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In eukaryotic and prokaryotic cells it is well known that the 2-oxo acids are oxidized via CoA- and NAD^+-dependent and lipoic acid-mediated oxidative decarboxylation reaction by 2-oxo acid dehy drogenase multienzyme complex family, which composed of multiple copies of three component enzymes: TDP (thiamin diphosphate) -depenent α-keto acid dehydrogenase (E1), dihydrolipoamide acyltransferase (E2) and FAD-containing dihydrolipoamide dehydrogenase (E3). This paper focused on the biochemical and molecular biological characterization of porcine and human TDP-dependent pyruvate and 2-oxoglutarate dehydrogenases [E1p (α2β2) and E1o (α2)]. Human E1pα, E1pβ and E1o cDNAs and their genomic DNAs have been cloned and characterized their structural organiza tiOnS. The E1o gene mapped on chromosome 7p13-p14. The E1o promoter of 5'-flanking region lacked both canonical TATA and CAAT boxes, activated only by 2-oxoglutarate and contained two cis-acting elements, which bind to nuclear factor. Human E1p defect has been diagnosed by enzymatical analyses, immunoblotting and sequence analyses of PCR products of all exons of Elpot and β genes. A rapid and microquantitative method for analyses of 2-oxo acids in serum and urine has been estab lished and underwent clinical use successfully.

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It is well established that vitamin D_3 is first metabolized in the liver to 25-hydroxyvitamin D_3 [25- (OH)D_3], then in the kidney to 1α, 25-dihydroxyvitamin D_3[1α,25(OH)_2D_3]before it acts on target tissues of vitamin D. Renal lot-hydroxylation of 25(OH)D_3 is a tightly regulated step in vitamin D metabolism. Recently, we cloned a full-length cDNA for renal mitochondrial 25(OH)D_3-1α-hydroxy lase from a vitamin D-deficient rat kidney cDNA library. The sequence analysis showed that the gene consisted of 2469 bp in length and contained an open reading frame encoding 501 amino acids. The deduced amino acid sequence showed a 53% similarity and 44% identity to the vitamin D_3-25-hy droxylase (CYP27), and it was renamed CYP27B1. Thus, it comprises a new subfamily of the CYP27 family. The expression of CYP27B1 mRNA was greatly increased in the kidney of hypocalcemic rats fed a vitamin D-deficient low Ca diet, suggesting that the expression of CYP27B1 is regulated at a transcriptional level. Administration of either parathyroid hormone (PTH) or cAMP into thyroparathyroidectomized (TPTX) vitamin D-deficient rats greatly increased the renal expression of CYP27B1 mRNA. This suggests that PTH is a major regulator of CYP27B1. However, administra tion of PTH did not induce the expression of CYP27B1 mRNA in the kidney of vitamin D-replete normocalcemic rats, indicating that the mechanism of regulation of CYP27B1 gene could be different between normocalcemic and hypocalcemic conditions. A single injection of calcitonin (CT) greatly increased the expression of CYP27B1 mRNA in the kidney of normocalcemic sham rats and normocalcemic TPTX rats generated by either PTH or CaC12 infusion. CT never stimulated the renal expression of CYP27B1 in hypocalcemic rats. Conversion of serum [3H]1α,25(OH)_2D_3 from[^3H]25- hydroxyvitamin D_3 in vivo was also greatly increased by the injection of CT into sham rats and normocalcemic TPTX rats, but not into hypocalcemic TPTX rats. These results suggest that CT, but not PTH, is a major regulator of the expression of the renal CYP27B1 gene under normocalcemic physiological conditions.

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