Isolation and properties of cytoplasmic alpha-glycerol 3-phosphate dehydrogenase from the pectoral muscle of the fruit bat, Eidolon helvum.
Cytoplasmic alpha-glycerol-3-phosphate dehydrogenase from fruit-bat-breast muscle was purified by ion-exchange and affinity chromatography. The specific activity of the purified enzyme was approximately 120 units/mg of protein. The apparent molecular weight of the native enzyme, as determined by gel filtration on Sephadex G-100 was 59,500 +/- 650 daltons; its subunit size was estimated to be 35,700 +/- 140 by SDS-polyacrylamide gel electrophoresis. The true Michaelis-Menten constants for all substrates at pH 7.5 were 3.9 +/- 0.7 mM, 0.65 +/- 0.05 mM, 0.26 +/- 0.06 mM, and 0.005 +/- 0.0004 mM for L-glycerol-3-phosphate, NAD(+), DHAP, and NADH, respectively. The true Michaelis-Menten constants at pH 10.0 were 2.30 +/- 0.21 mM and 0.20 +/- 0.01 mM for L-glycerol-3-phosphate and NAD(+), respectively. The turnover number, k(cat), of the forward reaction was 1.9 +/- 0.2 x 10(4)s(-1). The treatment of the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) under denaturing conditions indicated that there were a total of eight cysteine residues, while only two of these residues were reactive towards DTNB in the native enzyme. The overall results of the in vitro experiments suggest that alpha-glycerol-3-phosphate dehydrogenase of the fruit bat preferentially catalyses the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate.
Agboola
Femi Kayode
FK
Department of Biochemistry, Obafemi Awolowo University, Ile-Ife, Nigeria. fkagbo@oauife.edu.ng
Thomson
Alan
A
Afolayan
Adeyinka
A
D000255:Adenosine Triphosphate / Q000502:physiology; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002685:Chiroptera; D003593:Cytoplasm / Q000201:enzymology; D004099:Dihydroxyacetone Phosphate / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D050536:Glycerol-3-Phosphate Dehydrogenase (NAD+); D005993:Glycerolphosphate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005994:Glycerophosphates / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D010369:Pectoralis Muscles / Q000201:enzymology*; D010725:Phosphocreatine / Q000502:physiology
D000596:Amino Acids; D004791:Enzyme Inhibitors; D005994:Glycerophosphates; D010725:Phosphocreatine; D004099:Dihydroxyacetone Phosphate; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; C029620:alpha-glycerophosphoric acid; D005993:Glycerolphosphate Dehydrogenase; D050536:Glycerol-3-Phosphate Dehydrogenase (NAD+)