Dataset Information

Mouse UDP-GlcNAc: dolichyl-phosphate N-acetylglucosaminephosphotransferase. Molecular cloning of the cDNA, generation of anti-peptide antibodies and chromosomal localization.

ABSTRACT: A cDNA encoding UDP-GlcNAc-dolichyl-phosphate N-acetylglucosaminephosphotransferase (GPT; EC 2.7.8.15), an enzyme that catalyses the first step in the synthesis of dolichol-linked oligosaccharides, was isolated from mRNA prepared from mouse mammary glands. The cDNA contains an open reading frame that codes for a protein of 410 amino acids with a predicted molecular mass of 46.472 kDa. Mouse GPT has two copies of a putative dolichol-recognition sequence that has so far been identified in all eukaryotic enzymes which interact with dolichol, and four consensus sites for asparagine-linked glycosylation. It shows a high degree of conservation with yeast and hamster GPTs at the amino acid level. The mouse GPT cDNA recognized a single mRNA species of about 2 kb in mouse mammary glands when used as a probe in Northern blot analysis. An antiserum raised against a 15-residue peptide, derived from the predicted amino acid sequence of the cloned mouse cDNA, specifically precipitated the activity of GPT from solubilized mouse mammary gland microsomes, and detected a protein of about 48 kDa on Western blot. This size is in good agreement with that predicted from the cDNA sequence, and also with that (46 and 50 kDa) of purified bovine GPT. With the use of a panel of mouse/hamster somatic-cell hybrids and a specific probe derived from the 3'-non-coding region of the mouse cDNA, the GPT gene was mapped to mouse chromosome 17.

SUBMITTER: Rajput B

PROVIDER: S-EPMC1132892 | biostudies-other | 1992 Aug

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosamine-1-phosphate transferase (GPT) is the first enzyme in the dolichol pathway of protein N-glycosylation, and is implicated in the developmental programmes of a variety of eukaryotes. In the present study we describe the effects of all-trans-retinoic acid (RA) on the levels of GPT protein and enzymic activity, and on the transcription rate of the GPT gene, in mouse P19 teratocarcinoma cells. RA caused a dose-dependent and protein-synthesis-dependent induction of enzyme activity. The maximum induction of GPT activity (about 3-fold) required 2 days of exposure to 1 microM RA. Induced GPT activity also resulted in an increase in the rate of incorporation of [3H]mannose into Glc3Man9GlcNAc2. Enzymic activities paralleled GPT gene expression. The GPT gene was induced (2-fold) after 7 h of RA treatment. An approx. 3-fold increase in a 48 kDa GPT protein and approx. 4-fold increases in the levels of three GPT transcripts (1.8, 2.0 and 2.2 kb) were observed after 2 days of RA treatment. The enhanced levels of GPT protein and mRNAs began to decline 3 days after the initiation of differentiation, and GPT expression was down-regulated during cellular differentiation. GPT activity decreased about 2. 8-fold to a constant level in differentiated P19 cells. The results indicate that the RA-induced enzyme activity was mainly determined by increased transcription of the GPT gene. RA-treated P19 cells were about 4-fold more resistant to tunicamycin, a fungal antibiotic which inhibits GPT, than were control cells. In addition, GPT activity in membranes from RA-treated P19 cells exhibited approx. 4-fold increased resistance to tunicamycin compared with activity in membranes from untreated control cells, demonstrating that resistance to tunicamycin is correlated with induced GPT activity. Furthermore, increased GPT activity had regulatory significance with regard to the rate of incorporation of [3H]mannose into Glc3Man9GlcNAc2-P-P-dolichol and into glycoproteins. Together, the data provide additional insights into the hormonal regulation of GPT and present evidence that the RA-mediated induction of GPT has a regulatory impact on the dolichol pathway.

Dataset Information

Mouse UDP-GlcNAc: dolichyl-phosphate N-acetylglucosaminephosphotransferase. Molecular cloning of the cDNA, generation of anti-peptide antibodies and chromosomal localization.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets