Ultrastructural study on the stylar transmitting tissue in Japanese pear

Summary The stylar transmitting tissue in the mature pistil of the Japanese pear consists of its component cells and intercellular heterogeneous secretions. The cytoplasm of the periplasmic region contains two different organelles that are characteristic of floral bud development. One of these is the vesicle, which is derived from rough ER and transferred to the periplasmic region of the cell during an early stage of the floral bud. The other one is the lipid droplet, which reacts to polysaccharidic staining and is seen throughout floral bud development. The lipid droplets are closely associated with the Golgi bodies and seem to be dissolved in the vacuole. The materials found in the vacuoles appear to diffuse and pass through the cell walls as intercellular substances.     

Promotion of elongation and acid invertase activity in Phaseolus vulgaris L. internode segments by phenylacetic acid

Phenylacetic acid (PAA) significantly stimulated the elongation of isolated Phaseolus vulgaris internodal segments and prevented the decline in acid invertase specific activity observed in segments incubated in the absence of growth substances. Unlike IAA, which stimulated both elongation and invertase activity over a very wide range of concentrations (<10^-4 - 1 mol.m^-3; optimum 10^-2 mol.m^-3), the response to PAA was restricted to a much narrower range of concentrations (3 × 10^-2 - 1 mol.m^-3; optimum ca. 1–2 × 10^-1mol.m^-3). At the optimum concentration of PAA, the stimulation of both responses was about 63–75% of that induced by the optimum concentration of IAA. The differences in the concentration range and magnitude of the responses to IAA and PAA were not due to differences in uptake of the two compounds. The stimulation of elongation by both compounds was prevented by 3.6 × 10^-2mol.m^-3 cycloheximide (CH), and acid invertase activites were greatly reduced compared with samples treated with growth substances alone. A saturating concentration of the specific auxin efflux carrier inhibitor N-1-naphthylphthalamic acid (NPA) slightly promoted the growth of control segments, probably by reducing the loss of residual endogenous auxin to the incubation medium. The elongation induced by PAA at its optimum concentration was considerably greater than the elongation induced by NPA, indicating that PAA did not cause growth by preventing the loss of endogenous auxin from the segments. Elongation responses to combinations of IAA and PAA suggested that the compounds were acting additively and that they were affecting growth by the same mechanism.     

Purification and characterization of two extracellular endochitinases from Massilia timonae

Two extracellular chitinases (designated as Chi-56 and Chi-64) produced by Massilia timonae were purified by ion-exchange chromatography, ammonium sulfate precipitation, and gel-filtration chromatography. The molecular mass of Chi-56 was 56 kDa as determined by both SDS-PAGE and gel-filtration chromatography. On the other hand, Chi-64 showed a molecular mass of 64 kDa by SDS-PAGE and 28 kDa by gel-filtration chromatography suggesting that its properties may be different from those of Chi-56. The optimum temperature, optimum pH, pI, K_m, and V_max of Chi-56 were 55 °C, pH 5.0, pH 8.5, 1.1 mg mL⁻¹, and 0.59 μmol μg⁻¹ h⁻¹, respectively. For Chi-64, these values were 60 °C, pH 5.0, pH 8.5, 1.3 mg mL⁻¹, and 1.36 μmol μg⁻¹ h⁻¹, respectively. Both enzymes were stimulated by Mn²⁺ and inhibited by Hg²⁺, and neither showed exochitinase activity. The N-terminal sequences of Chi-56 and Chi-64 were determined to be Q-T-P-T-Y-T-A-T-L and Q-A-D-F-P-A-P-A-E, respectively.     

Roles of gibberellins in increasing sink demand in Japanese pear fruit during rapid fruit growth

Our previous work demonstrated that exogenous gibberellins (GAs) applications during rapid fruit growth significantly increases sink demand and results in a larger fruit in Japanese pear. In an attempt to unravel the mechanism of increased sink demand by applied GAs, the histology, cell wall components of the flesh, and carbon accumulation in the fruit were assessed for Japanese pear (Pyrus pyrifolia, cultivar ‘Kousui’), as were the activities of sucrose- and sorbitol-cleaving enzymes. Our results show that most vascular tissues occurred in core tissue with very little vascular tissue in the flesh. Application of a mixture of GA₃ + GA₄ in lanolin paste significantly increased the amount of ethanol-insoluble solids, e.g., total pectins, hemicellulose, and cellulose in the cell walls. There was a significantly increased sink demand (assessed by ¹³C accumulation in the fruit) by the applied GAs, and this increased sink strength was closely related to increased activities of cell wall-bound invertase in the core, neutral invertase and NAD-dependent sorbitol dehydrogenase in the flesh during rapid fruit growth. As well, concentrations of sorbitol and sucrose in the flesh were decreased by GA application, while glucose concentration increased. Most importantly, the fact that sink activity can be increased by GA application implies that endogenous GAs are likely to be important modulators for sugar metabolism. Hence, selecting for genotypes with elevated GA production in the growing fruit and increased activities of key enzymes for sugar metabolism could result in increased fruit size.     

The effect of shoot-bending on the amount of diffusible indole-3-acetic acid and its transport in shoots of Japanese pear

The amount of diffusible indole-3-acetic acid (IAA) in shoots ofJapanese pear (Pyrus pyrifolia) decreased when vertical shootswere bent at an angle of 45°. A significant decrease of diffusibleIAA was observed one day after shoot bending (DAB), and the degree ofthis decrease was larger in the apical region of the shoot than in thebasal region. The decrease caused by the shoot bending increased withthe duration of the treatment. The IAA amounts in the bent shoot in theapical, central, and basal segments on 1 DAB were58.2±6.4%, 92.6±7.6%, and79.1±7.1% of the control, while 43.7±4.1%,30.8±2.9%, and 39.4±2.5% on 14 DAB.Radiolabelled IAA transport velocity was also examined, but it was notinfluenced by the shoot angle in the apical region of the shoot.However, the IAA transport velocity in the basal region decreased. Itdropped first on 1 DAB, but it recovered to the control level 3 DAB,then it decreased again on 14 DAB. A large increase in ethyleneproduction was observed in the bent shoot, but it seemed transient anddid not continue for 14 days. These results suggest that the decrease ofdiffusible IAA amounts may be induced not by the decrease of IAAtransport velocity but by the production/supply of IAA in the apicalregion.     

Purification and characterization of an alkaline invertase from shoots of etiolated rice seedlings

One alkaline invertase and two acid invertase activities were detected in the shoots of etiolated rice ( Oryza sativa ) seedlings. The alkaline invertase (AIT) was purified to homogeneity through steps of ammonium sulphate fractionation, concanavalin A-Sepharose affinity chromatography (non-retained), DEAE-Sephacel chromatography and preparative electrophoresis. The pH optimum of AIT was 7.0 and the molecular mass, determined by gel filtration, was 240 kDa. It is apparently a homotetrameric enzyme (subunit molecular mass 60 kDa). The isoelectric point was 4.4 by isoelectric focusing. The best substrate of the enzyme was sucrose, with a K _m of 2.53 mM. The enzyme also hydrolysed raffinose, but not maltose or lactose, so it is a β-D-fructofuranosidase. It gave negative glycoprotein staining. Of the hydrolysis products, fructose was a competitive inhibitor and glucose was a non-competitive inhibitor. Treatment with an alkaline phosphatase could activate AIT, whereas other proteins such as BSA, concanavalin A and urease had no effect on the enzyme activity. The enzyme activity was inhibited by Tris, thiol reagents and heavy metal ions.     

Possible role of soluble invertase in the gibberellic acid berry-sizing effect in Sultana grape

It is well known that post-bloom applications ofgibberellic acid (GA₃) increase seedless grapeberry size by enhancing cell division, or cellenlargement, or both. As a consequence, total waterand sugar per berry are increased. Soluble invertaseis considered to be one of the key enzymes in theaccumulation of sugar in grape berries. To study apossible role of invertase in the GA₃berry-sizing effect, different rates of post-bloomGA₃ were applied to seedless grape cv. Sultanaand hexose concentration and invertase activity weremeasured. GA₃ stimulated both parameters as earlyas 24 and 32 h after applications, respectively.Moreover, the increment in sugar content and enzymeactivity remained throughout the growing of the berries period and, at ripening, increases in hexosescontent (102%) and invertase activity (60%) weredetected when GA₃ was applied at a rate of 45 ppm.At the same GA₃ rate the pericarp cellsdoubled in size. Furthermore, positive correlationswere found between berry-size, invertase activity andhexose content, suggesting that GA₃ stimulationof invertase could be one of the factors involved in theberry sizing-effect of GA₃.     

Meira nashicola sp. nov., a novel basidiomycetous, anamorphic yeastlike fungus isolated from Japanese pear fruit with reddish stain

Three undescribed strains of basidiomycetous, anamorphic yeastlike fungi were isolated from Japanese pear fruits with a reddish stain collected in Tottori Prefecture, Japan. The strains are classified in a single group and assigned to the genus Meira by conventional and chemotaxonomic studies. Sequence analyses of the D1/D2 domain of 26S rDNA and internal transcribed spacer (ITS) regions indicate that the strains represent a novel species with a close phylogenetic relationship to Meira geulakonigii and M. argovae. The name Meira nashicola sp. nov. is proposed for the strains (type strain PFS 002 = MAFF 230028 = CBS 117161).     

Purification and characterization of a non-S-RNase and S-RNases from styles of Japanese pear (Pyrus pyrifolia)

In this study we biochemically characterized stylar ribonucleases (RNases) of Japanese pear (Pyrus pyrifolia), which exhibits S-RNase-based gametophytic self-incompatibility. We separated the RNase fractions NS-1, NS-2, and NS-3 from stylar extracts of the cultivar Nijisseiki (S(2)S(4)). The RNase in each fraction was purified to homogeneity through a series of chromatographic steps. Chemical analysis of the proteins revealed that the basic RNases in the NS-2 and NS-3 fractions were the S(4)- and S(2)-RNases, respectively. Five additional S-RNases were purified from other cultivars. An acidic RNase in the NS-1 fraction was also purified from other cultivars, and identified as a non-S-allele-associated RNase (non-S-RNase). The non-S-RNase is composed of 203 amino acids, is non-glycosylated and is a N-terminal-pyroglutamylated enzyme of the RNase T(2) family. The substrate specificities and optimum pH levels of the non-S-RNase and S-RNases were similar. Interestingly, the specific activity of the non-S-RNase was 7.5-221-fold higher than those of the S-RNases when tolura yeast RNA was used as the substrate. The specific activity of the S(2)-RNase was 8.8-28.6-fold lower than those of the other S-RNases. These differences in specific activities among the stylar RNases are discussed.     

Partitioning of (13)C-photosynthate from spur leaves during fruit growth of three Japanese pear (Pyrus pyrifolia) cultivars differing in maturation date

BACKGROUND AND AIMS: In fruit crops, fruit size at harvest is an important aspect of quality. With Japanese pears (Pyrus pyrifolia), later maturing cultivars usually have larger fruits than earlier maturing cultivars. It is considered that the supply of photosynthate during fruit development is a critical determinant of size. To assess the interaction of assimilate supply and early/late maturity of cultivars and its effect on final fruit size, the pattern of carbon assimilate partitioning from spur leaves (source) to fruit and other organs (sinks) during fruit growth was investigated using three genotypes differing in maturation date. METHODS: Partitioning of photosynthate from spur leaves during fruit growth was investigated by exposure of spurs to (13)CO(2) and measurement of the change in (13)C abundance in dry matter with time. Leaf number and leaf area per spur, fresh fruit weight, cell number and cell size of the mesocarp were measured and used to model the development of the spur leaf and fruit. KEY RESULTS: Compared with the earlier-maturing cultivars 'Shinsui' and 'Kousui', the larger-fruited, later-maturing cultivar 'Shinsetsu' had a greater total leaf area per spur, greater source strength (source weight x source specific activity), with more (13)C assimilated per spur and allocated to fruit, smaller loss of (13)C in respiration and export over the season, and longer duration of cell division and enlargement. Histology shows that cultivar differences in final fruit size were mainly attributable to the number of cells in the mesocarp. CONCLUSIONS: Assimilate availability during the period of cell division was crucial for early fruit growth and closely correlated with final fruit size. Early fruit growth of the earlier-maturing cultivars, but not the later-maturing ones, was severely restrained by assimilate supply rather than by sink limitation.     

Purification and characterization of urease from dehusked pigeonpea (Cajanus cajan L) seeds

Urease has been purified from the dehusked seeds of pigeonpea (Cajanus cajan L.) to apparent electrophoretic homogeneity with approximately 200 fold purification, with a specific activity of 6.24 x10(3) U mg(-1) protein. The enzyme was purified by the sequence of steps, namely, first acetone fractionation, acid step, a second acetone fractionation followed by gel filtration and anion-exchange chromatographies. Single band was observed in both native- and SDS-PAGE. The molecular mass estimated for the native enzyme was 540 kDa whereas subunit values of 90 kDa were determined. Hence, urease is a hexamer of identical subunits. Nickel was observed in the purified enzyme from atomic absorption spectroscopy with approximately 2 nickel ions per enzyme subunit. Both jack bean and soybean ureases are serologically related to pigeonpea urease. The amino acid composition of pigeonpea urease shows high acidic amino acid content. The N-terminal sequence of pigeonpea urease, determined up to the 20th residue, was homologous to that of jack bean and soybean seed ureases. The optimum pH was 7.3 in the pH range 5.0-8.5. Pigeonpea urease shows K(m) for urea of 3.0+/-0.2 mM in 0.05 M Tris-acetate buffer, pH 7.3, at 37 degrees C. The turnover number, k(cat), was observed to be 6.2 x 10(4) s(-1) and k(cat)/K(m) was 2.1 x 10(7) M(-1) s(-1). Pigeonpea urease shows high specificity for its primary substrate urea.     

Simultaneous production of citric acid and invertase by Yarrowia lipolytica SUC+ transformants

Simultaneous production of citric acid (CA) and invertase by Yarrowia lipolytica A-101-B56-5 (SUC⁺ clone) growing from sucrose, mixture of glucose and fructose, glucose or glycerol was investigated. Among the tested substrates the highest concentration of CA was reached from glycerol (57.15 g/L) with high yield (Y_CA/S = 0.6 g/g). When sucrose was used, comparable amount of CA was secreted (45 g/L) with slightly higher yield (Y_CA/S = 0.643 g/g). In all cultures amount of isocitrate (ICA) was below 2% of total citrates. Considering invertase production, the best carbon source appeared to be sucrose (72 380 U/L). The highest yield of CA and invertase biosynthesis calculated for 1 g of biomass was obtained for cells growing from glycerol (9.9 g/g and 4325 U/g, respectively). Concentrates of extra- and intracellular invertase of the highest activity were obtained from sucrose as substrate (0.5 and 1.8 × 10⁶ U/L, respectively).     

Purification and characterization of soluble peroxidase from oil palm (Elaeis guineensis Jacq) leaf

Soluble peroxidase (POD) from oil palm leaf was purified by (NH(4))(2)SO(4) precipitation, anion exchange chromatography and molecular exclusion chromatography. The purification grade obtained was 429 yielding 54% of the enzyme activity. Electrophoresis of purified enzyme under denatured conditions revealed M(r) of 48+/-2 kDa. It has an optimum pH of 5 and it exhibited very high pH and thermal stabilities. K(m) for guaiacol, ABTS and pyrogallol were 3.96, 1 and 0.84 mM, respectively. Immunocytochemical localization studies showed that soluble POD was mainly located in the vascular bundles and epidermis of leaf.     

Purification and biochemical characterization of two novel antigens from Leishmania major promastigotes

The identification and characterization of antigens that elicit human T cell responses is an important step toward understanding of Leishmania major infection and ultimately in the development of a vaccine. Micropreparative SDS-PAGE followed by electrotransfer to a PVDF membrane and elution of proteins from the PVDF, was used to separate 2 novel proteins from L. major promastigotes, which can induce antibodies of the IgG2a isotype in mice and also are recognized by antisera of recovered human cutaneous leishmaniasis subjects. Fractionation of the crude extract of L. major revealed that all detectable proteins of interest were present within the soluble Leishmania antigens (SLA). Quantitation of these proteins showed that their expression in promastigotes is relatively very low. Considering the molecular weight, immunoreactivity, chromatographic and electrophoretic behavior in reducing and non-reducing conditions, these proteins are probably 2 isoforms of a single protein. A digest of these proteins was resolved on Tricine-SDS-PAGE and immunoreactive fragments were identified by human sera. Two immunoreactive fragments (36.4 and 34.8 kDa) were only generated by endoproteinase Glu-C treatment. These immunoreactive fragments or their parent molecules may be ideal candidates for incorporation in a cocktail vaccine against cutaneous leishmaniasis.     

Purification and characterization of three neutral extracellular isoperoxidases from rye leaves

Rye (Secale cereale L.) seedlings; contain two major flavone glucuronides, luteolin 7-O-diglucuronyl-4'-O-glucuronide (L3GlcUA) (1) and luteolin 7-O-diglucuronide (L2GlcUA) (2) in abundance in the apoplast of primary leaves; express a large number of peroxidase isoenzymes; and release H(2)O(2) into the apoplast during primary leaf development. We purified and characterized three neutral extracellular peroxidase isoenzymes (rPOXs N1, N2, and N3) that can oxidize L2GlcUA as a natural substrate. The isoelectric points and molecular weights of rPOXs N1, N2, and N3 were 6.1, 7.2, and 6.3, and 42, 37, and 51 kDa, respectively. The optimum pH of the rPOXs N1, N2, and N3 were 5.5, 5.5, and 8.5, respectively, and their optimum temperatures ranged from 45 to 50 degrees C for all isoenzymes. rPOXs N1, N2, and N3 recognized flavonoids with 3', 4'-OH groups as potential substrates, but not flavonoids with a glycosylated 4'-OH group or those without a 3'-OH group. The activities on phenol-type substrates were high in the order of guaiacol>catechol>o-cresol for all isoenzymes. rPOXs N1, N2, and N3 exhibited broad reactivity with endogenous hydrogen donors including luteolin glucuronides derived from the apoplast of rye primary leaves.     

Isolation and characterization of fruit vacuolar invertase genes from two tomato species and temporal differences in mRNA levels during fruit ripening

To determine the relationship between invertase gene expression and glucose and fructose accumulation in ripening tomato fruit, fruit vacuolar invertase cDNA and genomic clones from the cultivated species, Lycopersicon esculentum cv. UC82B, and a wild species, Lycopersicon pimpinellifolium, were isolated and characterized. The coding sequences of all cDNA clones examined are identical. By comparison to the known amino acid sequence of mature L. esculentum fruit vacuolar invertase, a putative signal sequence and putative amino-terminal and carboxy-terminal propeptides were identified in the derived amino acid sequence. Of the residues 42% are identical with those of carrot cell wall invertase. A putative catalytic site and a five-residue motif found in carrot, yeast, and bacterial invertases are also present in the tomato sequence. Minor differences between the nucleotide sequences of the genomic clones from the two tomato species were found in one intron and in the putative regulatory region. The gene appears to be present in one copy per haploid genome. Northern analysis suggests a different temporal pattern of vacuolar invertase mRNA levels during fruit development in the two species, with the invertase mRNA appearing at an earlier stage of fruit development in the wild species. Nucleotide differences found in the putative regulatory regions may be involved in species differences in temporal regulation of this gene, which in turn may contribute to observed differences in hexose accumulation in ripening fruit.     

Purification and characterization of recombinant human soluble guanylate cyclase produced from baculovirus-infected insect cells

Soluble guanylate cyclase (sGC) has been purified from 100 L cell culture infected by baculovirus using the newer and highly effective titerless infected-cells preservation and scale-up (TIPS) method. Successive passage of the enzyme through DEAE, Ni²⁺-NTA, and POROS Q columns obtained approximately 100 mg of protein. The sGC obtained by this procedure was already about 90% pure and suitable for various studies which include high throughput screening (HTS) and hit follow-up. However, in order to obtain enzyme of greater homogeneity and purity for crystallographic and high precision spectroscopic and kinetic studies of sGC with select stimulators, the sGC solution after the POROS Q step was further purified by GTP-agarose affinity chromatography. This additional step led to the generation of 26 mg of enzyme that was about 99% pure. This highly pure and active enzyme exhibited a M_r = 144,933 by static light scattering supportive of a dimeric structure. It migrated as a two-band protein, each of equal intensity, on SDS–PAGE corresponding to the α (M_r 77,000) and β (M_r 70,000) sGC subunits. It showed an A₄₃₀/A₂₈₀ = 1.01, indicating one heme per heterodimer, and a maximum of the Soret band at 430 nm indicative of a penta-coordinated ferrous heme with a histidine as the axial ligand. The Soret band shifted to 398 nm in the presence of an NO donor as expected for the formation of a penta-coordinated nitrosyl-heme complex. Non-stimulated sGC had k_cat/K_m = 1.7 × 10⁻³ s⁻¹ μM⁻¹ that increased to 5.8 × 10⁻¹ s⁻¹ μM⁻¹ upon stimulation with an NO donor which represents a 340-fold increase due to stimulation. The novel combination of using the TIPS method for co-expression of a heterodimeric heme-containing enzyme, along with the application of a reproducible ligand affinity purification method, has enabled us to obtain recombinant human sGC of both the quality and quantity needed to study structure–function relationships.     

Purification and characterization of pectate lyase from banana (Musa acuminata) fruits

Pectate lyase (PEL) has been purified by hydrophobic, cation exchange and size exclusion column chromatographies from ripe banana fruit. The purified enzyme has specific activity of 680 +/- 50 pkat mg protein(-1). The molecular mass of the enzyme is 43 kDa by SDS-PAGE. The pI of the enzyme is 8 with optimum activity at pH 8.5. Analysis of the reaction products by paper and anion exchange chromatographies reveal that the enzyme releases several oligomers of unsaturated galacturonane from polygalacturonate. The K(m) values of the enzyme for polygalacturonate and citrus pectin (7.2% methylation) are 0.40 +/- 0.04 and 0.77 +/- 0.08 g l(-1), respectively. PEL is sensitive to inhibition by different phenolic compounds, thiols, reducing agents, iodoacetate and N-bromosuccinimide. The enzyme has a requirement for Ca(2+) ions. However, Mg(2+) and Mn(2+) can substitute equally well. Additive effect on the enzyme activity was observed when any two metal ions (out of Mg(2+), Ca(2+) and Mn(2+)) are present together. The banana PEL is a enzyme requiring Mg(2+), in addition to Ca(2+), for exhibiting maximum activity.     

Purification and characterization of phosphoribulokinase from wheat leaves

Homogeneous phosphoribulokinase (PRK; ATP: d-ribulose-5-phosphate 1-phosphotransferase, EC 2.7.1.19) was isolated from wheat leaves with a specific activity of 15 kat mg^-1 protein. The purification included ammonium sulfate cuts, isoelectric precipitation, and hydrophobic and affinity chromatography on pentylagarose and Blue Sepharose CL 6B, respectively. Gel filtration of the purified enzyme yielded a 83000 Da protein. Subunits of about 42000 Da were estimated from sodium dodecyl sulfate-polyacrylamide gels. Wheat leaf PRK was stable for at least four weeks when stored at 4°C. Saturation curves for ribulose 5-phosphate (Ru5P) and ATP followed Michaelis-Menten kinetics (K _m values: K _{m Ru5P}=50–80 M; K _{m ATP}=70 M). The saturation curve for MgCl₂ was sigmoidal (half-maximal velocity <0.5 mM). The affinity for Ru5P, ATP and Mg²⁺ was not affected by pH changes comparable to pH shifts in the stroma. In contrast to chloroplast fructose-bisphosphatase (Zimmermann et al. 1976, Eur. J. Biochem. 70, 361–367) the affinity for ligands remained unchanged in the dithiothreitol-activated and in the non-activated state. The activity of PRK was increasingly sensitive to inhibition by 3-phosphoglyceric acid with decreasing pH below pH 8.0.Abbreviations DTT dithiothreitol - EDTA ethylenediamine-tetraacetic acid - PRK phosphoribulokinase - Ru5P ribulose-5-phosphate - SDS-PAGE sodium dodecyl sulfate-polyacryl-amide gel electrophoresis     

Purification and characterisation of soluble invertases from leaves of Arabidopsis thaliana

Multiple isoforms of -fructofuranosidase (invertase, EC 3.2.1.26) were identified in mature green leaves of the cruciferous plant Arabidopsis thaliana (L.) Heynh. There were four major and one minor isoforms of soluble acid invertase and an additional activity which could be released from the cell wall by buffers of high ionic strength. This study reports the separation and characterisation of three soluble isoforms following ammonium sulphate and polyethylene glycol 6000 precipitations, Concanavalin A, MonoQ ion exchange, Superose 12 sizeexclusion chromatography and chromatofocusing. These isoforms, designated INV1, INV2 and INV3, had isoelectric points of 4.75, 4.70 and 4.65 and a K _m for sucrose of 5, 12 and 5 mM, respectively. Each had a pH optimum of 5.5, exhibited optimal activity at 45 °C and used sucrose as the preferred substrate. All fractions containing these isoforms contained a 52-kDa polypeptide which was specifically detected by immunoblotting with an antibody raised against deglycosylated wheat invertase. The N-terminal amino-acid sequence of this polypeptide was homologous to acid invertases isolated from other plant species. The possible origin of isoforms of soluble acid invertase is discussed.Abbreviations PEG polyethylene glycol - pI isoelectric point - PMSF phenylmethylsulphonyl fluoride We wish to acknowledge the support of the British/Swiss Joint Research Programme and the Sheffield University Research Support Fund. X.T. was in receipt of an Overseas Research Scholarship and a University of Sheffield Research Scholarship. We wish to thank Dr A. Moir for his help in N-terminal amino-acid sequencing.