Micro-Sequence Analysis of Ovine Hypothalamic Corticotropin Releasing Factor

Spiess, Joachim; Rivier, Jean; Rivier, Catherine; Vale, Wylie

doi:10.1007/978-1-4612-5832-2_9

Cited by 10 publications

(5 citation statements)

References 12 publications

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“…Preparation of the peptide for sequencing included reduction and carboxymethylation (39). Introduction into the cup and sequencer programming were as previously reported (36,37). The products from each sequencer cycle were monitored by liquid scintillation counting.…”

Section: Radiosequencingmentioning

confidence: 99%

Cotranslational and posttranslational proteolytic processing of preprosomatostatin-I in intact islet tissue.

Noe¹,

Andrews²,

Dixon³

et al. 1986

The Journal of Cell Biology

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Preprosomatostatin-I (PPSS-I) is processed in anglerfish islets to release a 14-residue somatostatin (SS-14). However, very little is known regarding other processing events that affect PPSS-I. This is the first study to identify and quantify the levels of nonsomatostatin products generated as a result of processing of this somatostatin precursor in living islet tissue. The products of PPSS-I processing in anglerfish islet tissue were identified in radiolabeling studies using a number of criteria. These criteria included immunoreactivity, specific radiolabeling by selected amino acids, radiolabel sequencing, and chromatographic comparison to isolated, structurally characterized fragments of anglerfish PPSS-I using reverse-phase high performance liquid chromatography. Intact prosomatostatin-I (aPSS-I) was isolated from tissue incubated with [3H]tryptophan and [14C]leucine. Significant 14C radioactivity was observed in the products of 11 of the first 44 sequencer cycles in positions consistent with the generation of a 96-residue prosomatostatin. These results indicate that signal cleavage occurs after the cysteine located 25 residues from the initiator Met of PPSS-I, resulting in a signal peptide 25 amino acids in length. Nonsomatostatin-containing fragments of the precursor were also found in tissue incubated with a mixture of 3H-amino acids. Only a small quantity of the dodecapeptide representing residues 69-80 in the prohormone was found (10 nmol/g tissue). Two other fragments of aPSS-I, also observed to be present in low abundance, were found to correspond to residues 1-27 (16 nmol/g tissue) and to residues 1-67 (7 nmol/g tissue) of aPSS-I. No evidence for the presence of the fragment corresponding to residues 29-67 was found. However, large quantities of SS-14 were observed (287 nmol/g tissue), indicating that the major site of aPSS-I cleavage is at the basic dipeptide immediately preceding SS-14. Recovery of much lower levels of the nonsomatostatin fragments of aPSS-I suggests that prohormone processing at the secondary sites identified in this study occurs at a low rate relative to release of SS-14 from aPSS-I.

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Section: Radiosequencingmentioning

confidence: 99%

Cotranslational and posttranslational proteolytic processing of preprosomatostatin-I in intact islet tissue.

Noe¹,

Andrews²,

Dixon³

et al. 1986

The Journal of Cell Biology

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show abstract

mentioning

confidence: 99%

“…Peptides (0.5-2.0 nmol) were degraded in the presence of 3-4 mg of Polybrene in an automatic spinning cup sequencer (27) with a single-coupling singlecleavage program as described (28,29). Phenylthiohydantoin (> PhNCS)-conjugated amino acids were determined with RP-HPLC (coefficient of variation, 1-6%; minimal detectable amount, <10 pmol) (28). The main > PhNCS derivative of 5-hydroxylysine (Calbiochem) subjected to automatic Edman degradation and RP-HPLC was presumably > PhNCS-N8-phenylthiocarbamyl (PhNHCS)-hydroxylysine, which was eluted between > PhNCS-dehydrothreonine and > PhNCS-methionine with an absorption maximum at 262 nm.…”

mentioning

confidence: 99%

Processing of an anglerfish somatostatin precursor to a hydroxylysine-containing somatostatin 28.

Spiess¹,

Noe²

1985

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

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“…This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. (28,29) modified Beckman 890C spinning cup sequencer (27,30,31). Extensively purified Polybrene was added to the cup and precycled prior to peptide application.…”

Section: Methodsmentioning

confidence: 99%

“…Extensively purified Polybrene was added to the cup and precycled prior to peptide application. The phenylthiohydantoin (>PhNCS) derivatives of all frequently occurring amino acids, automatically generated in a converting flask, were identified and quantified by reverse-phase HPLC (31). Usually, small amounts of asparagine and glutamine were desamidated during Edman degradation under these conditions.…”

Section: Methodsmentioning

confidence: 99%

NH2-terminal amino acid sequence and peptide mapping of purified human beta-lipotropin: comparison with previously proposed sequences.

Spiess¹,

Mount²,

Nicholson³

et al. 1982

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

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Micro-Sequence Analysis of Ovine Hypothalamic Corticotropin Releasing Factor

Cited by 10 publications

References 12 publications

Cotranslational and posttranslational proteolytic processing of preprosomatostatin-I in intact islet tissue.

Cotranslational and posttranslational proteolytic processing of preprosomatostatin-I in intact islet tissue.

Processing of an anglerfish somatostatin precursor to a hydroxylysine-containing somatostatin 28.

NH2-terminal amino acid sequence and peptide mapping of purified human beta-lipotropin: comparison with previously proposed sequences.

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