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Abstract
The amino acid sequence of rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi, O. (1985) J. Biol. Chem. 260, 12410-12415) was determined by a combination of cDNA and protein sequencing. cDNA clones specific for this enzyme were isolated from a lambda gt11 rat brain cDNA expression library. Nucleotide sequence analyses of cloned cDNA inserts revealed that this enzyme consisted of a 564- or 549-base pair open reading frame coding for a 188- or 183-amino acid polypeptide with a Mr of 21,232 or 20,749 starting at the first or second ATG. About 60% of the deduced amino acid sequence was confirmed by partial amino acid sequencing of tryptic peptides of the purified enzyme. The recognition sequence for N-glycosylation was seen at two positions of amino acid residues 51-53 (-Asn-Ser-Ser-) and 78-80 (-Asn-Leu-Thr-) counted from the first Met. Both sites were considered to be glycosylated with carbohydrate chains of Mr 3,000, since two smaller proteins with Mr 23,000 and 20,000 were found during deglycosylation of the purified enzyme (Mr 26,000) with N-glycanase. The prostaglandin D synthetase activity was detected in fusion proteins obtained from lysogens with recombinants coding from 34 and 19 nucleotides upstream and 47 and 77 downstream from the first ATG, indicating that the glycosyl chain and about 20 amino acid residues of N terminus were not essential for the enzyme activity. The amino acid composition of the purified enzyme indicated that about 20 residues of hydrophobic amino acids of the N terminus are post-translationally deleted, probably as a signal peptide. These results, together with the immunocytochemical localization of this enzyme to rough-surfaced endoplasmic reticulum and other nuclear membrane of oligodendrocytes (Urade, Y., Fujimoto, N., Kaneko, T., Konishi, A., Mizuno, N., and Hayaishi, O. (1987) J. Biol. Chem. 262, 15132-15136) suggest that this enzyme is a membrane-associated protein.
Highlights
Chem. 260, 12410-12415) was determined by a combination of cDNA and protein sequencing. cDNA clones specific for this enzyme were isolated from a Xgtll rat brain cDNA expression li- Prostaglandin (PG)’D2is a majorPG produced in the brain brary
Nucleotide sequence analyses of cloned cDNA of rats [1]and humans [2] and shows several central actions inserts revealed that this enzyme consisted of a 564- such as inductionof hypothermia [3] and sleep [4,5, 27] and o r 549-base pair open reading framecoding for a 188- modulation of pain response [6]
The reaction was stopped by boiling for 5 min after addition of 10 pl of 10% SDS an2d5% w/v glycerol, and aliquots were withdrawn for SDS-PAGE [16]
Summary
When PAS stainwas performed on the nylon blot, the protein bands with M , 26,000 and 23,000 were positively stained, whereas that band with M , 20,000was brain PGD synthetase (7w) as used to screen a Xgtll rat braincDNA library (Clontech). The filter was incubated in PBS containing 5%skim milk for 1h a t negative (data not shown). All three protein bands were immunoreactive against the antibody for this enzyme (data notshown) These results showed that rat brain PGD synthetase was an N-glycosylated protein withtwo carbohydrate chains with a size responsible for M , 3,000 on SDS-PAGE, and suggested room temperature and incubated at 25 "C for 1h with anti-rat that this enzyme was synthesized and glycosylated in roughbrain PGDsynthetase serum ( X 2000 dilution in PBS containing 0.1% Tween 20 and 1%skim milk). Limiting dilution method.As control phages, Xgtll without any insert Isolation of Rat Brain PGD Synthetase cDNA Clones-By and a phage insert-coded cDNA of ovalbumin (XOv) were isolated
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