Further Investigations on Intestinal Hormonal Polypeptides

Mutt, Viktor

doi:10.1111/j.1365-2265.1976.tb03825.x

Cited by 117 publications

(27 citation statements)

References 46 publications

(29 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This second peptide precipitate (:1 kg) was subjected to gel filtration on a column of Sephadex G-25 in 0.2 M HOAc. PHI was found in the fractions that also contained other intestinal peptides, secretin, VIP, and cholecystokinin (16,17). These fractions were pooled and the peptides were precipitated by NaCl at saturation.…”

Section: Methodsmentioning

confidence: 99%

Isolation and characterization of the intestinal peptide porcine PHI (PHI-27), a new member of the glucagon--secretin family.

Tatemoto¹,

Mutt²

1981

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

334

View full text Add to dashboard Cite

A new peptide, designated PHI (PHI-27), has been discovered and isolated from porcine upper intestinal tissue by using a chemical method for finding peptide hormones and other active peptides. The method is based on chemical detection ofpeptides having the COOH-terminal a-amide structure, which is an unusual chemical feature of some peptide hormones and active peptides. Porcine PHI was found in the intestinal extract by the presence ofits COOH-terminal isoleucine amide structure. It consists of27 amino acid residues and has the following amino acid sequence: His-Ala-Asp-Gly-Val.Phe-Thr-Ser-Asp-Phe-Ser-Arg-LeuLou-Gly-Gln-Leu-Ser-Ala-Lys-Lys-Tyr-Leu-Glu-Ser-Lou-lle-NH2. Me2CO, CHC13, and EtOH for high-performance liquid chromatography (HPLC) were of spectroscopic grade; all other reagents were ofanalytical grade and were used without fiurther purification. The enzyme preparations used were subtilisin BPN' (EC 3.4.21.14; Sigma), trypsin (EC 3.4.21.4; N-tosylphenylalanine chloromethyl ketone-treated, Worthington), chymotrypsin (EC 3.4.21.1; Merk), and carboxypeptidase Y (EC 3.4.12. 1; Boehringer Mannheim).Chemical Assay ofPHI. PHI was determined by the amount ofisoleucine amide released after treatment ofthe sample with subtilisin BPN' by using .the following procedure, which was derived from a slight modification of the amide determination method previously described (3). Samples (<1 mg) in siliconized glass tubes (5 x 50 mm) were dissolved in 20 Adl-of Hepes, pH 7.5, containing 40 ,g ofsubtilisin BPN', and the tubes were sealed with parfilm and incubated at room temperature for 48 hr. At the end ofthe incubation, 1 nmol of y-aminobutyric acid amide was added to each tube as an internal standard. The reaction mixtures were evaporated to dryness under reduced pressure, and the residues were dissolved in 20-100 p1 of 0.1 M sodium bicarbonate/carbonate, pH 9.5, and treated with equal volumes of DnsCl (2-5 mg/ml) in Me2CO; the mixtures were incubated at 37C for 1 hr. The amount of DnsCl added should not greatly exceed the estimated content of DanCl-reactive groups in the sample to avoid side reactions due to modAbbreviations: PHI, PHI-27, the peptide (P) having NH2-terminal histidine (H) and COOH-terminal isoleucine (I) amide and 27 amino acid residues; VIP, vasoactive intestinal peptide; HPLC, high-performance liquid chromatography; TLC, thin-layer chromatography; Dns, dansyl. * A preliminary communication on this study has been published: Ta The publication costs ofthis article were defr-ayed in part by page charge payment. This article must -therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

show abstract

Section: Methodsmentioning

confidence: 99%

Isolation and characterization of the intestinal peptide porcine PHI (PHI-27), a new member of the glucagon--secretin family.

Tatemoto¹,

Mutt²

1981

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

334

View full text Add to dashboard Cite

show abstract

“…In a few cases it has been possible to use chromatographic methods of high resolution for direct physicochemical determination of the intact polypeptide hormones (1,2), but often the peptide patterns of the extracts are so complex that this approach may not be generally applicable. As pointed out previously (3,4), it may, however, in certain cases be possible to submit hormone-containing peptide mixtures to selective fragmentation procedures which produce characteristic fragments of known hormones and to analyze the characteristic fragments instead of whole polypeptide molecules. For many polypeptide hormones, such characteristic fragments could be (or include) the COOH-terminal amino acid a-amides which occur in such diverse hormonal polypeptides as a-melanotropin, calcitonin, cholecystokinin, gastrin, gonadoliberin, oxytocin, the pancreatic hexatriacontapeptide, secretin, substance P, thyroliberin, the vasoactive intestinal peptide (VIP), vasopressin, and others (5,6).…”

mentioning

confidence: 99%

Chemical determination of polypeptide hormones.

Tatemoto¹,

Mutt²

1978

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

183

View full text Add to dashboard Cite

The presence or absence of peptide hormones in tissue extracts may in certain cases be demonstrated by exposing the extracts to conditions under which characteristic fragments of the polypeptide molecule in question are formed and then analyzing for such fragments. An approximate quantitation of the hormones may also be achieved thereby. In the present work the COOH-terminal fragments of polypeptides containing characteristic a-amide groups were released enzymically and then converted into the fluorescent dansyl derivatives, which were identified by thin-layer chromatography. In this way the presence of secretin, cholecystokinin, and the vasoactive intestinal peptide in concentrates of porcine intestinal extracts were demonstrated by their COOH-terminal amide fragments: valine (or leucylvaline) amide, phenylalanine amide, and asparagine (or leucylasparagine) amide, respectively.The analytical methodology used in the present study may also be useful in devising simple and reliable chemical assay methods for the isolation of already known polypeptides and in the isolation of previously uncharacterized polypeptides from natural sources.Polypeptide hormones in tissue extracts are usually determined by either bioassay or radioimmunoassay. In view of the interactions between hormones in vivo, it is increasingly evident that bioassays of tissue extracts, which often contain several different hormones, may give ambiguous results about the presence and the concentrations of a specific hormone. Radioinimunoassay too may be complicated by the problem of crossreactivity.The determination of polypeptide hormones by chemical means has been, in most cases, unsuccessful in spite of the importance of chemical assays in other hormone determinations. In a few cases it has been possible to use chromatographic methods of high resolution for direct physicochemical determination of the intact polypeptide hormones (1, 2), but often the peptide patterns of the extracts are so complex that this approach may not be generally applicable. As pointed out previously (3,4), it may, however, in certain cases be possible to submit hormone-containing peptide mixtures to selective fragmentation procedures which produce characteristic fragments of known hormones and to analyze the characteristic fragments instead of whole polypeptide molecules. For many polypeptide hormones, such characteristic fragments could be (or include) the COOH-terminal amino acid a-amides which occur in such diverse hormonal polypeptides as a-melanotropin, calcitonin, cholecystokinin, gastrin, gonadoliberin, oxytocin, the pancreatic hexatriacontapeptide, secretin, substance P, thyroliberin, the vasoactive intestinal peptide (VIP), vasopressin, and others (5, 6).In this paper we show that the presence of secretin, cholecystokinin, and VIP in peptide concentrates from porcine intestinal extracts may be demonstrated by exposing the concentrates to conditions of enzymatic degradation that release the characteristic COOH-terminal amides of these hormones and then dansyl...

show abstract

“…A larger molecular form, CCK-58, has been extracted from dog intestine and partially characterized (6). Forms of CCK similar in size to CCK-12, CCK-8, and CCK- 4 have been characterized immunochemically in intestinal extracts (10,11).…”

Section: Introductionmentioning

confidence: 99%

“…Other molecular forms of CCK have been identified in intestinal extracts, brain, and plasma of various species (4-1 1). CCK-39 was characterized from hog intestine as a hexapeptide extension on the amino terminus of CCK-33 (4,5). A larger molecular form, CCK-58, has been extracted from dog intestine and partially characterized (6).…”

Section: Introductionmentioning

confidence: 99%