γ-Butyrobetaine hydroxylase in human kidney

Abstract

The activity of γ-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·g^-1 protein (range 32–70 nkat·g^-1 protein). 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 μkat·g^-1 protein but was contaminated to a minor degree by other proteins as judged by polyacrylamide gel electrophoresis. The K_m values for γ-butyrobetaine, 2-oxoglutarate and oxygen were 0.2 mmol/1, 0.3 mmol/1 and 5.5% (by volume in the gas phase). There was an absolute requirement for ferrous ion. Half-maximal activity was reached with 10 μmol/1 of Fe²⁺ in phosphate buffer (14 mmol/1) 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 γ-butyrobetaine as substrate. None of several compounds chemically related to 2-oxoglutarate, including oxaloacetate, stimulated γ-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.

Key-words:

• γ-butyrobetaine hydroxylase
• carnitine
• human kidney
• hydroxylases
• oxygenases