The optimal pH of both the membrane-bound and soluble Type I enzymes was 7.8, while that of soluble Type II was 6.8. The Km values for inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] of all three enzymes were similar (5-8 microM), but those for inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] were quite different, the Km values of membrane-bound and soluble Type I enzymes being 0.8 microM, while that of soluble Type II was 130 microM. These similarities between the membrane-bound and soluble Type I enzymes suggest that these two molecules may be the same protein, and that concentrations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, both of which are considered to play critical roles in the regulation of intracellular Ca2+-concentration, may be differently regulated by two functionally distinct enzymes.     

蔗糖酶活力与甜菜块根贮藏质量的关系

可溶性酸性蔗糖酶是决定甜菜块根贮藏质量的关键酶。贮藏期间其活力的提高是由于蛋白质重新合成所致。不良的贮藏条件使块根汁液pH降低,膜透性增加,这两种因素与可溶性酸性蔗糖酶活力成正相关,与贮藏质量成负相关。     

Inhibition of Potato Tuber Invertase by an Endogenous Inhibitor: EFFECTS OF SALTS, pH, TEMPERATURE, AND SUGARS ON BINDING

Binding between potato tuber invertase and its endogenous inhibitor followed second-order reaction kinetics. Binding rates were diminished by the presence of various inorganic salts, MgCl₂ being especially effective. This effect of MgCl₂ was used in binding rate studies by adding the salt with sucrose to reduce binding during assay of previously unbound activity. The optimal pH for binding was about 4.8, similar to the optimal pH for catalytic activity of invertase. The optimal temperature for binding was about 45 C, approximately 5 C less than the optimum for catalytic activity. Sucrose at concentrations as low as 2 millimolar slowed binding; reducing sugars had little or no effect on binding or on catalytic activity.     

Invertase Activity, Soluble Carbohydrates and Inflorescence Development in the Tomato (Lycopersicon esculentum Mill.)

The concentration of reducing sugars in the developing firstinflorescence of the tomato (Lycopersicon esculentum Mill.)increased steadily between the macroscopic appearance of theflower buds and the initial stages of fruit expansion. Overthis period sucrose concentrations remained relatively constant.The rise in reducing sugar concentration was accompanied byan increase in the activity of an acid invertase. In individualflower buds invertase activity rose to a maximum shortly beforeanthesis and declined sharply as the anthers dehisced. Increased planting densities and removal of source leaves reducedthe rate of dry matter accumulation by the first inflorescenceand increased the incidence of flower bud abortion. These changeswere correlated with reductions in reducing sugar concentrations,in reducing sugar/sucrose ratios and in acid invertase levels.Removal of young leaves at the shoot apex significantly increasedthe relative growth rate of the inflorescence and led to a substantialincrease in its invertase content. These treatments had relativelylittle effect on sucrose concentration in the inflorescence. The data are consistent with the operation of an invertase-mediatedunloading mechanism for transported sucrose at sinks in theflower buds. It is suggested that the retarded development ofthe first inflorescence and the high incidence of flower budabortion observed under conditions of reduced photoassimilateavailability are causally related to the decline in invertaseproduction in the flower buds. Possible mechanisms for the regulationof invertase synthesis in the flowers are discussed. Lycopersicon esculentum Mill, tomato, inflorescence development, invertase, sink activity     

Biochemical Mechanism for Regulation of Sucrose Accumulation in Leaves during Photosynthesis

It is not known why some species accumulate high concentrations of sucrose in leaves during photosynthesis while others do not. To determine the possible basis, we have studied 10 species, known to differ in the accumulation of sucrose, in terms of activities of sucrose hydrolyzing enzymes. In general, acid invertase activity decreased as leaves expanded; however, activities remaining in mature, fully expanded leaves ranged from low (<10 micromoles per gram fresh weight per hour) to very high (>100 micromoles per gram fresh weight per hour). In contrast, sucrose synthase activities were low and relatively similar among the species (4-10 micromoles per gram fresh weight per hour). Importantly, leaf sucrose concentration, measured at midafternoon, was negatively correlated with acid invertase activity. We propose that sucrose accumulation in vacuoles of species such as soybean and tobacco is prevented by acid invertase-mediated hydrolysis. Initial attempts were made to characterize the relatively high activity of acid invertase from mature soybean leaves. Two apparent forms of the enzyme were resolved by Mono Q chromatography. The two forms had similar affinity for substrate (apparent K_m [sucrose] = 3 millimolar) and did not interconvert upon rechromatography. It appeared that the loss of whole leaf invertase activity during expansion was largely the result of changes in one of the enzyme forms. Overall, the results provide a mechanism to explain why some species do not accumulate sucrose in their leaves. Some futile cycling between sucrose and hexose sugars is postulated to occur in these species, and thus, the energy cost of sucrose production may be higher than is generally thought.     

Purification and Characterization of Soluble (Cytosolic) and Bound (Cell Wall) Isoforms of Invertases in Barley (Hordeum vulgare) Elongating Stem Tissue

Three different isoforms of invertases have been detected in the developing internodes of barley (Hordeum vulgare). Based on substrate specificities, the isoforms have been identified to be invertases (β-fructosidases EC 3.2.1.26). The soluble (cytosolic) invertase isoform can be purified to apparent homogeneity by diethylaminoethyl cellulose, Concanavalin-A Sepharose, organomercurial Sepharose, and Sephacryl S-300 chromatography. A bound (cell wall) invertase isoform can be released by 1 molar salt and purified further by the same procedures as above except omitting the organo-mercurial Sepharose affinity chromatography step. A third isoform of invertase, which is apparently tightly associated with the cell wall, cannot be isolated yet. The soluble and bound invertase isoforms were purified by factors of 60- and 7-fold, respectively. The native enzymes have an apparent molecular weight of 120 kilodaltons as estimated by gel filtration. They have been identified to be dimers under denaturing and nondenaturing conditions. The soluble enzyme has a pH optimum of 5.5, K_m of 12 millimolar, and a V_max of 80 micromole per minute per milligram of protein compared with cell wall isozyme which has a pH optimum of 4.5, K_m of millimolar, and a V_max of 9 micromole per minute per milligram of protein.     

Soluble acid invertase determines the hexose-to-sucrose ratio in cold-stored potato tubers

Cold storage of potato (Solanum tuberosum L.) tubers is known to cause accumulation of reducing sugars. Hexose accumulation has been shown to be cultivar-dependent and proposed to be the result of sucrose hydrolysis via invertase. To study whether hexose accumulation is indeed related to the amount of invertase activities, two different approaches were used: (i) neutral and acidic invertase activities as well as soluble sugars were measured in cold-stored tubers of 24 potato cultivars differing in the cold-induced accumulation of reducing sugars and (ii) antisense potato plants with reduced soluble acid invertase activities were created and the soluble sugar accumulation in cold-stored tubers was studied. The cold-induced hexose accumulation in tubers from the different potato cultivars varied strongly (up to eightfold). Large differences were also detected with respect to soluble acid (50-fold) and neutral (5-fold) invertase activities among the different cultivars. Although there was almost no correlation between the total amount of invertase activity and the accumulation of reducing sugars there was a striking correlation between the hexose/sucrose ratio and the extractable soluble invertase activitiy. To exclude the possibility that other cultivar-specific features could account for the obtained results, the antisense approach was used to decrease the amount of soluble acid invertase activity in a uniform genetic background. To this end the cDNA of a cold-inducible soluble acid invertase (EMBL nucleicacid database accession no. X70368) was cloned from the cultivar Desirée, and transgenic potato plants were created expressing this cDNA in the antisense orientation under control of the constitutive 35S cauliflower mosaic virus promotor. Analysis of the harvested and cold-stored tubers showed that inhibition of the soluble acid invertase activity leads to a decreased hexose and an increased sucrose content compared with controls. As was already found for the different potato cultivars the hexose/sucrose ratio decreased with decreasing invertase activities but the total amount of soluble sugars did not significantly change. From these data we conclude that invertases do not control the total amount of soluble sugars in coldstored potato tubers but are involved in the regulation of the ratio of hexose to sucrose.The authors are grateful to Heike Deppner and Christiane Prüßner for tuber harvest and technical assistance during the further analysis. We thank Andrea Knospe for taking care of tissue culture, Birgit Schäfer for patient photographic work, Hellmuth Fromme and the greenhouse personnel for attending plant growth and development and Astrid Basner for elucidating the sequence of clone INV-19. The work was supported by the Bundesministerium für Forschung und Technologie (BMFT).     

Regulation of starch and protein accumulation in alfalfa (Medicago sativa L.) somatic embryos

Compared to seeds, somatic embryos accumulated relatively low levels and different types of storage carbohydrates. The regulation of starch accumulation was studied to determine its effects on desiccation tolerance and vigor of dry somatic embryos. Somatic embryos of Medicago sativa are routinely matured through three phases: 7 days of development; 10 days of phase I maturation, a rapid growth phase; and 10 days of phase II maturation, a phase leading to the acquisition of desiccation tolerance. The control of starch deposition was investigated in alfalfa somatic embryos by manipulating the composition of the phase I maturation medium with different levels of sucrose, abscisic acid, glutamine and different types of carbohydrates and amino acids. After phase II maturation, mature somatic embryos were collected for desiccation and subsequent conversion, or for biochemical analyses. Starch deposition occurred primarily during phase I maturation, and variations in the composition of this medium influenced embryo quality, storage protein and starch accumulation. A factorial experiment with two levels of glutamine × three levels of sucrose showed that increasing the sucrose concentration from 30 to 80 g/l increased embryo size and starch content, but had minimal effect on accumulation of storage proteins; glutamine also increased embryo size, but decreased starch content and increased accumulation of the high salt soluble S-2 (medicagin) storage proteins. ABA did not influence any of the parameters tested when included in phase I maturation at concentration up to 10 μM. Replicating sucrose with maltose, glucose, or glucose and fructose did not alter embryo size or starch accumulation (mg/g fresh weight), but replacement with fructose alone reduced embryo size, and replacement with glucose alone reduced germination. Suplementation with the amino acids, asparagine, aspartic acid and glutamine increased seedling vigor, but decreased the starch content of embryos. The data indicate that starch accumulation in somatic embryos is regulated by the relative availability of carbon versus nitrogen nutrients in the maturation medium. The quality of mature somatic embryos, determined by the rate of seedling development (conversion and vigor), correlated with embryo size, storage protein and free amino acid but not with starch. Therefore, further improvements in the quality of somatic embryo may be achieved through manipulation of the maturation medium in order to increase storage protein, but not starch deposition.     

Increased lung expansion alters the proportions of type I and type II alveolar epithelial cells in fetal sheep

Type I and type II alveolar epithelial cells (AECs) are derived from the same progenitor cell, but little is known about the factors that regulate their differentiation into separate phenotypes. An alteration in lung expansion alters the proportion type II AECs in the fetal lung, indicating that this may be a regulatory factor. Our aim was to quantify the changes in the proportion of type I and type II AECs caused by increased fetal lung expansion and to provide evidence for transdifferentiation of type II into type I cells. Lung tissue samples were collected from ovine fetuses exposed to increased lung expansion induced by 2, 4, or 10 days of tracheal obstruction (TO). The identities and proportions of AEC types were determined with electron microscopy. The proportion of type II cells was reduced from 28.5 +/- 2.2% in control fetuses to 9.4 +/- 2.3% at 2 days of TO and then to 1.9 +/- 0.8% at 10 days. The proportion of type I AECs was not altered at 2 days of TO (63.1 +/- 2.3%) compared with that of control cells (64.8 +/- 0.5%) but was markedly elevated (to 89.4 +/- 0.9%) at 10 days of TO. The proportion of an intermediate AEC type, which displayed characteristics of both type I and type II cells, increased from 5.7 +/- 1.3% in control fetuses to 23.8 +/- 5.1% by 2 days of TO and was similar to control values at 10 days of TO (7.7 +/- 0.9%). Our data show that increases in fetal lung expansion cause time-dependent changes in the proportion of AEC types, including a transient increase in an intermediate cell type. These data provide the first evidence to support the hypothesis that increases in fetal lung expansion induce differentiation of type II into type I AECs via an intermediate cell type.     

Rat brain and liver soluble phospholipase C: resolution of two forms with different requirements for calcium

Two forms of phospholipase C, hydrolyzing specifically inositol phospholipids, are resoluted and partially purified from rat brain as well as liver cytosol by DEAE-cellulose followed by heparin-Sepharose, Sephacryl S-400, and aminohexyl-Sepharose column chromatographies. With phosphatidylinositol as substrate, at pH 7.4 one is most active at 10(-6) M Ca2+ (Type I) whereas the other requires 10(-3) M Ca2+ (Type II). At pH 5.5 both Type I and II are active at 10(-3) M Ca2+ but essentially inactive at lower concentrations of this divalent cation. Both Type I and II hydrolyze preferentially polyphosphoinositides particularly at lower concentrations of Ca2+.