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dc.contributor.authorJedziniak, Judith A.en_US
dc.date.accessioned2019-05-07T19:26:40Z
dc.date.issued1966
dc.date.submitted1966
dc.identifier.otherb14629288
dc.identifier.urihttps://hdl.handle.net/2144/35459
dc.descriptionThesis (Ph.D.)--Boston Universityen_US
dc.descriptionPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.en_US
dc.description.abstractThe enzyme, deoxyribophosphoaldolase, reversibly cleaves deoxyribose- 5-P to glyceraldehyde-3-P and acetaldehyde. Until this exposition, it was not known to exist in human erythrocytes. However, the enzyme was postulated to occur in erythrocytes in order to account for the synthesis of glyceraldehyde-3-P observed in ghosts when deoxynucleosides were metabolized. Therefore, initial experiments were designed to detect acetaldehyde as an intermediate of deoxynucleoside metabolism by ghosts. Indeed, it was found in incubation mixtures containing deoxyinosine or deoxyadenosine as substrate. Accumulation of acetaldehyde was found to depend upon substrate concentration and no acetaldehyde was detected in the absence of substrate or in the presence of inosine or ribose-5-P. Acetaldehyde was identified spectrophotometrically by its reaction with yeast alcohol dehydrogenase and colorimetrically by its reaction with buffered semicarbazide solution. Confirmation of its identity was obtained by isolation of the 2,4 dinitrophenylhydrazone derivative of acetaldehyde aerated from incubation extracts. A direct relationship between acetaldehyde production and triosephosphate production to deoxyribose-5-P utilization by hemolysates prepared from human erythrocytes was shown. Conclusive evidence for the existence of deoxyribophosphoaldolase was obtained by isolating the enzyme from human erythrocytes. Two procedures were developed to isolate the enzyme. The first involved the use of Sephadex G-100 and DEAE-cellulose columns. The second procedure proved more fruitful and employed ammonium sulfate fractionation followed by elution from calcium phosphate gels. It was found that 26% of the original enzyme activity was recovered and purification was approximately 3,000 fold. Several characteristic properties of partially purified and purified preparations of the enzyme were studied. It was found that: 1. Enzyme activity decayed rapidly upon storage. Magnesium ion, cysteine HCl, beta-mercaptoethanol or reduced glutathione increased enzyme stability. In addition, the sulfhydryl containing compounds were able to partially reactivate previously inactivated enzyme. 2. The molecular weight of deoxyribophosphoaldolase was estimated by Sephadex gel fractionation to be slightly greater than 68,000, the molecular weight of hemoglobin. Because of its low affinity for DEAE-cellulose and high affinity for carboxy methyl cellulose, the enzyme appeared to be a very basic protein. 3. The reaction catalyzed by deoxyribophosphoaldolase favored cleavage of deoxyribose-5-P. At equilibrium, 60% of deoxyribose-5-P was converted to products. When acetaldehyde and glyceraldehyde-3-P were used as substrates, 40% of each was converted to deoxypentose product. 4. The enzyme showed a high specificity for each of its three substrates. No reaction of the enzyme occurred with ethyl alcohol, pyruvate, ribose-5-P, deoxyribose, lactate and dihydroxyacetone phosphate. 5. The enzyme reacted optimally at pH 6.5. 6. Deoxyribophosphoaldolase was activated by several carboxylic acids. The degree of activation was greater in the presence of citric acid than any dicarboxylic acid tested. An optimal activation of enzyme occurred when the concentration of citrate was varied between 3-15 umoles/ml. Citrate activation did not appear to reside in its ability to act as a chelator. Comparison of enzyme elution from Sephadex G-100 columns in the presence and absence of citrate suggested that citrate causes enzyme aggregation by a still unexplained mectanism. 7. The apparent Michaelis constants for deoxyribose-5-P cleavage were determined in the presence and absence of citrate and were found to be 6.4 x 10^-4 moles/liter and 24.0 x 10^-4 moles/liter respectively. The presence of deoxyribophosphoaldolase in human erythrocytes can clearly explain the production of triosephosphate and acetaldehyde from deoxynucleoside substrates and may, in fact, provide a major catabolic pathway for the deoxypentose moiety of these deoxynucleosides. The enzyme provides a simple mechanism for triosephosphate formation from deoxypentose and may be part of a pathway that converts deoxypentose phosphate to pentose phosphate.en_US
dc.language.isoen_US
dc.publisherBoston Universityen_US
dc.subjectBiochemistryen_US
dc.subjectEnzymologyen_US
dc.subjectDeoxyribose-phosphate aldolaseen_US
dc.subjectRed blood cellsen_US
dc.subjectHumanen_US
dc.titleDeoxyribophosphoaldolase of human erythrocytes: identification, purification and characterizationen_US
dc.typeThesis/Dissertationen_US
dc.description.embargo2031-01-01
etd.degree.nameDoctor of Philosophyen_US
etd.degree.leveldoctoralen_US
etd.degree.disciplineBiologyen_US
etd.degree.grantorBoston Universityen_US
dc.identifier.barcode11719025634686
dc.identifier.mmsid99181162890001161


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