A 17-kDa Nicotiana tabacum cell-wall peptide acts as an in-vitro inhibitor of the cell-wall isoform of acid invertase

When cell-wall invertase (CWI) from Nicotiana tabacum L. cell-suspension cultures, either non-transformed or transformed with Agrobacterium tumefaciens, was salt-eluted from intact cells and purified on Sulfopropyl-Sephadex (SPS) by pH-gradient elution, the enzyme lost about 50% of its activity during a 1-h incubation at pH 4.8. However, Western-blot analysis indicated no appreciable enzyme degradation. Re-chromatography of CWI peak fractions on SPS using NaCl-gradient elution showed the presence of a 17-kDa peptide (p17) in fractions with low CWI activity but strong CWI immunosignal (Weil and Rausch 1994, Planta 193, 430–437). When separating CWI from p17 by Concanavalin A (Con A)-Sepharose chromatography, inhibition could be restored by incubating the inhibitor-containing fraction with inhibitor-free CWI. More than 90% of CWI could be inhibited, suggesting that all CWI was susceptible to p17 binding. The presence of divalent metal ions (Ca²⁺, Mg²⁺, Zn²⁺) during pre-incubation of CWI with p17 reduced CWI inhibition substantially. Also, sucrose protected CWI against inhibition by p17 (half-maximum protection at 1.3 mM). Binding of p17 to CWI during a 1-h pre-incubation was pH-dependent, pH 4.5 causing maximum inhibition, whereas at pH 6.5 no inhibition was observed. Gel-permeation chromatography revealed that the native inhibitor acts as a monomer. Immunoprecipitation of CWI co-precipitated p17, confirming direct binding of p17 to CWI. When fractions containing CWI and p17 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blotting a diffuse immunosignal of 86–90 kDa was observed (in addition to the prominent CWI signal at 69 kDa). Equilibration of this zone with urea-containing sample buffer prior to a second SDS-PAGE run resulted in a strong immunosignal at 87 (± 2) kDa, suggesting that during one step in the formation of the p17-CWI complex the two polypeptides became firmly aggregated. The distribution of CWI and glucose-6-phosphate dehydrogenase activities between the cell-wall protein fraction and salt-eluted cells shows that cells retained their structural integrity, thus indicating co-localization of p17 and CWI in situ (Weil and Rausch 1994). We have purified p17 to homogeneity and its N-terminus has been sequenced, revealing no similarity to other known protein sequences. Possible physiological roles of p17 are discussed.Abbreviations Con A concanavalin A - CWI cell-wall invertase - 1-OMG methyl -d-glucopyranoside - p17 17-kDa peptide - PMSF phenylmethylsulfonyl fluoride - PR pathogenesis related This work was supported by a grant from the Deutsche Forschungsgemeinschaft. The antiserum against the deglycosylated carrot cell-wall invertase was a gift from Dr. Sturm (Friedrich-Miescher-Institut, Basel, Switzerland). The antiserum against acidic tobacco PR1 proteins was obtained from Dr. Lotan (Weizmann Institute of Science, Rehovot, Israel). The antiserum against tomato hsp17 was a gift from Prof. Nover (J.-W.-Goethe-Universität, Frankfurt, Germany).     

Changes in Sugar Content and Activities of Sucrose Metabolizing Enzymes in Roots and Nodules of Lentil

Activities of acid and alkaline invertases and sucrose synthase were determined in roots and nodules of lentil at various stages of development. Alkaline invertase and sucrose synthase were both involved in sucrose metabolism in the nodule cytosol, but there was only a small amount of acid invertase present. Activity of sucrose metabolizing enzymes in roots was significantly less than that observed in the nodules. Amongst sugars, sucrose was found to be the main component in the host cytosol. Lentil neutral invertase (LNI) was partially purified from nodules at 50 days after sowing (DAS). Two forms of invertase were identified, i.e., a major form of 71 kDa which was taken for enzyme characterization and a minor form of 270 kDa which was not used for further studies. The purified enzyme exhibited typical hyperbolic saturation kinetics for sucrose hydrolysis. It had a Km of 11.0 to 14.0 mM for sucrose depending upon the temperature, a pH optimum of 6.8 and an optimum temperature of 40 °C. Compared with raffinose and stachyose, sucrose was better substrate for LNI. The enzyme showed no significant hydrolysis of maltose and p-nitrophenyl--D-glucopyranoside, showing its true -fructosidase nature. LNI is completely inhibited by HgCl₂, MnCl₂ and iodoacetamide but not by CaCl₂, MgCl₂ or BaCl₂.     

Cloning and expression of the Bacillus circulans endo-β-1,3-1,4-glucanase gene (bglBC1) in Escherichia coli

A gene coding for the endo--1,3-1,4-glucanase of B. circulans ATCC21367 was cloned into Escherichia coli. The cloned enzyme hydrolyzed lichenan or barley -glucan to produce 3-O--cellobiosyl-d-glucose as a main product but was inactive with carboxymethyl cellulose, laminarin and xylan. The enzyme, M _r=28 kDa, remained within the cytoplasm of E. coli. A 771 bp open reading frame was in the 2 kb PstI fragment of the recombinant plasmid pLL200K. The deduced protein sequence consists of 257 amino acids and has a putative signal peptide of 26 amino acids. The amino acid sequence of the endo--1,3-1,4-glucanase showed 68 and 51% homology to previously reported endo--1,3-1,4-glucanases from Bacillus strain N-137 and B. brevis, respectively.     

The role of separate molecular domains in the structure of phytochrome from etiolated Avena sativa L.

The spectral properties of peptides generated from etiolated-Avana, 124-kDa (kilodalton) phytochrome by endogenous protease(s) have been studied to assess the role of the amino-terminal and the carboxyl-terminal domains in maintaining the proper interaction between protein and chromophore. The amino-terminal, 74-kDa chromopeptide, a degradation product of the far-red absorbing form of the pigment (Pfr), is shown to be spectrally similar to the 124-kDa, undegraded molecule. The minimum and maximum of the difference spectrum (Pr-Pfr) are 730 and 665 nm, respectively, and the spectral-change ratio is unity. Also, like undegraded, 124-kDa phytochrome, the 74-kDa peptide exhibits minimal dark reversion. These data indicate that the 55-kDa, carboxyl-terminal half of the polypeptide does not interact with the chromophore and may not have a role in the structureal integrity of the amino-terminal domain. The 64-kDa chromopeptide can be generated directly from the 74-kDa species by cleavage of 10 kDa from the amino terminus upon incubation of this species as Pr. Accompanying this conversion are changes in the spectral properties, namely, a shift in the difference spectrum minimum to 722–724 nm and a tenfold increase in the capacity for dark reversion. These data indicate that the 6–10 kDa, amino-terminal segment continues to function in its role of maintaining proper chromophore-protein interactions in the 74-kDa peptide as it does in the undegraded molecule. Conversely, removal of this segment upon proteolysis to the 63-kDa species leads to aberrant spectral properties analogous to those observed when this domain is lost from the full-length, 124-kDa molecule, resulting in the 118/114-kDa degradation products. The data also show that photoconversion of the 74-kDa chromopeptide from Pfr to Pr exposes proteolytically susceptible sites in the same way as in the 124-kDa molecule. Thus, the separated, 74-kDa amino-terminal domain undergoes a photoinducible conformational change comparable to that in the intact molecule.Abbreviations and symbols Da dalton - Pfr far-red-absorbing from of phytochrome - PMSF phenylmethylsulfonyl fluoride - Pr red-absorbing form of phytochrome - R red light - FR lar-red light - A _r/A _fr spectral change ratio - _max ^FR peak maximum (nm) of Pfr absorbance     

The carbohydrate moiety of the acid carboxypeptidase fromAspergillus saitoi

Acid carboxypeptidase fromAspergillus saitoi is a glycoprotein that contains both N-and O-linked sugar chains. The N-glycanase released high-mannose type oligosaccharides that were separated into eight components on HPLC. One, which had a unique structure of Man₁₁GlcNAc₂, was characterized. Mild alkali treatment of the carboxypeptidase, under conditions that effect -elimination, yieldedd-mannose. Deglycosylation of the carboxypeptidase with endo--N-acetylglucosaminidase and -mannosidase effected the reduction of the molecular mass from 72 kDa to 60 kDa. Partial changes of CD spectra of the native and the deglycosylated enzymes indicate that some conformational changes on the peptide of the enzyme occurred after deglycosylation. Other enzymatic properties, such as catalytic activity, pH, and thermal stability and resistivity to protease digestion, did not appear to change. Tunicamycin halted secretion of the carboxypeptidase extracellularly.     

Regulation of processing of a plant glycoprotein in the Golgi complex: A comparative study usingXenopus oocytes

The synthesis of phytohemagglutinin (PHA), the major seed lectin ofPhaseolus vulgaris, was investigated inXenopus oocytes injected with RNA isolated from developing bean cotyledons. As is the case for normal PHA, oocyte-synthesized PHA polypeptides were found to contain two asparagine-linked oligosaccharide chains, one of which was of the high-mannose type and the other one of the Golgi-modified type, being largely resistant to endo--N-acetylglucosaminidase H digestion and containing fucose. The modified oligosaccharide chain of oocyte-synthesized PHA appeared to be much larger and more heterogeneous with respect to the modified chain normally present on PHA. When the oocytes were injected with purified mRNA for PHA, isolated by hybrid-selection using a PHA complementary-DNA clone, the results were the same as those obtained by injecting total cotyledonary RNA. On the whole, these results indicate that plant glycoproteins are directed to the Golgi complex even when synthesized in an animal cell, and that correct sorting of the oligosaccharide chains to be processed is independent of the cell-type in which protein synthesis occurs. The form of processing is however cell-type specific.Abbreviations endo H endo H--N-acetylglucosaminidase H - ER endoplasmic reticulum - PHA phytohemagglutinin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Cloning and sequencing of a cDNA encoding the major storage proteins of Theobroma cacao

The major storage proteins, polypeptides of 31 and 47 kilodaltons (kDa), from the seeds of cocoa (Theobroma cacao L.), have been identified and partially purified by preparative gel electrophoresis. The polypeptides were both N-terminally blocked, but some N-terminal amino-acid sequence was obtained from a cyanogen bromide peptide common to both polypeptides, permitting the construction of an oligonucleotide probe. This probe was used to isolate the corresponding copy-DNA (cDNA) clone from a library made from poly(A)⁺ RNA from immature cocoa beans. The cDNA sequence has a single major open reading frame, that translates to give a 566-amino-acid polypeptide of M_r 65 612. The existence of a common precursor to the 31- and 47-kDa polypeptides of this size was confirmed by immunoprecipitation from total poly(A)⁺RNA translation products. The precursor has an N-terminal hydrophobic sequence which appears to be a typical signal sequence, with a predicted site of cleavage 20 amino acids after the start. This is followed by a very hydrophilic domain of 110 amino acids, which, by analogy with the cottonseed -globulin, is presumed to be cleaved off to leave a domain of approx. 47 kDa, very close to the observed size of the mature polypeptide. Like the hydrophilic domain of the cottonseed -globulin the cocoa hydrophilic domain is very rich in glutamine and charged residues (especially glutamate), and contains several Cys-X-X-X-Cys motifs. The cyanogen-bromide peptide common to the 47-kDa and 31-kDa polypeptides is very close to the proposed start of the mature domain, indicating that the 31-kDa polypeptide arises via further C-terminal processing. The polypeptide sequence is homologous to sequences of the vicilin class of storage proteins, previously found only in legumes and cotton. Most of these proteins have a mature polypeptide size of approx. 47 kDa, and are synthesised as precursors only slightly larger than this. Some, however, are larger polypeptides (e.g. -conglycinin from soybean is 72 kDa), usually due to an additional N-terminal domain. In cottonseed the situation appears to parallel that in cocoa in that the vicilin is synthesised as an approx. 70-kDa precursor and then processed to a 47-kDa (and in the case of cocoa also a 31-kDa) mature protein. In this context it is interesting that cotton is closer in evolutionary terms to cocoa than are the legumes, both cotton and cocoa being in the order Malvales.Abbreviations A absorbance - cDNA copy DNA - IgG immunoglobulin G - kb kilobase pairs - kDa kilodaltons - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacylamide gel electrophoresis The authors are very grateful to Dr R. Jennings of the Virology Department, Sheffield University Medical School, for help in raising antibodies.     

Control of wheat root growth. The effects of excision on growth,wall rheology and root anatomy

Excision and subsequent incubation of the apices (1 cm) of wheat (Triticum aestivum L.) seedling roots in simple media severely reduced elongation from 28 mm·(24 h)^-1 in intact roots to a maximum of 2 mm·(24 h)^-1 in excised roots. The reduction in growth was accompanied by a loss of cell turgor in the growing zone but was correlated with a hardening of the cell walls in this region. Rheological properties were measured as percent extensibility (both plastic and elastic) using a tensiometer, and as instantaneous volumetric elastic modulus ( _i) using the pressure probe. Excision decreased plastic and elastic properties with a half-time of some 60 min. Plastic extension was reduced from 2.5% to 0.9% and elastic from 4.8% to 2.6% for an 8-g load. By contrast, _i was increased by excision. The observed reduction in root elongation rate was accompained by a reduction in mature cell length from 240 m to 40 m and a shortening of the zone of cell expansion.Symbol _i instantaneous volumetric elastic modulus     

Purification and partial characterization of an acid β-fructosidase from sweet-pepper (Capsicum annuum L.) fruit

The present study describes the biochemical characteristics of an acid -fructosidase (EC 3.2.1.26) purified from the fruit of sweet pepper (Capsicum annuum L.). The soluble form, which constitutes more than 95% of the total activity at pH 4.5, hydrolyzes sucrose, raffinose, and stachyose. Its pH and temperature optima are 4.5 and 55 °C, respectively. Metal cations such as Ag⁺ and Hg²⁺ strongly inhibit its activity, suggesting the presence of at least one sulfhydryl group at the catalytic site. After purification of the enzyme by means of ammonium sulfate fractionation, gel chromatography (diethyl-aminoethyl-Sephacel, hydroxylapatite, concanavalin A-Sepharose), and preparative gel electrophoresis, the purified enzyme was shown to be a 42 kDa glycoprotein interacting specifically with concanavalin A. After complete chemical deglycosylation with trifluoromethanesulfonic acid, the molecular weight of the constitutive polypeptide was estimated to be 39 kDa. The enzyme glycans were characterized using both affino- and immunodetection. The enzyme has at least two N-linked oligosaccharide sidechains, one of the high-mannose type, and the other of the complex type. The high-mannose glycan has a low molecular weight (1 kDa), and is responsible for the interaction between the enzyme and concanavalin A. The complex-type glycan has an estimated molecular weight of 2 kDa. It contains one 1 2-linked xylose residue, probably one fucose residue 1 3-linked to the chitobiose unit, and no terminal galactose residue. The two glycans, associated to the 39 kDa polypeptide, constitute the acid -fructosidase of the sweet-pepper fruit.Abbreviations F -fructosidase - ConA concanavalin A - DEAE diethylaminoethyl - DTNB dithionitrobenzoic acid - endo F endo--N-acetylglucosamidase F - endo H endo--N-acetylglucosamidase H - NEM N-ethylmaleimide - PCMB parachloromercurobenzoate - PNGase glycopeptide-N-glycosidase - TFMS trifluoromethane sulfonic acid This work was partly supported by a grant from the Commission Permanente de Coopération Franco-Québécoise to L. Faye, and S. Yelle. D. Michaud was a recipient of a graduate scholarship from the Natural Science and Engineering Research Council of Canada.     

Uteroferrin contains complex and high mannose-type oligosaccharides when synthesized in vitro

Mature uteroferrin (Uf; M = 35,500) is a progesterone-induced acid phosphatase secreted by the pig uterus. It contains a single, unphosphorylated, high mannose-type oligosaccharide. Endometrial explants cultured in vitro secrete Uf with a M of 37,000 (37k Uf) having phosphorylated high mannose oligosaccharides. In this report we demonstrate that 37k Uf contains two N-linked oligosaccharides which are a mixture of complex and high mannose-type oligosaccharides. The complex-type glycopeptides are biantennary and a portion may be fucosylated on the GlcNac of the chitobiose core proximal to the peptide. Only a portion of the high mannose-type oligosaccharides are phosphorylated. The remainder appear to be typical Man_6-4GlcNac₂ oligosaccharides found on mature Uf.Abbreviations Uf Uteroferrin - ConA Concanavalin A - WGA Wheat Germ Agglutinin - endoH endo--N-acetylglucosaminidase H - SDS Sodium Dodecyl Sulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of SDS     

Specificity towards oligomannoside and hybrid type glycans of the endo-β-N-acetylglucosaminidase B from the basidiomy ceteSporotrichum dimorphosporum

We have previously shown that an endo--N-acetylglucosaminidase (EC 3.2.1.96) named Endo B, isolated from culture filtrates of the basidiomyceteSporotrichum dimorphosporum cleaves asialo-, and to some extent, monosialylated bi-antennary glycans of theN-acetyllactosamine type linked to the asparagine residue of peptide or protein moieties [Bouquelet S, Strecker G, Montreuil J, Spik G (1980) Biochimie 62:43–49]. In the present paper, the substrate specificity of the enzyme towards oligomannoside and hybrid type glycans has been analyzed. The results obtained indicate that ovalbumin glycopeptides containing four to seven mannose residues and bovine lactotransferrin glycopeptides containing four to nine mannose residues were completely hydrolyzed by the enzyme. The degree of cleavage was variable among hybrid type structures, since glycopeptides containing the following glycans: (Gal)₁(GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₄(GlcNAc)₂ were not hydrolyzed by the enzyme while the percentage of hydrolysis of a glycopeptide containing (GlcNAc)₂(Man)₅(GlcNAc)₂ glycan reached 90%. The bovine lactotransferrin was partially deglycosylated (40%) in the absence of non-ionic detergent while native ovalbumin glycoprotein was not hydrolyzed by the enzyme.The oligomannoside-and theN-acetyllactosamine-type degrading activities present in the culture filtrates were not separated at any step of the purification procedure. Both activities were eluted as a single component with an apparent molecular mass of 89 kDa suggesting that they are located on the same enzyme molecule.Endo B represents a powerful tool for removing oligomannoside-andN-acetyllactosamine-type glycans fromN-glycopeptides andN-glycoproteins. Moreover, advantages in the use of Endo B in a soluble form as well as in an immobilized form result in its high activity and in its stability to heat denaturation and storage.Abbreviations Gal d-galactose - Man d-mannose - GlcNAc N-acetyl-d-glucosamine - Con A concanavalin A - Asn asparagine - GLC gas liquid chromatography - TLC thin layer chromatography - Endo endo--N-acetylglucosaminidase - Endo B endo--N-acetylglucosaminidase isolated fromSporotrichum dimorphosporum - PBE polybuffer exchanger - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Different susceptibilities of complex-, hybrid- and high-mannose-type α1- inhibitor and α1-acid glycoprotein to endo-β-N-acetylglucosaminidase F and peptide:N-glycosidase F

Endo--N-acetylglucosaminidase F (endo F, EC 3.2.1.96) and peptide:N-glycosidase F (PNGase F, EC 3.2.2.18) fromFlavobacterium meningosepticum were used for the deglycosylation of ₁-proteinase inhibitor and ₁-acid glycoprotein carrying oligosaccharide side chains of the complex-, high-mannose- and hybrid-type. High-mannose-and hybrid-type glycoproteins were obtained by the incubation of rat hepatocyte primary cultures with 1-deoxymannojirimycin or swainsonine, respectively. It was found that endo F cleaves hybrid- and high-mannose-type ₁-proteinase inhibitor and ₁-acid glycoprotein at pH 4.5 as well as at pH 8.5 in the presence or absence of 1% octyl--d-glucopyranoside. Complex-type ₁-proteinase inhibitor or ₁-acid glycoprotein were not cleaved by endo F even in the presence of octyl--d-glucopyranoside.PNGase F was found to cleave complex-, hybrid- and high-mannose-type oligosaccharide side chains of ₁-proteinase inhibitor and ₁-acid glycoprotein at pH 4.5 and pH 8.5 in the presence of 0.75% octyl--d-glucopyranoside. The deglycosylation of both protein substrates was very poor without detergents.Abbreviations Endo F endo--N-acetylglucosaminidase F (EC 3.2.1.96) - PNGase F peptide:N-glycosidase F (EC 3.2.2.18) Dedicated to Prof. Dr. Wolfgang Gerok on the occasion of his 60th birthday     

Thylakoid-protein phosphorylation during the life cycle of Scenedesmus obliquus in synchronous culture

The phosphorylation of thylakoid proteins, which comprise apoproteins of the light-harvesting chlorophyll a/b-protein complex (LHCP), was investigated in vivo and in vitro during the development of Scenedesmus obliquus in synchronous cultures. The in-vitro and in-vivo protein phosphorylation exhibited a maximum activity in cells with maximum photosynthetic capacity (8th hour) and miximum activity in cells with minimum photosynthetic capacity (16th hour). The major phosphorylated polypeptides in vivo were the 24/25-kDa and 28–30-kDa apoprotein of the LHCP, a protein of about 32 kDa, and some smaller polypeptides within the range 10 to 20 kDa. In vitro, the main phosphoproteins were the 28–30-kDa apoprotein and the protein characterized by an apparent molecular weight of 32 kDa. Pulse-chase experiments in vivo established that the latter had the fastest radioactivity turnover of the thylakoidal phosphoproteins.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHCP light-harvesting chlorophyll a/b-protein complex - PSII photosystem II Dedicated to Prof. Erwin Bünning on the occasion of his 80^th birthday     

Self-assembly properties of the proteinaceous coat secreted by the “slime” variant of Neurospora crassa

The proteinaceous extracellular material (PEM) synthesized by the cells of the slime strain of Neurospora crassa (see Martinez et al. 1989) was solubilized by treatment with urea or guanidine. Removal of these chemicals by dialysis, caused reassembly of the solubilized proteins into material with the same microscopic appearance as the original PEM. Polypeptide patterns from both native and reassembled structures were identical. Dialysis-mediated reassembly of the solubilized proteins appeared to be dependent on both concentration of the soluble macromolecules and time. Gel chromatography of PEM solubilized with different agents revealed two discrete populations of complexes with molecular masses of 1,500 and 500 kDa respectively. These were able to reassemble into lamellar structures with a variable degree of efficiency.Abbreviations ConA Concanavalin A - Fe-ConA ferritin-labeled Concanavalin A - Endo H endo--N-acetylglucosaminidase H - PMSF phenyl methyl sulphonyl fluoride     

Avena sativa L. contains three phytochromes,only one of which is abundant in etiolated tissue

Phytochrome from leaves of light-grown oat (Avena sativa L. cv. Garry) plants is characterized with newly generated monoclonal antibodies (MAbs) directed to it. The results indicate that there are at least two phytochromes in green oat leaves, each of which differs from the phytochrome that is most abundant in etiolated oat tissue. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with reference to 124-kilodalton (kDa) phytochrome from etiolated oats, the two phytochromes from green oats have monomer sizes of 123 of 125 kDa. Immunoblot analysis of SDS, sample buffer extracts of lyophilized, green oat leaves indicates that neither the 125-kDa nor the 123-kDa polypeptide is a degradation product arising after tissue homogenization. Of the two, the 123-kDa phytochrome appears to be the predominant species in light-grown oat leaves. During SDS-PAGE in the presence of 1 mM Zn²⁺, 123-kDa phytochrome undergoes a mobility shift corresponding to an apparent mass increase of 2 kDa. In contrast, the electrophoretic mobility of 125-kDa phytochrome is unaffected by added Zn²⁺. Some MAbs that recognize 123-kDa phytochrome fail to recognize 125-kDa phytochrome and vice versa, indicating that these two phytochromes are not only immunochemically distinct from 124-kDa phytochrome, but also from each other. It is evident, therefore, that there are at least three phytochromes in an oat plant: 124-kDa phytochrome, which is most abundant in etiolated tissue, plus 123-and 125-kDa phytochromes, which predominate in light-grown tissue.Abbreviations Da Dalton - HA hydroxyapatite - MAb monoclonal antibody - PAb polyclonal antibody preparation - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate This research was supported by the U.S. Department of Energy (contract DE-AC-09-81SR10925 to L.H.P.). We thank Dr. Alan Jones, Department of Biology, University of North Carolina, Chapel Hill, USA, for kindly providing rabbit antiserum 4032, and Mrs. Donna Tucker and Mrs. Danielle Neal for their technical assistance.     

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.     

Cytosolic and cell-wall-bound acid invertases from leaves of Urtica dioica L.: a comparison

Two different forms of acid invertase (EC 3.2.1.26) were extracted from expanding leaves of the stinging nettle (Urtica dioica L.). One form was soluble and could be localized within the cytosol, whereas the other was ionically bound to the cell-wall and could not be detected in protoplasts. Both forms were purified, the latter to homogeneity. Western blotting with antibodies against the pure enzyme from cell walls was positive with the cell-wall enzyme but negative with the soluble form of acid invertase. Both forms are glycoproteins with identical molecular weights of 58 kDa. The K_m values for sucrose (raffinose) are 5 mM (4.8 mM) for the soluble and 1.2 mM (3.6 mM) for the cell-wall-bound enzyme. The pH optimum of the latter is slightly more acidic (4.5) than that of the soluble invertase (5.5). Both forms could easily be distinguished by their isoelectric points which were determined at pH 4.6 for the soluble and pH 9.3 for the wall-bound enzyme. When extraction and purification were carried out in the absence of protease inhibitors, both acid invertases showed microheterogeneity (multiple forms). However, with benzamidine and phenylmethylsulfonylfluoride as protease inhibitors each invertase produced only one protein band upon isoelectric focusing and gel electrophoresis, respectively.Abbreviations B benzamidine - Con A concanavalin A - FPLC fast protein liquid chromatography - IEF isoelectric focusing - kDa kilodalton - pI isoelectric point - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - SDS sodium dodecyl sulfate This work was supported by the Deutsche Forschungsgemeinschaft within the scope of the Sonderforschungsbereich 137.     

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.     

Effects of Size of Carbohydrate Chain on Protease Digestion of Aspergillus niger Endo-β-1,4-glucanase

Three different carbohydrate-depleted enzymes were prepared from an endo-β-l,4-glucanase of Aspergillus niger IF031125 by treatment with endo-β-N-acetylglucosaminidase or α-mannosidase. They were purified by Concanavalin A-Sepharose affinity and DEAE ion-exchange column chromatographies. The molecular sizes of these enzymes had been decreased from 40 kDa containing 9.0% carbohydrate to 39, 38, and 37kDa with carbohydrate at 4.5, 1.3, and 0.8% (wt/wt), respectively. The native and these carbohydrate-depleted enzymes were compared in their enzymatic properties, and it was found that they were identical in their catalytic activities and both thermal and pH stabilities. However, the 37-kDa enzyme was more susceptible to proteolysis by Savinase, proteinase K, and Pronase E. On the other hand, the specific protease trypsin showed no such effect on activity of all enzymes. These results suggested that the core structure of the asparagine-linked sugar chain, which consisted of three monosaccharide residues, contributed to the high stability of the endo-β-l,4-glucanase against protease digestion.     

Comparative study of stability of soluble and cell wall invertase from Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae is the most significant source of enzyme invertase. It is mainly used in the food industry as a soluble or immobilized enzyme. The greatest amount of invertase is located in the periplasmic space in yeast. In this work, it was isolated into two forms of enzyme from yeast S. cerevisiae cell, soluble and cell wall invertase (CWI). Both forms of enzyme showed same temperature optimum (60°C), similar pH optimum, and kinetic parameters. The significant difference between these biocatalysts was observed in their thermal stability, stability in urea and methanol solution. At 60°C, CWI had 1.7 times longer half-life than soluble enzyme, while at 70°C CWI showed 8.7 times longer half-life than soluble enzyme. After 2-hr of incubation in 8?M urea solution, soluble invertase and CWI retained 10 and 60% of its initial activity, respectively. During 22?hr of incubation of both enzymes in 30 and 40% methanol, soluble invertase was completely inactivated, while CWI changed its activity within the experimental error. Therefore, soluble invertase and CWI have not shown any substantial difference, but CWI showed better thermal stability and stability in some of the typical protein-denaturing agents.