The amino acid sequence of chymodenin, a hormone-like peptide from porcine duodenum, is identical to cytochrome C-oxidase, peptide VII

The amino acid sequence of chymodenin, a hormone-like peptide from porcine duodenum is reported. The molecule is known to rapidly alter the proportions of digestive enzymes secreted by the rabbit pancreas in vivo and in vitro, by selection of the specific intra-pancreatic source from which the preset mixture of digestive enzymes is secreted. The sequence is identical to that of cytochrome C-oxidase peptide VII (cCoVII) from bovine heart, with the exception of a substitution of threonine for alanine at position 6 and a second substitution of alanine for threonine at position 71. Disulfide bridges link positions 29-64 and 39-53. cCoVII-chymodenin has a pentapeptide (-Ala-Glu-Gly-Thr-Phe-) near the carboxy-terminus which is immediately preceded by an -Arg-Arg- sequence in the porcine and bovine sequences of cCoVII. This peptide is identical to a pentapeptide found close to the amino terminus of the hormones gastric inhibitory peptide (GIP) and glucagon-like peptide I. The identity to cCoVII means chymodenin as isolated is itself unlikely to be a gastrointestinal hormone. However, the partial commonality of sequence with the glucagon-secretin family immediately adjacent to a pro-hormone-like activation site, and the specific actions on the exocrine pancreas, means that the molecule probably mimics the natural actions of an as-yet uncharacterized member of the glucagon family, which exerts a unique action on exocrine pancreatic secretion.     

Studies on cytochrome c oxidase, VII. Isolation and chemical characterization of polypeptide VII.

Polypeptide VII of cytochrome c oxidase was isolated and purified by gel filtration on Bio-Gel P-10 in 10% acetic acid. Automatic Edman degradation of this peptide chain was not successful, because it is blocked at the N-terminus. The amino acid analysis shows a relatively high content of hydrophilic residues (54%). On the basis of this analysis and the apparent molecular weight by sodium dodecyl sulfate gel electrophoresis and gel filtration, a chain length of about 80 residues was calculated. Among the tryptic peptides one blocked heptapeptide was found. Cleavage of this peptide with thermolysin gave two peptide fragments, one of which was not retained on a cation exchange resin. Mass spectrometric sequence determination of this peptide revealed the structure Ac-Ala-Glu-Asp for the N-terminus of polypeptide VII. Treatment with carboxypeptidase A at two different pH values showed that the C-terminal amino acid is isoleucine and the penultimate amino acid is lysine.     

Isolation of a porcine intestinal peptide with C-terminal somatostatin

A peptide showing somatostatin-like immunoreactivity has been isolated from acid extracts of porcine upper small intestine. Purification was followed by radioimmunoassay, high pressure liquid chromatography, thin layer chromatography and isotachophoresis. Preliminary chemical characterization shows that it has N-terminal serine and that it is composed of somatostatin extended from its N-terminus by an additional peptide.     

Purification and characterization of chymodenin. A hormone-like peptide from porcine duodenum

Chymodenin, a hormone-like duodenal peptide which rapidly alters the proportions of secreted pancreatic digestive enzymes to a mixture relatively richer in chymotrypsinogen than that found in basal secretion, has been purified to homogeneity. The starting material was an acidic methanol-soluble, neutral pH-insoluble fraction of an acetic acid extract of porcine duodenum; the purification consisted of cation-exchange chromatography on SP-Sephadex and CM-Sephadex in ammonium bicarbonate gradients, and gel filtration on Sephadex G-75 in dilute acetic acid. The yield of material was followed by radioimmunoassay. Homogeneity was determined from chymodenin's behavior in disc gel electrophoresis in an acidic counter-migration-of-dye system, sodium dodecyl sulfate-urea gels, gel filtration, dansyl-Edman reaction, reversed-phase high pressure liquid chromatography, isotachophoresis, and sedimentation equilibrium ultracentrifugation. The electrophoretic mobility, the molecular weight of 9,000-10,000, and the biological activity differed from those of other gastrointestinal peptide hormones. The amino acid composition was unique. Chymodenin is the first purified hormone-like substance reported capable of altering the composition of the mixture of secreted digestive enzymes, independent of the stimulation of massive pancreatic protein output.     

Further analysis of the polypeptide subunits of yeast cytochrome c oxidase. Isolation and characterization of subunits III, V, and VII

By using a modified purification procedure in which we have substituted detergent exchange gel filtration for DEAE-cellulose or hydroxylapatite chromatography (Mason, T. L., Poyton, R. O., Wharton, D. C., and Schatz, G. (1973) J. Biol. Chem. 248, 1346-1354), we have isolated yeast cytochrome c oxidase preparations which are low in contaminating polypeptides and which have been successfully used for the large scale purification of subunits. Subunits have been purified from this preparation by a simple two-step procedure which involves: 1) the release of subunits IV and VI from an "insoluble" core composed of subunits I, II, III, V, and VII; and 2) gel filtration of the "core" subunits in the presence of sodium dodecyl sulfate. Molecular weights of the isolated subunits, obtained from sodium dodecyl sulfate gel retardation coefficients (KR) derived from Ferguson plots, were: I, 54,000; II, 31,000; III, 29,500; IV, 14,500; V, 12,500; VI, 9,500; VII, 4,500. In their purified state all subunits, except for subunit V, exhibited electrophoretic behavior similar to that exhibited by unpurified subunits in sodium dodecyl sulfate-dissociated holoenzyme preparations. As purified, subunit V exhibits a slightly smaller apparent molecular weight than its counterpart in the holoenzyme. Amino acid analysis of the isolated subunits revealed that subunit III, a mitochondrial translation product, contained 41.9% polar amino acids, whereas subunits V and VII, cytoplasmic translation products, each contained 47.7% polar amino acids. These results extend and support our previous finding that the mitochondrially translated subunits of yeast cytochrome c oxidase are more hydrophobic than the cytoplasmically translated subunits.     

Studies on cytochrome c oxidase, XII. Isolation and primary structure of polypeptide VIb from bovine heart

The isolation and complete sequence analysis of the cytoplasmically synthesized polypeptide VIb from bovine heart cytochrome c oxidase is described. The protein is a stoichiometric constituent of the respiratory complex IV. Its primary structure is deduced from N-terminal sequencing and overlapping peptides obtained from tryptic cleavage and specific cleavage at arginyl and tryptophyl peptide bonds. The polypeptide chain consists of 84 amino acids from which a Mr of 9419 is derived. It has a relatively high content of histidine and proline and contains a single cysteine. A hydrophobic sequence of 20 amino acids points to a membrane-penetrating structure similar to that found in polypeptides I, II, III, IV and VIIIa, VIIIb, VIIIc of the bovine oxidase. The sequence of VIb is tissue-specific, it contributes to the formation of nuclear coded isoenzymes of cytochrome c oxidase. The protein thus may be involved in a tissue-specific regulation of cellular respiration.     

Studies on cytochrome c oxidase, VIII. The amino acid sequence of polypeptide VII.

The sequence determination of polypeptide VII from beef heart cytochrome c oxidase is described. The amino acid sequence is deduced from overlapping tryptic peptides and peptides obtained after cleavage with Staphylococcus aureus protease. The protein consists of 85 amino acids corresponding to a Mr of 10026, in agreement with a value of 9500 obtained by sodium dodecyl sulfate gel electrophoresis. The amino acid sequence around the only methionine present is very similar to sequences around the invariant heme binding methionine of the cytochrome c family. This similarity suggests that the protein is one of the heme bindings subunits of the oxidase.     

Natural purified porcine gastric inhibitory polypeptide (GIP) stimulates exocrine pancreas secretion due to contamination with a cholecystokinin-like substance

The effects of purified natural gastric inhibitory polypeptide-enterogastrone III (GIP-EG III) and a fraction which is further purified by high pressure liquid chromatography (GIP-HPLC) were investigated on the endocrine and exocrine isolated perfused pancreas of rats. At the dose of 5 ng/ml used for both GIP preparations, only GIP-EG III significantly stimulated volume and amylase secretion of the exocrine pancreas. The response of insulin release to stimulation by GIP-EG III or GIP-HPLC was not significantly different. In the presence of cholecystokinin-octapeptide (CCK-8) at a concentration which gave half-maximal stimulation of amylase secretion, GIP-EG III almost doubled the response of the exocrine pancreas, whereas GIP-HPLC had no additional effect. CCK-8 alone significantly increased total insulin output under hyperglycemic conditions. We conclude that porcine GIP purified by gel chromatography contains a CCK-like substance which can be removed by further purification on high pressure liquid chromatography without affecting the insulinotropic activity. Some of the reported effects of GIP could be due to contamination.     

Studies on cytochrome c oxidase, X. Isolation and amino-acid sequence of polypeptide VIIIb

The isolation and sequence determination of the cytoplasmically synthesized polypeptide VIIIb from beef heart cytochrome c oxidase is described. Several methods for isolating polypeptide VIIIb with gelchromatographic technics are presented. The complete amino-acid sequence is deduced from a N-terminal sequencer run, overlapping tryptic peptides and peptides obtained after tryptophan specific cleavage with cyanogen bromide in heptafluorobutyric acid/formic acid. The small protein consists of 46 amino acids and has a molecular mass of 4 962 Da. The existence of a hydrophobic segment with a length of 20 residues characterizes it as a membrane penetrating protein. The stoichiometry of this polypeptide in the functional monomer of cytochrome c oxidase (complex IV) is 2 and is thus different from all the other polypeptides constituting the respiratory complex IV. The function of this component of the terminal oxidase is as yet unknown.     

Isolation and partial charcterization of a cytochrome b complex and cytochrome oxidase from yeast mitochondria.

A cytochrome b complex and cytochrome oxidase have been purified 14- and 20-fold respectively from yeast submitochondrial particles by a simple procedure involving their spontaneous precipitation from a deoxycholate extract. The recovery of both proteins was almost quantitative. The specific heme contents were 11 and 8 nmoles/mg protein for the cytochrome b complex and cytochrome oxidase respectively and both were spectrally pure. Sodium dodecyl sulfate gel electrophoresis resolved the cytochrome b complex into seven distinct subunits with molecular weights 42,000, 33,000, 27,500, 23,000, 15,500, 13,000 and 10,500. Cytochrome oxidase contained five bands with molecular weights 42,000, 26,500, 21,000, 14,000 and 10,500.     

Preparation of polypeptide subunits of cytochrome oxidase from Neurospora crassa.

Cytochrome oxidase was purified from Neurospora crassa by ammonium sulfate fractionation in the presence of bile salts. The enzyme preparations contained 10-13 nmol of heme a per mg of protein; no other hemoproteins could be detected. Dodecylsulfate gel electrophoresis resolved the enzyme complex into seven major bands, representing seven polypeptide subunits. A procedure is described that allows the isolation of these enzyme subunits on a large scale starting from a single batch of oxidase preparation. It involves dissociation of the enzyme complex by dodecylsulfate and subsequent separation of the obtained polypeptides by chromatography in the presence of various dodecylsulfate concentrations. Purification of subunits 3, 4, 5, 6 and 7 was achieved by column chromatography using molecular sieves (Sephadex G-100, Bio Gel P-60) and hydroxylapatite. For the purification of subunits 1 and 2 an electrophoretic separation on a preparative polyacrylamide gel was required. The advantages and disadvantages of the separation procedure of the enzyme polypeptides are discussed. As a special point of interest, the conservation of antigenic determinants of the polypeptide chains during the dodecylsulfate treatment is considered.     

The polypeptide composition of cytochrome oxidase from the bovine heart muscle

The beef heart cytochrome oxidase (EC 1.9.3.1) has been purified by hydrophobic chromatography. The enzyme has been resolved into 11 different polypeptides by SDS/urea gel-electrophoresis.     

Isolation and characterization of a novel peptide amide from porcine brain.

Peptide with C-terminal tyrosine amide was isolated from porcine brain by acid extraction and sequential steps of reverse phase HPLC. Microsequence, amino acid and mass spectral analyses revealed the structure: Ac-Ala-Ser-Glu-Lys-Arg-Pro-Ser-Glu-Arg-His-Gly-Ser-Lys- Tyr-amide. Since this peptide had the identical sequence to N-terminus of porcine myelin basic protein (pMBP) 1-14, we have designated porcine myelin peptide amide 14 (pMPA14). The final HPLC step yielded 20 micrograms of homogeneous peptide preparation from 20 kg brain tissue. Unlike other amidated peptides, pMPA14 may be produced by non enzymatic mechanism or unknown amidating enzyme. This unique amidation seems to occur exclusively to MBP in the brain.     

Metals and activity of bovine heart cytochrome c oxidase are independent of polypeptide subunits III, VII, a, and b

Bovine heart cytochrome c oxidase, depleted of polypeptide subunits by alkaline detergent treatment, was characterized with respect to metal content, optical spectral properties, and oxidase activity. Treatment with 1.0% Triton X-100 at pH 9.5 followed by anion-exchange chromatography caused removal of subunit III, subunit VII, and polypeptides a and b. The metal atom stoichiometries of the control and the polypeptide-depleted enzyme were in both cases 2.5Cu/2Fe/1Zn/1Mg with metal-to-protein ratios significantly greater in the latter. The treated enzyme exhibited a red shifted oxidized Soret maximum and bound carbon monoxide upon reduction. Activity was markedly decreased by the treatment but was restored to control levels by incubation with 0.3% Tween 80 at pH 6.0. Therefore, subunit III, subunit VII, polypeptide a, and polypeptide b do not contain Cu, Fe, Zn, or Mg and are not essential for reduction of O2 by ferrocytochrome c.     

Isolation of a heme binding subunit from bovine heart cytochrome C oxidase.

A subunit which retains heme has been isolated and purified up to a homogenous form on polyacrylamide gel electrophoretic column in the presence of sodium dodecyl sulfate and β-mercaptoethanol from cytochrome oxidase. The separation of the subunit does not rely on any detergent except cholate used in the preparation of cytochrome oxidase. The purification involves a reaction with pyridine, pH precipitation, and DEAE-cellulose column chromatography. The purified subunit has a molecular weight of 11,600 daltons and contains more than 40 nmol Fe per mg protein; the lower iron content than the calculated value is apparently due to the loss of heme $\text{a}$ in the course of the purification. The subunit is freely soluble in aqueous solution at neutral pH to give a dark green color. Spectral properties and amino acid composition of this subunit have been studied.     

Isolation and characterization of a variant form of vasoactive intestinal polypeptide

A variant form of the vasoactive intestinal polypeptide (VIP) has been isolated. It was found to consist of a molecule which instead of the C-terminal asparagine amide of VIP has a C-terminal extension of Gly-Lys-Arg. This VIP variant displaces VIP in a VIP receptor assay, reacts with N-terminally-directed antisera in a VIP radioimmunoassay and possesses VIP-like bioactivity in an assay measuring pancreatic juice secretion in the cat.     

Isolation of a brain peptide identical to the intestinal PHI (peptide HI)

The isolation of a brain peptide identical to the intestinal peptide PHI (peptide HI) is described. The peptide was isolated from porcine brain extract using a chemical assay method based on its C-terminal isoleucine amide structure. The complete amino acid sequence of the peptide was found to be: His-Ala-Asp-Gly-Val-Phe-Thr-Ser-Asp-Phe-Ser-Arg-Leu-Leu-Gly-Gln-Leu-Ser-Ala- Lys-Lys-Tyr-Leu-Glu-Ser-Leu-Ile-NH2. This sequence is identical to the intestinal peptide thus demonstrating PHI to be a brain-gut peptide. The role of PHI in the central nervous system as a neurotransmitter or neuromodulator is discussed.