Serine proteases are known to perform a wide range of functions essential to life; however there has to be some form of control mechanism in place. One of the many control mechanisms is their specific inhibition by protein proteinase inhibitors. Proteinase inhibitors in plants, present in their seeds, participate in defense mechanisms and their production is induced by herbivory or wounding. Plant proteinase inhibitors have been reported to inhibit a variety of serine proteinases, including enzymes of the blood coagulation cascade. In this study, various indigenous seed extracts were screened for potential serine proteinase inhibition. Acacia schweinfurthii was selected as a potential inhibitor that inhibited trypsin and factor X. The AS inhibitor was successfully purified to homogeneity by precipitating with 80 percent (v/v) acetone and the sequential chromatographic steps including ion-exchange chromatography, size exclusion chromatography, affinity purification on a trypsin-agarose column and RP-HPLC. Reducing SDS-PAGE conditions revealed an inhibitor of two polypeptide chains A and B of approximate molecular weights 16 and 10 kDa, respectively, and under non-reducing conditions, 25 kDa was observed. The inhibitor was shown to inhibit trypsin, chymotrypsin and factor X indicating the dynamic nature of the reactive site. An enzyme: inhibitor ratio of 1:1, and a Ki of 3.45nM was determined for the AS inhibitor on trypsin, and the inhibitor also weakly inhibit chymotrypsin. AS inhibitor and STI inhibited factor X with a Ki values of 13.7nM and 77.5μM respectively. Amino acid analysis revealed Mmin values of the A- and B- chain of 15,000 and 7,800, respectively. The effect of seed extracts on the activated partial thrombin time (APTT) and prothrombin time (PT) was tested. No prolongation of the PT was obtained. For the crude extracts of AK and AS, IC200 values of 4.6 and 189.62 μg/mL, were respectively obtained. For the purified fractions of STI, AS and AK, IC200 values of 51.5, 114.31 and 893.8 μg/ml were observed, respectively. Keywords: proteinase inhibitors, Acacia species, trypsin inhibitor, FX inhibitor.

published_or_final_version / Dentistry / Master / Master of Dental Surgery

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.

No description available.

No description available.

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.

No description available.

The major serine proteinase inhibitors Kunitz and Bowman-Birk-type trypsin are key anti-nutrients responsible for the low nutritional value of raw soy cake, the by product of oil expression from soybean. Traditionally, proteinase inhibitors are eliminated from soy cake through intensive heating, which is highly costly. The long term goal is to generate soybean seeds devoid of trypsin inhibitors through tissue culture and genetic modification of soybean. The RNAi technology has been selected in this study as a technique for down-regulation or silencing these two major serine trypsin inhibitors. Conserved regions, which have been identified by searching NCBI and EMBL database, were targeted for down regulation. Seed specific promoters were also isolated to drive the expression of hairpin constructs designed to down-regulate selected conserved regions of the inhibitors in soybean seeds. RNAi silencing constructs were designed for use in soybean transformation. Ultimately, a tissue culture and transformation protocol for a local soybean variety PAN 512 was established for transformation with two designed RNAi constructs. Suitability of selected promoters was tested by attaching promoters to the gus gene and evaluating specificity of seed expression after soybean transformation using the Agrobacterium tumefaciens strain EHA101. Future work will focus on further optimisation of the transformation protocol and generation of transformed plants carrying the designed silencing vectors. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Plant Science / unrestricted

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 117-138).