Rashid, Hamida Khalid (1998) PURIFICATION AND CHARACTERIZATION OF DIHYDROPTERIDINE REDUCTASE AND TETRAHYDROBIOPTERIN METABOLISM IN MAMMALIAN TISSUES. PhD thesis, University of the Punjab, Lahore.
Dihydropteridine reductase (DHPR) was isolated from rat liver by affinity chromatography using sodium-1, 2-naphthaquinone-4-sulphonate as a ligand. The enzyme was isolated in two forms; monomeric and dimeric-DHPR. The percentage recoveries of the activities of monomeric and dimeric forms of DHPR were 19.9 and 43.8%, respectively. Dimethyl tetrahydropterin (DMPH4) and reduced nicotinamide adenine dinucleotide (NADH) were found to be substrates. The monomeric-DHPR had Km values of 7.8 x 10-5M and 8.3 x 10-5 M for DMPH4and NADH. The dimeric-DHPR had Km values of 1.1 x 10-5 M and 2.8 x 10-5 M of for DMPH4and NADH. The molecular weights of the monomeric and the dimeric enzymes were found to be 26,000 and 52,000 respectively. Ca++ bound to the monomeric DHPR was eluted by passing through a sephadex G-15 column. DHPR activities in both the forms were inhibited by the metal ions AI+++, Sc+++, Ga+++ , Bi+++ , Pb ++ , Mn++ and Cd++. The inhibition effect of these metals was generally more pronounced on the dimeric DHPR. Ca++ and Mg++ activated the monomeric-DHPR. Calcium was bound to the monomeric form of DHPR and converted it into the dimeric form. The order of inhibition of dimeric-DHPR by various organic compounds was adrenochrome> 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) > 6-OH-DOPA > 6-OH-dopamine > triamterene. Tamoxifen did not inhibit DHPR activity. Triamterene was found to be competitive inhibitor for both the monomeric and dimeric-DHPR for DMPH4 while mixed type inhibitor was observed for NADH. Adrenochrome was a mixed inhibitor for NADH and competitive inhibitor for DMPH4 for both the forms of DHPR. 6-OH-DOPA was found to be a mixed inhibitor for both monomeric and dimeric-DHPR with both substrates. 6-OH-dopamine was found to be a mixed inhibitor for both monomeric and dimeric-DHPR with respect to DMPH4 and non-competitive inhibitor for both the monomeric-and dimeric-DHPR for NADH. MPTP showed competitive inhibition with respect to NADH and mixed inhibition respect to DMPH4 for both forms of DHPR. Presence of different levels of dopamine in the assay mixtures of both monomeric and dimeric-DHPR gave sigmoid velocity versus substrate concentration curves for both DMPH4 and NADH as substrates. Tetrahydrobiopterin (8H4) levels in the rat brain were determined using reverse phase high performance liquid chromatography. It was found that the level of dihydrobiopterin (BH2) increased significantly and that of BH4 was reduced by the metal ions- Pb ++, AI+++, Sc+++ and Ga+++ and the neurotoxic compounds 6-hydroxydopa and MPTP. Inhibition of the DHPR activity by these reagents lowered the levels of BH4 in brain cells. Human subjects with normal activity of DHPR may therefore display mild mental retardation and other neurological disorders when exposed to large amount of these toxic metals and neurotoxic compounds. Human subjects with low activity of DHPR may have slightly reduced I.Q. scores.
|Item Type:||Thesis (PhD)|
|Uncontrolled Keywords:||Dihydropteridine reductase, Tetrahydrobiopterin Metabolism, Mammalian tissues, DHPR, Dimethyl tetrahydropterin, Nicotinamide, Adenine dinucleotide (NADH), Nonomeric-DHPR, Retardation, Neurological disorders, Pteridine, Tetrahydrobiopterin, Dihydropteridine,|
|Subjects:||Physical Sciences (f) > Chemistry(f2)|
|Deposited By:||Mr Ghulam Murtaza|
|Deposited On:||16 Jun 2006|
|Last Modified:||04 Oct 2007 20:59|
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