No description available.

The distribution of isozymes of triosephosphate isomerase was normal in all human tissues examined. This finding argues against the existence of tissue-specific isozymes. Normal distributions of isozymes were also found in patients with cri-du-chat syndrome. Thus it is unlikely that a gene for triosephosphate isomerase is located on the short arm of chromosome five in man. When triosephosphate isomerases from a wide range of species were examined by starch gel electrophoresis, definite evolutionary patterns were found. Kinetic studies were conducted on human triosephosphate isomerase under conditions simulating the intracellular environment of the erythrocyte. Calculations using the kinetic parameters obtained indicate that even in triosephosphate isomerase deficiency disease, enough enzyme activity remains that the rate of glycolysis should not become inhibited.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 104-107).

No description available.

published_or_final_version / Botany / Master / Master of Philosophy

Glucose phosphate isomerase (GPI) occurs in different bovine tissues as multiple, catalytically active isozymes which can be resolved by polyacrylamide gel electrophoresis and isoelectric focusing. GPI from bovine heart was purified to homogeneity and each of the isozymes was resolved. Four of the five isozymes were characterized with regard to their physical, chemical and catalytic properties in order to establish their possible physiological significance and to ascertain their molecular basis. The isozymes exhibited identical native (118 Kd) and subunit (59 Kd) molecular weights but had different apparent pi values of 7.2, 7.0, 6.8 and 6.6. Structural analyses showed that the amino terminus was blocked and the carboxyl terminal sequence was -Glu-Ala-Ser-Gly for all four isozymes. The most basic isozyme was more stable than the more acidic isozymes (lower pi values) at pH extremes, at high ionic strength, in the presence of denaturants or upon exposure to proteases. Kinetic constants, such as turnover number, Km and Ki values, were identical for all isozymes. Identical amino acid composition and peptide mapping by chemical cleavage at methionine and cysteine residues of the isozymes suggest a postsynthetic modification rather then a genetic origin for the in vivo isozymes. When the most basic isozyme was incubated in vitro under mild alkaline conditions, there was a spontaneous generation of the more acidic isozymes with electrophoretic properties identical to those found in vivo. The simultaneous release in ammonia along with the spontaneous shift to more acidic isozymes and changes in the specific cleavage of the Asn-Gly bonds by hydroxylamine of the acidic isozyme indicates deamidation as the probable molecular basis. In summary the isozymes appear to be the result of spontaneous, postsynthetic modifications involving the addition of an equal number of negative charges and are consistent with the deamidation process.

In the stamen (male reproductive tissue) of petunia 'Hurrah' flowers, the occurrence of SOD (superoxide dismutase) provided an effective anti-oxidative mechanism against superoxide production. Superoxide production and SOD activities at five developmental stages showed a positive correlation. The highest superoxide production and SOD activity in different parts of the stamen (anther, filament and pollen) were at stages with high metabolic activity: (i) during growing buds (in anthers and filaments) (ii) when flowers with predehiscent anthers were fully open (in pollen). In all parts of the stamen, SOD activity was the lowest at stage five (fully open flowers with dehiscent anthers), superoxide production was also lower at this stage with the exception of the pollen. The highest SOD activity was localized in anthers with the pollen, suggesting that the filaments only have a structural support function. SOD was examined on a native PAGE with regard to the isozymes present within the stamen of five developmental stages. Three isozymes, which were identified as Mn SOD, Fe SOD and Cu/Zn SOD by reactions with inhibitors, were commonly found at five developmental stages in crude extracts of anthers, filaments and pollen. The developmental stages with stronger isozyme bands on the native PAGE were consistent with the stages with higher SOD activities, and the Mn SOD and Fe SOD isozyme bands were more intense than Cu/Zn SOD bands, suggesting the activities of Mn SOD and Fe SOD in the crude extracts were much higher than Cu/Zn SOD. SOD from 1,000 stamens of dehiscent mature flowers was partially purified using ammonium sulphate fractionation and DEAE cellulose column chromatography. The purified bound fraction contained only one SOD isozyme on a native PAGE, which was shown to be a Mn SOD, as it is sensitive to neither hydrogen peroxide nor cyanide. The specific activity of the purified SOD was 66.5 U/mg and the yield of total activity was 3.0%. The progress of enzyme purification was monitored using SDS-PAGE and the bound fraction contained two major polypeptide bands. The purified enzyme activity was optimal in the range of neutral pH, but it was the highest at pH 7.8. Through incubation at various pH levels for 24 hours, favourable stability of the purified fraction was confirmed around a pH range of 7 to 8.5. The purified enzyme retained 87% of its initial activity at -20 ? after one month of storage, but at 4 ? only 38% of the initial activity remained after the same period of storage.

Superoxide Dismutase (SOD) activity in cell-free extracts prepared from healthy mature flowers of Petunia hybrida (variety 'Hurrah') was studied. The SOD activity in the crude extracts was stable for more than one month when stored at -20 oC. It was found that pH 7.8 is optimal for SOD activity. Different flower tissues of petunia (stigma, style and ovary) at various stages of development were extracted and analysed for SOD activity. SOD activity was found to be significantly highest in the ovary tissue of dehiscent petunia flowers. Three SOD isozymes were detected after crude extracts of the different female reproductive tissues of petunia flowers were analysed on a non-denaturing polyacrylamide gel electrophoresis system. Based on a difference in the sensitivity of the SOD isoforms to H2O2 and KCN, it is suggested that Mn-SOD, Fe-SOD and Cu/Zn-SOD were present in the crude extracts of the female reproductive tissues of petunia flowers. The response of the female reproductive parts of petunia flowers was also tested under water deficiency and high temperature (35 oC) stress. The SOD activity seemed to increase more in response to the high temperature than the water deficiency stress. Intense blue staining was observed from developing younger buds, and much lower formazan deposition was detected at the later stage. This indicates the lower O2- produced during later stages mainly due to increasing SOD synthesis. DEAE cellulose chromatography was successfully used to partially purify SOD from the ovaries of petunia flowers. The characteristics of the partially purified enzyme fraction were found to be very similar to those of the crude extracts.

Glucose phosphate isomerase (GPI) was purified from human placenta utilizing cross-linked spherical particle phosphocellulose. In three steps, GPI could be purified approximately 5500 fold with greater than 50% recovery. The purified enzyme exhibited four bands upon non-denaturing PAGE and isoelectric focusing (IEF) when stained with GPI specific activity stain. The four isozymes were isolated by preparative IEF. The isoelectric points of the isozymes were determined. Sodium dodecyl sulfate (SDS) gel electrophoresis showed two types of subunits with different molecular weights. Structural analyses showed both types of subunits had blocked amino termini. Other properties of the isozymes and subunits, including immunological reactivity, pH stability, peptide mapping and amino acid composition, were also established.

In the stamen (male reproductive tissue) of petunia 'Hurrah' flowers, the occurrence of SOD (superoxide dismutase) provided an effective anti-oxidative mechanism against superoxide production. Superoxide production and SOD activities at five developmental stages showed a positive correlation. The highest superoxide production and SOD activity in different parts of the stamen (anther, filament and pollen) were at stages with high metabolic activity: (i) during growing buds (in anthers and filaments) (ii) when flowers with predehiscent anthers were fully open (in pollen). In all parts of the stamen, SOD activity was the lowest at stage five (fully open flowers with dehiscent anthers), superoxide production was also lower at this stage with the exception of the pollen. The highest SOD activity was localized in anthers with the pollen, suggesting that the filaments only have a structural support function. SOD was examined on a native PAGE with regard to the isozymes present within the stamen of five developmental stages. Three isozymes, which were identified as Mn SOD, Fe SOD and Cu/Zn SOD by reactions with inhibitors, were commonly found at five developmental stages in crude extracts of anthers, filaments and pollen. The developmental stages with stronger isozyme bands on the native PAGE were consistent with the stages with higher SOD activities, and the Mn SOD and Fe SOD isozyme bands were more intense than Cu/Zn SOD bands, suggesting the activities of Mn SOD and Fe SOD in the crude extracts were much higher than Cu/Zn SOD. SOD from 1,000 stamens of dehiscent mature flowers was partially purified using ammonium sulphate fractionation and DEAE cellulose column chromatography. The purified bound fraction contained only one SOD isozyme on a native PAGE, which was shown to be a Mn SOD, as it is sensitive to neither hydrogen peroxide nor cyanide. The specific activity of the purified SOD was 66.5 U/mg and the yield of total activity was 3.0%. The progress of enzyme purification was monitored using SDS-PAGE and the bound fraction contained two major polypeptide bands. The purified enzyme activity was optimal in the range of neutral pH, but it was the highest at pH 7.8. Through incubation at various pH levels for 24 hours, favourable stability of the purified fraction was confirmed around a pH range of 7 to 8.5. The purified enzyme retained 87% of its initial activity at -20 ? after one month of storage, but at 4 ? only 38% of the initial activity remained after the same period of storage.