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No description available.

Full-fat soybeans require heat treatment to denature trypsin inhibitors which interfere with proper digestion of the soybean protein. This study examines the heat treatment of soybeans in a continuous particulate-medium conduction processor. / Whole soybeans at 13% and 23% moisture were roasted at salt temperatures of 225, 250 and 275$ sp circ$C for residence times of 15, 30 and 60 seconds. The soybeans reached temperatures ranging from 107$ sp circ$C to 134$ sp circ$C, after which they were maintained in an insulated container for 0, 5, 10 or 15 minutes. Salt temperature, residence time, and moisture content were found to significantly affect final temperature, temperature change, moisture loss and total heat transfer. Moisture losses in the processor were very high, indicating that combining the processes of heat treatment and drying may provide a high combined efficiency. / Analysis of trypsin inhibitor activity and soluble protein indicated that all three factors likely affect soybean quality, and the soybean quality achieved in these tests is likely to be sufficient for livestock feeding, although this remains to be confirmed through feeding trials.

Effective inhibition of starch digestion in vivo may diminish glucose formation and absorption by the small intestine and increase the amount of undigested starch reaching the colon. The enzyme involved in the digestion of starch, alpha-amylase, has been identified and crystallized several years ago. Controversy exists as to whether effective inhibition can decrease starch digestion sufficiently to result in weight loss. The objective of this project is (a) to isolate and characterize alpha-amylase inhibitors from Phaseolus species (b) to synthesize and characterize known inhibitors in an effort to understand their mechanism of action. / The supernatant from ground beans was subjected to reverse phase chromatography. The separated peaks were lyophilized and assayed for alpha-amylase inhibitory activity. The inhibitor with the highest activity (peak 6) was repurified and fully characterized. It was exposed to physiological amounts of endoproteases to check its stability. / A known inhibitor of alpha-amylase was synthesized and studied. Its binding constant has not been previously reported. (Abstract shortened by UMI.)

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No description available.

The following thesis examines the effects of ionic strength and solution composition on the thermal stability and. activity of bovine erythrocyte superoxide dismutase (BESOD). An indirect assay procedure was employed. The oxidation of xanthine, catalyzed by xanthine oxidase, served as a generator of superoxide radicals (02) and ferricytochrome c functioned as a scavenger of these radicals. The assays were monitored and recorded spectrophotometrically. Percent superoxide dismutase activity remaining after a 30 minute heat treatment and 241 hours after the heat treatment were determined. As the concentration and computed ionic strength values of the ionic solutions increased, the percent BESOD activity remaining after 30 minutes at 80 C decreased. Recovery of BESOD activity after 24 hours was minimal in all ionic solutions. BESOD denaturation was apparently similar in the chloride and sulfate solutions, but much greater in the phosphate solutions. Mathematical interpretation confirmed the similar relationships between chloride and sulfate solutions, and the much greater effects of phosphate solutions on thermal denaturation of SOD. At concentrations of 10-1 M or less, thermal denaturation of SOD was greatest, implying the appearance of a denaturation threshold. A specific phosphate effect may be apparent and was described by two models: 1- complexation of Cut + ions with phosphate anions, and 2- conformational alteration due to charge repulsion between molecular strands of the SOD molecule. Both of these models could be possible explanations as to the much greater thermal denaturation observed in the phosphate solutions.

Steroid sulfatase (STS) catalyzes the desulfation of biologically inactive sulfated steroids to yield biologically active desulfated steroids and is currently being examined as a target for therapeutic intervention for the treatment of breast and other steroid-dependent cancers. A series of 17-arylsulfonamides of 17-aminoestra-1,3,5(10)-trien-3-ol were prepared and evaluated as inhibitors of STS. Introducing n-alkyl groups into the 4´-position of the 17-benzenesulfonamide derivative resulted in an increase in potency with the n-butyl derivative exhibiting the best potency with an IC50 of 26 nM. A further increase in carbon units (to n-pentyl) resulted in a decrease in potency. Branching of the 4´-n-propyl group resulted in a decrease in potency while branching of the 4´-n-butyl group (to a tert-butyl group) resulted in a slight increase in potency (IC50 = 18 nM). Studies with 17-benzenesulfonamides substituted at the 3´- and 4´-positions with small electron donating and electron withdrawing groups revealed the 3´-bromo and 3´-trifluoromethyl derivatives to be excellent inhibitors with IC50’s of 30 and 23 nM respectively. The 17-2´-naphthalenesulfonamide was also an excellent inhibitor (IC50 = 20 nM) while the 17-4´-phenylbenzenesulfonamide derivative was the most potent inhibitor with an IC50 of 9 nM. Kinetic studies with 3´-bromo derivative revealed it to be a non-competitive inhibitor and so these types of inhibitors might be capable of binding at the active site and also at a secondary site outside the active site. The amide analogs of some of these compounds were found not to be as potent inhibitors as the sulfonamides. Introducing a nitro group or fluorine atom into the 4-position of the 17-arylsulfonamide inhibitors resulted in an increase in potency. Some of these compounds are the most potent reversible STS inhibitors ever reported with apparent Ki’s as low as 1 nM. 3-O-Sulfation of these compounds did not significantly alter their potency. It is not known if 3-O-sulfated derivatives were acting as inhibitors or reversible suicide inhibitors. Docking studies were performed on selected inhibitors to gain insight into how they might interact with STS. Selected 17-arylsulfonamide inhibitors were sent to the NCI (USA) for in vitro screening with a panel of 60 human tumor cell lines (NCI-60 panel). Almost all of the compounds exhibited GI50’s in the 1 to 10 M range with all 60 cell lines and so were only moderately potent in terms of their ability to inhibit the growth. None of the compounds stood out in terms of their ability to inhibit the growth of any breast cancer, prostate cancer or any other cancer cell line studied. The thiadiazolidinedione group was proposed as a sulfate mimic for obtaining STS inhibitors. A new approach to the synthesis of 3-aminoestrone was achieved as part of an attempt to prepare the thiadiazolidinedione target. 3-O-Sulfamoylation of one of the 17-arylsulfonamide inhibitors was attempted using a variety of reaction conditions but was unsucce

Over the previous quarter century the vine weevil (Otiorhynchus sulcatus) has become a pest of horticultural and agricultural plants. The vine weevil is a polyphagous coleopteran insect and is able to attack over one hundred different plant species. Its spread has been limited by its lack of flight but modern world trade in live container grown plants has spread the insect to new habitats. Damage to plants caused by vine weevil is two fold, with the larvae destroying root balls while the adults attack the, leaves. The larval stage, in particular is difficult to treat with conventional insecticides unless environmentally undesirable soil treatments are used. The current lack of defence against the vine weevil has opened the door for methods of crop protection through the generation of genetically modified plants. The design of an efficient GM approach to control the vine weevil requires a sound knowledge of the insect’s digestive enzymes, which may be used as potential targets for insecticidal proteins. This approach was achieved for the vine weevil through analysis of active digestive proteases in the insects gut and the identification of suitable proteinase inhibitors which would reduce the overall level of protein hydrolysis. Using this method it was discovered that the vine weevil contained both serine and cysteine proteases in addition to a range of other digestive hydrolases. This biochemical data was supported by a molecular approach to isolate cDNA clones associated with the insect's digestive tract. Using a gut specific cDNA library clones encoding a cathepsin B protease, two trypsin proteases, a pectinesterase, a lipase and a cellulase were isolated and characterised. The cellulase isolated from vine weevil has been shown to originate from the insect genome as shown through Southern Blot analysis and sequencing across several intronic regions. Evidence presented herein shows that the vine weevil gut extract hydrolyses both cellulose and cellobiose. Similar results were observed with recombinant protein expressed in the eukaryotic yeast P.pastoris. Furthermore data presented here shows that the vine weevil has the full complement of enzymes needed for the complete digestion of crystalline cellulose, which was until recently believed to be the sole domain of several species of bacteria and yeast. In addition a cDNA clone encoded a vine weevil endogenous chitinase was isolated from the cDNA library. This chitinase cDNA and one encoding the proteinase inhibitor Oryzacystatin-I were used to generate transgenic tobacco plants which have been shown to express the transgene. These transgenic plants are the first step in developing a strategy for plant protection against vine weevil based on genetic modification.

Studies of the purification, characterization and primary structure of protein inhibitors of trypsin and -amylase from seeds of Job's Tears (Coix lachryma-jobi) were undertaken. The major trypsin inhibitor from seeds of Coix was purified by heat treatment, fractional precipitation with ammonium sulphate, ion-exchange chromatography, gel filtration and preparative reversed-phase HPLC. The complete amino acid sequence was determined by analysis of peptides derived from the reduced and S- carboxymethylated protein by digestion with trypsin, chymotrypsin and the S.aureus V8 protease. The polypeptide contained 64 amino acids with a high content of cysteine. The sequence exhibited strong similarity with a number of Bowman-Birk inhibitors from legume and cereal seeds. A protein inhibitor of locust gut ζ-amylase was purified from seeds of Coix using ammonium sulphate precipitation, affinity chromatography on Red Sepharose and reversed-phase HPLC. It consisted of two major isomers, each a dimer of two identical or closely similar subunits of M(_r) about 26 400. These two isomers also had very similar amino acid compositions. The major isomer showed no inhibitory activity against amylases from other sources: human saliva, porcine pancreas, B. subtilis. A. oryzae and barley malt. The manual DABITC/PITC method was used to determine about half of the amino acid sequence of the major isoform. This showed a high degree of similarity with previously reported sequences of endochitinase enzymes from several species (tobacco, potato, barley, bean). Endochitinase activity was demonstrated by following the release of radioactivity from [(^3)H] chitin. As far as can be ascertained from the literature this is the first characterization of a plant protein with activity as an enzyme and as an enzyme inhibitor. Preliminary molecular studies were also carried out, including the isolation and in vitro translation of mRNA fractions from developing seeds of Coix.