Gamma-butyrobetaine Hydroxylase Research Articles

Tissue distribution of carnitine biosynthetic enzymes in man.

The distribution in human tissues of enzymes which convert epsilon-N-trimethyl-L-lysine to L-carnitine was studied. Existing methodology was modified and new procedures were developed to measure enzyme activities. Epsilon-N-Trimethyl-L-lysine was converted to gamma-butyrobetaine in three enzymatic steps (hydroxylation at carbon 3, aldol cleavage between carbons 2 and 3 to yield glycine and gamma-trimethylaminobutyraldehyde, and subsequent oxidation of the aldehyde) in all tissues studied (liver, brain, kidney, heart and skeletal muscle), but gamma-butyrobetaine was hydroxylated to form L-carnitine only in liver, kidney and brain. Gamma-Butyrobetaine hydroxylase (4-trimethylaminobutyrate, 2-oxoglutarate: oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1) activity in liver was dependent on the age of the subject. The activity rose from 12% in infants to 100% of the adult mean by age 15 years. No age dependence could be demonstrated for the other three enzymes studied.

A simplified method for the measurement of gamma-butyrobetaine hydroxylase activity.

A method for the assay of gamma-butyrobetaine hydroxylase activity is described. The procedure is based on the measurement of 3H2O formed from [2,3-3H]gamma-butyrobetaine. The formation of 3H2O was essentially linear with time of incubation and enzyme concentration. Despite a significant isotope effect that causes the extent of hydroxylation to be underestimated, an appropriately determined correction factor permits one to relate quantitatively the degree of detritiation to the amount of carnitine formed. The assay is simple, rapid, specific, accurate, highly reproducible, and relatively sensitive. Its reliability and convenience represent an improvement over existing methods based on the tedious and time-consuming enzymatic radioisotopic determination of the carnitine formed or on the coupled decarboxylation of [1-14C]alpha-ketoglutarate, a method that cannot be used in crude extracts.

Purification and properties of gamma-butyrobetaine hydroxylase from Pseudomonas sp AK 1.

gamma-Butyrobetaine hydroxylase (4-trimethylaminobutyrate, 2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1) has been isolated from Pseudomonas sp AK 1 by ion-exchange, adsorption, and molecular-sieving chromatography. The preparation was homogeneous as judged from electrophoresis in agarose and polyacrylamide gels, isoelectric focusing, and equilibrium sedimentation. The molecular mass was 95 kdaltons as determined by sedimentation equilibrium centrifugation. From electrophoresis in polyacrylamide gel the molecular mass was estimated to 92 kdaltons, from gel filtration through columns of Sephadex G-200 to 86 kdaltons, and from gel filtration through thin layers of Sephadex G-150 and G-200 to 82 kdaltons. Calculation of molecular mass from Stokes radius, sedimentation coefficient, and partial specific volume gave a value of 96 kdaltons, and from the sedimentation coefficient, 93 kdaltons. Gel filtration through Sephadex G-200 in 6 M guanidinium chloride and electrophoresis in polyacrylamide gel containing 3.5 mM sodium dodecyl sulfate resulted in single bands with mobilities corresponding to molecular masses of 39 and 37 kdaltons, respectively, indicating that the enzyme is composed of two polypeptides chains with similar size. NH2-terminal amino acid sequencing in three cycles resulted in two amino acids in each cycle (Ala + Asn, Ala + Ile, Ala + Ile). The Stokes radius was 3.8 nm, corresponding to a diffusion coefficient of 5.7 X 10(-7) cm2/s. A sedimentation coefficient of 5.8 X 10(-13) s and a frictional ratio of 1.26 was found. The partial specific volume was 0.729 mL/g at 20 degrees C as calculated from amino acid analysis. The isoelectric point was 5.1, as determined by isoelectric focusing analysis. The light absorption in the ultraviolet and visible regions was that of a protein without light-absorbing prosthetic groups. The absorption coefficient at 280 nm of a 1.0% solution at pH 6.5 was 12.6. Amino acid analysis by ion-exchange chromatography showed a half-cystine content of 19 mol per 95 kg of protein (23 residues/1000). Thirteen sulfhydryl groups were found by colorimetric analysis before as well as after reduction with NaBH4, indicating absence of disulfide bonds. Less than 0.1 mol of iron was found per mol of enzyme.