An unusual partial HPRT deficient mutant, HPRT Cape Town was observed to have a low activity in erythrocyte lysates at high concentrations of the purine substrates, hypoxanthine and guanine. This substrate inhibition was not observed with the substrate PPRP. The low activity was not associated with changes in the Km or Vmax for any of the substrates (Steyn and Harley, 1984). The kinetics of the proband's enzyme was studied in lymphoblast extracts. The characteristic substrate inhibition was observed which showed that this phenomenon was not confined to erythrocytes but was a more generalized phenomenon. This result implies that the decreased HPRT activity observed in the proband is due to substrate inhibition by the purine bases. The HPRT enzyme is coded for by a gene which is located on the X chromosome (Pai et al., 1980). The proband's daughter was therefore studied in order to determine the cause of the mutation. It was not known whether the substrate inhibition was the result of a mutation in the gene coding for the enzyme, a mutation which results in altered post-translational modification or the absence or alteration of factors influencing normal HPRT kinetics. The daughter's transformed lymphoblasts exhibited growth patterns in selective media that resembled those of her father. The daughter's enzyme prepared from lymphoblast extracts exhibited the characteristic substrate inhibition. These results suggest that this cell line results from the selection of a clone or clones which have suppressed the function of the X chromosome carrying the maternal and presumably normal HPRT allele. The daughter's enzyme prepared from erythrocyte lysates exhibited intermediate enzyme activity between that of the proband and a normal control. This result suggests that the daughter is an obligate heterozygote and that the defect is due to a mutation in the HPRT gene itself. The defect was studied at the gene level. No difference was observed in the banding patterns of the proband's DNA and control DNA which were digested with various restriction enzymes and hybridized to ³²p-labelled HPRT cDNA. The size of the HPRT mRNA of the proband was the same as the control. These results imply that there is no major gene alteration; this is expected since the proband only has a partial deficiency of the enzyme. The HPRT cDNA was subcloned into a riboprobe vector, pGEM-3. The T7 promoter was used to transcribe antisense RNA strands which were then hybridized to the proband's RNA and control RNA. No difference was observed in the size of the protected fragment. This result does not exclude the possibility of a point mutation as the cause of the defect in HPRT Cape Town.

No description available.

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

Call number: LD2668 .T4 1986 V47 / Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program

by Tam Yan Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 102-114). / Abstract --- p.1 / Acknowledgement --- p.3 / Table of Contents --- p.5 / List of Tables --- p.7 / List of Figures --- p.8 / List of Plates --- p.8 / Chapter Chapter1 --- Introduction --- p.9 / Chapter Chapter2 --- Literature Review / Chapter 2.1 --- The study of enzyme polymorphism --- p.11 / Chapter 2.2 --- The zymogram technique --- p.20 / Chapter 2.3 --- Molecular approaches to systematic studies --- p.24 / Chapter 2.4 --- Genetic variation and systematic studies of decapod Crustacea --- p.29 / Chapter 2.5 --- Taxonomy of Penaeus and Metapenaeus --- p.33 / Chapter Chapter3 --- Materials and Methods / Chapter 3.1 --- Sample collection and storage --- p.43 / Chapter 3.2 --- Extract preparation --- p.44 / Chapter 3.3 --- Starch gel preparation --- p.45 / Chapter 3.4 --- Starch gel electrophoresis --- p.46 / Chapter 3.5 --- Gel slicing --- p.47 / Chapter 3.6 --- Enzyme staining --- p.47 / Chapter 3.7 --- Data collection --- p.48 / Chapter 3.8 --- Data analysis --- p.49 / Chapter Chapter4 --- Results / Chapter 4.1 --- Genetic interpretation of zymograms --- p.65 / Chapter 4.2 --- Conformity to Hardy-Weinberg equilibrium distribution --- p.67 / Chapter 4.3 --- Level of genetic variability --- p.67 / Chapter 4.4 --- Interspecific and intergeneric genetic differentiation --- p.68 / Chapter Chapter5 --- Discussion / Chapter 5.1 --- Enzyme polymorphism in Penaeus and Metapenaeus --- p.84 / Chapter 5.2 --- Level of genetic variability --- p.85 / Chapter 5.3 --- Phylogenetic relationships --- p.91 / Chapter 5.4 --- Biochemical and molecular systematic studies of Penaeus and Metapenaeus --- p.96 / Chapter Chapter6 --- General Conclusions --- p.99 / References --- p.102

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

Thesis (MSc. (Biochemistry)) -- University of Limpopo, 2013 / Hydroxynitrile lyases (HNLs) are enzymes that catalyse enantioselective cleavage of the substrate in a reaction and are also used as important industrial biocatalysts for the synthesis of chiral cyanohydrins. The aim of the study was to screen indigenous South African plants for potential hydroxynitrile lyase activity, purify and biochemically characterise the active hydroxynitrile lyase(s) from the selected plants. Several indigenous plants were randomly collected, identified and screened for HNL activity. The plant parts (leaves, seeds or fruits) were processed using established experimental protocols in order to obtain the crude enzyme extracts. The enzymatic conversion of benzaldehyde and potassium cyanide to mandelonitrile was optimised and consequently used for the screening of HNL activity. Enzyme activity was detected in the crude enzyme extracts of Kalanchoe spp and Senecio spp and these were then designated as Ks and Sb, respectively. Ammonium sulphate fractionation, DEAE Toyopearl 650M and Concanavalin A chromatography techniques were then used in the purification process of the active crude enzyme extracts. Subsequently, two purified active fractions were isolated from each plant species with molecular masses estimated at 64.64 kDa and 64.06 kDa for the KsHNL enzymes and 70.60 kDa and 74.04 kDa for SbHNL enzymes. The optimum temperature and pH of all the isolated enzymes were determined as 50°C and pH 5, respectively. The experimental Km and Vmax values of the enzymes were respectively determined to be 0.33 and 0.73 mM and 1.238 and 1.948 μM/min for KsHNL; while that for SbHNL enzymes were 5.86 and 0.22 mM and 9.741 and 1.905 μM/min. The effect of additives and metal ions (viz., DTT, DEP, mercury chloride, magnesium chloride and zinc chloride) was determined. The experimental data obtained alluded to the notion that both KsHNL and SbHNL enzymes may contain the cysteine and serine residues next to their active sites and that a histidine residue may be involved in the catalytic activities of both the isolated KsHNL enzymes and one of the SbHNL enzymes. All the isolated enzymes from the two plant species did not seem to contain an FAD group. These findings compared favourably to the theoretical type II HNLs, although with a slight difference in that they displayed high molecular weights. Kalanchoe spp and Senecio spp are the two indigenous South African plants that were found to contain active HNLs. The isolated HNLs from the two plants have a potential to be xv purified to homogeneity, cloned and overexpressed into robust recombinant enzymes that can be used for large scale industrial applications.

Call number: LD2668 .T4 1984 S87 / Master of Science

Sandra Jane Chapman. / "October 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 163-178). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.