Biomass production of chlorophyllase of the marine alga (Phaeodactylum tricornutum) at the exponential and stationary stages was performed. The results demonstrated that the biomass yield at the stationary stage was three times that of the exponential stage. A procedure for the extraction and purification of chlorophylls from fresh spinach leaves, used as substrate, was also developed. Chlorophyllase was extracted from photosynthetic membranes of the disrupted cells and partially purified. The purification procedure resulted into 70-fold increase in enzyme activity. Further purification of the partially purified enzyme was performed, using preparative isoelectric focusing on Rotofor-Cell System. Three enzyme fractions, FI$ sp prime$, FII$ sp prime$, and FIII$ sp prime$ were separated, however, most of enzyme activity (84%) was located in fraction FII$ sp prime$. The partially purified chlorophyllase was further purified by native preparative gel electrophoresis on Prep-Cell System, which resulted into a single active fraction. The purified enzyme fraction was then subjected to further purification, using automated Fast Protein Liquid Chromatography (FPLC) System, on ion-exchange Mono Q HR 5/5 column. The purification procedure resulted into two well separated isozymes, FI$ sp prime$ and FII$ sp prime$. Enzyme fraction (FI$ sp prime$) showed the highest enzymatic activity compared to FII$ sp prime$. The homogeneity of each fraction was demonstrated by a single protein band on SDS-PAGE. The molecular weights of these fractions FI$ sp prime$ and FII$ sp prime$ were 67 kD and 66kD, respectively. The optimum pH for chlorophyllase activity fractions FI$ sp prime$, and FII$ sp prime$ were 8.0 and 8.3, respectively. The enzymatic fraction FI$ sp prime$ showed higher activity towards commercial purified chlorophyll b when it was compared to that with the crude chlorophyll, partially purified chlorophyll and commercial purified chlorophyll a. However, enzymatic fraction FI

No description available.

Typescript. / Photocopy. [S.l. : s.n., 1976]. 29 cm. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1976. / Bibliography: leaves 231-243. / Microfilm / xiii, 243 leaves ill

Typescript. / Thesis (Ph. D.)--University of Hawaii, 1964. / Bibliography: leaves [80]-86. / vi, 86 l mounted illus

Typescript. / Thesis (Ph. D.)--University of Hawaii, 1969. / Bibliography: leaves 92-96. / viii, 96 l illus

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1975. / Bibliography: leaves 188-204. / xiii, 204 leaves ill. (some col.)

The cellular levels of a number of proteins have been found to be regulated by the ubiquitin-proteasome pathway. In this pathway, proteins are covalently tagged (&ldquo / ubiquitinated&rdquo / ) by ubiquitin, which acts as a signal for degradation by the proteasome. A number of key cellular processes, including cell-cycle progression, transcription and DNA repair, are regulated in this way. In recent years a number of cellular proteins resembling ubiquitin in structure or function, the so-called ubiquitin-like proteins, have been identified. Ubiquitin-like proteins can be divided into two classes-the so-called &ldquo / ubiquitin-like modifiers&rdquo / , which consist of a single domain that structurally resembles ubiquitin, and &ldquo / ubiquitin-domain&rdquo / proteins, which are multi-domain proteins, which include domains that resemble ubiquitin.<br /> <br /> This thesis describes the recombinant expression, purification and full backbone assignment of the human DWNN domain, a novel ubiquitin-like domain. The DWNN domain occurs at the N-terminus of RBBP6, a protein that has been shown to interact with p53 and Rb as well as to be involved in mRNA processing and apoptosis. A bacterial expression system was used to overexpress the DWNN domain as a GST fusion protein. The domain was labelled with 15N and 13C to perform triple-resonance heteronuclear NMR experiments, from which full backbone assignments were obtained.<br /> <br /> Although full structure determination of the DWNN domain falls outside the scope of this thesis, the backbone assignments formed the basis for the subsequent structure determination, which confirmed that the DWNN domain is indeed a novel ubiquitin-like domain. The RBBP6 protein may therefore represent a novel E3 ubiquitin ligase that plays a role in regulating the cellular levels of p53 and Rb.

<font face="Arial">This study represented the first structural investigation of any type III glutamine synthetase (GS). The GS, GlnA, from the medically important opportunistic human pathogen Bacteroides fragilis was studied with a view to better understanding the function and regulation of this important enzyme. </font>

Pollen grains are ubiquitous triggers of allergic asthma and seasonal rhinitis. Proteolytic enzymes in pollen as well as other sources are capable of disrupting airway epithelial integrity in vivo and in vitro. This provides a plausible mechanism for the initiation of sensitisation of the respiratory immune system to inhaled pollen allergens, comparable to that suggested for Group 1 allergens from house dust mites and cat dander, which are known to possess intrinsic proteolytic activity. This thesis explores the relationship between pollen allergens and proteolytic enzymes. It describes the different strategies used for the characterisation, purification and identification of immunogenic and proteolytic proteins in the complex mixtures of pollen diffusates. The peptidases in the diffusates of Kentucky blue grass, ryegrass and Bermuda grass pollens were characterised by a sensitive fluorescence assay and gelatin zymography. Among these, Bermuda grass pollen demonstrated the presence of a serine peptidase at Mr ~30,000 Da, which corresponded to an intense band by Western blotting using a monoclonal antibody to the timothy grass (Phleum pratense) group 1 allergen, Phl p 1. Purification of this enzyme from Bermuda grass was complicated by the low levels of the enzyme present in the diffusate, as well as by its autohydrolysis. Partial purification of the serine peptidase activity by affinity chromatography using Concanavalin A Sepharose demonstrated that the diffusate contained a trypsin-like peptidase, detected by the fluorescent assay, in addition to the ~30,000 Da serine endopeptidase, detected on gelatin zymography. Proteomic analysis of the ~30,000 Da protein using one- and two-dimensional electrophoresis and mass spectrometry identified it as the major pollen allergen of Bermuda grass, Cyn d 1. The studies reported here provide, for the first time, evidence that a pollen allergen may possess intrinsic proteolytic activity. This activity may play a role in the initiation of airway inflammation and allergic sensitisation.

Bibliography: leaves 104-116 / iv, 116 leaves, [11] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1987