Previous investigations of two direct methyltransferase systems in winter wheat have shown these systems to be operating at different times during the germination period and have suggested interconversions between these systems. S-Methyl-L-methionine, a methyl donor involved in these systems, could be transferring methyl groups to nucleic acid bases, histones, or pectic substances.In this study the translocation of labeled methyl groups from S-methyl-L-methionine-i4CH3 into nucleic acids, histones, or pectic substances was examined. Absorption of S-methyl-L-methionine-14CH3 was established and uptake of the labeled methyl group was indicated in the seed parts for 1, 2, and 3 days germination, and in the root and shoot for 3 days germination. Autoradiographic analysis of labeled tissue suggests translocation of the methyl group to meristematic regions and to the walls of the elongating cells in the region of cell elongation in root tissue.

Two direct methyltransferase systems in winter wheat have been reported using partially purified enzyme extracts. In order to gain further understanding of these enzymes, such classical enzyme investigations as pH range and optimum pH, the calculation of average activation energies, and inhibition investigations need to be undertaken using more highly purified enzymes. The purpose of this investigation was to develop procedures for further purification of S-methyl-Lmethionine:homocysteine methyltransferase in order that future researchers might undertake such studies.

The role of the labile methyl groups of betaine in the transmethylation reactions in the barley plants was investigated using carbon-14 methyl labelled betaine. The N-methyl groups of N-methyl tyramine, hordenine and choline were found to arise from betaine. Betaine was also administered to castor bean seedlings and the alka­loid ricinine isolated. It was found that its N- and 0-mthyl groups did not arise from betaine methyl. The origin of the methylenedioxy and N-methyl groups of the alkaloid protopine was also investigated by the tracer technique. Carbon-14 methyl labelled L-methionine, carbon-14 labelled choline and carbon-14 labelled sodium formate were fed to Dicentra species and the extent and position of labelling of the protopine molecule determined by degradation / Thesis / Master of Science (MS)

The role of the labile methyl groups of betaine in the transmethylation reactions in the barley plants was investigated using carbon-14 methyl labelled betaine. The N-methyl groups of N-methyl tyramine, hordenine and choline were found to arise from betaine. Betaine was also administered to castor bean seedlings and the alkaloid ricinine Isolated. It was found that Its N- and 0- methyl groups did not arise from betaine methyl. The origin of the methylenedioxy and N-methyl groups of the alkaloid protopine was also investigated by the tracer technique. Carbon-14 methyl labelled L- methionine, carbon-14 methyl labelled choline and carbon- 14 labelled sodium formate were fed to Dicentra species and the extent and position of labelling of the protopine molecule determined by degradation / Thesis / Master of Science (MS)

No description available.

Thesis (Ph. D.)--Institute of Paper Chemistry, 1983. / Includes bibliographical references (p. 113-122).

<p>Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive disorder characterized by degeneration of motor neurons in the cortex, brainstem and spinal cord. The patients usually die within 3-5 years after onset. The full etiology of ALS is unknown and many hypotheses have been proposed to explain the neurodegeneration. However, basic mechanisms of cellular function such as transmethylation and polyamine metabolism have not been extensively studied in ALS. Transmethylation reactions are very important in the synthesis of substrates such as proteins, neurotransmitters, DNA and RNA. The polyamines, putrescine, spermidine and spermine, are involved in essential functions such as cellular growth, proliferation and differentiation.</p><p>An initial study in this thesis concerned the process of neuronal death (apoptosis) in ALS spinal cord. The results showed increased levels of an apoptosis-stimulating protein and increased levels of DNA fragmentation indicative of an apoptotic process in the tissue. A comparative study of MAT-enzyme activity in spinal cord from different mammalian species was undertaken to provide a background for future studies on transmethylation and neurodegeneration. Transmethylation reactions were found altered in erythrocytes from males with ALS but not in spinal cord from ALS patients as compared to controls. An adaptation of previously described polyamine assays was made for the study of polyamines in ALS spinal cord. The method was validated and applied for polyamine analysis in human materials of different characteristics. Determination of polyamines in control and ALS spinal cords showed no major differences. However, in female ALS patients, significantly increased spermidine and spermine levels were observed in ventral horn regions. These gender-related alterations in transmethylation and polyamine metabolism are of interest since there is a male preponderance for the disease.</p><p>The lack of major differences in polyamine levels between ALS and control spinal cord suggests a maintained regulation of polyamines at the end stage of this neurodegenerative disease.</p>

Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive disorder characterized by degeneration of motor neurons in the cortex, brainstem and spinal cord. The patients usually die within 3-5 years after onset. The full etiology of ALS is unknown and many hypotheses have been proposed to explain the neurodegeneration. However, basic mechanisms of cellular function such as transmethylation and polyamine metabolism have not been extensively studied in ALS. Transmethylation reactions are very important in the synthesis of substrates such as proteins, neurotransmitters, DNA and RNA. The polyamines, putrescine, spermidine and spermine, are involved in essential functions such as cellular growth, proliferation and differentiation. An initial study in this thesis concerned the process of neuronal death (apoptosis) in ALS spinal cord. The results showed increased levels of an apoptosis-stimulating protein and increased levels of DNA fragmentation indicative of an apoptotic process in the tissue. A comparative study of MAT-enzyme activity in spinal cord from different mammalian species was undertaken to provide a background for future studies on transmethylation and neurodegeneration. Transmethylation reactions were found altered in erythrocytes from males with ALS but not in spinal cord from ALS patients as compared to controls. An adaptation of previously described polyamine assays was made for the study of polyamines in ALS spinal cord. The method was validated and applied for polyamine analysis in human materials of different characteristics. Determination of polyamines in control and ALS spinal cords showed no major differences. However, in female ALS patients, significantly increased spermidine and spermine levels were observed in ventral horn regions. These gender-related alterations in transmethylation and polyamine metabolism are of interest since there is a male preponderance for the disease. The lack of major differences in polyamine levels between ALS and control spinal cord suggests a maintained regulation of polyamines at the end stage of this neurodegenerative disease.

This thesis documents the subcellular localization and protein-protein interactions of two methyl recycling enzymes. These two enzymes, adenosine kinase (ADK) and S-adenosyl-L-homocysteine hydrolase (SAHH), are essential to sustain the hundreds of S-adenosyl-L-methionine (SAM)-dependent transmethylation reactions in plants. Both ADK and SAHH are involved in the removal of a competitive inhibitor of methyltransferases (MTs), S-adenosyl-L-homocysteine (SAH), that is generated as a by-product of the each transfer of a methyl group from SAM to a substrate. This research focused on understanding how SAH is metabolized in distinct cellular compartments to maintain MT activities required for plant growth and development. Localization studies using green fluorescent protein (GFP) fusions revealed that both ADK and SAHH localize to the cytoplasm and the nucleus, and possibly to the chloroplast, despite the fact that the primary amino acid sequence of neither protein contains detectable targeting signals. This suggested the possibility that these methyl-recycling enzymes may be targeted by specific protein-protein interactions. Moreover, deletion analysis of SAHH1 indicated that the insertion region (IR) of 41 amino acids (Gly150-Lys190), which is present only in plants and parasitic protozoan SAHHs among eukaryotes, is essential for nuclear targeting. This result suggested that the surface-exposed IR loop may serve as a binding domain for interactions with other proteins that may direct SAHH to the nucleus. To investigate protein-protein interactions, several methods were performed including co-immunoprecipitation, bimolecular fluorescence complementation, and pull-down assays. These results not only revealed that ADK and SAHH possibly interact through the IR loop of SAHH in planta, but also suggested that this interaction is either dynamic or indirect, requiring a cofactor/another protein(s) or post-translational modifications. Moreover, possible interactions of both ADK and SAHH with a putative Arabidopsis mRNA cap methyltransferase (CMT), which is localized predominantly in the nucleus, were also confirmed. These results support the hypothesis that the nuclear targeting of both SAHH and ADK can be mediated by the interaction with CMT. In addition, purification of Strep-tagged SAHH1 expressed in Arabidopsis identified a novel interaction between SAHH and aspartate-semialdehyde dehydrogenase (ASDH), an enzyme that catalyzes the second step of the aspartate-derived amino acid biosynthesis pathway. Analysis of ASDH-GFP fusions revealed that ASDH localizes to the chloroplast and the stromule-like structure that emanates from chloroplasts. Moreover the mutation in three amino acids (Pro164-Asp165-Pro166) located within the IR loop of SAHH disrupted its binding to ASDH which affected the plastid localization of SAHH, suggesting that the interaction between SAHH and ASDH is required for plastid-targeting of SAHH. Taken together, this thesis demonstrated that the localization of ADK and SAHH in or between compartments is possibly mediated by specific protein interactions, and that the surface-exposed IR loop of SAHH is crucial for these interactions.

This thesis documents the subcellular localization and protein-protein interactions of two methyl recycling enzymes. These two enzymes, adenosine kinase (ADK) and S-adenosyl-L-homocysteine hydrolase (SAHH), are essential to sustain the hundreds of S-adenosyl-L-methionine (SAM)-dependent transmethylation reactions in plants. Both ADK and SAHH are involved in the removal of a competitive inhibitor of methyltransferases (MTs), S-adenosyl-L-homocysteine (SAH), that is generated as a by-product of the each transfer of a methyl group from SAM to a substrate. This research focused on understanding how SAH is metabolized in distinct cellular compartments to maintain MT activities required for plant growth and development. Localization studies using green fluorescent protein (GFP) fusions revealed that both ADK and SAHH localize to the cytoplasm and the nucleus, and possibly to the chloroplast, despite the fact that the primary amino acid sequence of neither protein contains detectable targeting signals. This suggested the possibility that these methyl-recycling enzymes may be targeted by specific protein-protein interactions. Moreover, deletion analysis of SAHH1 indicated that the insertion region (IR) of 41 amino acids (Gly150-Lys190), which is present only in plants and parasitic protozoan SAHHs among eukaryotes, is essential for nuclear targeting. This result suggested that the surface-exposed IR loop may serve as a binding domain for interactions with other proteins that may direct SAHH to the nucleus. To investigate protein-protein interactions, several methods were performed including co-immunoprecipitation, bimolecular fluorescence complementation, and pull-down assays. These results not only revealed that ADK and SAHH possibly interact through the IR loop of SAHH in planta, but also suggested that this interaction is either dynamic or indirect, requiring a cofactor/another protein(s) or post-translational modifications. Moreover, possible interactions of both ADK and SAHH with a putative Arabidopsis mRNA cap methyltransferase (CMT), which is localized predominantly in the nucleus, were also confirmed. These results support the hypothesis that the nuclear targeting of both SAHH and ADK can be mediated by the interaction with CMT. In addition, purification of Strep-tagged SAHH1 expressed in Arabidopsis identified a novel interaction between SAHH and aspartate-semialdehyde dehydrogenase (ASDH), an enzyme that catalyzes the second step of the aspartate-derived amino acid biosynthesis pathway. Analysis of ASDH-GFP fusions revealed that ASDH localizes to the chloroplast and the stromule-like structure that emanates from chloroplasts. Moreover the mutation in three amino acids (Pro164-Asp165-Pro166) located within the IR loop of SAHH disrupted its binding to ASDH which affected the plastid localization of SAHH, suggesting that the interaction between SAHH and ASDH is required for plastid-targeting of SAHH. Taken together, this thesis demonstrated that the localization of ADK and SAHH in or between compartments is possibly mediated by specific protein interactions, and that the surface-exposed IR loop of SAHH is crucial for these interactions.