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The relative contributions of the three main intermediates of indole-3-acetic acid (IAA) biosynthesis from L-tryptophan (L-Trp); indole-3-pyruvate (IPyA), tryptamine (TNH2) and indole-3-acetaldoxime (IAOX), were investigated in vivo in tomato shoots. Initially, L-Trp, D-Trp, IPyA, TNH2 and IAA were purified from shoots, identified by full-scan mass spectrometry and their concentrations measured using gas chromatography with an electron capture detector. High specific activity [5-3H]IAOX and [5-3H]IPyA were synthesized from L-[5-3H]Trp and used as internal standards. Purification of endogenous IPyA was enabled by forming a stable pentafluorobenzyl oxime derivative in the crude plant extract. The respective endogenous concentrations of L-Trp, D-Trp, TNH2, IPyA and IAA were found to be 2,520, 103, 146.3, 5.9 and 8.5 ng g-1 f. wt. However, IAOX could not be identified as a natural constituent of tomato shoots by full-scan GC-MS. Secondly, incubation of tomato shoots for 6, 10 and 21 h in 30% 2H2O was used as a means of labelling IAA and its putative precursors in vivo. L-Trp, D-Trp, TNH2, IPyA and IAA were then extracted and purified and the 2H content measured by combined gas chromatography-mass spectrometry. These indole compounds were labelled rapidly with up to four 2H atoms. Direct comparison of the number and the amount of 2H atoms incorporated (pattern) was obtained from the mass spectral data on the common m/z 130 ion and its isotope peaks. IAA and L-Trp demonstrated an increase in 2H label with up to 17% and 21% of their molecules labelled at 10 h respectively. This was followed by a significant decrease in 2H label at 21 h to 12% for both L-Trp and IAA. This decrease in 2H label was attributed to an increase in protein catabolism, following shoot excision, resulting in the dilution of free L-Trp pool(s) with unlabelled L-Trp from which IAA is biosynthesized. This is reflected in the observed 1.6 to 1.8 fold increase of free L-Trp from 10 to 21 h. In contrast, tryptamine demonstrated a continual increase in 2H label with an average of 8, 20 and 28% of the molecules labelled at 6, 10 and 21 h respectively, suggesting that TNH2 and IAA were synthesized from separate Trp pools. In addition, the relatively slow rate at which 2H is incorporated into tryptamine would not be sufficient to account for the rate at which IAA becomes labelled. However, IPyA demonstrated a rapid increase in 2H with 22% and 37% of its molecules labelled at 6 and 10 h respectively. From the rate at which IPyA was labelled with 2H and the concentration of IPyA in tomato shoots a rate of synthesis for IPyA in tomato shoots was estimated which was sufficient to provide most of the shoot IAA requirements. Furthermore, the extent to which IAA and IPyA were labelled relative to that of total L-Trp would imply that a smaller more rapidly metabolised pool(s) of L-Trp was the precursor of these compounds. The rate and extent that D-Trp was labelled was consistently less than that of IAA precluding it as a possible precursor of IAA. These results indicate that in tomato shoots IAA is biosynthesized from a rapidly metabolized sub-pool(s) of L-trptophan predominantly via IPyA.

The relative contributions of the three main intermediates of indole-3-acetic acid (IAA) biosynthesis from L-tryptophan (L-Trp); indole-3-pyruvate (IPyA), tryptamine (TNH2) and indole-3-acetaldoxime (IAOX), were investigated in vivo in tomato shoots. Initially, L-Trp, D-Trp, IPyA, TNH2 and IAA were purified from shoots, identified by full-scan mass spectrometry and their concentrations measured using gas chromatography with an electron capture detector. High specific activity [5-3H]IAOX and [5-3H]IPyA were synthesized from L-[5-3H]Trp and used as internal standards. Purification of endogenous IPyA was enabled by forming a stable pentafluorobenzyl oxime derivative in the crude plant extract. The respective endogenous concentrations of L-Trp, D-Trp, TNH2, IPyA and IAA were found to be 2,520, 103, 146.3, 5.9 and 8.5 ng g-1 f. wt. However, IAOX could not be identified as a natural constituent of tomato shoots by full-scan GC-MS. Secondly, incubation of tomato shoots for 6, 10 and 21 h in 30% 2H2O was used as a means of labelling IAA and its putative precursors in vivo. L-Trp, D-Trp, TNH2, IPyA and IAA were then extracted and purified and the 2H content measured by combined gas chromatography-mass spectrometry. These indole compounds were labelled rapidly with up to four 2H atoms. Direct comparison of the number and the amount of 2H atoms incorporated (pattern) was obtained from the mass spectral data on the common m/z 130 ion and its isotope peaks. IAA and L-Trp demonstrated an increase in 2H label with up to 17% and 21% of their molecules labelled at 10 h respectively. This was followed by a significant decrease in 2H label at 21 h to 12% for both L-Trp and IAA. This decrease in 2H label was attributed to an increase in protein catabolism, following shoot excision, resulting in the dilution of free L-Trp pool(s) with unlabelled L-Trp from which IAA is biosynthesized. This is reflected in the observed 1.6 to 1.8 fold increase of free L-Trp from 10 to 21 h. In contrast, tryptamine demonstrated a continual increase in 2H label with an average of 8, 20 and 28% of the molecules labelled at 6, 10 and 21 h respectively, suggesting that TNH2 and IAA were synthesized from separate Trp pools. In addition, the relatively slow rate at which 2H is incorporated into tryptamine would not be sufficient to account for the rate at which IAA becomes labelled. However, IPyA demonstrated a rapid increase in 2H with 22% and 37% of its molecules labelled at 6 and 10 h respectively. From the rate at which IPyA was labelled with 2H and the concentration of IPyA in tomato shoots a rate of synthesis for IPyA in tomato shoots was estimated which was sufficient to provide most of the shoot IAA requirements. Furthermore, the extent to which IAA and IPyA were labelled relative to that of total L-Trp would imply that a smaller more rapidly metabolised pool(s) of L-Trp was the precursor of these compounds. The rate and extent that D-Trp was labelled was consistently less than that of IAA precluding it as a possible precursor of IAA. These results indicate that in tomato shoots IAA is biosynthesized from a rapidly metabolized sub-pool(s) of L-trptophan predominantly via IPyA.

Despite individual autoimmune diseases being relatively rare, collectively these diseases afflict 8 % of the population according to the American Autoimmune Related Diseases Association. With over 75 % of those affected being women, autoimmune disease has been recognised, by the World Health Organisation and the US National Institutes of Health, as a major global women's health issue. One third of autoimmune sufferers have a rheumatological disorder, which commonly affect the joints, muscle, skin, salivary glands and kidneys. Antibodies against nuclear antigens are a serological hallmark of these diseases. Detection of these antibodies is used in the diagnosis and prognosis of the disease. The sensitivity and specificity of the test, of which the antigen is a key component, is pivotal to correct disease diagnosis and management. The relationship between circulating autoantibodies and the target antigen is complex. Improving the effectiveness of a test to assist in diagnosis and prognosis comes from characterisation and understanding these complex relationships. This thesis compares bovine spliceosomal U1 small nuclear ribonucleoprotein particle (U1 snRNP) complex with its human equivalent, and examines the validity of using this bovine derived autoantigen in the diagnosis of the human autoimmune diseases, systemic lupus erythematosus and mixed connective tissue disease. Differences between bovine and human U1 snRNP composition were characterised using a combination of electrophoretic, immunoassay and mass spectrometry techniques. Although the U1C protein could not be identified in bovine U1 snRNP, all other specificities were present. U1A remained intact, whilst the U1 snRNP specific 68K protein was dephosphorylated and a large C-terminal domain was removed, such that 68K migrated as a 30-36 kDa cluster on SDS-PAGE. Bovine SmD proteins, present in U1 and non-U1 snRNPs, were unaffected, whereas, SmB'/B was truncated to a 12 kDa peptide, which interestingly, was no longer reactive with anti-RNP sera in western blot. The recognition of human SmB'/B protein by anti-RNP sera in western blot was further examined. A technique was developed to immunoaffinity purify tryptic digests of SmB'/B which could then be analysed by mass spectrometry. Interestingly, the human replication element protein (HREP) was tentatively identified, rather than SmB'/B as expected. It may be possible, therefore, that anti-RNP sera may be reacting with a protein other than SmB'/B. To examine the contribution of the individual U1 snRNP proteins to anti-RNP and anti-Sm sera reactivities, a method was developed to dissociate bovine U1 snRNP and to purify the individual component antigens. It was demonstrated both empirically and through anecdotal feedback from a commercial diagnostic kit producer that patient sera respond better to purified Sm-free 68K than the recombinant 68K antigen. The effect of commercial processing of bovine thymus, the source for U1 snRNP antigen, was determined. In this study, variables that may be controlled during processing, such as temperature, protease activity and pH, were investigated. Hydrolysis of the intact human 68K protein with the necrotic protease, cathepsin L, produced 38 and 25 kDa fragments, whereas exposure to ambient temperature and low pH produced 32 kDa peptide fragments similar to those observed in purified bovine 68K. It was therefore proposed that 68K protein may undergo autocatalytic hydrolysis during necrotic cell death. Thorough characterisation of the bovine spliceosomal U1 snRNP proteins has not only validated their use as diagnostic reagents in autoimmune disease but also provided some insight into the inactivation of U1 snRNP function during early cell death.