ABSTRACT
The methanol poisoning by oral intake or skin contact occurs occasionally, which may have serious consequences including blindness and/or death. Methanol and its metabolites, formaldehyde and formic acid, are associated with metabolic acidosis, visual dysfunction and neurological symptoms. At present, the mechanism of methanol poisoning primarily focuses on the cell hypoxia, the alteration of structure and biological activity induced by free radical and lactic acid. Meanwhile, methanol poisoning causes changes in the balance between the production of free radicals and antioxidant capacity and in the proteases-protease inhibitors system, which lead to a series of disturbances.
Subject(s)
Animals , Humans , Acidosis/chemically induced , Formaldehyde/poisoning , Formates/poisoning , Free Radicals/metabolism , Methanol/poisoning , Nervous System/pathology , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Proteins/metabolism , Vision Disorders/pathologyABSTRACT
Deoxyhypusine synthase catalyzes the first step in the posttranslational synthesis of an unusual amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF-5A) precursor protein. We earlier observed that yeast recombinant deoxyhypusine synthase was phosphorylated by protein kinase C (PKC) in vitro (Kang and Chung, 1999) and the phosphorylation rate was synergistically increased to a 3.5-fold following treatment with phosphatidylserine (P.Ser)/diacylglycerol (DAG)/ Ca2+, suggesting a possible involvement of PKC. We have extended study on the phosphorylation of deoxyhypusine synthase in vivo in different cell lines in order to define its role on the regulation of eIF5A in the cell. Deoxyhypusine synthase was found to be phosphorylated by endogenous kinases in CHO, NIH3T3, and chicken embryonic cells. The highest degree of phosphorylation was found in CHO cells. Moreover, phosphorylation of deoxyhypusine synthase in intact CHO cells was revealed and the expression of phosphorylated deoxyhypusine synthase was significantly diminished by diacyl ethylene glycol (DAEG), a PKC inhibitor, and enhanced by phorbol 12-myristate 13-acetate (PMA) or Ca2+/DAG. Endogenous PKC in CHO cell and cell lysate was able to phosphorylate deoxyhypusine synthase and this modification is enhanced by PMA or Ca2+ plus DAG. Close association of PKC with deoxyhypusine synthase in the CHO cells was evident in the immune coprecipitation and was PMA-, and Ca2+/phospholipiddependent. These results suggest that phosphorylation of deoxyhypusine synthase was PKC-dependent cellular event and open a path for possible regulation in the interaction with eIF5A precursor for hypusine synthesis.
Subject(s)
Animals , Chick Embryo , Female , Mice , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Cell Line , Cricetinae , Phosphorylation , Protein Binding , Protein Kinase C/antagonists & inhibitorsABSTRACT
The biosynthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl)-lysine] occurs in the eIF-5A precursor protein through two step posttranslational modification involving deoxyhypusine synthase which catalyzes transfer of the butylamine moiety of spermidine to the epsilon-amino group of a designated lysine residue and subsequent hydroxylation of this intermediate. This enzyme is exclusively required for cell viability and growth of yeast (Park, M.H. et al., J. Biol. Chem. 273: 1677-1683, 1998). In an effort to understand structure-function relationship of deoxyhypusine synthase, posttranslational modification(s) of the enzyme by protein kinases were carried out for a possible cellular modulation of this enzyme. And also twelve deletion mutants were constructed, expressed in E. coli system, and enzyme activities were examined. The results showed that deoxyhypusine synthase was phosphorylated by PKC in vitro but not by p56lck and p60c-src. Treatment with PMA specifically increased the relative phosphorylation of the enzyme supporting PKC was involved. Phosphoamino acid analysis of this enzyme revealed that deoxyhypusine synthase is mostly phosphorylated on serine residue and weakly on threonine. Removal of Met1-Glu10 (deltaMet1-Glu10) residues from amino terminal showed no effect on the catalytic activity but further deletion (deltaMet1-Ser20) caused loss of enzyme activity. The enzyme with internal deletion, deltaGln197-Asn212 (residues not present in the human enzyme) was found to be inactive. Removal of 5 residues from carboxyl terminal, deltaLys383-Asn387, retained only slight activity. These results suggested that deoxyhypusine synthase is substrate for PKC dependent phosphorylation and requires most of the polypeptide chains for enzyme activity except the first 15 residues of N-terminal despite of N- and C-terminal residues of the enzyme consist of variable regions. Copyright 2000 Academic Press.
Subject(s)
Humans , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Oxidoreductases Acting on CH-NH Group Donors/genetics , Amino Acid Motifs , Amino Acid Sequence , Escherichia coli/genetics , Fungal Proteins/metabolism , Fungal Proteins/genetics , Molecular Sequence Data , NAD/metabolism , Phosphorylation , Promoter Regions, Genetic , Protein Kinase C/metabolism , Recombinant Proteins/metabolism , Recombinant Proteins/genetics , Sequence Deletion , Sequence Homology, Amino Acid , Threonine/metabolism , Yeasts/enzymologyABSTRACT
The two key enzymes, methylenetetrahydrofolate reductase and methionine synthase involved in methionine synthesis from homocysteine were studied in atherogenic diet fed mice. Methylenetetrahydrofolate reductase activity was elevated while methionine synthase was impaired in atherogenic diet fed group. Impaired methionine synthase activity would adversely affect the methionine synthesis from homocysteine, resulting in a rise in the homocysteine levels, which are atherogenic. This is reflected by the increased levels of very low density and low density lipoprotein cholesterol values and a higher ratio for total cholesterol to high density lipoprotein cholesterol.
Subject(s)
5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase/metabolism , Animals , Diet, Atherogenic , Folic Acid Deficiency/enzymology , Homocysteine/blood , Male , Methylenetetrahydrofolate Reductase (NADPH2) , Mice , Mice, Inbred C57BL , Oxidoreductases Acting on CH-NH Group Donors/metabolismABSTRACT
Monoamine axodation has been studied in different organs of adult rats. Activity of monoamine dehydrogenase (MADH) has been measured both aerobically and anaerobically. Brain is the organ where maximum activity of MADH has been observed while in ovary the minimal enzyme activity has been noticed. In the absence of air, the activity proceeded at faster rate whereas MAO activity cannot take place in absence of oxygen. Oxygen can not be replaced by other electron acceptors like NTC. However, in some organs like pancreas, the formazan production was almost negligible. Aerobically moderate MADH activity was observed in case of ileum and testis. On the other hand, spleen, brain, testis and lung homogenates showed moderate amount of tetrazolium salts reduction in absence of air. This indicates the differential nature of the MADH activities in aerobic and anaerobic condition. High activity of monoamine oxidase (MAO) has been observed in liver. Brain and pancreas were also found good organs for MAO activity, but liver homogenate failed to reduce tetrazolium salt. Only dialysed liver homogenate in the presence of tryptamine, demonstrated moderate activity of MADH. Relative activity of both the enzymes has been studied. The organwise distribution pattern of MAO and MADH appeared quite different.