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.     

Purification and Characterization of Soluble and Membrane-Associated DNA Polymerases from a Virus PBCV-1 Infected Green Alga Chlorella NC64A

DNA polymerases were purified several hundred-fold from the10 000 x g soluble (polymerase I) and particulate (polymeraseIII) fractions prepared from virus PBCV-1 infected ChlorellaNC64A extracts. Both DNA polymerases exhibited optimal activitywith activated calf thymus DNA at pH 8.5. DNA polymerase I required3.0 mol m^–3 MgSO₄ and 150 to 250 mol m^–3 KCl foroptimum activity whereas, DNA polymerase III required 2.0 molm^–3 MgSO₄ and 150 mol m^–3 KCl. Both enzymes wereinhibited by pyrophosphate, actinomycin D, ethidium bromide,dideoxythymidine triphosphate, and N-ethylmaleimide but wererelatively insensitive to aphidicolin. DNA polymerase I differedfrom DNA polymerase III in its response to cations (particularlyNH₄Cl), elution from a DEAE cellulose column, and molecularweight. Key words: Algal virus, DNA polymerase, Chlorella     

Changes in activity and isoenzymes of invertase in cucumber leaves due to nitrogen status

When 20 ppm ammonium nitrogen was supplied to nitrogen starvedcucumbers, acid and alkaline invertase activities increased6 and 2 fold on a fresh weight basis, respectively. Two acidinvertase isozymes, I and II, were detected in cucumber leavesby DEAE cellulose chromatography and Sephadex G-100 gel filtration.Their activities were found to be reversed in nitrogen-starvedand -sufficient cucumber leaves, isozyme II being the majorisozyme in nitrogen sufficient plant. Isozyme II had nearlyhalf the Km value of I for sucrose, and hydrolyzed raffinoseless than half as much as I did, although they showed the nearlysame pH optimum at 5.0 and similar heat stability. These resultsare discussed in relation to the regulatory role of acid invertasein cucumber leaves under different nitrogen status. (Received March 26, 1976; )     

Sucrose metabolism during tobacco callus growth

Activities of soluble and insoluble invertases and sucrose synthetase in tobacco callus increased significantly within the first 3 days of culture. After this period soluble invertase activity declined, while the activities of the insoluble invertase and the sucrose synthetase were relatively unchanged.     

Invertases of carnation petals. Partial purification, characterization and changes in activity during petal growth

Carnation ( Dianthus caryophyllus L. cv. White Sim) petals contained two distinct invertases (EC 3.2.1.26) based on chromatographic behavior on DEAE-cellulose. Both are soluble in 20 m M sodium phosphate buffer (pH 6.5) and exhibit acid pH optimum of 5.5. Extraction of a cell wall preparation from petals with 1 M NaCl released little additional activity. Furthermore, only traces of activity remained associated with the NaCl-extracted cell wall preparation. One of the soluble invertases, representing over 75% of the total activity, was partially purified by (NH₄)₂SO₄ fractionation and sequential chromatography over diethylaminoethyl-cellulose, concanavalin-A sepharose and polyacrylamide P-200. The enzyme was purified 38-fold with a recovery of 12%. It had an apparent native molecular weight of 215 kDa. The partially purified invertase is a β-fructofuranosidase (EC 3.2.1.26) based on its specificity for sucrose. The K_m for sucrose was 3.3 m M . Accumulation of reducing sugars and increased invertase activity during expansive petal growth indicates that sucrose is the major source of carbon for petal growth.     

Organ‐specific localization and molecular properties of three soluble invertases from Lilium longiflorum flower buds

Three soluble invertase (EC 3.2.1.26) isoforms from Easter lily ( Lilium longiflorum Thunb. cv. Nellie White) flower buds were purified to apparent homogeneity. Non‐denaturing PAGE showed one band for all three invertases that corresponded to the invertase activity. SDS‐PAGE of purified invertase I gave a single band at 78 kDa, whereas invertases II and III gave three bands at 54, 52 and 24 kDa. Antibodies against tomato fruit acid invertase and Urtica dioica leaf acid invertase recognized all three invertase isoforms, whereas antibodies against wheat coleoptile acid invertase recognized only 56‐ and 54‐kDa bands of invertases II and III. Antibodies against wheat coleoptile invertase recognized the 54‐ and 52‐kDa proteins from crude extracts of all flower organs, and a 72‐kDa protein in both leaf and bulb scale extracts. All three invertases bound to Con‐A peroxidase. Deglycosylation of invertase I with glycopeptidase F was complete and resulted in a peptide of 75 kDa. Invertases II and III were deglycosylated partially by glycopeptidase F and resulted in proteins of 53, 51, 50 and 22 kDa. Invertase I was localized only in anther and filament, whereas the other two isoforms were present in all flower organs.     

Import of Sucrose and its Partitioning in the Synthesis of Galactomannan and Raffinose-oligosaccharides in the Developing Guar (Cyamopsis tetragonolobus) Seed

Import of sucrose and its transformation to galactomannan andraffinose-oligosaccharides have been studied in the developingguar seed. The amount of galactomannan gradually increased withthe ageing of the seed. During the entire period of pod development,sucrose constituted the major portion of the free sugars inthe seed (both endosperm and cotyledons) as well as in the podwall. Besides myo-inositol, the free sugars detected in thedeveloping endosperm and cotyledons were glucose, fructose,raffinose and stachyose. Some compounds, possibly glycosides(RG values higher than that of fructose), were also detectedin the endosperm. In the later stages of seed development, therelative proportion of raffinose in the free sugars increased,reaching 50% of the total free sugars in 77-d-old cotyledons.With pod maturity, the activities of soluble acid and boundacid invertases in the pod wall increased manifold with a concomitantdecline in the non-reducing sugar content. These enzymes seemto be involved in the mobilization of sucrose from this fruitingstructure into the seed. An increased synthesis of raffinose-oligosaccharidesboth in the endosperm and cotyledons was associated with highactivities of soluble acid invertase (pH 4.8) and sucrose-UDPglucosyl transferase in these tissues. Feeding uniformly labelled¹⁴C-sugars to the detached intact pods as well as to the isolatedendosperm and cotyledons resulted in labelling of all endogenousfree sugars and galactomannan. The uptake and incorporationinto galactomannan of ¹⁴C was stimulated by Co²⁺, Mn²⁺ and Mg²⁺.Except for mannose, a major proportion of the ¹⁴C from glucose,fructose and sucrose appeared in sucrose in both endosperm andcotyledons indicating a fast reconstitution of sucrose in situ.Based on the present results, a possible mode of transformationof sucrose to galactomannan and raffinose-oligosaccharides hasbeen proposed. Key words: Sucrose, galactomannan, raffinose-oligosaccharides, invertase, sucrose-UDP glucosyl transferase, ¹⁴C-incorporation, guar seed     

Properties of Invertases in Sugar Storage Tissues of Citrus Fruit and Changes in Their Activities during Maturation

Both acid and alkaline invertases were present in immature juice sacs of satsuma mandarin (Citrus‘Unshu Marcovitch”) fruit, in which sugar content was low. Maturing and mature juice sacs, in which sugar content increased steadily with time, were characterized by the presence of alkaline invertase and the absence of acid invertase. When the immature juice sacs were homogenized with 0.2 M sodium phosphate-citrate buffer (pH 8.0), almost all of the acid invertase activity was found in the solubilized fraction, whereas almost all of the alkaline invertase activity was present in the insoluble fraction. The distribution of alkaline invertase between the solubilized and insoluble fractions changed with the development of fruit. The acid invertase had a molecular weight of 69,000, optimum pH of 4.8–5.3, and K_m value for sucrose of 7.3 mM. The alkaline invertase had a molecular weight of 200,000, pH optimum of 7.2–7.7, and K_m value of 35.7 mM. The hydrolysing activities of both enzymes for raffinose were considerably less than those for sucrose. The alkaline invertase had lower activity for raffinose than the acid invertase.     

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.     

Properties of the Invertases of Cultured Sycamore Cells and Changes in Their Activity during Culture Growth

When cultured sycamore cells are homogenised in a phosphate-citrate buffer at pH 7.0 and the homogenate centrifuged two fractions are obtained both of which show the presence of an acid (opt. pH 4.0–4.5) and a neutral (opt. pH 7.0–7.4) invertase. The activity of the insoluble pellet appears to be located in its cell wall fragments. The acid and neutral invertases of the soluble fraction can be separated by fractional precipitation with (NH₄SO₄. The activities of these enzymes are low in stationary phase cells but they increase following subculture to reach peaks of activity towards the end of the period of most active cell growth and division and then decline again as the cells begin to enter stationary phase. The activities of both enzymes are higher in the cell wall than in the soluble fraction and the acid invertase reaches higher levels of activity than the neutral enzyme in both fractions. When cells are subcultured there occurs within a few hours an increase in the acid invertase and a decline in the neutral invertase activity in the cell wall fraction and a decline in the acid invertase of the soluble fraction prior to the large net increases in the activities of both enzymes in both locations which occurs as the cells embark upon cell division. The pattern of changes in the invertase activities through the growth cycle of batch propagated cultures is similar whether the cells are grown in sucrose, or glucose, or sucrose plus glucose; the highest levels of activities were recorded in the glucose-grown cells. The total yield of invertase activities and the distribution of activities between the soluble and cell wall fractions of the homogenates are affected by the pH of the extraction medium (within the range pH 4.0–8.0). It has not proved possible to completely remove the invertases from the cell wall fraction; upwards of 50 % of the acid invertase was recovered from this fraction by treatment with Triton-X followed by urea, but these treatments inactivated a high proportion of the neutral enzyme. These findings are compared with other studies on the activity and intra-cellular distribution of plant invertases and the possible roles of these enzymes discussed.     

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.     

Sucrose and Nitrogen Supplies Regulate Growth of Maize Kernels

The growth of maize (Zea mays L.) kernels depends on the availabilityof carbon (C) and nitrogen (N) assimilates supplied by the motherplant and the capacity of the kernel to use them. Our objectiveswere to study the effects of N and sucrose supply levels ongrowth and metabolism of maize kernels. Kernel explants of Pioneer34RO6 were culturedin vitro with varying combinations of N (5to 30 m M) and sucrose (117 to 467 m M). Maximum kernel growthwas obtained with 10 m M N and 292 m M sucrose in the medium,and a deficiency of one assimilate could not be overcome bya sufficiency of the other. Increasing the N supply led to increasesin the kernel sink capacity (number of cells and starch granulesin the endosperm), activity of certain enzymes (soluble andbound invertases, sucrose synthase, and aspartate aminotransaminase),starch, and the levels of N compounds (total-N, soluble protein,and free amino acids), and decreased the levels of C metabolites(sucrose and reducing sugars). Conversely, increasing the sucrosesupply increased the level of endosperm C metabolites, freeamino acids, and ADPG-PPase and alanine transaminase activities,but decreased the activity of soluble invertase and concentrationsof soluble protein and total-N. Thus, while C and N are interdependentand essential for accumulation of maximum kernel weight, theyappear to regulate growth by different means. Nitrogen supplyaids the establishment of kernel sink capacity, and promotesactivity of enzymes relating to sucrose and nitrogen uptake,while sucrose regulates the activities of invertase and ADPG-PPase.Copyright 1999 Annals of Botany Company Zea mays, maize,, invertase, ADPG-PPase, media composition, sucrose, nitrogen, C/N.     

Soluble invertase expression is an early target of drought stress during the critical,abortion-sensitive phase of young ovary development in maize

To distinguish their roles in early kernel development and stress, expression of soluble (Ivr2) and insoluble (Incw2) acid invertases was analyzed in young ovaries of maize (Zea mays) from 6 d before (-6 d) to 7 d after pollination (+7 d) and in response to perturbation by drought stress treatments. The Ivr2 soluble invertase mRNA was more abundant than the Incw2 mRNA throughout pre- and early post-pollination development (peaking at +3 d). In contrast, Incw2 mRNAs increased only after pollination. Drought repression of the Ivr2 soluble invertase also preceded changes in Incw2, with soluble activity responding before pollination (-4 d). Distinct profiles of Ivr2 and Incw2 mRNAs correlated with respective enzyme activities and indicated separate roles for these invertases during ovary development and stress. In addition, the drought-induced decrease and developmental changes of ovary hexose to sucrose ratio correlated with activity of soluble but not insoluble invertase. Ovary abscisic acid levels were increased by severe drought only at -6 d and did not appear to directly affect Ivr2 expression. In situ analysis showed localized activity and Ivr2 mRNA for soluble invertase at sites of phloem-unloading and expanding maternal tissues (greatest in terminal vascular zones and nearby cells of pericarp, pedicel, and basal nucellus). This early pattern of maternal invertase localization is clearly distinct from the well-characterized association of insoluble invertase with the basal endosperm later in development. This localization, the shifts in endogenous hexose to sucrose environment, and the distinct timing of soluble and insoluble invertase expression during development and stress collectively indicate a key role and critical sensitivity of the Ivr2 soluble invertase gene during the early, abortion-susceptible phase of development.     

Multiple forms of invertase in developing oat internodes

Three different invertases are found in the developing internodes of oat (Avena sativa cv. Victory). Two soluble invertases (I and II) are separable on diethylaminoethylcellulose and Sephadex columns. They are further distinguished by their kinetic constants, heat stability, and differences in stability and apparent activity optima in response to pH treatments. Relative activities of the two soluble isozymes change considerably during the developmental stages examined. Invertase I activity rises early and begins to fall after maximal activity is reached at 6 hours of incubation. This early increase in activity accompanies the period of most rapid growth rate of the internode. Invertase II activity does not increase significantly during the first 6 hours of internode extension, but rapidly rises to a maximum activity at 16 hours, then declines. The third form of invertase, bound invertase (III), is present in both immature and mature stem tissue. Its activity increases (by 6 hours) during immature growth stages, decreases considerably with maturation, and remains relatively constant in mature tissue.     

Immobilization of cane invertase on bentonite

Sugar-cane invertase (β-d-fructofuranoside fructohydrolase, EC 3.2.1.26) immobilized on bentonite clay in 0.05 m acetate buffer, pH 4.5, has been shown to be capable of hydrolysing sucrose. The bentonite-invertase (BI) complex gave 55.5% retention of enzyme activity on the surface. A further 17 and 22% increase in retention of enzyme activity was obtained using the covalent linking agents, cyanuric chloride and thionyl chloride, giving bentonite-cyanuric chloride-invertase (BCCI) and bentonite-thionyl chloride-invertase (BTCI) complexes. Concentrations of acetate buffer >0.2 M disrupt the bentonite-invertase complexes. The immobilized invertase complexes showed high temperature optima (60–65°C) and high thermal stability compared to the free enzyme. The pH profiles of the free and immobilized enzyme were the same. The rate of hydrolysis of sucrose was increased using immobilized enzymes, which required a higher substrate concentration than the free enzyme. The insoluble enzyme conjugate-carrier complexes when used for sucrose hydrolysis in a batch process showed 53.1 (BI), 57.4 (BCCI) and 59.6% (BTCI) conversions, respectively, in 12 h, compared to 42.3% conversion in 24 h with the free enzyme. The immobilized invertase complexes can be used for sucrose inversion for about five cycles. The application of this immobilization procedure may help in the removal of invertase from cane juice to reduce sugar losses in industry.     

Acid and alkaline invertases in suspension cultures of sugar beet cells

Alkaline invertase was induced during the initiation of suspension cultures of single cells from leaf explants of sugar beets in Murashige-Skoog liquid medium which contained benzyladenine. This activity was barely detectable in the leaves themselves. In suspension cultures, the presence of both acid and alkaline invertases was detected; alkaline invertase was only present in the cytoplasm of the cultured cells, whereas acid invertase was present in the cytoplasm and cell walls, and was also detected in the culture medium. The cell wall contained at least three types of acid invertase; two of these activities were solubilized by saline (saline-released) and EDTA (EDTA-released), respectively, and the third remained tightly associated with the cell wall. Saline-released and EDTA-released invertases from the cell wall showed the significant differences in their properties: the saline-released enzyme had the highest affinity for sucrose among the invertases tested, and was easily bound to cell walls, to DNA, and to a cation exchanger, unlike the EDTA-released enzyme. Sucrose is the source of carbon for plant cells in suspension culture and is probably degraded in the cell wall by the saline-released invertase, which had the highest activity and the highest affinity for sucrose. Hexose products of this degradation would be transported to cytoplasm. Soluble invertase, EDTA-released invertase from the cell wall, and one of two extracellular invertases behaved similarly upon chromatography on DEAE-cellulose. They had similar activity profiles with changing pH, and similar K_m values for sucrose. Thus it appears that they are identical. Two extracellular invertases found in the growth medium of the suspension cultures were probably identical with those in the soluble fraction of callus and seedlings of sugar beets, because they showed similar behaviors during chromatography on DEAE-cellulose, and had similar activity profiles with changing pH and K_m values for sucrose.     

Purification and characterization of three soluble invertases from barley (Hordeum vulgare L.) leaves.

Three soluble isoforms of invertase (beta-fructofuranosidase; EC 3.2.1.26) were purified from 7-d-old primary leaves of barley (Hordeum vulgare L.). Invertase I, a monomeric protein of 64 kD, was purified to apparent homogeneity as shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Invertases IIA and IIB, multimeric proteins with molecular masses of the 116 and 155 kD, were purified 780- and 1370-fold, respectively, but were not yet homogeneous. Extracts of epidermal strips of leaves contained only invertase IIB. The specific activity of invertase was more than 100-fold higher in the epidermis than in the mesophyll. All three isoforms were acidic invertases, with pH optima of around 5.0 and little activity in the alkaline range. Invertase I had a Km for sucrose of 8.1 mM, and invertases IIA and IIB had much lower values of 1.0 and 1.7 mM, respectively. Invertase I was more than 2-fold more resistant than the other two invertases to the inhibitors HgCl2 and pyridoxal. All three constitutive invertases were found to act also as sucrose-sucrose fructosyltransferases when supplied with high concentrations of sucrose, forming 1-kestose as principal product. However, the fructosyltransferase activity of all three enzymes was inhibited by pyridoxal in the same way as their invertase activity. This characteristic clearly differentiates them from the inducible sucrose-sucrose fructosyltransferase of barley leaves, the activity responsible for the initial steps of fructan biosynthesis, which has previously been shown to be insensitive to pyridoxal.     

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.     

Characterization and distribution of invertase activity in developing maize (Zea mays) kernels

Invertase ( β -fructofuranoside fructohydrolase, EC 3.2.1.26) activity in developing maize ( Zea mays L. inbred W64A) was separated into soluble and particulate forms. The particulate form was solubilized by treatment with 1 M NaCl or with other salts. However, CaCl₂ inhibited invertase activity, and neither detergents nor 0.5 M methyl mannoside were effective in solubilizing the invertase activity. The soluble and particulate invertases were both glycoproteins, both had pH optima of 5.0 and K_m values for sucrose of 2.83 and 1.84 m M , respectively. The apparent molecular weight of salt-solubilized invertase was 40 kDa. Gel filtration of the soluble invertase showed multiple peaks with apparent molecular weights ranging from 750 kDa to over 9 000 kDa. Histochemical staining of cell wall preparations for invertase activity suggested that the particulate invertase is associated with the cell wall. Also, nearly all the invertase activity was localized in the basal endosperm and pedicel tissues, which are sites of sugar transport. No invertase activity was found in the upper endosperm, the embryo or in the placento-chalazal tissue. In contrast, sucrose synthase (EC 2.4.1.13) activity was found primarily in the embryo and the upper endosperm, which are areas of active biosynthesis of storage compounds.     

Invertase activity and the kinetin-stimulated enlargement of detached radish cotyledons

Cytokinin treatment is known to promote expansion of light-grown excised radish (Raphanus sativus L. cv Crimson Giant) cotyledons. This expansion, at least in part, seems to be related to an increased accumulation of osmotically active reducing sugars. Kinetin treatment did not cause increased levels of isocitrate lyase activity over the controls, but stimulated increased levels of two invertase forms, designated types I and II. Type I was soluble and type II was insoluble after homogenization in 10 millimolar tris(hydroxymethyl)aminomethane-HCl (pH 7.0). Both types were soluble after homogenization in 300 millimolar NaCl. At low salt concentration, type II was retained on a diethylamioethyl-cellulose column and type I was not. Type II was then eluted from the column at high salt concentration. Types I and II exhibited pH optima of 5.3 and 4.3, Michaelis constants of 4.96 and 1.23 millimolar sucrose, and molecular weights of 65,000 and 57,000 daltons, respectively. The kinetin promotion of reducing sugar accumulation may be related to increased levels of the two invertase forms, but is probably not a result of direct cytokinin-stimulated glyoxysomal activity.