Biomedical subjects
Publications and source records attributed to M Carlquist.
The isolation and primary structure of a novel gastrointestinal peptide, designated valosin, is described. The peptide was purified from porcine upper gut extracts using an HPLC and N-terminal sequence screening strategy which depends on chromatographic and structural characteristics as isolation criterion. The amino acid sequence of this peptide consists of 25 amino acid residues:
The primary structures of the 2 polypeptide chains (HTH alpha and HTH beta) of the homeostatic thymus hormone (HTH) were determined. The entire structures were found to be identical to those of histones H2A and H2B, respectively, without evidence for sub-types, proteolytic processings, or other peptide fragments. The results show that suggestions for new extranuclear and hormone-like histone functions apply to HTH preparations with intact protein chains of the H2 histones.
The total composition, the N-terminal amino acid sequence, and the amino acid sequences of four internal regions have been determined for the ribonucleotide reductase large subunit, protein B1, prepared from a recombinant lambda-lysogenic Escherichia coli K strain, which overproduces the enzyme 30-50-fold. The data have been compared with those previously reported for B1 prepared from a thymine-starved E. coli B strain and with the indirectly derived primary structure of B1 recently reported from the nucleotide sequence of the E. coli K nrdA gene. Two major differences to these results were found. First, the B1 polypeptides started with initiator Met-1 (45%), Asn-2 (30%) or Gln-3 (15%), demonstrating a different type of N-terminal heterogeneity than that found earlier. Secondly, the total amino acid composition as derived from hydrolyzed protein B1 differed substantially from the amino acid composition derived from the nucleotide data. This has the consequence that Cys, Arg, Thr and possibly Val and Ser appear more frequently whereas Asx, Glx, Tyr and possibly Gly appear less frequently in the nucleotide-derived data as compared to direct protein hydrolysates. We suggest usage of other reading frames in the approximate area of residues 630-700 of the primary structure of the nrdA gene to compensate for these discrepancies and for the relatively high incidence of uncommon codons in the reading frame proposed for this area of the gene. Such changes have implications on the previously assigned putative active-site region of protein B1.
A variant form of the heptacosapeptide amide secretin, with C-terminal -Val-Gly-Lys-Arg instead of valine amide, has been isolated from porcine upper intestinal tissue. Unexpectedly, this triacontapeptide exhibited a substantially higher bioactivity than the heptacosapeptide amide.
The primary structures of two novel forms of cholecystokinin, isolated from bovine upper intestine are reported. The two peptides are composed of 33 and 39 amino acid residues, respectively, the larger being an N-terminally extended form of the shorter peptide. The primary structure of the 39 amino acid peptide is: (Formula: see text) This amino acid sequence differs from the porcine hormone at positions 13 and 15, which are Val and Met, respectively, in pig, the same amino acid substitutions have previously been found to occur also in dog.
A novel form of gastric inhibitory polypeptide (GIP), later also referred to as glucose-dependent insulinotropic polypeptide, has been isolated from bovine upper intestine. The purification was monitored by a recently developed radioreceptor assay, specific for GIP, using membrane preparations from hamster beta-cell tumors. A combination of ion-exchange and reverse-phase high-performance liquid chromatography was used in the isolation which resulted in homogeneous bovine GIP. Bovine GIP is, like porcine GIP, composed of 42 amino acid residues. The sequence is: Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-Arg- Gln-Gln - Asp-Phe-Val-Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Ser-Asp-Trp-Ile-His- Asn-Ile - Thr-Gln, which differs from that of the previously characterized porcine GIP by having isoleucine instead of lysine at position 37. Upon proteolytic digestion of GIP with the staphylococcal V8 protease and with enterokinase, two fragments are formed in each case, corresponding to GIP1-3, GIP4-42, and GIP1-16, GIP17-42, respectively.
A novel form of the polypeptide termed PHI (peptide HI with N-terminal histidine and C-terminal isoleucine amide) has been isolated from bovine upper intestine. This bovine peptide was obtained in a 40 times higher yield than the corresponding polypeptide isolated from porcine intestine. Bovine PHI is, like porcine PHI, composed of 27 amino acid residues. The complete amino acid sequence of the bovine peptide is His-Ala-Asp-Gly-Val-Phe-Thr-Ser-Asp-Tyr-Ser-Arg-Leu-Leu-Gly-Gln-Leu-Ser- Ala- Lys-Lys-Tyr-Leu-Glu-Ser-Leu-Ile-NH2. This sequence differs from porcine PHI at position 10 and from human PHI at positions 10, 12 and 27. The amino acid residue exchange between porcine and bovine PHI makes the latter more similar to the vasoactive intestinal polypeptide (VIP), gastric inhibitory polypeptide (GIP), glucagon and the growth-hormone-releasing factor (GRF).
The isolation of the human form of PHI (peptide HI) is described. The peptide was purified from human colonic extracts by using a chemical method for the detection of its C-terminal amidated structure. Human PHI consists of 27 amino acid residues and the complete amino acid sequence is: His-Ala-Asp-Gly-Val-Phe-Thr-Ser-Asp-Phe-Ser-Lys-Leu-Leu-Gly-Gln-Leu-Ser- Ala-Lys-Lys-Tyr-Leu-Glu-Ser-Leu-Met-NH2. The differences between the structures of porcine and human PHI are at position 12 (Arg/Lys replacement) and at position 27 (Ile/Met).
The polypeptide termed somatomedin A (SMA) was isolated from outdated human plasma by a new purification procedure, not using acid ethanol extraction. Fractions containing SMA were monitored by a placenta radioreceptorassay and a radioimmunoassay for SMA. The purification method utilized a microcomputer-controlled chromatography system, yielding both SMA (identified as insulin-like growth factor 1 (IGF-1) or a deamidated derivative) and insulin-like growth factor 2 (IGF-2). The first step of CM-Affigel blue adsorbed at neutral pH the majority of somatomedins detectable by the radioreceptorassay for SMA. Exclusion chromatography on Sephadex G-50 in 0.1 M acetic acid separated this active material from albumin and NaCl. Separation between SMA and IGF-2 was achieved on two different cation-exchange columns, but not in the final high-performance liquid chromatography step. The isoelectric points, determined by chromatofocusing, were 8.0 for SMA and 6.2 for IGF-2. The amino acid compositions of the two isolated peptides were indistinguishable from the known compositions of IGF-1 and IGF-2. Sequence analysis up to position 39 of the peptide with a pI of 6.2 also proved identity with IGF-2 for all positions examined. The peptide with a pI of 8.0, corresponding to SMA, was degraded directly as well as after CNBr cleavage. The results show that it is identical to IGF-1, with the possible exception of acid/amide assignment, which could correspond to a deamidation. If occurring in the native preparation before analysis, it could explain the chromatographic properties and isoelectric point of SMA versus IGF-1 isolated by other techniques.
A polypeptide with secretin-like bioactivity has been isolated from upper small intestinal porcine tissue by ion-exchange and reversed-phase high-performance liquid chromatography (HPLC). The purification was followed by determination of biological activity. Its elution position in the ion-exchange HPLC indicated that it was less basic than secretin. Amino acid analysis showed that it contained an additional glycine residue as compared to secretin. Digestions by trypsin and subtilisin established that the polypeptide was a variant form of secretin in which the previously known secretin is extended C-terminally by a glycine residue.
Galanin (GAL), a 29 amino acid peptide, was recently isolated from the small intestine of pigs. In the present study an antiserum towards porcine GAL has been developed and used for radioimmunoassay and immunohistochemical studies. We now report that GAL-like immunoreactivity occurs in wide-spread systems in the rat CNS and intestine.
A 32-residue peptide has been isolated from extracts of porcine upper intestine. Amino acid sequence determination showed that the peptide is a fragment of hemoglobin, corresponding to the C-terminal part of the beta-chain. The region in the beta-chain which precedes the isolated fragment is hydrophobic (8 Leu/Val/Ile) and has no charges at 10 preceding positions. It therefore, to some extent, resembles the structure of 'signal sequences' which may suggest a specific cleavage site in hemoglobin beta-chains.
A polypeptide in preparations of 'large' form (Mr approximately 14000) material with inhibin-like activity (inhibiting FSH secretion) has been isolated from human seminal plasma. Its amino acid composition, cleavage pattern with CNBr, N-terminal sequence, and properties on reverse-phase high-performance liquid chromatography establish this inhibin-like preparation to be homogeneous. The polypeptide contains close to 130 residues, has a free N-terminal serine residue, a methionine residue in position 19, and a dibasic structure (Arg-Lys) in positions 16-17.
A peptide hormone was extracted from the porcine right atrium following a bioassay for differential vasorelaxant effects on smooth muscle strips from aorta and renal and inferior mesenteric arteries. The isolation procedure included several steps of gel-permeation and ion-exchange chromatography, and high performance liquid chromatography. During the isolation procedure, other peptides of smaller molecular weight were also found, which, in relation to cardiodilatin-126 (CDD-126), are shorter at their N-terminal. Among these, CDD-88 has also been isolated and characterized, and has been established as a prominent member of the cardiac hormone family. The N-terminal and C-terminal segments of the 126 amino acid-containing molecule were synthesized and used to raise region-specific antibodies. The natural peptide was then localized within myoendocrine cells of the right atrium where specific atrial granules are located. Renal effects of cardiodilation were studied in conscious dogs and showed strong diuretic and natriuretic activities. According to our functional studies, cardiodilatin-126 and cardiodilatin-88 possess qualities of a significant hormone family regarding the regulation of extracellular fluid volume and blood pressure.
High affinity binding sites have been found in membrane preparations from hamster beta-cell tumors by using radiolabeled gastric inhibitory polypeptide (125I-GIP). HPLC of 125I-GIP resulted in two major peaks (A III and B III), with identical specific binding. It was verified that peaks A III and B III stimulate insulin release from the isolated perfused rat pancreas to an extent at least equal to that obtained with unlabeled GIP at 10(-9) M. Natural GIP competitively inhibited the binding of 125I-GIP in the range of 10(-10) -10(-6) M and half-maximal inhibition was observed at 1.9 +/- 0.19 X 10(-9) M GIP. The number of high affinity sites was 219 +/- 8 fmol/mg protein and the dissociation constant was 2.05 +/- 0.1 X 10(-9) M. None of 10 regulatory peptides tested exhibited any effect on the 125I-GIP binding at concentrations in the range of 10(-6) -10(-4) M. Consequently, saturable, high affinity and specific binding sites for the GIP have been found and characterized in the plasma membranes of beta-cells. This model can be of use in studying the interaction of GIP with its preponderant target tissue.
Hemoglobin from the tropic lizard Uromastix hardwickii was isolated. Chain separations were studied, and the whole carboxymethylated globin was cleaved with trypsin. Peptides were pre-fractionated by exclusion chromatography and finally purified by reversed phase high-performance liquid chromatography. Amino acid sequence analysis permitted ordering of peptides in alpha- and beta-chains by homology with known structures in other hemoglobins. Results show large structural variations (about 50% homology between Uromastix and viper alpha-chains) and suggest chain heterogeneity with the presence of at least two types of both the alpha- and beta-chains in the preparations.
An immunosorbent method using antiglutaredoxin-Sepharose was developed for purification of glutaredoxin in high yield from a mutant strain of Escherichia coli K 12 lacking thioredoxin reductase (C 10-17). The primary structure of the protein was determined by analyses of [14C]carboxymethylated glutaredoxin and its proteolytic fragments obtained by digestions with trypsin, clostripain, chymotrypsin and staphylococcal Glu-specific extracellular protease. The single active-center disulfide has the structure-Cys-Pro-Tyr-Cys-, with the half-cystine residues located at positions 11 and 14 in the polypeptide chain. In total the protein was deduced to have 85 residues corresponding to a molecular weight of 9674 for the reduced form of glutaredoxin, making it one of the smallest known enzymes (a glutathione-disulfide transhydrogenase). The half-cystines are identically spaced and similarly positioned in the N-terminal part of the protein when compared with a corresponding functionally active disulfide/dithiol in thioredoxins. Glutaredoxin is also distantly homologous with thioredoxins from phage T4 and E. coli, but extensive differences, even around the redox-active disulfide, distinguish glutaredoxin from the thioredoxins. Allowing for deletions in the glutaredoxin sequence (or insertions in the T4 thioredoxin sequence) at four places, there are identical residues at 25 positions of the 77 compared (= 32% identity). The results establish that glutaredoxin belongs to the same superfamily of small redox proteins as the thioredoxins. The structures are, however, subject to large changes, only four positions have residues identical among all presently analyzed forms. The fluorescence of reduced and oxidized glutaredoxin demonstrates an increase in the quantum yield of the tyrosine emission upon reduction with dithiothreitol. Differences in the spectra support the presence of tyrosine adjacent to the redox-active disulfide bridge. They also confirm that glutaredoxin lacks the disulfide-adjacent tryptophan residues of E. coli thioredoxin. There are known to be great differences between the bacterial E. coli and phage T4 forms of thioredoxin. The glutaredoxin structure is most similar to the phage type, both with respect to size of the polypeptide chain and to actual sequence. From the structural results and the previously known functional similarities it appears possible that the phage thioredoxin may have evolved from an early glutaredoxin gene. The mixed properties are compatible with this conclusion, the superfamily assignment, and the differences in biological activity.
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.