A glutaminyl cyclase (QC) that is probably involved in the biosynthesis of pyroglutamyl peptides such as gonadotropin-releasing hormone and thyrotropin-releasing hormone has been purified to homogeneity from bovine anterior pituitary. On the basis of N-terminal sequence analysis, a 2088-base-pair cDNA done was isolated from a bovine anterior pituitary library. From the nucleotide sequence of this clone, the primary structure of a 330-residue protein and a preceding 31-residue prepropeptide sequence was deduced. By transfection of COS-7 monkey cells with a QC cDNA/pCDM8 vector construct, QC activity was expressed. Hybridization with mRNAs of various bovine tissues revealed expression of QC mainly in brain tissue.For the pyroglutamyl peptides thyrotropin-releasing hormone (TRH) and gonadotropin-releasing hormone (GnRH) the N-terminal pyroglutamyl modification is required for biologic activity (1, 2). The details ofthe biosynthesis ofthese hormones have not been elucidated. In the primary structures of the precursors to TRH (3, 4) and GnRH (5), sequences corresponding to glutaminyl analogs of TRH and GnRH (C-terminally deamidated and extended by glycine) are flanked by pairs of basic amino acids. On the basis of these structures it is probable that glutaminyl peptides are intermediates in the propeptide peptide conversion. Although the cyclization of N-terminal glutamine residues can occur under nonenzymatic conditions, especially under the catalytic influence of phosphate ions (6), a previous study has demonstrated (7) that tissue extract loses almost all cyclization activity after short exposure to heat. Therefore (and on the basis of other data) we have concluded that glutaminyl peptides are converted to pyroglutamyl peptides in an enzymatic reaction by glutaminyl cyclase (QC) (7). QCs have been identified in plants (8) and in mammalian tissues (7, 9) such as pituitary, hypothalamus, other parts of the brain, adrenal medulla, and B lymphocytes. Investigation of the biologic function of these enzymes has been hampered by the lack of structural data and specific antibodies, mainly because at least the mammalian enzymes are rather labile and present only in very low concentrations in the tissues tested. We report here the purification to homogeneity and DNA sequence determination* of a mammalian QC as well as confirmation ofthe identity of the cloned sequence by expression in COS-7 cells. On the basis of these findings it will now be possible to investigate in transfection experiments the biologic significance of this enzyme for posttranslational processing, the enzyme's maturation along the secretory pathway, its transcriptional regulation, and its mechanism of action. MATERIALS AND METHODSTissue Extracts. From the local slaughterhouse, 1085 pituitaries were freshly obtained and dissected. The anterior lobes (wet weight, 1979 g) were homogenized in batches qf 50-100 glands with a Polytron homogenizer (Brinkmannj, isotonically extracted, and centrifuged at 600 and 45,000 X g as described (7). The bottom par...

Four DNaseI hypersensitive (HS) chromatin regions were found in the uteroglobin locus located at -3.7, -2.4, -0.1 and +4.1 kb with respect to the transcription start site of the gene. The three sites upstream of the gene are only detected in the hormonally stimulated endometrium and disappear after hormone withdrawal, whereas the site at +4.1 is also found in tissues that do not express uteroglobin. In the -2.4 HS region, which is strictly dependent on progesterone treatment, three DNaseI sites are clustered within a 240 bp DNA segment that contains 20 imperfect repeats of an octanucleotide motif. Upstream of the uteroglobin gene there are three regions containing binding sites for the glucocorticoid and the progesterone receptors, located at -3.7, -2.6/-2.7 and -2.4. The -2.4 region contains two binding sites for the hormone receptors flanking the central HS site. In footprinting experiments with naked DNA binding of the receptor also renders this site more susceptible towards digestion with DNaseI. The -2.6/-2.7 region contains three binding sites for the hormone receptors located 140 bp upstream of the HS -2.4. While the -3.7 HS is also located within a receptor binding fragment, there is no binding of the hormone receptors to the promoter region. Thus, interaction of the receptor with DNA sequences far upstream from the promoter alters the chromatin conformation of neighbouring sequences and results in transcriptional activation.

[3H]Triamcinolone acetonide was used to tag covalently specific glucocorticoid receptors by photoaffinity labelling at lambda greater than or equal to 320 nm. Receptors of wild‐type mouse lymphoma cells and two glucocorticoid resistant mutants of “nuclear transfer deficient” (nt‐) and “increased nuclear transfer” (nti) phenotypes, respectively, were used. Wild‐type and nt‐ receptors yielded radiolabelled polypeptide bands of mol. wt. 98 000 as revealed by gel electrophoresis under denaturing conditions and fluorography. In contrast, the nti receptor had a mol. wt. of 42 000. Partial proteolysis of the wild‐type receptor with alpha‐chymotrypsin resulted in a fragment of mol. wt. 39 000 which still contained the steroid binding site but had increased affinity for DNA indistinguishable from that of the nti receptor. Chymotrypsin thus removed a domain from the wild‐type receptor polypeptide which is involved in modulating DNA binding. The same domain is missing from the nti receptor.

No abstract

No abstract