Purification and properties of calf liver γ-butyrobetaine hydroxylase

Kondo, Atsushi; Blanchard, John S.; Englard, Sasha

doi:10.1016/0003-9861(81)90374-x

Cited by 38 publications

(23 citation statements)

References 33 publications

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“…Because a single protein of 43 kDa was present in the final purification sample and the MALDI-TOF analysis of the blue native PAGE sample demonstrated that the dimer consisted of a single protein, TMLH appears to be homodimer. The last enzyme of carnitine biosynthesis, ␥-butyrobetaine hydroxylase, has considerable homology with TMLH and has also been reported to function as a homodimer (37)(38)(39). Analysis of the non-redundant data base with the BLASTp algorithm using rat TMLH as query, only retrieved ␥-butyrobetaine hydroxylase sequences from several organisms.…”

Section: Discussionmentioning

confidence: 99%

Molecular and Biochemical Characterization of Rat ε-N-Trimethyllysine Hydroxylase, the First Enzyme of Carnitine Biosynthesis

Vaz

Ofman

Westinga

et al. 2001

Journal of Biological Chemistry

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⑀-N-Trimethyllysine hydroxylase (EC 1.14.11.8) is the first enzyme in the biosynthetic pathway of L-carnitine and catalyzes the formation of ␤-hydroxy-N-⑀-trimethyllysine from ⑀-N-trimethyllysine, a reaction dependent on ␣-ketoglutarate, Fe 2؉ , and oxygen. We purified the enzyme from rat kidney and sequenced two internal peptides by quadrupole-time-of-flight mass spectroscopy. The peptide sequences were used to search the Expressed Sequence Tag data base, which led to the identification of a rat cDNA of 1218 base pairs encoding a polypeptide of 405 amino acids with a calculated molecular mass of 47.5 kDa. Using the rat sequence we also identified the homologous cDNAs from human and mouse. Heterologous expression of both the rat and human cDNAs in COS cells confirmed that they encode ⑀-N-trimethyllysine hydroxylase. Subcellular fractionation studies revealed that the rat enzyme is localized exclusively in mitochondria. Expression studies in yeast indicated that the rat enzyme is synthesized as a 47.5-kDa precursor and subsequently processed to a mature protein of 43 kDa, presumably upon import in mitochondria. The Michaelis-Menten constants of the purified rat enzyme for trimethyllysine, ␣-ketoglutarate, and Fe 2؉ were 1.1 mM, 109 M, and 54 M, respectively. Both gel filtration and blue native polyacrylamide gel electrophoresis analysis showed that the native enzyme has a mass of approximately 87 kDa, indicating that in rat ⑀-N-trimethyllysine hydroxylase is a homodimer.

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Section: Discussionmentioning

confidence: 99%

Molecular and Biochemical Characterization of Rat ε-N-Trimethyllysine Hydroxylase, the First Enzyme of Carnitine Biosynthesis

Vaz

Ofman

Westinga

et al. 2001

Journal of Biological Chemistry

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“…In this respect, P4H is typical of the majority of enzymes in this family in being a monomer of M r 30 000-40 000 [6], although there are exceptions, including mammalian prolyl 4-hydroxylase (α # β # ; M r 240 000) [5] and γ-butyrobetaine hydroxylase (α # ; 92 000-96 000) [15]. A preliminary investigation into the dependence of P4H activity on each of the assay components was carried out.…”

Section: Initial Characterization Of P4h and The Influence Of Ascorbamentioning

confidence: 99%

Purification and initial characterization of proline 4-hydroxylase from Streptomyces griseoviridus P8648: a 2-oxoacid, ferrous-dependent dioxygenase involved in etamycin biosynthesis

Lawrence¹,

Sobey²,

Field³

et al. 1996

Biochemical Journal

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Proline 4-hydroxylase is a 2-oxoacid, ferrous-ion-dependent dioxygenase involved in the biosynthesis of the secondary metabolite etamycin. The purification, in low yield, of proline 4-hydroxylase from Streptomyces griseo iridus P8648 to near apparent homogeneity and its initial characterization are reported. In most respects proline 4-hydroxylase is a typical member of the 2-oxoacid-dependent dioxygenase family. It is monomeric (M r approx. 38 000) (by gel filtration on Superdex-G75) and has typically strict requirements for ferrous ion and 2-oxoglutarate. The enzyme was inhibited by aromatic analogues of 2-oxoglutarate. -Proline-uncoupled turnover of 2-

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“…Therefore, many studies reported the enhancement of g-BBD and TMLD activity upon the addition of VC in a dose-dependent manner using tissue extracts or partially purified enzymes. [18][19][20][21][22][26][27][28] In the absence of VC, however, g-BBD activity was detected by adding glutathione peroxidase and glutathione (GSH) to the reaction mixture, 29) although this test was not performed for TMLD.…”

mentioning

confidence: 99%

Vitamin C Is Not Essential for Carnitine Biosynthesis in Vivo: Verification in Vitamin C-Depleted Senescence Marker Protein-30/Gluconolactonase Knockout Mice

Furusawa

Sato

Tanaka

et al. 2008

Biological & Pharmaceutical Bulletin

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Carnitine is an essential cofactor in the transport of long-chain fatty acids into the mitochondrial matrix and plays an important role in energy production via b b-oxidation. Vitamin C (VC) has long been considered a requirement for the activities of two enzymes in the carnitine biosynthetic pathway, i.e., 6-N-trimethyllysine dioxygenase and g g-butyrobetaine dioxygenase. Our present study using senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize VC in vivo, led to the conclusion that this notion is not true. After weaning at 40 d of age, SMP30/GNL KO mice were fed a diet lacking VC and carnitine, then given water containing 1.5 g/l VC (VC(؉) mice) or no VC (VC(؊) mice) for 75 d. Subsequently, total VC and carnitine levels were measured in the cerebrum, cerebellum, liver, kidney, soleus muscle, extensor digitorum longus muscle, heart, plasma and serum. The total VC levels in all tissues and plasma from VC(؊) SMP30/GNL KO mice were negligible, i.e., Ͻ2% of the levels in SMP30/GNL KO VC(؉) mice; however, the total carnitine levels of both groups were similar in all tissues and serum. In addition, carnitine was produced by incubated liver homogenates from the VC-depleted SMP30/GNL KO mice irrespective of the presence or absence of 1 mM VC. Collectively, these results indicate that VC is not essential for carnitine biosynthesis in vivo.

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Purification and properties of calf liver γ-butyrobetaine hydroxylase

Cited by 38 publications

References 33 publications

Molecular and Biochemical Characterization of Rat ε-N-Trimethyllysine Hydroxylase, the First Enzyme of Carnitine Biosynthesis

Molecular and Biochemical Characterization of Rat ε-N-Trimethyllysine Hydroxylase, the First Enzyme of Carnitine Biosynthesis

Purification and initial characterization of proline 4-hydroxylase from Streptomyces griseoviridus P8648: a 2-oxoacid, ferrous-dependent dioxygenase involved in etamycin biosynthesis

Vitamin C Is Not Essential for Carnitine Biosynthesis in Vivo: Verification in Vitamin C-Depleted Senescence Marker Protein-30/Gluconolactonase Knockout Mice

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