L-glycerol-3-phosphate dehydrogenase from the insect Ceratitis capitata. Purfication, physicochemical and enzymic properties.
Soluble L-glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate: NAD+ 2-oxidoreductase, EC 1.1.1.8) from the mediterranean fruit fly Ceratitis capitata has been purified 130-fold with an overall yield of about 40%. The final preparation had a specific activity of about 200 mumol NADH/min/mg protein. The enzyme preparation has been shown to be homogeneous throughout disc gel electrophoresis, dodecyl sulphate gel electrophoresis, isoelectric focusing and ultracentrifugation. The Km values for dihydroxyacetone phosphate, NADH, L-glycerol-3-phosphate and NAD+ were respectively 0.33, 0.018, 0.74 and 0.26 mM. L-glycerol-3-phosphate dehydrogenase from the insect had a maximal activity around pH 6.6 for the oxidation of NADH and pH 10.0 for the reduction of NAD+. It was stable from pH 6.0 to pH 9.0 at 20 degrees C for 1 h and remained active after incubating at 30 degrees C for 30 min at pH 6.6. The enzyme was completely inactivated by incubating at 60 degrees C for 5 min. Enzyme stability versus ionic strength as well as the dependence of the reaction velocity on temperature are also reported. The active enzyme was found to have a minimum molecular weight of approx. 63 000. Molecular weight determinations by sodium dodecyl sulphate gel electrophoresis gave subunit weights of 33 500. The isoelectric point of the protein was determined by electrofocusing and found to be 5.75 +/- 0.05. The extinction coefficient at 278 nm was calculated by dry weight measurements to be E1cm 1mg/ml = 0.42 +/- 0.1. Sedimentation velocity studies on ultracentrifuge indicated a dependence of the sedimentation coefficient on the enzyme concentration. The amino acid composition of the enzyme was determined. The protein has no free N-terminal residue and the digestion with carboxypeptidases gave the C-terminal sequence: -ala-gly-ser. All these data are discussed in relation to the properties of the enzyme from other sources.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004175:Diptera / Q000201:enzymology*; D005993:Glycerolphosphate Dehydrogenase / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009994:Osmolar Concentration; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D000596:Amino Acids; D009243:NAD; D005993:Glycerolphosphate Dehydrogenase