Biomedical subjects

J D Hulmes

Publications and source records attributed to J D Hulmes.

47 records · Page 3Linked to original sources

PubMed1986

Cholecystokinin-associated COOH-terminal peptides are fully sulfated in pig brain.

A radioimmunoassay was developed to detect the cholecystokinin (CCK)-associated nonapeptide (CAP-9) that forms the COOH terminus of pig preproCCK. This peptide (Ser-Ala-Glu-Glu-Tyr-Glu-Tyr-Thr-Ser) is presumably produced at the time that the tyrosine-sulfated octapeptide CCK8(s) is cleaved from preproCCK. Radioimmunoassay of a dried methanol extract of pig brain revealed no detectable CAP-9 immunoreactivity, whereas acid desulfation of the dried methanol extract prior to radioimmunoassay resulted in easily measurable concentrations of CAP-9 immunoreactivity. Two peptides, CAP-9 and des-Ser9-CAP-9, were purified from a methanol extract of 8 kg of commercially obtained whole pig brains. Amino acid analysis showed that each peptide has both tyrosines sulfated. Thus, the likely sequence of CCK post-translational processing events is sulfation of the three tyrosines in the COOH terminus of preproCCK followed by peptide cleavage and appearance of CCK8(s) and CAP-9(s,s).

Amino Acid Sequence↗

PubMed1986

Guinea pig glucagon differs from other mammalian glucagons.

Mammalian glucagon is thought to be highly conserved. Glucagons from pig, cow, human, rat, and hamster have identical amino acid sequences, whereas the amino acid contents of rabbit and camel glucagons are consistent with this 29-amino acid sequence. It had earlier been reported that guinea pig (GP) glucagon contains 40 amino acids. In the current study, glucagon was purified from two GP pancreata by a series of three HPLC steps after acid-alcohol extraction and acetone precipitation. GP glucagon is a 29-amino acid peptide that differs from other mammalian glucagons by substitution of Gln for Asp in position 21, Leu for Val in position 23, Lys for Gln in position 24, Leu for Met in position 27, and Val for Thr in position 29. In view of the marked changes in the COOH-terminal of GP glucagon, receptor binding studies were performed using both rat and GP liver membranes. Labeled synthetic porcine glucagon has similar binding in the two systems and its binding is inhibited to a similar degree by synthetic porcine glucagon, whereas GP glucagon is 10-fold less potent at inhibiting binding in both systems. This suggests that glucagon receptor binding sites in the GP are evolutionarily more conserved than is GP glucagon.

Amino Acid Sequence↗

PubMed1985

Guinea pig "little" gastrin is a hexadecapeptide.

Gastrin heptadecapeptides (gastrins I and II which differ in the presence of sulfate on the tyrosine of the latter) have been purified and sequenced from several mammalian species including pig, dog, cat, sheep, cow, human and, more recently, rat. This report describes the purification of "little" gastrin from guinea pig (GP) antra. GP antra were defatted with acetone and the acetone cakes were extracted with 0.1M NH4HCO3. The extract was concentrated by adsorption to DE53 anion exchange cellulose and the peak eluates were fractionated on a Sephadex G50F column. The peptides were brought to final purity by 3 successive HPLC steps. The GP sequence compared to other species is shown: (formula: see text) Thus GP "little" gastrins I and II are hexadecapeptides due to a deletion of a glutamic acid in the region 6-9 from the N-terminus.

Amino Acid Sequence↗

PubMed1985

Purification of dog VIP from a single animal.

VIP, a potent vasodilator peptide, is reported to be identical in pig, cow, human and rat but to differ in four amino acids in chicken. This report describes the purification of dog VIP from the small intestine of a single animal. The purification method is based on tissue extraction with a sequence of organic solvents. The extracted VIP is concentrated onto cation-exchange cellulose and brought to purity by three HPLC steps. A 30% final yield of pure VIP was obtained from the original extract. Dog VIP was found to have the following sequence: His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala -Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn. Thus the amino acid sequence of dog VIP is identical with all the mammalian VIP's which have been reported. This suggests that a high degree of conservation throughout the molecule may be required for VIP bioactivity.

Amino Acid Sequence↗

PubMed1985

Guinea pig has a unique mammalian VIP.

Mammalian vasoactive intestinal peptide (VIP) has been reported to be identical in four species. This report describes the extraction of guinea pig (GP) intestinal VIP, its purification and sequence. Frozen intestines were extracted in five volumes of methanol and the methanol cakes reextracted with acid. VIP in the acid extract was concentrated onto ion-exchange cellulose and was brought to final purity through a series of HPLC steps. GP VIP differs from other mammalian VIP's by four amino acid substitutions: (sequence in text) This is further evidence that the GP gastroenteropancreatic axis has a unique evolutionary separation from other mammals.

Amino Acid Sequence↗

PubMed1985

Purification and partial sequencing of bovine liver alkaline phosphatase.

Bovine liver alkaline phosphatase has been purified to homogeneity by procedures that include reverse-phase HPLC. The pure enzyme has an apparent Mr of 160,000 and is composed of what appears to be two identical monomers of Mr 82,000. About 80% of the material yielded the amino-terminal sequence Leu-Val-Pro-Glu-Lys-Glu-Lys-Asp-Pro-?-Tyr-?-Arg-Asp-Gln-Ala-Gln. The minor component was extended at the amino terminus by two residues that have not yet been identified, i.e., ?-?-Leu-Val-Pro-Glu-Lys-Glu-Lys-Asp-Pro-?-Tyr-?-Arg-Asp-Gln-Ala-Gln.

Alkaline Phosphatase↗

PubMedDate not supplied

Purification and sequencing of a rat intestinal 22 amino acid C-terminal CCK fragment.

Fractionation on Sephadex G50 gel of methanol extracts of rat intestine revealed two molecular forms of cholecystokinin (CCK) of about equal immunopotency: one form has an elution volume between CCK33 and CCK12; the other elutes in the salt region as does authentic CCK8. Purification and sequencing have demonstrated that the smaller molecular form is CCK8 with a sequence identical to the pork and sheep CCK8's that had previously been sequenced. Purification and sequencing of the larger molecular form reveals that it is a 22 amino acid C-terminal CCK fragment identical with pig CCK22 except that glycine instead of serine is present at the nineteenth residue from the C-terminus. This sequence is consistent with that predicted by cloned cDNA encoding preprocholecystokinin from a rat medullary thyroid carcinoma. CCK22 has not previously been reported to be a prominent molecular form in either pig or dog intestines.

Amino Acid Sequence↗

PubMedDate not supplied

Unique cholecystokinin peptides isolated from guinea pig intestine.

Fractionation on Sephadex G50 gel of methanol extracts of guinea pig intestine reveals two molecular forms of cholecystokinin (CCK) of about equal abundance. One elutes at the position of CCK8 while the other elutes at a position intermediate between CCK33 and CCK8. Purification and sequencing of these peptides identify them as CCK8 and CCK22, respectively. Guinea pig CCK8 differs from other mammalian CCK octapeptides isolated thus far in that there is a valine substituted for methionine at position 6 from the C-terminus. In addition to the substitution in CCK8, serine is substituted for asparagine in position 22, glycine for serine in position 19, and asparagine for serine in position 15 from the C-terminus compared to the pig sequence. HPLC separation on a C18 column yields two peaks each of CCK8 and of CCK22 in pig intestinal tissue obtained from a commercial supplier. The two CCK8 peptides have identical amino acid sequences as do the two CCK22 peptides. The CCK22 peptides are equally bioactive in the guinea pig pancreatic acinar cell assay but are about 10-fold less potent than synthetic CCK8(s). One of the guinea pig CCK8 peptides is fully bioactive whereas the other is about 50-fold less potent compared to synthetic CCK8(s).

Amino Acid Sequence↗

PubMedDate not supplied

Sequences of gastrins purified from a single antrum of dog and of goat.

Heptadecapeptide gastrins (G17) have been purified and sequenced from a variety of species. However, progastrin (G34) sequences have been determined only for pig and human from purified peptides and for rat from cDNA. Since G34 in most species accounts for only approximately 5% of total antral gastrin, micropurification techniques must be employed to avoid the need for large quantities of antral tissue. Efficient purification methodology yielded 1.5 and 1.3 nmol of G34 from the antrum of a single goat and of a single dog, respectively. The N-terminal pyroglutamyl residues were enzymatically removed and the peptides were sequenced through to the proximity of their COOH-termini. The COOH-terminal sequences of goat and dog G34 were confirmed by sequencing the corresponding deblocked G17 from each animal. The previously published dog G17 sequence was shown to be incorrect. The sequences for dog and goat G34 are: Dog less than ELGLQGPPQLVADLSKKQGPWMEEEEAAYGWMDF# Goat less than ELGLQDPPHMVADLSKKQGPWVEEEEAAYGWMDF# Dog and goat gastrins differ in 3 sites in the 17 amino acid NH2-terminus and only a single site in G17 (the sites of differences are underlined). The ratio for sulfated to non-sulfated antral G17 is 9:1 for the goat and 1:9 for the dog.

Amino Acid Sequence↗

PubMedDate not supplied

Guinea pig pancreatic polypeptide: structure and pancreatic content.

Guinea pig (GP) pancreatic polypeptide (PP) has been purified from an acid-alcohol extract of 6 GP pancreata by a series of 3 HPLC steps. The sequence for GP PP as compared with that of beef and human is shown: (Sequence: see text). In a single GP pancreas weighing 2.4 g the total PP content was 1.0 nmol and the total glucagon content 61 nmol; in a single dog pancreas weighing 35 g the total PP was 385 nmol and the total glucagon 81 nmol. The relatively low content of PP in GP pancreas is consistent with the fact that the GP lacks a ventral pancreas, the region in which PP is found in highest concentration. The high glucagon content of GP pancreas is consistent with that reported in earlier studies.

Amino Acid Sequence↗

PubMedDate not supplied

Identity of tumour necrosis factor and the macrophage-secreted factor cachectin.

In mammals, several well-defined metabolic changes occur during infection, many of which are attributable to products of the reticuloendothelial system. Among these changes, a hypertriglyceridaemic state is frequently evident, resulting from defective triglyceride clearance, caused by systemic suppression of the enzyme lipoprotein lipase (LPL). We have found previously that macrophages secrete the hormone cachectin, which specifically suppresses LPL activity in cultured adipocytes (3T3-L1 cells). When originally purified from RAW 264.7 (mouse macrophage) cells, cachectin was shown to have a pI of 4.7, a subunit size of relative molecular mass (Mr) 17,000 and to form non-covalent multimers. A receptor for cachectin was identified on non-tumorigenic cultured cells and on normal mouse liver membranes. A new high-yield purification technique has enabled us to determine further details of the structure of mouse cachectin. We now report that a high degree of homology exists between the N-terminal sequence of mouse cachectin and the N-terminal sequence recently determined for human tumour necrosis factor (TNF). Purified cachectin also possesses potent TNF activity in vitro. These findings suggest that the 'cachectin' and 'TNF' activities of murine macrophage conditioned medium are attributable to a single protein, which modulates the metabolic activities of normal as well as neoplastic cells through interaction with specific high-affinity receptors.

Adipose Tissue↗