ABSTRACT
The original novel UGT1 complex locus previously shown to encode six different UDP-glucuronosyltransferase (transferase) genes has been extended and demonstrated to specify a total of 13 isoforms. The genes are designated UGT1A1 through UGT1A13p with four pseudo ones. UGT1A2p and UGT1A11p through UGT1A13p have either nucleotide deletions or flawed TATA boxes and are therefore pseudo. In the 5' region of the locus, the 13 unique exons 1 are arranged in a tandem array with each having its own proximal TATA box element and, in turn, are linked to four common exons to allow for the independent transcriptional initiation to generate overlapping primary transcripts. Only the lead exon in the nine viable primary transcripts is predicted to undergo splicing to the four common exons generating mRNAs with identical 3' ends and transferase isozymes with an identical carboxyl terminus. The unique amino terminus specifies acceptor-substrate selection, and the common carboxyl terminus apparently specifies the interaction with the common donor substrate, UDP-glucuronic acid. In the extended region, the viable TATA boxes are either A(A)TgA(AA)T or AT14AT; in the original locus the element for UGT1A1 is A(TA)7A and TAATT/CAA(A) for all of the other genes. UGT1A1 specifies the critically important bilirubin transferase isoform. The relationships of the exons 1 to each other are as follows: UGT1A2p through UGT1A5 comprises a cluster A that is 87-92% identical, and UGT1A7 through UGT1A13p comprises a cluster B that is 67-91% identical. For the two not included in a cluster, UGT1A1 is more identical to cluster A at 60-63%, whereas UGT1A6 is identical by between 48% and 56% to all other unique exons. The locus was expanded from 95 kb to 218 kb. Extensive probing of clones beyond 218 kb with coding nucleotides for a highly conserved amino acid sequence present in all transferases was unable to detect other exons 1. The mRNAs are differentially expressed in hepatic and extrahepatic tissues. This locus is indeed novel, indicating the least usage of exon sequences in specifying different transferase isozymes that have an expansive substrate range.
Subject(s)
Glucuronosyltransferase/genetics , Multigene Family , Base Sequence , Chromosomes, Artificial, Bacterial , Cloning, Molecular , Cosmids , DNA Primers/genetics , Exons , Gene Library , Humans , Isoenzymes/genetics , Liver/enzymology , Molecular Sequence Data , Pseudogenes , RNA Caps/genetics , RNA Splicing , RNA, Messenger/genetics , Sequence Deletion , Sequence Homology, Nucleic Acid , TATA BoxABSTRACT
A soluble nitric oxide synthase (NOS) activity was purified 2800-fold from Leishmania donovani, the causative parasite of visceral leishmaniasis, by two-step affinity and anion-exchange chromatography. The purified enzyme ran as a prominent band of 110 kDa on SDS-PAGE whereas gel filtration experiments estimated the native molecular mass to be 230 +/- 20 kDa indicating that the native enzyme exists as a dimer. The enzyme activity required NADPH and was blocked by EGTA. The enzyme kinetics, cofactor requirements, inhibition studies and Western blot analysis with brain anti-NOS antibody suggest its similarity with mammalian NOS isoform I.
Subject(s)
Leishmania donovani/enzymology , Nitric Oxide Synthase/isolation & purification , Animals , Dimerization , Egtazic Acid/pharmacology , Enzyme Inhibitors/pharmacology , Isoenzymes/chemistry , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Kinetics , Mammals , Molecular Weight , NADP/metabolism , Nitric Oxide Synthase/chemistry , Nitric Oxide Synthase/metabolismABSTRACT
Methylparathion and Benthiocarb inhibition of N2 fixation in the cyanobacterium Nostoc muscorum was reversed by Ca(2+) at 1 mM but not at 0.1 mM. The concentration of intracellular Ca(2+) was relatively high in the presence of these pesticides when 1 mM Ca(2+) was also present, indicating that intracellular Ca(2+) may participate in protecting nitrogenase activity against Methylparathion and Benthiocarb.
