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.     

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 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.     

Invertase and sucrose synthase activity, carbohydrate status and endogenous IAA levels during Citrus leaf development

Levels of activity of the sucrose catabolizing enzymes, acid invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13), were measured during development of new leaves of Citrus sinensis (L.) Osbeck cv. Shamouti. Soluble acid invertase showed a peak activity of 32 nkat (g fresh weight)⁻¹ at ca 60% of full leaf expansion and rapidly declined toward and after full expansion. There was no concomitant increase in an insoluble form of the enzyme. Sucrose synthase activity, measured in the synthesis direction, declined from 33% of full leaf expansion [10 nkat (g fresh weight)⁻¹] 10, and following, full expansion. Highest sucrose synthase activity, measured in the cleavage direction, was 6 nkat (g fresh weight)⁻¹ and showed little change during development. Acid invertase has a K_m of 5 m M for sucrose, while sucrose synthase had a K_m of 118 m M for sucrose. Changes in acid invertase activity correlated with changes in the reducing sugar:sucrose ratio. These results suggest that soluble acid invertase activity is the primary enzyme responsible for sucrose catabolism in the expanding Citrus leaf. Changes in leaf expansion rate and invertase activity did not correlate positively with changes in endogenous free IAA level, as determined by enzyme linked immunoassay.     

Effect of nitrogen concentration on fructan and fructan metabolizing enzymes in young chicory plants (Cichorium intybus)

Witloof chicory seeds ( Cichorium intybus L. var. foliosum cv. Flash) were sown in acid-washed vermiculite in a controlled environment growth chamber. Plants received a nitrogen poor ("N-poor": 0.2 m M NH₄NO₃) but otherwise complete medium, or a nitrogen rich ("N-rich": 2 m M NH₄NO₃) medium. After 1 month of growth the fructan concentration in the "N-poor" plants was about five times higher and also the activity of sucrose:sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99) was twice as high as in "N-rich" plants. The activities of the catabolic enzymes fructan 1-exohydrolase (1-FEH; EC 3.2.1.80) and acid invertase (EC 3.2.1.26) were higher in the "N-rich" plants where significant energy was invested in root and leaf growth. After one month of growth, part of the "N-poor" plants were switched to the "N-rich" medium. One day after this switch, a sharp decrease in sucrose and glucose concentration was observed in the roots. During the following days, both the activities of 1-SST and fructan:fructan 1-fructosyl transferase (1-FFT; EC 2.4.1.100) decreased and the 1-FEH and invertase activities increased. These changes were correlated with a decrease in fructan concentration. Ten days after the switch, glucose and sucrose concentrations increased again and fructan synthesis resumed. During this period 1-SST activity increased and 1-FEH activity decreased. Apparently 1-SST, 1-FFT and 1-FEH simultaneously control fructan in young chicory roots. The rather unexpected finding that 1-FEH activity, which was believed to occur only in older material, can be induced in very young roots indicates that this enzyme can be induced at any physiological stage.     

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.     

Purification, Biochemical and Immunological Characterization of Acid Invertases from Apple Fruit

The soluble acid invertase (SAI) and cell wall-bound invertase (CWI) were purified from apple fruit to apparent electrophoretic homogeneity. Based on sequencing, substrate specificity, and immunoblotting assay, the purified enzymes were identified to be two isoforms of acid invertase (β-fructosidase; EC 3.2.1.26). The SAI and CWI have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kDa composed of 50 kDa subunits. The SAI has a lower Km value for sucrose and higher Km for raffinose compared with CWI. These acid invertases differ from those in other plants in some of their biochemical properties, such as the extremely high Km value for raffinose, no hydrolytic activity for stachyose, and a mixed form of inhibition by fructose to their activity. The antibodies directed against the SAI and CWI recognized, from the crude extract, three polypeptides with a molecular mass of 50, 68, and 30 kDa, respectively.These results provide a substantial basis for the further studies of the acid invertases in apple fruit.     

Characterization of hexose kinases from camellia and lily pollen grains

Extracts from Camellia japonica, Lilium longiflorum and L. lancifolium pollen grains showed a far higher kinase activity with fructose than with glucose. Fructokinase (EC 2.7.1.4) and hexokinase (EC 2.7.1.1) preparations were obtained by partial fractionation of the extracts by DEAE-cellulose chromatography; the former was practically inactive with glucose. All had a pH optimum at 7.0–8.0 and required Mg¹⁺ ions for activity with optima at 0.5-1 m M and 1-2 m M for fructokinase and hexokinase activities, respectively. Fructokinases had K_m of 0.2-0.4 m M for fructose and similar affinities for ATP and UTP, and were inhibited by fructose above 1 m M . Hexokinases had a higher affinity for glucose than for fructose and a lower affinity for UTP compared to ATP. In camellia pollen most of hexose kinase activities were found to be due to fructokinase. These results are discussed in relation to stimulation of camellia pollen tube growth by oligosaccharides susceptible to invertase (EC 3.2.1.26).     

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.     

Unusual regulatory properties of sucrose-phosphate synthase purified from soybean (Glycine max) leaves

Sucrose-phosphate (SPS) from source leaves of soybean ( Glycine max (L.) Merr. cv. Ransom II) was purified 74-fold to a final specific activity of 1.8 U (mg protein)¹. The partially purified preparation was free from phosphoglucoseisomerase (EC 5.3.1.9), pyrophosphatase (EC 3.6.1.1), phosphoenolpyruvate-phosphatase (EC 3.1.3.-), phosphofructokinase (EC 2.7.1.11), and uridine diphosphatase (EC 3.6.1.6), and was used for characterization of the kinetic and regulatory properties of the enzyme. The enzyme showed hyperbolic saturation kinetics for both fructose-6-phosphate (K_m=0.57 m M ) and UDPGlucose (UDPG) (K_m=4.8 m M ). The activity of SPS was inhibited by the product UDP. In vitro this inhibition could be partially overcome by the presence of Mg²⁺. Inorganic orthophosphate was only slightly inhibitory (35% inhibition at 25 m M phosphate). Glucose-6-phosphate (up to 20 m M ) had no effect on activity, and did not show any significant interaction with phosphate inhibition. A range of potential effectors was tested and had no effect on SPS activity: Glucose-1-phosphate, fructose-1, 6-bisphosphate, α-glycero-phosphate, dihydroxyacetone-phosphate, 3-phosphoglyceric acid, (all at 5 m M ), sucrose at 100 m M and pyrophosphate at 0.1 m M . The apparent lack of allosteric regulation of soybean SPS makes this enzyme markedly different from SPS previously characterized from spinach and maize.     

Sucrose‐cleaving enzymes and carbohydrate pools in Lilium longiflorum floral organs

The activities of soluble invertase (EC 3.2.1.26), cell wall invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13) were determined in Easter lily ( Lilium longiflorum Thunb. cv. Nellie White) floral organs during flower development. These enzyme activities were correlated with dry weight gains and carbohydrate pools to investigate the importance of their expression in maintaining sink strength of floral organs. In the early stages of flower bud development, anthers exhibited the highest rates of dry weight gain and activity of sucrolytic enzymes. Once anther growth was completed, the dry weight gain of tepal, filament, stigma and style increased with a concomitant increase in hexose concentrations and invertase activity. Although all three enzymes capable of catalyzing sucrose cleavage were present in every flower organ of L. longiflorum , soluble invertase was the predominant enzyme in all flower organs except stigma where cell wall invertase dominated. Soluble invertase activity was highly correlated with dry weight gain in most of the flower organs.     

Translocation of 14C, carbohydrate content and activity of the enzymes of sucrose metabolism in rose petals temperatures

Both export of ¹⁴C from the source leaves of roses (Rosa × hybrida cv. Golden Times) and import of ¹⁴C to the petals were reduced by plant exposure to low night temperature. However, the import was affected to a greater extent than the export. During all stages of flower bud development the concentration of reducing sugars in petals of roses grown at reduced night temperature was lower than in petals of plants grown at higher night temperature. There was no significant difference in starch content in response to the night temperature, and the content of starch decreased toward complete flower bud opening. The concentration of sucrose in flowers at the low night temperature remained low during all stages of flower bud development, while at the high night temperature the concentration of sucrose increased during flower bud development, reaching a peak at the stage when petals start to unfold. At both temperatures the concentration of sucrose declined at complete flower opening. The activity of sucrose synthase (EC 2.4.1.14) was inhibited by low temperature in young rose shoots more than in the petals, while the activity of acid invertase (EC 3.2.1.26) was affected similarly in both tissues by the temperature treatments.     

Measurement of enzymes related to sucrose metabolism in permeabilized Chlorella vulgaris cells

Sucrose phosphate synthase (UDP-glucose: D-fructose-6-phosphate-2-glucosyl transferase, EC 2.4.1.14), sucrose synthase (UDP-glucose: D-fructose-2-glucosyl transferase, EC 2.4.1.13) and invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) were measured in toluene permeabilized cells of Chlorella vulgaris Beijerinck. All three activities were detected at all stages of the growth curve; sucrose synthase and sucrose phosphate synthase showed a zone of maximum activity, while invertase increased with time of growth. Sucrose phosphate synthase and sucrose synthase (sucrose synthesis direction) were stimulated by divalent cations and inhibited by UDP. This inhibition could be reversed by Mg²⁺ or Mn²⁺. Sucrose phosphate synthase activity was inhibited by inorganic phosphate and was enhanced by glucose-6-phosphate, but was insensitive to sucrose. Arbutine decreased sucrose synthase activity in both directions. Sucrose cleavage was inhibited by divalent cations and by pyrophosphate. The effects on the enzyme activities of the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), gibberellic acid, abscisic acid and kinetin in the growth medium were investigated. Sucrose synthase activity was practically unaffected by all plant hormones tested, except for the presence of kinetin which stimulated the activity. Sucrose phosphate synthase activity was increased by both kinetin and abscisic acid. The effect of the latter was partially reversed by the presence of gibberellic acid. 2,4-D and kinetin were potent stimulators of invertase activity.     

Purification and characterization of an endoamylase from tulip (Tulipa gesneriana) bulbs

Amylase activity extracted from tulip ( Tulipa gesneriana L. cv. Apeldoorn) bulbs that had been stored for 6 weeks at 4°C was resolved to 3 peaks by anion-exchange chromatography on diethylaminoethyl-Sephacel. These 3 amylases exhibited different relative mobilities during non-denaturing polyacrylamide gel electrophoresis (PAGE). The most abundant amylase form (amylase I) was purified to apparent homogeneity using hydrophobic interaction chromatography, gel filtration and chromatofocusing. The apparent molecular mass of the purified amylase was estimated to be 51 kDa by sodium dodecyl sulfate-PAGE and 45 kDa by gel filtration chromatography. The purified amylase was determined to be an endoamylase (EC 3.2.1.1) based on substrate specificity and end-product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55°C. The apparent K_m value with soluble starch (potato) was 1.28 mg ml⁻¹. The presence of Ca²⁺ increased the activity and thermal stability of the enzyme. The presence of dithiothreitol enhanced the activity, while β -mercaptoethanol and reduced glutathione had no significant effect. When pre-incubated in the absence of the substrate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) partially inhibited the enzyme. α -cyclodextrins or β -cyclodextrins had no effect on enzyme activity up to 10 m M . In addition to CaCl₂, CoCl₂ slightly enhanced activity, while MgCl₂ and MnCl₂ had no significant effect at a concentration of 2 m M . ZnCl₂, CuSO₄, AgNO₃ and EDTA partially inhibited enzyme activity, while AgNO₃ and HgCl₂ completely inhibited it at 2.0 m M .     

Sucrose accumulation in developing peach fruit

Uptake of ¹⁴C-sugars and activities of sucrose metabolizing enzymes were determined in order to study the mechanism(s) of sucrose accumulation in developing peach fruit. Mesocarp of young peach fruit contained glucose and fructose but little sucrose. Starting 88 days after anthesis (DAA) the sucrose concentration increased greatly. The mechanism of sucrose accumulation was studied by measuring ¹⁴C-sucrose and ¹⁴C-glucose uptake rates at three different stages of fruit development, and by assaying weekly the activity of enzymes involved in the hydrolysis and/or synthesis of the soluble sugars. Uptake of 0.5–100 m M ¹⁴C-sucrose and ¹⁴C-glucose by mesocarp tissue slices showed a complex pattern at the first stage of fruit development (62 DAA). During the subsequent growth stages the pattern of sugar uptake changed and was approximately monophasic at the third stage of fruit development.
At 10 m M , glucose was taken up more rapidly than sucrose at the first and second stage of fruit development. Uptake was partially inhibited by the uncoupler carbonylcyanide m -chlorophenylhydrazone (CCCP) at 25 μ M. These results, together with the presence of a putative extracellular invertase, suggest an apoplastic route for sucrose uptake which is dependent, at least in part, on energy supply.
Activities of sucrose hydrolyzing enzymes (insoluble acid invertase, soluble acid invertase, neutral invertase, sucrose synthase) were high in young fruits and declined sharply with fruit development concomitantly with accumulation of sucrose. The storage of the sugar was not accompanied by a rise in synthetic activities (sucrose synthase, sucrose phosphate synthase), suggesting that sucrose could, at least in part enter the carbohydrate pool directly.     

Purification, Biochemical and Immunological Characterization of Acid Invertases from Apple Fruit

The soluble acid invertase (SAI) and cell wall-bound invertase (CWI) were purified from apple fruit to apparent electrophoretic homogeneity. Based on sequencing, substrate specificity, and immunoblotting assay, the purified enzymes were identified to be two isoforms of acid invertase (β-fructosidase; EC 3.2.1.26). The SAI and CWI have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kDa composed of 50 kDa subunits. The SAI has a lower Km value for sucrose and higher Km for raffinose compared with CWI. These acid invertases differ from those in other plants in some of their biochemical properties, such as the extremely high Km value for raffinose, no hydrolytic activity for stachyose, and a mixed form of inhibition by fructose to their activity. The antibodies directed against the SAI and CWI recognized, from the crude extract, three polypeptides with a molecular mass of 50, 68, and 30 kDa, respectively.These results provide a substantial basis for the further studies of the acid invertases in apple fruit.     

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.     

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.     

The occurrence of inorganic pyrophosphate: d-fructose-6-phosphate 1-phosphotransferase in higher plants. I. Initial characterization of partially purified enzyme from Sanseviera trifasciata leaves

Among 30 plant species examined, the PPi-phosphofructokinase (EC 2.7.1.90) was found in leaves of 21 plants. Some of the plants exhibit no activity of ATP-dependent phosphofructokinase but display only activity of PPi-phosphofructokinase. A partly purified preparation of PPi-phosphofructokinase with specific activity of 8.4 Hmol (mg protein)⁻¹ min⁻¹ was obtained from Sanseviera trifasciata leaves. The enzyme is restricted to the cytoplasm, it exhibits pronounced substrate specifity, requires Mg²⁺ ions, is inhibited by AMP, PEP, methylenediphosphonate and stabilized by mercaptoethanol. At pH 7.8 with 1.5 m M MgCl₂ the following K_M values were observed: pyrophosphate, 0.58 m M ; fructose 6-phosphate, 0.8 m M . The K_M values for substrates of reverse reaction (pH 7.3; 2 m M MgCl₂) are of the same order of magnitude: 0.83 m M for fructose 1,6-diphosphate, and 0.14 m M for orthophosphate. The molecular weight of the studied enzyme is about 125 000 dalton as estimated by gel filtration.