Yu-Ching E. Pan

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 VIPs 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.

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).

Cholecystokinin octapeptides purified from chinchilla and chicken brains.

Cholecystokinin octapeptides (CCK8s) have been purified from methanol extracts of 30 chinchilla and 50 chicken brains containing 9.3 nmol and 8.5 nmol of the peptides respectively. Immunoreactive CCK was concentrated on a DEAE trisacryl column and purification effected by two successive HPLC steps. The peptides were each shown to have a sulfated tyrosine. The sequences of the two peptides are compared with the corresponding CCK8s of pig and guinea pig (GP). Chinchilla & GP: D Y V G W M D F; Chicken & pig: D Y M G W M D F. Since chinchilla insulin resembles other mammalian insulins more than does GP insulin, it is of particular interest that the CCK8s of these two species are identical and raises the question as to whether other brain-gut peptides of the chinchilla, which is a New World mammal as is the GP, would resemble those of the GP or the corresponding peptides of Old World mammals.

Identification and NH2-terminal amino acid sequence of three insulin-like growth factor-binding proteins in porcine serum

Three distinct species of IGFBP in porcine serum were identified by NH2-terminal amino acid sequence analysis. The IGFBPs identified include pIGFBP-2 (34 kDa), three isoforms of pIGFBP-3 (43, 40 and 30 kDa) and two isoforms of pIGFBP-4 (30 and 26 kDa). The three isoforms of pIGFBP-3 were found to have a common NH2-terminal amino acid sequence, as were the two isoforms of pIGFBP-4. These results indicate that porcine serum contains a truncated form of IGFBP-3 and two forms of pIGFBP-4, similar to those previously isolated from human and rat serum. Furthermore, the presence of a truncated form(s) of the GH-dependent IGFBP-3 in porcine serum suggests that elucidating its origin and function may be important in understanding how IGFBPs affect the somatogenic actions of GH.

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 VIPs which have been reported. This suggests that a high degree of conservation throughout the molecule may be required for VIP bioactivity.

Structural analysis of recombinant soluble human interleukin-2 receptor primary structure, assignment of disulfide bonds and core IL-2 binding structure

A purified soluble and functional form of recombinant human interleukin-2 receptor, engineered and expressed in Chinese hamster ovary cells, was structurally characterized. The primary sequence of this 224 amino acid recombinant protein which lacks most of the carboxy-terminal transmembrane and cytoplasmic portions of the intact protein was established by sequence analyses. The disulfide bonds were assigned by comparative peptide mapping of the reduced and non-reduced peptide digests. As in the case of natural interleukin-2 receptor they occur between cysteines 3-147, 46-104, 131-163, and 28/30-59/61. Based on assignment of the disulfide bonds, a structural model of the interleukin-2 receptor for interleukin-2 binding is proposed.

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.

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.

Collaborative Trial Analyses of ABRF-91AAA

Publisher Summary The ABRF studies to date have examined the recovery, accuracy, and precision of amino acid analysis using either synthetic peptides, purified proteins, or a protein hydrolysate. These studies concentrate on the sensitivity of analysis with hydrolyse and analyses being performed in the 0.1–5 μg range. The accuracy of analyses has generally been in the 80–90% range. This chapter discusses an attempt at investigating the quality of analyses under conditions where laboratories might feel more comfortable and where ample supply of sample was available. Three general aspects were explored. The first two were a realistic assessment of the accuracy of absolute quantitation and of the accuracy of relative yields of residues obtainable at present. The third aspect was the analysis of the problem amino acids tryptophan and cystine/cysteine.

“Fishing out” ligand binding proteins from protein mixtures by a two-dimensional gel electrophoresis system utilizing native PAGE followed by SDS-PAGE

Publisher Summary This chapter presents a two-model system that demonstrates the potential of the 2D approach for the identification of a ligand binding protein in a mixture. For a complicated protein mixture, the resolution of the native polyacrylamide gel electrophoresis (PAGE) alone is not sufficient to separate all components from the protein/ligand complex. The 2D method improves the resolution of all species, while establishing the binding ability for the components of interest. These proteins can be recovered onto polyvinylidene difluoride (PVDF) for further analyses. This method does not require large amounts of sample and is capable of capturing a binding protein out of an unpurified protein mixture. The proteins involved in complex formation can be identified by comparing the 2D profiles of (1) the protein mixture with ligand added, (2) the protein mixture without ligand added, and (3) the non-complexed ligand control. The use of sequential native and sodium dodecyl sulfate (SDS)-PAGE helps in protein concentration and purification. The non-denaturing environment of native PAGE helps to maintain the protein/ligand complexes. A ligand binding protein present in a mixture can be identified at the microscale level with minimal sample manipulation.