A variant of ornithine aminotransferase from mouse small intestine

Abstract

The ornithine aminotransferase (OAT) activity of mouse was found to be highest in the small intestine. The mitochondrial OAT from mouse small intestine was purified to homogeneity by the procedures including heat treatment, ammonium sulfate fractionation, octyl-S e p h a r o s e chromatography, and Sephadex G-150 gel filtration. Comparing to the amino acid sequence of mouse hepatic OAT, six N-terminal amino acid r e s i d u e s have been deleted in intestinal OAT. However, t h e subsequent sequence was identical with that of hepatic OAT. The molecular weights of both intestinal and hepatic OAT were estimated as 46 kDa by SDSgel electrophoresis and as 92 kDa by gel filtration, indicating that both native OATs are dimeric. Biochemical properties of intestinal OAT, such as molecular weight, pH optimum and K m values for Lornithine and -ketoglutarate, were similar to those of hepatic OAT. However, intestinal OAT was more labile than hepatic OAT to tryptic digestion. Keywords: ornithine aminotransferase; mouse; small intestine IntroductionOrnithine aminotransferase (OAT; L-ornithine:2-oxo-acid aminotransferase; EC 2.6.1.13) is a pyridoxal phosphaterequiring enzyme which catalyzes the reversible transamination of L-ornithine and a-ketoglutarate to glutamate and glutamic-γ-semialdehyde (its cyclized form being Δ 1 -pyrroline-5-carboxylate), and the latter product can be reversibly converted to no undeeline (Peraino and Pitot, 1963). OAT is expressed in nearly all mammalian tissues, including liver, kidney, brain, skeletal muscle, and eyes. OAT plays a role in arginine catabolism, proline biosynthesis, or de novo ornithine biosynthesis, depending on the tissue and the physiological circumstances (Mestichelli et al., 1979; Merril and Pitot,1983). Liver OAT is suggested to be involved in the ornithine synthesis for urea cycle while the kidney OAT participates in ornithine degradation (Herzfeld and Knox, 1968;Volpe et al. , 1969). Matsuzawa et al. (1994) suggested that the intestinal OAT may be involved in the ornithine supply to the liver, with the reversal of OAT reaction. In human, a genetic deficiency of OAT causes gyrate atrophy, an autosomal recessive degenerative disease of the choroid and retina of the eye that leads to blindness ( Valle et al., 1977;Kobayashi et al., 1995).OAT is known as a mitochondrial matrix enzyme and has been purified from various tissue sources, including rat liver, kidney, and brain (Matsuzawa et al., 1968;Sanada et al., 1970;Deshmukh, 1984) as well as human liver (Ohura et al., 1982). The mitochondrial OAT has been shown to be synthesized as a large precursor molecule with N-terminal leader peptide on cytoplasmic ribosome, which is then processed and becomes associated with the mitochondrion (Mueckler and Pitot, 1985). The amino acid sequence of OAT precursor protein was predicted from the nucleotide sequence of cDNA (Mueckler and Pitot, 1985;Inaga et al., 1986;Ramesh et al., 1986;Giometti et al., 1992). Recently it was reported that human kidney OAT had the same nucleotide sequenc...

Overexpression of ornithine aminotransferase: consequences on amino acid homeostasis

“…It should be highlighted that the apparent K m for α-ketoglutarate was more than fivefold lower than that for ornithine, showing that overexpressed hOAT, like mOAT ( 24 ) , has a higher affinity for α-ketoglutarate. Thus, the highly conserved OAT DNA sequence ( 43 ) would lead to highly similar enzymic properties, in agreement with previous literature data ( 44 , 45 ) .

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Proteomic Evaluation of the Natural History of the Acute Radiation Syndrome of the Gastrointestinal Tract in a Non-human Primate Model of Partial-body Irradiation with Minimal Bone Marrow Sparing Includes Dysregulation of the Retinoid Pathway

“…Two other key enzymes that regulate small molecules were also among the dysregulated proteins: OAT and PTGS1. OAT is an ornithine aminotransferase that is involved in the intestinal synthesis of citrulline and arginine (Lim et al 1998). It is highly expressed in the villus epithelium and was downregulated at all timepoints after radiation consistent with the depletion of citrulline in jejunum and plasma that was also observed in this natural history study (Kumar, Wang et al also in this issue).…”

Ornithine Aminotransferase, an Important Glutamate-Metabolizing Enzyme at the Crossroads of Multiple Metabolic Pathways

“…Although it is not possible to tell from the crystal structure whether the hexamer is of biological relevance or merely an effect of crystal packing, there is experimental evidence for the existence of hexamers of rat liver OAT in solution approaching crystallization conditions [61]. Assemblies of dimers or tetramers have been observed in electron microscopic studies on pig kidney OAT [62], and oligomers are detected in high concentrations of mouse liver OAT [63]. The oligomerization state seems to change the kinetic properties of the enzyme [30], and may partly explain the differences observed between tissues, although the kinetic studies performed up till now have never shown any sign of cooperation between the various subunits (the kinetics seems to be of the Michaelis Menton type, see above).…”