ADRI is a transcription factor required for activation of the glucose-repressible alcohol dehydrogenase 2 (ADH2) gene in Saccharomyces cerevisiae. ADRI has two zinc finger domains between amino acids 102 and 159, and it binds to an upstream activation sequence (UASI) in the ADH2 promoter. A functional dissection of ADR1 was performed by using a series of amino-and carboxy-terminal deletion mutants of ADR1, most of which were fused to the Escherichia coli I(-galactosidase. These deletion mutants were assayed for binding to UAS1 in vitro, for the ability to activate ADH2 transcription in vivo, and for level of expression. Deletion of ADR1 amino acids 150 to 172 and 76 to 98 eliminated DNA binding in vitro, which accounted for the loss of transcriptional activation in vivo. Results with the former deletion mutant indicated that both of the ADR1 zinc fingers are necessary for sequence-specific DNA binding. Results with the latter deletion mutant suggested that at least part of the sequence between amino acids 76 to 98, in addition to the two finger domains, is required for high-affinity DNA binding. The smallest fusion protein able to activate ADH2 transcription, containing ADRI amino acids 76 to 172, was much less active in vivo than was the longest fusion protein containing amino acids I to 642 of ADR1. In addition, multiple regions of the ADRI polypeptide (including amino acids 40 to 76, 260 to 302, and 302 to 505), which are required for full activation of ADH2, were identified. An ADRI-l-galactosidase fusion protein containing only the amino-terminal 16 amino acids of ADRI was present at a much higher level than were larger fusion proteins, which suggested that the sequences within ADRI influence the expression of the gene fusion.Eucaryotic transcription factors are important in cell growth and differentiation. The few transcription factors that have been analyzed in detail appear to consist of discrete functional units, including a nuclear targeting signal, a DNAbinding domain, a transcription activation region, and a regulatory domain (14,30,34,36). The DNA-binding domain can often be identified by similarity to known DNA-binding motifs (3,22). The transcriptional activation domain in GCN4 and one in GAL4 appear to be rich in acidic amino acids (13,17,23,24), but whether this is a general feature of activation regions remains to be determined.ADR1 is a transcription factor that activates expression of the glucose-repressed alcohol dehydrogenase 2 (ADH2) gene in Sacchar-onivc es (ce) evisiae. Posttranslational modification of ADR1 (5), perhaps by phosphorylation (11), in response to the presence or absence of glucose apparently controls the activity of this protein as a transcription factor. ADR1 consists of 1,323 amino acids and contains two zinc finger domains between amino acids 102 and 159 (15, 29). Genetic evidence indicates that both zinc finger domains are essential for ADR1 activity (4). An ADR1-3-galactosidase fusion protein containing amino acids 17 to 229 of ADRI binds to an upstream activator seque...

Rat brain enkephalin aminopeptidase was purified to apparent electrophoretic homogeneity. Enzyme activity was monitored during the purification by using ([3,5-3H2]Tyr)-Met-enkephalin and Tyr-beta-naphthylamide as substrates. It was shown that the enzyme activities resulting in hydrolysis of the tyrosine residue of ([3,5-3H2]Tyr)Met-enkephalin and formation of beta-naphthylamine from Tyr-beta-naphthylamide copurified. The homogeneous enzyme had a specific activity of 10.5 mumol of beta-naphthylamide hydrolyzed min-1 mg-1. Hydrolysis of Met-enkephalin yielded the products L-tyrosine and the tetrapeptide Gly-Gly-Phe-Met. Subsequent removal of glycine from Gly-Gly-Phe-Met was not observed with the purified enzyme. The homogeneous aminopeptidase has an apparent molecular weight of 115000 on Sephadex G-200 and a molecular weight of 102000 as determined by electrophoresis in the presence of sodium dodecyl sulfate. The enkephalin-degrading enzyme had a pH optimum of 6.5-7.0 and exhibited maximal activity at 40 degrees C. Enzyme activity was inhibited by metal chelators, and it was found that 1 mol of Zn2+ was associated with 1 mol of enzyme (102000 Mr). The enzyme hydrolyzes various neutral and basic amino acid beta-naphthylamides but will not utilize acidic, D-amino acid, or N-terminal-blocked amino acid beta-naphthylamides as substrates.

We report the nucleotide sequence of a precursor to somatostatin that upon proteolytic processing may give rise to a hormone of 22 amino acids. The nucleotide sequence of a cDNA from the channel catfish (Ictalurus punctatus) encodes a precursor to somatostatin that is 105 amino acids (Mr,11,500). The cDNA coding for somatostatin-22 consists of 36 nucleotides in the 5' untranslated region, 315 nucleotides that code for the precursor to somatostatin-22, 269 nucleotides at the 3' untranslated region, and a variable length ofpoly(A). The putative preprohormone contains a sequence ofhydrophobic amino acids at the amino terminus that has the properties of a "signal" peptide. A connecting sequence ofapproximately 57 amino acids is followed by a single ArgArg sequence, which immediately precedes the hormone. Somatostatin-22 is homologous to somatostatin-14 in 7 of the 14 amino acids, including the Phe-Trp-Lys sequence. Hybridization selection ofmRNA, followed by its translation in a wheat germ cell-free system, resulted in the synthesis of a single polypeptide having a molecular weight of approximately 10,000 as estimated on NaDodSO4/polyacrylamide gels.Somatostatin has been isolated from ovine hypothalamus (1) as a tetradecapeptide (somatostatin-14). The peptide inhibits the secretion of growth hormone (1), insulin, and glucagon (2). Large forms of somatostatin or somatostatin-like immunoreactive material have been demonstrated in extracts ofrat pancreas and stomach (3), ovine (4) and porcine (5, 6) hypothalami, and rat (7) and mouse (8) brain. A somatostatin of28 amino acid residues has been isolated from porcine intestine (9) and porcine (10) and ovine (11) hypothalami, and its sequence has been determined (9-11). Biosynthetic pulse-chase experiments and cell-free translations have shown that there are at least two fish preprosomatostatins, which have molecular weights estimated to be 10,000-15,000 (refs. 12-15 and references cited in ref. 12).Nucleotide sequences coding for precursors to somatostatin-14 from the anglerfish (16, 17) and the channel catfish (18) have been reported. In addition to somatostatin-14, the channel catfish harbors a peptide of 22 amino acids that differs in amino acid sequence from somatostatin-14 (19).During the construction and screening of a cDNA library prepared from channel catfish (Ictalurus punctatus) pancreatic mRNA, a unique recombinant plasmid having a restriction endonuclease cleavage pattern different from that of the cDNA encoding the precursor to somatostatin-14 was discovered. We report here the nucleotide sequence ofthis cDNA, which codes for a precursor to somatostatin-22. (24). The sequences of both strands of a single restriction fragment were determined. The strands of more than 95% of all restriction fragments were successfully separated. DNA sequence analysis was performed as described by Maxam and Gilbert (25). MATERIALS ANDRNA Hybridization. Five micrograms of poly(A)+ RNA isolated from the catfish Brockmann bodies was subjected to electrophoresis on a 1.5...

Thyrotrophin-releasing hormone (TRH)-immunoreactive peptides were extracted from rat prostate and divided into two groups by mini-column cation exchange chromatography. The amounts of the peptides in each group were determined by radioimmunoassay with a TRH antiserum. The unretained peptides which lacked a basic group and the retained peptides which possessed a basic group were further purified by high-performance liquid chromatography. The unretained fraction was found to contain a series of TRH-immunoreactive peptides, one of which corresponded chromatographically to synthetic pGlu-Glu-Pro amide and another to pGlu-Phe-Pro amide. None of the TRH-immunoreactive peptides in either fraction exhibited the chromatographic behaviour of TRH. Additional evidence for the absence of TRH gene expression in the prostate was obtained by Northern blot analysis and by application of polymerase chain reaction amplification, which failed to reveal TRH mRNA. Furthermore the preproTRH-derived peptide, preproTRH(53-74), could not be detected by radioimmunoassay. The influence of thyroid status was investigated on the levels of the TRH-like peptides in the prostate. Adult rats were treated chronically with thyroxine (T4) or propylthiouracil (PTU) and the concentrations of the TRH-immunoreactive peptides were determined by chromatography and radioimmunoassay. Treatment with T4 caused the levels of the neutral and acidic TRH-like peptides to fall to approximately one-third of the levels in the controls. No significant difference from the controls was seen in the concentrations of the peptides in the prostates of rats rendered hypothyroid by administration of PTU. The results demonstrate that rat prostate contains TRH-immunoreactive peptides which are not derived from the TRH gene.(ABSTRACT TRUNCATED AT 250 WORDS)

The hypothalamic peptide hormone TRH is also found in other tissues, including the thyroid. While TRH may be regulated by T3 in the hypothalamus, other regulators of TRH have not been identified and the regulation of TRH in nonhypothalamic tissues is unknown. We recently demonstrated the biosynthesis of TRH in the CA77 neoplastic thyroidal C cell line. We studied the regulation of TRH by dexamethasone in this cell line because glucocorticoids have been postulated to inhibit TSH secretion by decreasing TRH in the hypothalamus. Furthermore, TRH in the thyroid inhibits thyroid hormone release. Thus by regulating thyroidal TRH, glucocorticoids could also directly affect thyroid hormone secretion. Treatment of CA77 cells for 4 days with dexamethasone produced dose-dependent increases in both TRH mRNA and cellular and secreted TRH. Increases in TRH mRNA and peptide levels could be seen with 10(-9) M dexamethasone. A 4.8-fold increase in TRH mRNA and a 4-fold increase in secreted peptide were seen with 10(-7) M dexamethasone. Dexamethasone treatment did not increase beta-actin mRNA levels or cell growth. These results suggest that glucocorticoids may be physiological regulators of TRH in normal C cells. In addition to their inhibitory effects on TSH, glucocorticoids may decrease thyroid hormone levels by increasing thyroidal TRH. Since the glucocorticoid effects on C cell TRH are the converse of what is expected for hypothalamic TRH, glucocorticoid effects in these two tissues may be mediated by different regulators.