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This paper presents the first purification of the mitochondrial branched‐chain amino acid aminotransferase (BCATm) from sheep placenta. It is a homodimer with an apparent subunit molecular mass of 41 kDa. The enzyme differs from those of the rat and human as it appears to form at least one intermolecular disulfide bond. The sheep BCATm cDNA (1.4 kb) encodes a mature polypeptide of 366 amino acids with a calculated molecular mass of 41 329 Da and a partial mitochondrial targeting sequence of seven amino acids. The sheep BCATm sequence shares higher identity with other mammalian BCATm isoenzymes (82–85%) than with the cytosolic isoenzymes (60%). By Northern blot analysis, a message of 1.7 kb was detected in sheep placenta and skeletal muscle. Measurements of BCAT activity, mRNA and BCATm protein in sheep placenta and skeletal muscle revealed that BCATm is the sole BCAT isoenzyme expressed in placenta, whereas it contributes 57 and 71% of the BCAT activity in tensor fascia latae and masseter muscles from weaned lambs respectively. Skeletal muscle, the main site of branched‐chain amino acid transamination, exhibits significantly lower BCAT activity in sheep than in rat. Our results suggest that the low BCATm mRNA level probably accounts for the low BCAT activity in sheep skeletal muscle, and that the metabolic scheme for branched‐chain amino acid catabolism is specific to each species. [ABSTRACT FROM AUTHOR]

Objectives: Kuadro is a new wet-reagent biochemistry analyser that is available for in-house use. The aim of this study was to compare the results produced by this analyser with those obtained by a wet-reagent analyser (KoneLab 30i) that served as the reference instrument. Methods: Blood samples from 80 clinical cases (40 dogs and 40 cats) were analysed for urea, creatinine, total proteins, albumin, glucose, cholesterol, alanine aminotransferase, alkaline phosphatase, total bilirubin, amylase, total calcium, and phosphate using both the reference and Kuadro instruments. Results: Kuadro performed very well on canine and feline samples, showing clinically acceptable correlations (r value) (canine/feline) for urea (0·99/0·97), creatinine (0·99/0·98), total proteins (0·95/0·97), albumin (0·98/0·90), glucose (0·99/0·99), cholesterol (0·99/0·99), alanine aminotransferase (1·00/0·99), alkaline phosphatase (0·96/0·99), total bilirubin (1·00/1·00), amylase (0·98/0·96), and phosphate (0·91/0·92). The correlation for total calcium measurements was clinically unacceptable (0·78/0·83). Clinical Significance: Urea, creatinine, albumin, glucose, cholesterol, alanine aminotransferase, and phosphate Kuadro values can be used interchangeably with those generated by the reference analyser. Alkaline phosphatase, amylase, and total proteins Kuadro values cannot be used interchangeably, as Kuadro overestimated alkaline phosphatase and amylase and underestimated total protein measurements. Kuadro significantly underestimated total calcium concentrations due to the shorter incubation time used by the Kuadro analyser compared with the incubation time used in the reference analyser assay system. [ABSTRACT FROM AUTHOR]

The analogues 5-phospho-pyridoxal-aminooxyacetate and 4-vinyl-pyridoxal 5-phosphate inhibit reduced 4-aminobutyrate aminotransferase reconstituted with pyridoxat 5-phosphate, but they do not affect the catalytic activity of the holoenzyme. The binding of the 5-phosphopyridoxal adducts to the holoenzyme was monitored by measuring the fluorescence properties of the protein and the ligands. The presence of nonequivalent binding sites in reduced samples of enzyme containing 1 mol and 1.8 mol P-pyridoxyl residues/mol dimer was detected by steady and nanosecond fluorescence spectroscopy. The spectroscopic properties (fluorescence lifetime τ = 1 ns, polarization = 0.36 and quantum yield = 0.01) of the first molecule of 5-phosphoapyridoxyl are different from the properties of the second molecule of 5-phospho-pyridoxyl covalently bound to the enzyme (τ = 2.1 ns, P = 0.16, Qτ = 0.1). The spectroscopic properties of pyridoxal 5-phosphate and 5-phospho-pyridoxal-aminooxyacetate bound to different sites on the holoenzyme can be used to deduce proximity relationships between the catalytic sites of 4-aminobutyrate aminotransferase. On the basis of resonance energy transfer measurements, it is proposed that a distance of 27 Å (2.7 nm) separates the two catalytic binding sites. [ABSTRACT FROM AUTHOR]