No description available.

No description available.

No description available.

Shoots excised from pea seedlings (Pisum sativum) at different stages of growth (2, 9, 13 and 21 day old) were supplied with [U-19C] adenine. Incorporation of radioactivity into the resulting metabolites was traced and measured. In the 2 day old shoots, adenine was converted to the adenosine nucleotides AMP, ADP and A1P and incorporated into nucleic acids. In 9, 13 and 21 day old shoots radioactivity was mostly incorporated into the purine catabolites xanthine and allantoin, and to a lesser extent into adenosine nucleotides a very small proportion of the radioactivity was incorporated into hypoxanthine and inosine. These experiments were repeated with 13 and 21 day old seedlings in the presence of allopurinol which fed, simultaneously, with [U-19C] adenine to the plants. Marked changes in the pattern of incorporation of the precursor into its metabolites were observed. Incorporation of radioactivity into allantoin and xanthine decreased, whereas incorporation into inosine, hypoxanthine and the adenosine nucleotides increased. It is concluded from the results obtained that allopurinol blocks the reactions leading to formation of xanthine and uric acid. Attempts to detect traces of radioactive uric acid, IMP and guanine derivatives in the presence of absence of allopurinol failed. In the expectation of directly demonstrating xanthine oxidase activity in seedlings of P. sativum and Lens eacutenta, enzymic extracts of these seedlings were made and incubated with [U-14C] adenine, [8-19C] hypoxanthine. However, whereas the adenine was converted to hypoxanthine and AMP, the hypoxanthine was recovered unchanged.

The cellulolytic enzyme complex of Humicola insolens has been fractionated, by column chromatography, into four components: (i) cellobiase (ii) a CM-cellulase (iii) component C1 and (iv) component C2. A scheme was devised for the isolation of component C2 relatively free from its main contaminant, CM-cellulase. In this scheme the fractionation of concentrated culture filtrate by chromatography on columns of cellulose powder was followed by repeated fractionation on columns of DEAL cellulose. The C2 component so isolated released a range of products from bacterial cellulose and, less extensively, from cotton. These products were glucose and a range of cellodextrins up to cellopentose. The cellotriose, cellotettose and cellopentose were degraded to glucose and cellobiose in the presence of CM-cellulase. Component C2 also attacked insoluble dextrins and short fibres but not glucose, cellobiose or cellotriosz. The main products from cotton were short fibres, but these were not extensively produced by purified component C2 unless CM-cellulase was added. The purified component C2 was unstable and the synergistic response with CM-cellulase could be achieved by replacing CM-cellulase with bovine serum albumin. Swelling-factor (S-factor) activity was a property of both component C2 and CM-cellulase. Short fibre formation was decreased in the absence of oxygen but the formation of soluble products from cotton or bacterial cellulose was not affected. Short fibre formation was inhibited by CM-cellulase but not much by glucose or cellobiose. The interrelationship of these effects is discussed and a scheme for the solubilisation of cotton is given.

The lectin Concanavalin A is processed <i>in planta </i>by a splicing-mediated circular permutation of its initial single-chain precursor, pro-Concanavalin A. Active (carbohydrate-binding) protein conformations can be purified by dextran-affinity chromatography. This thesis demonstrates <i>in vitro</i> splicing by asparaginyl endopeptidase of two cofolded polypeptide fragments (A- and B-chains) corresponding to Concanavalin A precursors. This ligation of A- and B-chains was enzyme-, temperature- and pH-dependent. The 9-residue extension at the C-terminus of A-chain is essential for splicing. To test whether correct cofolding and an intervening spacer are required for maturation, recombinant A- and B-chains without spacer sequence were purified and refolded separately. Asparaginyl endopeptidase was again absolutely required for ligation of non-cofolded A- and B-chains. Correct folding is crucial to form an active structure, but is less important for enzyme-mediated splicing. Protein splicing of two-chain forms of precursors was clearly evident from the large decrease in electrophoretic mobility of ligated product. However, interpretation of proteolytic cleavage patterns was difficult. <i>In vivo</i> maturation could be reconstituted <i>in vitro</i> using recombinant pro-Concanavalin A (single-chain, active) with asparaginyl endopeptidase. Enzyme could also splice synthetic peptides corresponding to processing-sequences of precursor proteins. Two short peptides were ligated to form a new longer peptide as indicated by reverse-phase chromatography. It was generally observed that increasing the pH from 5 to 7.5 changed the balance away from proteolytic cleavages (hydrolysis) and towards protein (or peptide) ligation (aminolysis). Promotion of splicing at higher pH indicates that availability of the unprotected (nucleophilic) form of the attacking terminal a-amino group is a major factor in determining product formation. Other factors (substrate conformation, concentrations, temperature and incubation time) may also influence the outcome.

The role of Ca<SUP>2+</SUP> in the interaction between lucerne (<i>Medicago sativa</i> L.) and the wilt fungus, <i>Verticillium albo-atrum</i> R & B has been examined. Elicitor prepared from culture-filtrates of <i>V.albo-antrum</i> reduced the incorporation of <SUP>45</SUP>Ca<SUP>2+</SUP> into protoplasts of lucerne. Elicitor and extracellular Ca<SUP>2+</SUP> caused an efflux of <SUP>45</SUP>Ca<SUP>2+</SUP> from protoplasts preloaded with <SUP>45</SUP>Ca<SUP>2+</SUP>, the elicitor causing the increase in efflux through the activity of an orthovanadate sensitive pump. Antagonists of Ca<SUP>2+</SUP> ion channels were used to show that a constant cycling of Ca<SUP>2+</SUP> occurs across the plasma membrane. Dibutyryl 3'5' cAMP and the calmodulin antagonist compound R24571 had no effect on the cycling of <SUP>45</SUP>Ca<SUP>2+</SUP> suggesing cAMP and calmodulin are not directly involved in the mechanism of cycling. A Ca<SUP>2+</SUP> dependent, calmodulin independent ATPase was identified which was sensitive to inhibition by sodium orthovanadate and the Ca<SUP>2+</SUP> ion channel blocker verapamil, which could be responsible for the efflux of <SUP>45</SUP>Ca<SUP>2+</SUP> across the plasma membrane. Tissues of lucerne were examined for the presence of receptor elements that could be components of a Ca<SUP>2+</SUP> second messenger system. Calmodulin and a calmodulin dependent 3',5' cAMP phosphodiesterase were identified. The 3',5' cAMP phosphodiesterase associated with a column of immobilised calmodulin, was inhibited by compound R24571 and was stimulated by bovine calmodulin. This enzyme provides a link between the Ca<SUP>2+</SUP> and the cAMP second messenger systems. Elicitor was shown to alter the pattern and increase the rate of phosphorylation of proteins from lucerne cell suspension cultures. A protein kinase activity that was stimulated by diacyl glycerol and phosphatidyl serine in the presence of Ca<SUP>2+</SUP> was associated with a protein fraction prepared by phosphatidyl serine affinity chromatography. The preparation cross-reacted with monoclonal antibodies raised to mammalian Protein kinase C. Two Ca<SUP>2+</SUP> dependent protein kinases and a calmodulin dependent protein kinase were identified in soluble extracts from lucerne. A Ca<SUP>2+</SUP> dependent protein kinase activity was also identified in a plasma membrane preparation. All four kinase activities phosphorylated proteins of lucerne.

Two clones of Agrostis stolonifera, (salt tolerant and non tolerant) were assessed for responses to NaCl and MgCl<SUB>2</SUB>-stress. Elevated supplements of Ca<SUP>2+</SUP> and M<SUP>2+</SUP> were added to the growth medium in conjunction with the imposed stress, in order to ascertain any amelioration conferred by these ions. Whole plant and callus tissues were investigated. The effect of NaCl and MgCl<SUB>2</SUB>-stress on growth, ionic relations and accumulation of nitrogenous compounds, sugars and chlorophyll was investigated. The salt marsh clone was more tolerant than the inland towards NaCl and MgCl<SUB>2</SUB>. Under the imposed stresses the salt marsh clone exhibited greater growth rates and showed a comparatively greater ability to utilize Ca<SUP>2+</SUP> and Mg<SUP>2+</SUP> in an ameliorative capacity. Whole plant tissues showed significant loss of Mg<SUP>2</SUP> (roots) and K<SUP>+</SUP> (roots and shoots) in response to NaCl. Ca<SUP>2+</SUP> and Mg<SUP>2+</SUP> ameliorated this to some extent. Both clones exhibited enhanced proline and QAC accumulation in response to salinity, however, sugar and total α-amino and concentrations did not show this significant trend. Callus tissue responses showed a parallel with those of the whole plant, with respect to salinity induced reduction of growth rate, K^+ loss and proline accumulation. However, in contrast, NaCl and MgCl_2-stressed callus tissues accumulated increased concentrations of total sugars. Soil sample solutions from Llanrhidian Salt Marsh were analysed over a twelve month period and compared with the relevant climatological data. High Mg^2+, Ca^2+ and K^+ concentrations coincided with periods of low rainfall. High Cl^- and Na^+ concentrations correlated well with low rainfall and elevated ambient temperatures.

No description available.

No description available.