Purification and Partial Characterization of Tomato Extensin Peroxidase.

Early plant defense response is characterized by elevation of activity of peroxidases and enhanced insolubilization of hydroxyproline-rich glycoproteins, such as extensin, in the cell wall. The insolubilization process (cross-linking between soluble extensin precursor molecules) is catalyzed by extensin peroxidases. We have ionically eluted extensin peroxidases from intact water-washed suspension-cultured tomato (hybrid of Lycopersicon esculentum Mill. and Lycopersicon peruvianum L. [Mill.]) cells and purified them to homogeneity by molecular sieve and cation-exchange chromatography. Four ionic forms of peroxidase (PI,PII,EPIII, and EPIV) were resolved; only the latter two cross-linked tomato soluble extensin. The molecular weight (34,000-37,000), amino acid composition, and isoelectric point (9.0) of the extensin peroxidases were determined. Substrate specificities of the enzymes were investigated: soluble extensin and potato lectin (a hydroxyproline-rich glycoprotein with a domain that strongly resembles extensin) were cross-linked by only two forms of the enzyme, whereas bovine serum albumin, aldolase, insulin, a number of other marker proteins, and proteins eluted from tomato cells (except extensin) could not be cross-linked. We have also isolated a yeast elicitor that enhances total peroxidase activity and extensin insolubilization within 1 h of challenge in cultured cells of tomato. A highly sensitive enzyme-linked immunosorbent assay technique using polyclonal antiserum raised against soluble tomato extensin was used to demonstrate extensin insolubilization in vivo. A tomato cell-wall peroxidase that cross-links extensin has been purified and may have a role in plant defense.

Effect of ispaghula husk on the faecal output of bile acids in healthy volunteers.

Faecal bile acids are associated with both colorectal cancer and serum cholesterol levels. We investigate whether dosing with ispaghula husk affects the faecal bile acid weights and concentrations in healthy adults. Sixteen healthy volunteers consumed 7.0 g/day ispaghula husk, containing 5.88 g/day Englyst-determinable dietary fibre, for the middle 8 weeks of a 12-week period. Stool samples were collected, analysed for faecal bile acids and their form and dry weight determined. Correlations between the faecal bile acids, the stool parameters and the dietary intake were tested. Ispaghula husk treatment significantly lowers faecal lithocholic and isolithocholic acids and the weighted ratio of lithocholic acids to deoxycholic acid. These effects revert towards their initial states at the end of the treatment period. These changes in the faecal bile acid profiles indicate a reduction in the hydrophobicity of the bile acids in the enterohepatic circulation.

A proposal for the structuring of water.

In spite of much work, many of the properties of water remain puzzling. A fluctuating network of water molecules, with localised icosahedral symmetry, is proposed to exist derived from clusters containing, if complete, 280 fully hydrogen-bonded molecules. These are formed by the regular arrangement of identical units of 14 water molecules that can tessellate locally, by changing centres, in three-dimensions and interconvert between lower and higher density forms. The structure allows explanation of many of the anomalous properties of water including its temperature-density and pressure-viscosity behaviour, the radial distribution pattern, the presence of both pentamers and hexamers, the change in properties and 'two-state' model on supercooling and the solvation properties of ions, hydrophobic molecules, carbohydrates and macromolecules. The model described here offers a structure on to which large molecules can be mapped in order to offer insights into their interactions.

Magnetic, immobilised derivatives of enzymes.

A novel, water, insoluble, stable coating has been attached to various metals, which is capable of covalent attachment to enzymes. The coating (aminobenzoic acid-formaldehyde resin) can be attached to nickel, cobalt, tin, iron, and aluminium. beta-D-Glucosidase has been attached to the coated metals by diazotisation of the coating. The stability and usefulness of the resulting, metallic enzyme-preparations is discussed with special emphasis on the value of ferromagnetic supports for enzymes.

Dietary fibre, physicochemical properties and their relationship to health.

Dietary carbohydrates that escape digestion and absorption in the small intestine include non-digestible oligosaccharides (carbohydrates with a degree of polymerisation between three and ten), resistant starch and non-starch polysaccharides. The physiological effects of this heterogeneous mixture of substrates are partly predictable on the basis of their physicochemical properties. Monosaccharide composition and chain conformation influence the rate and extent of fermentation. Water-holding capacity affects stool weight and intestinal transit time. Viscous polysaccharides can cause delayed gastric emptying and slower transit through the small bowel, resulting in the reduced rate of nutrient absorption. Polysaccharides with large hydrophobic surface areas have potentially important roles in the binding of bile acids, carcinogens and mutagens. Ispaghula is capable of binding bile acids through a large number of weak binding sites on the polysaccharide structure, and having greatest effect on the potentially more harmful secondary bile acids deoxycholic acid and lithocholic acid.

Bile acids, fibre and colon cancer: the story unfolds.

Are the changes in faecal bile acid concentrations the cause of colorectal cancer or one of its effects? This is an area of controversy mainly due to the lack of a clear explanation as to how the bile acid concentrations are controlled under different circumstances. This review presents an outline of the evidence that bile acids are both a causal factor in colorectal cancer and that their concentrations are affected by it in a synergistic manner. It also offers an explanation of how some dietary fibre protects against colorectal cancer.

Analysis of bile acids and their conjugates using high-pH anion-exchange chromatography with pulsed amperometric detection.

Bile acids and their conjugated forms may be separated by anion-exchange chromatography in alkaline media (0.9 M sodium acetate, 0.1 M sodium hydroxide, 15% v/v acetonitrile) on a CarboPac PA-100 column. The effluent was monitored at high sensitivity, with detection limits of less than 10 microM, using a pulsed amperometric detector. Free bile acids and their glyco- and tauro-conjugated forms were separated and detected within 40 min under isocratic conditions.

The use of ninhydrin as a reagent for the reversible modification of arginine residues in proteins.

A simple technique was developed for the specific reversible modification of guanidino groups in proteins involving reaction with ninhydrin. The extent of the reaction is easily determined non-destructively by spectrophotometric analysis. The reagent can also be used for the titration of sterically unhindered thiol groups in proteins.

The structures of the carbohydrate moieties of the alpha subunit of human chorionic gonadotrophin.

The alpha subunit of human chorionic gonadotrophin was reduced with dithiothreitol followed by carboxymethylation with iodoacetic acid. The modified glycoprotein was hydrolysed with trypsin to give various peptides, the identities of which were established, and glycopeptides. The glycopeptides were separated by gel filtration and ion-exchange chromatography; they were subjected to component analysis and were found to represent the two carbohydrate moieties in the parent glycoprotein. Sequential removal with glycoside hydrolases of monosaccharide units from the glycopeptides demonstrated (1) that galactose, mannose, glucosamine (2-amino-2-deoxyglucose) and neuraminic acid (5-amino-3,5-dideoxy-glycero-galacto-2-nonulosonic acid) residues possess the D configurations, (2) that the glucosamine units are N-acetylated and (3) the order of the monosaccharide units in the chain, the neuraminic acid units being furthest from the peptide backbone of the subunit and substituting the D-galactose units. Methylation analysis of the glycopeptides by adaptation of the Hakomori technique demonstrated that: (4) D-galactose, D-mannose and N-acetylglucosamine (2-acetamido-2-deoxy-D-glucose) units exist in the pyranose forms; (5) the D-galactopyranose units are linked in the 1 and 6 positions; (6) the D-mannopyranose units exist in several forms, one in a terminal non-reducing position, one as 1,2-linked residues and some as 1,6-linked branch points; (7) the N-acetylglucosamine units are 1,6-linked. On the basis of the results of methylation and enzymic analysis, structures are proposed for the carbohydrate moieties and the assignments are compared with other data previously obtained by periodate-oxidation studies [Kennedy et al. (1974) Carbohydr. Res. 36, 369-377].

Structural investigation of the carbohydrate element of human pituitary follicle-stimulating hormone by methylation analysis.

A preparation of human follicle-stimulating hormone has been subjected to methylation analysis by using the methyl sulphinyl carbanion-dimethyl sulphoxide-methyl iodide method. The hydrolysis products of the methylated glycoprotein were reduced, acetylated, analysed by gas-phase chromatography and mass spectrometry and identified by comparison with standards. Methylation analysis demonstrated that (1) the d-galactose, mannose, fucose and 2-amino-2-deoxyglucose units exist in the pyranose forms, (2) the 2-amino-2-deoxyglucose units are N-acetylated, (3) the fucopyranose units occupy terminal non-reducing positions, (4) the d-galactopyranose units are linked in the 1- and 2-positions, (5) the mannopyranose units exist in three forms, some as terminal non-reducing residues, some as 1,6-linked residues and some as 1,3,4-linked branch points, and (6) the 2-acetamido deoxyglucopyranose units are 1,6-linked. These structural assignments are compared with other data previously obtained for the carbohydrate moieties of follicle-stimulating hormone.

Determination of bile acids in human faecal samples using supercritical fluid extraction and high-performance liquid chromatography.

A supercritical fluid extraction (SFE) method for the extraction of bile acids from faeces is described. HPLC with pulsed amperometric detection was used to examine and confirm the recovery of bile acids. The analytes were extracted within a period of 75 min using supercritical carbon dioxide at a pressure of 34.5 MPa and a temperature of 90 degrees C. In developing this method the following parameters were investigated: temperature, pressure, and extraction time. Two alternative methods of sample preparation were also investigated with a view to reducing the overall analysis time. The method was validated for the major primary and secondary bile acids found in faeces. It was found that the overall mean +/- SD recoveries were 102.1+/-7.92%, 111.6+/-9.91%, 112.1+/-9.92% and 113.7+/-9.92% for dry samples and 108.5+/-15.77%, 110.0+/-7.22%, 115.9+/-11.11% and 106.6+/-9.16% for wet samples with respect to cholic, deoxycholic, chenodeoxycholic and lithocholic acid. The SFE is an alternative to the traditional methods available. The extraction is relatively easy to conduct and does not utilise as much glassware, solvents or time.

Interaction of concanavalin A with native and denatured forms of jackbean alpha-D-mannosidase.

Tetrameric alpha-D-mannosidase from jackbean is a glycoprotein containing at least one mannosylated oligosaccharide. In the native enzyme, the oligosaccharide is sterically masked from interaction with either endoglucosaminidase H or concanavalin A. Denaturation into subunits permits endoglucosaminidase hydrolysis and removal of the oligosaccharide. The mannosyl residues are attached only to the heavy type of subunit. Removal of the oligosaccharide(s) from the denatured heavy subunit requires the joint action of both alpha-D-mannosidase and N-acetyl-beta-D-glucosaminidase.

Endopeptidase-24.11 purified from pig intestine is differently glycosylated from that in kidney.

Endopeptidase-24.11 (EC 3.4.24.11) was purified from pig intestinal microvilli by immunoadsorbent chromatography, using antibodies raised to kidney endopeptidase-24.11. In many respects, the kidney and intestinal enzymes were indistinguishable, but some structural differences were demonstrated. In particular, the detergent form of the intestinal enzyme had an apparent subunit Mr of 95000, which, on treatment with trypsin, fell to a value of 89000, identical with that of the kidney form. The intestinal enzyme contained 3-4% more carbohydrate and many more fucose residues than that from kidney. Although these results show that post-translational processing was different in the two cell types, the possibility that the primary translation products also differed cannot be excluded.

Deglycosylation by trifluoromethanesulphonic acid of endopeptidase-24.11 purified from pig kidney and intestine.

Endopeptidase-24.11, an integral microvillar membrane enzyme, exists in differently glycosylated forms when purified from pig kidney and intestine [Fulcher, Chaplin & Kenny (1983) Biochem. J. 215, 317-323]. When these glycoproteins, and another form of the kidney enzyme prepared from the Yucatan dwarf strain of piglet, were treated, under controlled conditions, with trifluoromethanesulphonic acid, the proteins were freed of carbohydrate and all had the same apparent subunit Mr (77 000) even though the untreated forms varied from Mr 89 000 to Mr 95 000.

Cytochrome b-245 from human neutrophils is a glycoprotein.

Cytochrome b-245 is a glycoprotein. It runs as a broad band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and its apparent Mr varies with the concentration of acrylamide. It stained positively with Schiff reagent and with silver stains after oxidation with periodic acid. It preferentially bound the lectin of Phaseolus vulgaris (type III), and cleavage of carbohydrate with endoglycosidase F resulted in a sharp band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 55000 G.l.c. analysis of carbohydrate showed this to account for about 15% of the Mr and N-acetylglucosamine and galactose to be the major sugars.

Interactions of soya-bean agglutinin with purified glycoconjugates and soya-bean seed components.

A radioaffinity assay for lectin binding to receptors was developed and characterized by using the interactions between soya-bean agglutinin and four glycoconjugates, namely thyroglobulin, galactomannan, fetuin and asialofetuin. On application of the assay to soya-bean extracts a wide range of seed components were found to have the capacity to interact with soya-bean agglutinin. These included both trichloroacetic acid-soluble and trichloroacetic acid-insoluble glycoconjugates and two classes of particulate matter distinguished by their differential solubility in Triton X-100.