Pre - proparathyroid hormone identified by cell - free translation of messenger RNA from hyperplastic human parathyroid tissue.

Habener, J F; Kemper, Byron; Potts, John T.; Rich, Alexander

doi:10.1172/jci108210

Cited by 40 publications

(17 citation statements)

References 28 publications

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“…The amino-terminal pre peptide of the human precursor has been little studied, although the assignment ofthe methionines was made by radiomicro-sequence analysis of [3S]methioninelabeled human preproPTH prepared in a cell-free protein synthesizing system (22). The amino acid sequence of the aminoterminal pre peptide can be deduced from the nucleotide sequence of the human preproPTH mRNA.…”

Section: Resultsmentioning

confidence: 99%

Nucleotide sequence of cloned cDNAs encoding human preproparathyroid hormone.

Hendy¹,

Kronenberg²,

Potts³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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172

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We have cloned cDNA copies of human preproparathyroid hormone in Escherichia coli after insertion of doublestranded DNA into the Pst I site of plasmid pBR322 using the poly(dG).poly(dC) homopolymer extension technique. Recombinant plasmids coding for preproparathyroid hormone were identified by filter hybridization assay using as a probe 32P-labeled bovine preproparathyroid cDNA. Nucleotide sequence analysis of five recombinant plasmids permitted the assignment of 74 nucleotides of the 5' noncoding region, the entire coding region of 345 nucleotides, and the entire 3' noncoding region of 348 nucleotides of the mRNA. The coding sequence predicts the previously unknown "pre" amino acid sequence and clarifies the hormone's amino acid sequence, which has been disputed. The 5' noncoding region contains an AUG codon followed by a UGA stop codon before the authentic initiator codon. The 3' noncoding region is 120 nucleotides longer than in bovine preproparathyroid mRNA and contains two A-A-U-A-A-A sequences, potential signals for polyadenlylation.Parathyroid hormone (PTH), an 84-amino acid polypeptide, is a major regulator of the level of blood calcium. It is biosynthesized as a 115-amino acid precursor, preproparathyroid hormone (preproPTH). Kronenberg et aL (1) and Kemper et aL (2) have described the cloning and sequence analysis of DNA copies of bovine preproPTH mRNA. In order to study further the regulation ofPTH biosynthesis in man, we have synthesized and amplified, by cloning in Escherichia coli, DNA copies ofhuman preproPTH mRNA. Nucleotide sequence analysis ofthe cloned cDNAs has revealed almost the entire primary structure of the mRNA.MATERIALS AND METHODS Enzymes. Reverse transcriptase was provided byJ. W. Beard (Life Sciences, St. Petersburg, FL). T4 polynucleotide kinase was purchased from Boehringer Mannheim and New England Nuclear. DNA polymerase I (large fragment) and restriction endonucleases were from New England BioLabs. S1 nuclease and lysozyme were from Sigma; terminal transferase and restriction endonucleases were from Bethesda Research Laboratories (Rockville, MD).Bacteria and Nucleic Acids. E. coli xy1776 and pBR322 were obtained as described (1). Human parathyroid mRNA was prepared as follows. Human parathyroid adenomas and hyperplastic tissue obtained at surgery performed to cure hyperparathyroidism were frozen in liquid nitrogen. Polyadenylylated RNA was prepared from 5 g of this tissue by phenoVchloroform/isoamyl alcohol extraction followed by oligo(dT)-cellulose chromatography as described (3).Synthesis of Poly(dC)-Tailed Double-Stranded DNA. DNA complementary in sequence to PTH mRNA was synthesized and made double-stranded as described (1). Poly(dC) homopolymer extensions were added by using terminal transferase as described (4). Similarly, poly(dG) homopolymer extensions were added to Pst I-cut pBR322 DNA.Transfection of E. colixVX776. Poly(dG)-tailed pBR322 (300 ng) and 20 ng of poly(dC)-tailed parathyroid DNA were annealed in 0.1 M NaCl/l0mM Tris-HCl, pH 7.6/0.1mM EDTA for 2 mi...

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Section: Resultsmentioning

confidence: 99%

Nucleotide sequence of cloned cDNAs encoding human preproparathyroid hormone.

Hendy¹,

Kronenberg²,

Potts³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

172

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“…It is also theorized that on binding to the endoplasmic reticulum, the nascent hormone is inserted into the endoplasmic reticulum and the signal-sequence is split from the NH2 terminus of the prohormone by a signal peptidase (25). Furthermore, processing is believed to take place by the action of "trypsin-like" activity, which fragments the prohormone at sites containing basic amino acid residues (1)(2)(3)(4)(5)25). Subsequently, the basic amino acids are removed, and hormones containing a free COOH-terminus are then ready for release.…”

Section: Discussionmentioning

confidence: 99%

Glycine-directed peptide amidation: presence in rat brain of two enzymes that convert p-Glu-His-Pro-Gly-OH into p-Glu-His-Pro-NH2 (thyrotropin-releasing hormone).

Kizer¹,

Busby²,

Cottle³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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To study the possibility of glycine-directed amidation in rat brain, we synthesized the substrate p-GluHis-Pro-Gly-OH. Adult and neonatal rat brain and adult rat pituitary were sonicated, frozen and thawed, and fractionated by gel permeation chromatography, and fractions from each tissue were assayed for enzymatic activity capable of converting this model substrate into thyrotropin-releasing hormone.We report the presence in rat brain and rat pituitary of two enzymes catalyzing conversion of p-Glu-His-Pro-Gly-OH into thyrotropin-releasing hormone. Based on the differing chemical and physical properties of these two enzymes and their differing affinities for a number of p-Glu-His-Pro-aa-OH analogs (in which aa = glycine, f-alanine, -butyric acid, and &-aminovaleric acid), we conclude that there are two distinct enzymatic processes for the terminal amidation of peptides in brain and that COOH-terminal extensions other than glycine are capable of directing COOH-terminal amidation.

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“…An unusual feature found was the presence of methionyl-methionyl at the amino terminus and the presence of 5 methionines within the first 14 amino acids. teins are physiological precursors, as we have suggested, for Pre-ProPTH (Kemper et al, 1974;Habener et al, 1975) inasmuch as the precursor must contain the sequence of the final mature protein species, as well as an additional sequence of amino acids. In this report, we compare the tryptic peptides containing methionine and lysine of Pre-ProPTH with those of PTH and ProPTH, and report the partial sequence analysis of the amino terminus of Pre-ProPTH.…”

mentioning

confidence: 80%

“…This method of post-translational cleavage may also occur with larger precursor viral proteins (Jacobson et al, 1970;Boime and Leder, 1972) and the possible precursors for the myeloma light chain (Milstein et al, 1972) and human placental lactogen (Boime et al, 1975). The conversion of Pre-ProPTH to ProPTH or PTH has not been demonstrated in vitro under conditions that allow the conversion of ProPTH to PTH (Habener et al, 1975). The initial cleavage of the Pre-ProPTH specific sequence is apparently necessary before the Arg-Ala bond, which is normally very sensitive to tryptic cleavage (Cohn et al, 1972), is cleaved, resulting in the conversion of ProPTH to PTH.…”

Section: Discussionmentioning

confidence: 99%

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Pre-proparathyroid hormone: Analysis of radioactive tryptic peptides and amino acid sequence

Kemper¹,

Habener²,

Ernst³

et al. 1976

Biochemistry

121

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The amino acid sequence of bovine pre-proparathyroid hormone has been partially determined by analysis of the polypeptide labeled selectively with radioactive amino acids. Analysis of tryptic peptides containing methionine or lysine indicated that parathyroid hormone, proparathyroid hormone, and pre-proparathyroid hormone had several common peptides. Two lysine-containing peptides present in proparathyroid hormone but not in parathyroid hormone were also present in pre-proparathyroid hormone. In addition, pre-proparathyroid hormone contained several additional lysine- and methionine-containing peptides not present in parathyroid hormone or proparathyroid hormone. Analysis by repetitive Edman degradation of the polypeptide labeled with lysine, methionine, and other amino acids indicated that pre-proparathyroid hormone contained 25 additional amino acids at the amino terminus of proparathyroid hormone; the identities of 17 of the 25 amino acids have been established. An unusual feature found was the presence of methionyl-methionyl at the amino terminus and the presence of 5 methionines within the first 14 amino acids.

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Pre - proparathyroid hormone identified by cell - free translation of messenger RNA from hyperplastic human parathyroid tissue.

Cited by 40 publications

References 28 publications

Nucleotide sequence of cloned cDNAs encoding human preproparathyroid hormone.

Nucleotide sequence of cloned cDNAs encoding human preproparathyroid hormone.

Glycine-directed peptide amidation: presence in rat brain of two enzymes that convert p-Glu-His-Pro-Gly-OH into p-Glu-His-Pro-NH2 (thyrotropin-releasing hormone).

Pre-proparathyroid hormone: Analysis of radioactive tryptic peptides and amino acid sequence

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