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  • in vitro glycation of human serum albumin by Dihydroxyacetone and Dihydroxyacetone Phosphate
    Biochemical and Biophysical Research Communications, 2012
    Co-Authors: Champika Seneviratne, George W Dombi, Joel A Dain
    Abstract:

    Amino groups in proteins can non-enzymatically react with reducing sugars to generate a structurally diverse group of compounds referred to as advanced glycation end products (AGEs). The in vivo formation of AGEs contributes to some of the complications of diabetes including atherosclerosis, cataract formation, and renal failure. The formation of AGEs is dependent on both sugar and protein concentrations. Increases in temperature, pH, and exposure time of sugars to the proteins also play a significant role in the rate of AGE formation. This study focuses on the use of a combination of analytical techniques to study the in vitro AGE formation of HSA with Dihydroxyacetone Phosphate (DHAP), a ketose generated during glycolysis, and its dephosphorylated analog, dihydroxy acetone (DHA), commonly used as a browning reagent in skin tanning preparations. The extent of AGE formation was affected by DHAP and DHA concentrations and by the duration of HSA exposure to these glycating agents. Increases in temperature and pH sped the glycation process and enhanced the formation of the AGEs of HSA. MALDI-TOF mass spectroscopic data provided a reliable result to evaluate the extent of the AGE formation.

  • in vitro nonenzymatic glycation of guanosine 5 triPhosphate by Dihydroxyacetone Phosphate
    Analytical and Bioanalytical Chemistry, 2008
    Co-Authors: Yuyuan Li, Menashi A Cohenford, Udayan Dutta, Joel A Dain
    Abstract:

    Dihydroxyacetone Phosphate (DHAP) is a glycolytic intermediate that has been found to be significantly elevated in the erythrocytes of diabetic patients and patients with triosePhosphate isomerase deficiency. DHAP spontaneously breaks down to methylglyoxal, a potent glycating agent that reacts with proteins and nucleic acids in vivo to form advanced glycation endproducts (AGEs). Like methylglyoxal, DHAP itself is also a glycating metabolite, capable of condensing with proteins and altering their structure or function. The objective of this investigation was to evaluate the susceptibility of nucleotides to nonenzymatic attack by DHAP, and to determine the factors influencing the rate and extent of nucleotide glycation by this sugar. Of the four nucleotide triPhosphates (ATP, CTP, GTP and UTP) that were studied, only GTP was reactive, forming a wide range of UV and fluorescent products with DHAP. Increases in temperature and nucleotide concentration enhanced the rate and extent of GTP glycation by DHAP and promoted the heterogeneity of AGEs. Capillary electrophoresis, HPLC, and mass spectrometry allowed for a thorough analysis of the glycated products and demonstrated that the reaction of DHAP with GTP occurred via the classical Amadori pathway.

  • In vitro nonenzymatic glycation of guanosine 5′-triPhosphate by Dihydroxyacetone Phosphate
    Analytical and Bioanalytical Chemistry, 2008
    Co-Authors: Yuyuan Li, Menashi A Cohenford, Udayan Dutta, Joel A Dain
    Abstract:

    Dihydroxyacetone Phosphate (DHAP) is a glycolytic intermediate that has been found to be significantly elevated in the erythrocytes of diabetic patients and patients with triosePhosphate isomerase deficiency. DHAP spontaneously breaks down to methylglyoxal, a potent glycating agent that reacts with proteins and nucleic acids in vivo to form advanced glycation endproducts (AGEs). Like methylglyoxal, DHAP itself is also a glycating metabolite, capable of condensing with proteins and altering their structure or function. The objective of this investigation was to evaluate the susceptibility of nucleotides to nonenzymatic attack by DHAP, and to determine the factors influencing the rate and extent of nucleotide glycation by this sugar. Of the four nucleotide triPhosphates (ATP, CTP, GTP and UTP) that were studied, only GTP was reactive, forming a wide range of UV and fluorescent products with DHAP. Increases in temperature and nucleotide concentration enhanced the rate and extent of GTP glycation by DHAP and promoted the heterogeneity of AGEs. Capillary electrophoresis, HPLC, and mass spectrometry allowed for a thorough analysis of the glycated products and demonstrated that the reaction of DHAP with GTP occurred via the classical Amadori pathway.

Patrick Page - One of the best experts on this subject based on the ideXlab platform.

Casimir Blonski - One of the best experts on this subject based on the ideXlab platform.

Amiya K. Hajra - One of the best experts on this subject based on the ideXlab platform.

  • Dihydroxyacetone Phosphate acyltransferase
    Biochimica et Biophysica Acta, 1997
    Co-Authors: Amiya K. Hajra
    Abstract:

    Abstract In this article the properties, assay, distribution, subcellular localization, deficiency in congenital peroxisomal disorders, purification and physiological functions of Dihydroxyacetone Phosphate acyltransferase (EC 2.3.1.42) are reviewed.

  • purification of Dihydroxyacetone Phosphate acyltransferase from guinea pig liver peroxisomes
    Archives of Biochemistry and Biophysics, 1993
    Co-Authors: Keith O Webber, Amiya K. Hajra
    Abstract:

    Abstract Dihydroxyacetone Phosphate acyltransferase (EC 2.3.1.42), a peroxisomal enzyme which initiates the biosynthesis of glycerolipids (especially the ether-linked glycerolipids) in higher eukaryotes, has been purified by over 3250-fold from guinea pig liver. Initial stages of purification entailed isolation of liver peroxisomes by a combination of differential and density-gradient centrifugation. Dihydroxyacetone Phosphate acyltransferase was solubilized from peroxisomal membranes with 3-[3-cholamidopropyl)dimethylammonio]-1-propane sulfonate at moderate ionic strength (0.15 M NaCl). The solubilized enzyme was further purified by a regimen of size-exclusion chromatography, cation-exchange chromatography, and hydroxylapatite chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of different fractions during the purification of the enzyme, a 69-kDa protein band copurified with the enzyme activity, indicating that the monomeric enzyme may have a M r of 69,000. This was verified by further purifying the enzyme by chromatofocusing, when a single 69-kDa band was observed on SDS-PAGE. The M r of Dihydroxyacetone Phosphate acyltransferase determined by gel filtration is 90 kDa. The V max of the purified enzyme was 4 pmol acylDihydroxyacetone Phosphate (acylDHAP) formed per minute per milligram protein and the K m (DHAP) is 70 μM when assayed at saturating concentrations of palmitoylCoA. Free coenzyme A inhibits the acyltransferase reaction with an inhibition constant ( K i ) of approximately 0.76 mM. To date, this is the most highly purified DHAP acyltransferase(>3200-fold) of mammalian origin.

  • Dihydroxyacetone Phosphate acyltransferase
    Methods in Enzymology, 1992
    Co-Authors: Keith O Webber, Amiya K. Hajra
    Abstract:

    Publisher Summary Dihydroxyacetone Phosphate acyltransferase (DHAPAT) catalyzes the transfer of the fatty acid moiety from long-chain acyl-coenzyme A (acyl-CoA) to the free hydroxyl group of Dihydroxyacetone Phosphate. This reaction initiates the synthesis of the ether-linked glycerolipids and the more common glycerol ester lipids. DHAPAT is an integral membrane-bound protein located on the luminal side of animal cell peroxisomes. Its enzyme activity is measured as the amount of lipophilic (at low pH) radioactivity formed from [ 32 P]DHAP in the presence of palmitoyl-CoA and enzyme. The purification of DHAPAT can be divided into three processes: (1) isolation of peroxisomes, (2) solubilization of the peroxisomal membranes, and (3) chromatographic purification of DHAPAT. The solubilized DHAPAT is purified to near homogeneity by a multistep regimen of both low-pressure and high-pressure column chromatography. A number of genetic diseases involving peroxisomal disorders are prenatally or postnatally diagnosed by the decreased activity of DHAPAT in aminocytes, chorionic villi, leukocytes, or in cultured skin fibroblasts obtained from the patients.