Sodium-calcium exchanger is initially expressed in a heart-restricted pattern within the early mouse embryo.

Although the sodium-calcium exchanger (NCX1) is encoded by a single gene, it is widely expressed in both fetal and adult tissues and functions in many diverse physiological processes to maintain intracellular calcium homeostasis. In order to determine whether NCX1 is also ubiquitously expressed in the early mouse embryo, in situ hybridization and RT-PCR were used to determine the spacio-temporal expression of NCX1. Our results indicate that NCX1 expression is present within the 7.75-8.0 dpc cardiogenic plate before the first heartbeat, and that NCX1 is initially expressed in a heart-restricted pattern within the early mouse embryo. However, in more developed embryos (11.0 dpc and older) NCX1 is expressed in other tissues.

The catalytic mechanism of kynureninase from Pseudomonas fluorescens: evidence for transient quinonoid and ketimine intermediates from rapid-scanning stopped-flow spectrophotometry.

The reaction of Pseudomonas fluorescens kynureninase with L-kynurenine and L-alanine has been examined using rapid-scanning stopped-flow spectrophotometry. Mixing kynureninase with 0.5 mM L-kynurenine results in formation of a quinonoid intermediate, with lambdamax = 494 nm, within the dead time (ca. 2 ms) of the stopped-flow mixer. This intermediate then decays rapidly, with k = 743 s-1, and this rate constant is reduced to 347 s-1 in [2H]H2O, suggesting that protonation of this intermediate by a solvent exchangeable proton takes place. Rapid quench experiments demonstrate that covalent changes in the cofactor occur, as pyridoxal 5'-phosphate is converted to pyridoxamine 5'-phosphate in about 30 mol % within 5 ms after mixing. Under single turnover conditions in the reaction of kynureninase with l-kynurenine, a transient shoulder absorbing at 335 nm is observed that may be a pyruvate ketimine intermediate. In contrast, the reaction of kynureninase with 0.5 mM l-kynurenine in the presence of 10 mM benzaldehyde results in the formation of a quinonoid intermediate (k = 67.4 s-1) with a very strong absorbance peak at 496 nm. The reaction of L-alanine with kynureninase exhibits the rapid formation (386 s-1 at 0.1 M) of an external aldimine intermediate absorbing at 420 nm, followed by slower formation of a quinonoid intermediate with a peak at 500 nm (k = 2.5 s-1). The 420 nm peak then decays slowly with concomitant formation of a peak at 320 nm corresponding to a pyruvate ketimine. These data demonstrate that quinonoid and ketimine intermediates are catalytically competent in the reaction mechanism of kynureninase, and provide additional support for our proposed mechanism for kynureninase from steady-state kinetic studies [Koushik, S. V., Sundararaju, B., and Phillips, R. S. Biochemistry 1998, 37, 1376-1382].

The catalytic mechanism of kynureninase from Pseudomonas fluorescens: insights from the effects of pH and isotopic substitution on steady-state and pre-steady-state kinetics.

The effects of pH and isotopic substitution of substrate and solvent on the reaction of kynureninase from Pseudomonas fluorescens have been determined. The pH dependence of kcat/Km for L-kynurenine is bell-shaped, with apparent pKa's of 6.25 +/- 0.05 on the acidic limb and 8.9 +/- 0.1 on the basic limb, and with a pH-dependent value of kcat/Km of 2 x 10(5) M-1 s-1. The pH dependence of kcat/Km for 3-hydroxykynurenine is also bell-shaped, with apparent pKa's of 6.49 +/- 0.07 and 8.55 +/- 0.09, and with a pH-dependent value of 2.5 x 10(3) M-1 s-1. The kcat for L-kynurenine decreases at acidic pH values, with an apparent pKa of 6.43 +/- 0.06 and a pH-dependent value of 7 s-1. The solvent kinetic isotope effect on kcat for the reaction of kynurenine in [2H]H2O is 6.56 +/- 0.59, whereas there is no normal kinetic isotope effect on kcat/Km, at pH 8.1. The proton inventory of kcat fits very well to the Gross-Butler equation, with x = 0.825 +/- 0.08, suggesting that only a single proton is transferred in the rate-determining step. In contrast, there is no significant kinetic isotope effect on either kcat or kcat/Km with alpha-[2H]-L-kynurenine as the substrate. There is a "burst" of anthranilate (0.7 mol/mol of enzyme) formed in the pre steady state of the reaction of kynureninase, with a rate constant of 54 s-1 which is not affected by [2H]H2O. The partition ratio of alanine to pyruvate formation is 2.3 x 10(4) in H2O and 6.9 x 10(3) in [2H]H2O. Taken together, these data indicate that the rate-limiting step in the reaction of kynureninase occurs subsequent to the first irreversible step, which is anthranilate release, is general base catalyzed, and involves transfer of only a single proton. On the basis of these observations, we propose that the rate-limiting step in the reaction of kynureninase is C-4' deprotonation of the pyruvate pyridoxamine 5'-phosphate ketimine intermediate.

Cloning, sequence, and expression of kynureninase from Pseudomonas fluorescens.

We have cloned the gene encoding kynureninase from Pseudomonas fluorescens using a restriction site polymerase chain reaction technique (RS-PCR) (G. Sarkar, R. T. Turner, and M. E. Bolander PCR Methods Appl. 2, 318-322, 1993) and expressed the enzyme in Escherichia coli DH5a F'. The kynureninase gene has an open reading frame (ORF) of 1251 base pairs that codes for a protein of 416 amino acids with a calculated molecular weight of 45,906. The protein purified from P. fluorescens has N-terminal threonine and an observed molecular weight of 45,787 by electrospray mass spectrometry, suggesting that the N-terminal methionine is removed by posttranslational processing. The complete gene was obtained by PCR and inserted into pTZ18U. The resultant plasmid was used to transform E. coli DH5alpha F', and these cells overexpressed kynureninase to about 37% of total soluble protein. The isolated recombinant protein has molecular weight and Km values identical to those of the native protein from P. fluorescens. The amino acid sequence exhibits 29% identity with those of rat and human kynureninases and 32% identity with the amino acid sequence translated from a Saccharomyces cerevisiae ORF. Alignment of the four sequences shows a highly conserved region which corresponds to the pyridoxal-5'-phosphate (PLP) binding site of rat kynureninase. Based on this alignment, we predict that Lys227 and Asp212 in P. fluorescens kynureninase are involved in pyridoxal-5'-phosphate binding. P. fluorescens kynureninase also exhibits significant homology to the nifS gene product, cysteine desulfurase, and to eucaryotic serine/pyruvate aminotransferases, suggesting that it is a member of subgroup IV of the aminotransferase family of PLP-dependent enzymes.