Sepharose CL Research Articles

Acid degradation of starch. The effect of acid and starch type

Starch from cereals (wheat, maize and finger millet) pulses (chick pea and green gram), tuber (potato) and root (tapioca) was modified with different acids (0.5 N, 1.5 h, 50°C). Molecular weight (number average, M ̄ n ) of these starches decreased after modification, H 3PO 4 causing the least and HCl and HNO 3 the highest reduction. Gel permeation chromatography of native starches using Sepharose CL 4B gave mainly two fractions. Fraction I (Fr. I), a higher molecular weight component eluting in the void volume and Fraction II (Fr. II), a lower molecular weight component that entered the gel and eluted at higher elution volumes. After acid modification, the carbohydrate content of Fr. II increased while that of Fr. I decreased. The magnitude of the effect for different acids followed the same pattern as was the case for molecular weight. Very high increase in the total carbohydrate content in Fr. II was seen in cereal starches followed by pulses, root and least by tuber. The λ max values of the peak of Fr. I increased in cereal and millet starches after modification by 9 to 14 nm, but either remained the same or decreased to some extent in other starches. The peak of Fr. II of modified starches had similar K av to that of the respective native starch suggesting that the degraded portion had a molecular size similar to that of Fr. II of the native starch. However, the λ max of the peak of Fr. II decreased after modification indicating that degraded portion of Fr. I which entered the gel and eluted with the peak of Fr. II was branched.

Immobilized 4-aminophenyl 1-thio-β- d-galactofuranoside as a matrix for affinity purification of an exo-β- d-galactofuranosidase

An alternative and fast method for the purification of an exo-β- d-galactofuranosidase has been developed using a 4-aminophenyl 1-thio-β- d-galactofuranoside affinity chromatography system and specific elution with 10 mM d-galactono-1,4-lactone in a salt gradient. A concentrated culture medium from Penicillium fellutanum was chromatographed on DEAE–Sepharose CL 6B followed by chromatography on the affinity column, yielding two separate peaks of enzyme activity when elution was performed with 10 mM d-galactono-1,4-lactone in a 100–500 mM NaCl salt gradient. Both peaks behaved as a single 70 kDa protein, as detected by SDS-PAGE. Antibodies elicited against a mixture of the single bands excised from the gel were capable of immunoprecipitating 0.2 units out of 0.26 total units of the enzyme from a crude extract. The glycoprotein nature of the exo-β- d-galactofuranosidase was ascertained through binding to Concanavalin A–Sepharose as well as by specific reaction with Schiff reagent in Western blots. The purified enzyme has an optimum acidic pH (between 3 and 6), and K m and V max values of 0.311 mM and 17 μmol h −1 μg −1 respectively, when 4-nitrophenyl β- d-galactofuranoside was employed as the substrate.

Characterization of secretory acetylcholinesterase from Setaria cervi microfilariae: a potential antigen for diagnosis of human filariasis.

Acetylcholinesterase (AChE) is released to the external medium when microfilariae (m.f.) of Setaria cervi, a bovine filarial parasite, are maintained in vitro. Intense enzyme staining at amphids, excretory pores, anal vesicle and phasmids suggest an active secretion of AChE from m.f. Excretory-secretory products of m.f. displayed two electromorphic variants of AChE when resolved by 6% nondenaturing PAGE. The two isoforms of AChE (A and B) were separated on the basis of charge by DEAE sepharose CL 6B column following gel filtration. The two isoforms showed differing kinetic properties with respect to substrate specificity and inhibitor sensitivity. Anti-Nippostrongylus brasiliensis AChE antibodies cross-reacted with the affinity purified secretory AChE in ELISA. Immunoblotting of purified AChEs with cross-reacting anti-AChE antibodies revealed the presence of an approximately 75 kD protein in the isoenzyme A and an approximately 45 kD protein in B, whereas both proteins were present in the enzyme purified via affinity chromatography on edrophonium sepharose column.

Non-proteolytic activation of human prorenin by anti-prorenin prosegment (pf#1: 1P-15P) antiserum.

Recombinant human prorenin was activated by incubation with anti-prorenin prosegment (L1PPTDTTTFKRIFLKR15P) antiserum at 4 degrees C. This activation was dependent on the concentration of the antiserum and incubation time. After the activation no molecular weight alteration of prorenin was observed by immunoblotting analysis. A peptide of L1PPTDTTTF8P as well as L1PPTDTTTFKRIFLKR15P potently interfered with the activation. Most of the activated prorenin bound to Protein A Sepharose CL 4B. The Km and Vmax values of the activated prorenin were 0.2 microM and 23.7 micrograms Ang I/ml/h, respectively, which were similar in level to those of mature renin obtained by trypsinization.

Purified polyclonal anti‐phenylurea antibodies for an improved immunoaffinity chromatography

A one‐step technique for the purification of isoproturon antibody has been developed to overcome the common elution problems encountered when high‐affinity polyclonal antibodies are used in immunoaffinity columns. Because of the non‐uniform affinity, desorption of the analyte can be problematic. Two herbicide derivatives were coupled to Sepharose CL solid support using a flexible linker, β‐lactoblobulin. Using a structurally‐related chlortoluron ligand showed a lower affinity to the target antibody rather than the isoproturon derivative ligand and resulted in an improved capacity of the affinity chromatography gel. The recovery of the specifically adsorbed antibodies was achieved using a carboxylic derivative of the analyte isoproturon in a water‐ethylene glycol buffer (70/30). The purification efficiences of this procedure were characterized by dot blot, fast protein liquid chromatography and ELISA. The chlortoluron‐β‐lactoglobulin purified IgG showed a six times higher titer than the pure isoproturon‐specific IgG fraction obtained using a commercial IgG purification gel. This one‐step purification procedure was reproducible in isolating the pesticide‐specific antibodies. Immobilization of such antibodies on activated silica afforded a high capacity immunosorbent which retained an average of 8.2 µg of isoproturon for 20 mg of immobilized antibodies per gram of silica.

Ig-Therasorb immunoadsorption for selective removal of human immunoglobulins in diseases associated with pathogenic antibodies of all classes and IgG subclasses, immune complexes, and fragments of immunoglobulins.

Therasorb immunoadsorption (IA), by selectively eliminating pathogenic substances from the circulation, allows for successful therapy of previously insufficiently treatable diseases. Molecules (specific polyclonal sheep antibodies) coupled to a matrix (Sepharose CL 4B) selectively bind plasma components in extracorporeal circulation. This procedure has been established in the treatment of various diseases. Examples are familial hypercholesterolemia (LDL-Therasorb) and selected autoimmune diseases (Ig-Therasorb). Ig-Therasorb IA has been performed in a variety of clinical indications, primarily in the treatment of autoimmune diseases. In most cases, Ig-Therasorb IA has been applied in patients who have failed to respond to conventional therapy with a high rate of clinical improvement. In defined groups of patients with autoimmune diseases and alloantibodies, immunoadsorption can now be considered an established therapeutic means. The fast and efficient removal of immunoglobulins obviously exceeds the efficiency of conventional plasma exchange by far. Autoimmune diseases could be induced by balanced and nonbalanced immunity. The importance of autoantibodies remains unclear, but the efficacy of Ig-Therasorb IA suggests a key role for them. In addition to the established indications for removal of immunoglobulins, there may be a number of promising new indications.

Micellar Lipoproteins as the Possible Storage and Translocation Form of Intracellular Diacylglycerol

Previous work indicated that diacylglycerol (DG) molecules translocate across the cytoplasm of mammalian cells, a process relevant to the signalling role of this lipid as protein kinase C activator. Here we investigated the possible mechanism underlying DG translocation. We examined the interaction between 1,2-di-[1-14C]oleoyl-sn-glycerol and rat liver cytosol (rlc) using assays based on Lipidex-1000 and on coelution on Sepharose CL 6B. We measured high DG binding activity and found that it resides in cytosolic proteins and not in cytosolic lipids. Chromatography of rlc proteins on Sepharose CL 6B showed profiles in which the activity measured by either method coincided. Further, we showed that the DG-rlc protein interaction results in the stabilization of DG in a micellar form, eluting in the void volume of Sepharose CL 6B. Such stabilized micelles are reminiscent of insect lipophorins and may represent a new, thus far unrecognized, mode of lipid transport within living cells.

Dysregulation of proteoglycan production by intrahepatic biliary epithelial cells bearing defective (delta-f508) cystic fibrosis transmembrane conductance regulator.

Hepatic dysfunction in cystic fibrosis (CF) has been attributed to accumulation of viscous mucoid secretions in intrahepatic bile ducts. The purpose of our study was to compare glycoconjugate secretion by intrahepatic biliary epithelial (IBE) cells derived from normal livers and livers of CF patients with the delta F508 mutation of the cystic fibrosis transmembrane conductance regulator (CFTR). Confluent cells were incubated with 3H-glucosamine (GlcN) for 16 hours, and radiolabeled macromolecules were analyzed for the amount and type of glycoconjugates. Incorporation of 3H-GlcN into macromolecular glycoconjugates was two- to threefold higher in CF cells versus normals, as was uptake of 3H-Glcn into the cytoplasm of CF cells. Gel exclusion chromatography on Sepharose Cl 4B revealed that the secreted glycoconjugates from CF cells eluted entirely in the excluded fraction (molecular weight > 2 x 10(6)), while, in the normal cells, 60% of the glycoconjugates eluted as lower-molecular-weight species. The high-molecular-weight glycoconjugates in both CF and normal cells were identified as chondroitin sulfates, as evidenced by susceptibility to beta elimination, chondroitinase digestion, and amino acid composition. Western blotting of IBE cell secretions with a polyclonal antibody to chondroitin sulfate revealed proteoglycan bands at 100 and 210 kd. Our results indicate that secretion of chondroitin sulfate is markedly increased in CF biliary epithelium in vitro compared with non-CF cells. Increased uptake of precursor 3H-GlcN may contribute to enhanced glycosylation of chondroitin sulfate in CF cells.

Carbohydrate solutilization of tomato locule tissue cell walls: Parallels with locule tissue liquefaction during ripening

Carbohydrate solubilization and glycosidase activities were investigated in tomato (Lycopersicon esculentum Mill. cv. Solar Set) locule cell walls to identity processes involved in the liquefaction of this tissue. Cell walls were prepared from the locule tissue of fruit at the immature green, mature green, and breaker stages of development. Enzymically active walls incubated in dilute buffer released high molecular mass pectins, oligomeric carbohydrate, and the neutral sugars rhamnose, glucose, galactose, arabinose, xylose, and mannose. The release was sustained for at least 50 h at 34°C and was inhibited more than 50% by 1 mM Hg2+. Pectins released from the cell walls of locule tissue at progressive stages of liquefaction were similar in molecular mass and showed no evidence of downshifts on a Sepharose CL–2B–300 column during prolonged incubation. A cell‐free protein extract prepared from the locule tissue of mature‐green fruit promoted a net release of polymeric and monomeric carbohydrates from high‐temperature inactivated cell walls. Polygalacturonase activity was not detected in locule protein although glycosidases including β‐mannosidase (EC 3.2.1.25), α‐ and β‐galactosidases (EC 3.2.1.22–23), β‐arabinosidase (EC 3.2.1.56) and β‐glucosidase (EC 3.2.1.21) were present. Pectinmethylesterase (EC 3.1.1.1 1) activity was detected at the immature‐green stage but declined to negligible levels in mature‐green and breaker locule tissue. Parallels between the in vitro solubilization of carbohydrate from locule tissue cell walls and the changes occurring during locule liquefaction are discussed.

Differential expression of membrane-anchored proteoglycans in rabbit articular chondrocytes cultured in monolayers and in alginate beads. Effect of transforming growth factor- β1

Cell-surface proteoglycans (PGs) were extracted with Triton X-100 from rabbit articular chondrocytes cultured in monolayers and in alginate beads. They were first purified on DEAE-Trisacryl columns and the proportion of hydrophobic PGs was determined by both Octyl-Sepharose chromatography and partitioning in Triton X-114. These two methods revealed that the proportion of hydrophobic PGs was higher in monolayer culture system as compared to alginate beads (24 and 15%, respectively). Characterization of the PGs by Sepharose CL 6B gel filtration followed by electrophoresis indicated that the PGs isolated from monolayers were composed of three chondroitin sulfate (CS) PGs (core proteins of 180, 100 and 50 kDa) and a heparan sulfate (HS) PG (core protein of 60 kDa). In the alginate system, CSPGs with core proteins of 180, 45 and 32 kDa were observed, but no HSPG was present. In parallel, the effect of TGF- β on the distribution of membrane-associated PGs was studied. The results showed that the synthesis of cell-surface PGs was stimulated by TGF- β in monolayers whereas it was inhibited in alginate beads, but the amount of hydrophobic PGs was not altered by the growth factor. These data clearly indicate that TGF- β induces a differential expression of the PG families present at the cell surface. Taken together, the results reveal the complex regulation of cell-surface PG distribution, which obviously depends on the culture method used and suggest that rabbit articular chondrocytes may differentially respond to extracellular ligands according to their morphological state and environment.

Identification of rat alpha1 macroglobulin as an inhibitor of rat Galbeta1-4GlcNAc alpha2-6 sialyltransferase.

Rat serum was found to contain an inhibitor of pure alpha2-6 sialyltransferase (EC 2.4.99.1). The inhibitor was a high Mr protein isolated by molecular filtration on Sephadex G100, followed by anion exchange chromatography on Sephadex DEAE A25, then separation on Sepharose CL 4B, and finally by isoelectric focusing. Electrophoretic separation and subsequent N-terminal sequence analysis of the active inhibitor preparation showed the presence of two protein components, identified as rat C-reactive protein, and rat alpha1 macroglobulin. Pure rat CRP did not inhibit alpha2-6 sialyltransferase. Treatment of the inhibitor preparations with monospecific antibodies against rat alpha1 macroglobulin blocked inhibitory activity in a dose-dependent manner. The results present strong evidence that alpha1 macroglobulin is capable of acting as an inhibitor of alpha2-6 sialyltransferase. No inhibition of galactosyltransferase (EC 2.4.1.38) could be detected, indicating that the interaction with alpha1 macroglobulin may have specificity among the glycosyltransferases. The entrapment of alpha2-6 sialyltransferase by alpha1 macroglobulin presented here occurs in vitro and will require further in vivo investigations to determine the precise physiological significance. Independent of the physiologic significance is the finding that this interaction occurs in vitro, which, pending elucidation of the precise mechanism and specificity, may provide a new tool for investigations into the functional significance of sialylation, and potentially for use or design of new inhibitors of therapeutic value in physiologic conditions involving altered levels of sialylation.

Purification and partial characterization of two thioredoxins from Streptomyces aureofaciens.

Thioredoxins are low molecular weight proteins, which participate in a wide spectrum of biochemical reactions. Two thioredoxins from Streptomyces aureofaciens 3239 have been purified to homogeneity by a sequence of chromatography steps including chromatography on Sephacryl S-300, Phenyl Sepharose CL 4B and MonoQ HR 5/5. Thioredoxin activity clearly separates into two protein fractions on MonoQ HR 5/5 chromatography. Molecular weights determined by chromatography on Superose 12 HR 10/30 and sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed M(r) approximately 10,500 for thioredoxin 1 (TR1) and M(r) approximately 11,000 for thioredoxin 2 (TR2). The isoelectric points of the two thioredoxins are different pI = 4.7 for TR1 and 5.6 for TR2, respectively. Both were effectively reduced with NADPH in reaction catalyzed by Streptomyces aureofaciens thioredoxin reductase. The specific activity of viewly for discovered TR2 is about 1/4 of the specific activity of TR1. Both thioredoxins activate spinach NADPH-malate dehydrogenase. Activation of this enzyme by TR2 is only half effective than by TR1. The stability of TR1 is high and similar to thioredoxins from other organisms unlike the activity of TR2 which is decreased during purification. The proteins diversed in their contents in exponentially growing mycelium.

Interaction of a rat intestinal brush border membrane glycoprotein with type-1 fimbriae of Salmonella typhimurium.

Type-1 fimbriated Salmonella typhimurium was found to adhere to rat intestinal brush border membrane in a mannose sensitive manner. The maximum binding of the purified fimbriae observed with the rat illeal enterocytes was inhibited by 69.2% in presence of D-mannose. Brush border membrane from rat illeum was isolated, delipidified, solubilised and fractionated by affinity chromatography on type-1 fimbriae coupled Sepharose CL 4B column. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of the material eluted from the column with D-mannose revealed a single band of molecular weight 60 kDa. The direct binding of this affinity eluted glycoprotein to the purified type-1 fimbriae was demonstrated by a modified Western blot experiment. Our findings suggest that the 60 kDa glycoprotein may serve as a receptor for the type-1 fimbriae in the rat intestinal brush border membrane and thereby may help in mediating bacterial adherence to the host epithelium.

Expression of mucin genes in ulcerative colitis.

Conference Article| November 01 1995 Expression of mucin genes in ulcerative colitis NEIL MYERSCOUGH; NEIL MYERSCOUGH 1Department of Medicine Labs, Bristol Royal Infirmary, Bristol BS2 8HW Search for other works by this author on: This Site PubMed Google Scholar BRYAN WARREN; BRYAN WARREN *Dept Cellular Pathology, John Radcliffe Hospital, Oxford OX3 9DU Search for other works by this author on: This Site PubMed Google Scholar MARGARET GOUGH; MARGARET GOUGH 1Department of Medicine Labs, Bristol Royal Infirmary, Bristol BS2 8HW Search for other works by this author on: This Site PubMed Google Scholar ANTHONY CORFIELD ANTHONY CORFIELD 1Department of Medicine Labs, Bristol Royal Infirmary, Bristol BS2 8HW Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1995) 23 (4): 536S. https://doi.org/10.1042/bst023536s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation NEIL MYERSCOUGH, BRYAN WARREN, MARGARET GOUGH, ANTHONY CORFIELD; Expression of mucin genes in ulcerative colitis. Biochem Soc Trans 1 November 1995; 23 (4): 536S. doi: https://doi.org/10.1042/bst023536s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1995 Biochemical Society1995 Article PDF first page preview Close Modal You do not currently have access to this content.

Comparative Study of the Adjuvanticity of FIA and Phosphatidylcholine Liposomes for Purified Mycobacterial RNA

AbstractIn the present study, RNA isolated from Mycobacterium t ubercu losis H,_Ra cells was further purified on sepharose CL-'tB which yielded three fractions i.e. F., F and F,. Out of these, F, was major fraction containing least amount of protein contamination (2.3%). A comparative study of the immunoreactivity of these three fractions indicated that F. and F2 were more immunoreactive than F,. Animals immunized with F. fraction emulsified with FIA or encapsulated in egg phosphatidylcholine (EPC) liposomes induced comparable humoral and cell mediated immune (CMI) responses. Further, immunization with this fraction resulted in significant protection as revealed by high survival rate and decreased bacterial load in the infected organs as compared to nonimmunized animals on challenge with Mt uberculosi s H. Rv. Protection provided by F. using FIA and liposomes as adjuvants was quite comparable, indicating that liposomes can replace FIA for mycobacterial antigens.

Preparation of cell-affinity adsorbents by immobilization of peptides to Sephadex G-10.

In this study, affinity adsorbents for the binding of activated blood platelets and endothelial cells were prepared from Sephadex G-10 by immobilization of peptides, derived from the cell-adhesive amino acid sequence RGD (arginine-glycine-aspartic acid). Derivatization of Sephadex G-10 was accomplished by sequential coupling of specific dipeptides (RG and DV) (V = valine) and by coupling of the RGD-containing hexapeptide GRGDSP (S = serine, P = proline). Two types of gel were prepared by sequential coupling, designated as G10 (acetone) and G10 (dimethylformamide) (G10(DMF)), containing peptides which had been coupled to the outer side of the beads and throughout the porous beads, respectively. The binding capacity of the prepared Sephadex-derivatives amounted up to 2 x 10(9) human blood platelets per millilitre GRGDV-Sephadex at immobilized peptide concentrations, that were in the nanomole range (per millilitre packed gel) and which differed a factor 10 between G10 (acetone) and G10 (DMF). In a second series of experiments, different amounts of the hexapeptide GRGDSP were coupled to carboxylated Sephadex G-10 and carboxylated Sepharose CL 6B. The binding of human umbilical vein endothelial cells to the resulting materials was studied. Up to 10(6) endothelial cells attached per ml GRGDSP-derivatized hydrogel at peptide concentrations of 15 nmol GRGDSP/ml Sephadex and at +/- 300 nmol GRGDSP/ml Sepharose. Substitution of the arginine residue of the RGD-sequence by glutamine abolished the cell-binding activity of the immobilized peptide towards activated blood platelets but not towards endothelial cells. From the results of this study it can be concluded that small peptides can be coupled to the outer side of the porous Sephadex beads, resulting in high effective ligand densities for cell-affinity applications. In this respect, Sephadex G-10, derivatized according to 'the acetone method' is a good alternative for polystyrene and other solid phase materials.

Rapid Purification of Yeast Cytoplasmic Fumarate Reductase Affinity Chromatography on Blue Sepharose CL–6B

The rapid and effective purification of soluble fumarate reductase from baker's yeast achieved by Blue Sepharose CL-6B chromatography. Cibacron Blue F3GA, the chromophore of Blue Sepharose, inhibited the activity of fumarate reductase. The enzyme bound to the column was selectively eluted by flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) or riboflavin. The purified enzyme was essentially homogeneous as indicated by polyacrylamide gel electrophoresis under nondenaturing conditions and under denaturing conditions in sodium dodecylsulfate. By this procedure, the enzyme could be rapidly purified with high yield from yeast cells.

Characterization of the autologous antibodies that opsonize erythrocytes with clustered integral membrane proteins

In earlier studies we presented evidence that the clustering of the integral membrane protein, band 3, can serve as a signal for immune recognition and clearance of senescent or abnormal erythrocytes from circulation. In this study, we have exploited the capacity of 1 mmol/L Zn+2 to mildly and reversibly cluster band 3 in situ to characterize the nature of the autologous antibodies specific for the clustered state. We report that the autologous IgG elute almost exclusively in a high molecular weight complex with other proteins when C12E8 detergent extracts of Zn clustered membranes are chromatographed on Sepharose CL- 6B. The complex was also seen to contain complement component C3, hemoglobin, and a cross-linked oligomer of band 3. Autologous IgG and complement were virtually absent from all other fractions. When the band 3 clusters were disaggregated by removal of the Zn+2, the autologous IgG eluted from the erythrocyte surface. Collection of this IgG and use of the antibody in immunoblots of erythrocyte membranes showed that the band 3 monomer, dimer, and oligomers were the major antigenic species. Except for a minor unidentified band at approximately 78,000 d, no other proteins were significantly stained. Curiously, band 3 showed an uneven staining pattern, with oligomers and the leading edge of the monomers appearing more intensely than expected from their abundances in the Coomassie blue-stained gels. Typing of the same autologous IgG with monoclonal antibodies specific for the different subclasses of IgG showed the presence of only subtypes 2 and 3. Taken together, these data suggest that a specific population of autologous IgG recognizes sites of integral membrane protein clustering (a common lesion in senescent and abnormal red blood cells) and that the antigen within these clusters involves an aggregated state of band 3.

Polyuronides in Avocado (Persea americana) and Tomato (Lycopersicon esculentum) Fruits Exhibit Markedly Different Patterns of Molecular Weight Downshifts during Ripening.

Avocado (Persea americana) fruit experience a rapid and extensive loss of firmness during ripening. In this study, we examined whether the chelator solubility and molecular weight of avocado polyuronides paralleled the accumulation of polygalacturonase (PG) activity and loss in fruit firmness. Polyuronides were derived from ethanolic precipitates of avocado mesocarp prepared using a procedure to rapidly inactivate endogenous enzymes. During ripening, chelator (cyclohexane-trans-1,2-diamine tetraacetic acid [CDTA])-soluble polyuronides increased from approximately 30 to 40 [mu]g of galacturonic acid equivalents (mg alcohol-insoluble solids)-1 in preripe fruit to 150 to 170 [mu]g mg-1 in postclimacteric fruit. In preripe fruit, chelator-extractable polyuronides were of high molecular weight and were partially excluded from Sepharose CL- 2B-300 gel filtration media. Avocado polyuronides exhibited marked downshifts in molecular weight during ripening. At the postclimacteric stage, nearly all chelator-extractable polyuronides, which constituted from 75 to 90% of total cell wall uronic acid content, eluted near the total volume of the filtration media. Rechromatography of low molecular weight polyuronides on Bio-Gel P-4 disclosed that oligomeric uronic acids are produced in vivo during avocado ripening. The gel filtration behavior and pattern of depolymerization of avocado polyuronides were not influenced by the polyuronide extraction protocol (imidazole versus CDTA) or by chromatographic conditions designed to minimize interpolymeric aggregation. Polyuronides from ripening tomato (Lycopersicon esculentum) fruit extracted and chromatographed under conditions identical with those used for avocado polyuronides exhibited markedly less rapid and less extensive downshifts in molecular weight during the transition from mature-green to fully ripe. Even during a 9-d period beyond the fully ripe stage, tomato fruit polyuronides exhibited limited additional depolymerization and did not include oligomeric species. A comparison of the data for the avocado and tomato fruit indicates that downshifts in polyuronide molecular weight are a prominent feature of avocado ripening and may also explain why molecular down-regulation of PG (EC 3.2.1.15) in tomato fruit has resulted in minimal effects on fruit performance until the terminal stages of ripening.

Studies on the Structure of Pea Starches. Part 3: Amylopectin of Smooth Pea Starch

Abstractβ‐Limit dextrins were isolated from smooth pea starch. It was shown by gel chromatography that the sample contained smaller limit dextrins with a degree of polymerization (D.P.) of 40–4000 in addition to high‐molecular‐weight material. The smooth pea starch and the β‐limit dextrins were hydrolysed by the α‐amylase of B. amyloliquefaciens and the molecular weight distribution of the reaction mixtures was analysed on Sepharose CL 6B. Initially the reaction was fast, but at later stages branched dextrins with 10–60 were only very slowly hydrolysed further. In addition, linear dextrins with d.p. 2–10 were formed continously. Based on the branched dextrins produced from the β‐limit dextrins a model of the structure of the amylopectin component in smooth pea starch is suggested, in which clusters of defined sizes are regularly interconnected.