Biomedical subjects

D W Hum

Publications and source records attributed to D W Hum.

57 records · Page 4Linked to original sources

PubMed1988

Primary structure of a human trifunctional enzyme. Isolation of a cDNA encoding methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase.

A DNA clone complementary to the messenger RNA encoding the human trifunctional enzyme 5,10-methylenetetrahydrofolate dehydrogenase-5,10-methenyl-tetrahydrofolate cyclohydrolase-10-formyltetrahydrofolate synthetase has been isolated from a lambda gt10 library. In vitro transcription-translation of the 3.1-kilobase cDNA clone yields a protein of 101 kDa, which is of identical size and exhibits the same immunoreactivity as the enzyme purified from human liver. A coding region of 2805 base pairs in the cDNA encodes a protein of 935 amino acids. The initiator methionine is absent from the purified enzyme and the amino-terminal 30 amino acids derived by automated sequence analysis are identical (arginine at position 18 was not identified) with that deduced from the nucleotide sequence. The amino acid sequence of the human enzyme shows extensive homology with that of the yeast enzyme, although the amino-terminal bifunctional dehydrogenase-cyclohydrolase domain is less homologous than the carboxyl-terminal synthetase domain. A region was identified which probably serves as a link between these two major domains of the human enzyme. The synthetase domain contains two regions that are homologous to consensus sequences for an ATP-binding site.

Amino Acid Sequence↗

PubMed1985

In vitro synthesis of the trifunctional protein, methylenetetrahydrofolate dehydrogenase--methenyltetrahydrofolate cyclohydrolase--formyltetrahydrofolate synthetase, in normal and transformed cells.

The trifunctional eucaryotic protein, methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase, catalyzes three consecutive steps in the interconversion of folate derivatives which are required for thymidine and purine synthesis. The protein was synthesized in vitro using a reticulocyte lysate cell-free translation system. The immunoprecipitated translation product programmed by normal rat liver and rat brain mRNA was identical in size to that programmed by mRNA from transformed cells (HeLa, Morris hepatamas 5123D and 3924A). The protein synthesized by normal rat liver slices or Reuber H35 hepatoma cells in culture was similar in size to the translation product, suggesting that there is no significant posttranslational modification in vivo that alters the molecular weight. Differences in the level of synthesis were observed among the tissues examined. Since no consistent pattern emerged, however, it appears that transformation per se does not alter the structure or expression of this polypeptide. Nonetheless, a two- to three-fold increase in synthesis by Morris hepatoma 5123D may be an important biological observation with respect to studying the regulation of this protein that is critical for nucleotide synthesis.

Aminohydrolases↗

PubMedDate not supplied

Characterization of UDP-glucuronosyltransferases active on steroid hormones.

In recent years, the enzymes which are involved in the formation of DHT in steroid target tissues have been well investigated, however, enzymes responsible for the catabolism and elimination of steroids in these tissues, in particular the uridine diphospho-glucuronosyltransferase (UGT) family of enzymes, have received much less attention. We have recently demonstrated that human and monkey are unique in having high plasma levels of C19 steroid glucuronides. These circulating conjugates have been proposed to reflect the peripheral conversion of adrenal and gonadal C19 steroids to potent androgens, especially DHT. In humans, the presence of steroid UGT activities is found in the liver and several extrahepatic tissues including the prostate, mammary gland and ovary. In addition, UGT activities were observed in breast and prostate tumor cell lines such as MCF-7 and LNCaP, respectively. In agreement with the presence of steroid conjugating enzymes in extrahepatic tissues, UGT cDNA clones, which encode steroid conjugating proteins, have been isolated from libraries constructed from human and monkey prostate mRNA. The presence of UGT transcripts and proteins in extrahepatic tissues in both species, as determined by Northern blot, ribonuclease protection, specific RT-PCR, in situ hybridization, Western blot and immunocytochemistry analysis, indicate the relevance of steroid glucuronidation in tissues other than the liver. Knowing that both the human prostate and the human prostate cancer LNCaP cell line express steroid metabolizing proteins, including UGT enzymes, regulation of UGT mRNA and protein levels, as well as promoter activity was studied in these cells. The results demonstrate a differential regulation between the two highly related isoforms UGT2B15 and UGT2B17, where only the expression of UGT2B17 was affected following treatments of LNCaP cells with androgens, growth factors or cytokines. Steroid conjugation by UGT enzymes is potentially involved in hormone inactivation in steroid target tissues, thus modifications in UGT expression levels may influence hormonal responses.

Animals↗