Gamma-butyrobetaine hydroxylase in human kidney.
The activity of gamma-butyrobetaine hydroxylase [4-trimethylaminobutyrate: oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1] was determined in different parts of a human kidney removed at surgery and in five perfused human cadaver kidneys. The activity in the 100,000 g supernatant fraction of a homogenate of whole kidneys was 48 nkat X g-1 protein (range 32-70 nkat X g-1protein). The cortex and outer medulla had four to six times higher activity than the inner medulla. A 60-fold purification from the soluble fraction of kidney homogenates with a 40% recovery was achieved by ammonium sulphate fractionation followed by DEAE-cellulose and hydroxylapatite chromatography. The enzyme had a specific activity of 2.4 mukat X g-1 protein but was contaminated to a minor degree by other proteins as judged by polyacrylamide gel electrophoresis. The Km values for gamma-butyrobetaine, 2-oxoglutarate and oxygen were 0.2 mmol/l, 0.3 mmol/l and 5.5% (by volume in the gas phase). There was an absolute requirement for ferrous ion. Half-maximal activity was reached with 10 mumol/l of Fe2+ in phosphate buffer (14 mmol/l) at pH 6.5. With a reaction time of 30 min ascorbate and catalase stimulated the reaction seven- and fivefold, respectively. Optimal pH value for the reaction was 6.2-6.5 in phosphate buffer. Decarboxylation of 2-oxoglutarate in the presence of 4-trimethylaminocrotonate or 4-dimethylaminobutyrate was 40 and 20%, respectively, of that with gamma-butyrobetaine as substrate. None of several compounds chemically related to 2-oxoglutarate, including oxaloacetate, stimulated gamma-butyrobetaine hydroxylation when tested in the absence of 2-oxoglutarate. We conclude that the requirements of the human kidney enzyme are similar to those previously reported for this enzyme from other sources.
D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D003653:Decarboxylation; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D050563:gamma-Butyrobetaine Dioxygenase
D006899:Mixed Function Oxygenases; D050563:gamma-Butyrobetaine Dioxygenase