Biomedical subjects

G Gafvelin

Publications and source records attributed to G Gafvelin.

29 records · Page 2Linked to original sources

PubMed1993

Different sec-requirements for signal peptide cleavage and protein translocation in a model E. coli protein.

We describe a secretory E. coli protein with a novel phenotype: signal peptide cleavage is largely unaffected whereas chain translocation is efficiently blocked under conditions where SecA, a central component of the secretory machinery, is rendered non-functional, and we have traced this phenotype to the presence of a mildly hydrophobic segment located approximately 30 residues downstream of the signal peptide. When this segment is deleted, normal SecA-dependent signal peptide cleavage and chain translocation is observed; when its hydrophobicity is increased, it becomes a permanent membrane anchor with cleavage of the signal peptide and membrane insertion both being SecA-independent. These findings suggest that the initial insertion of the signal peptide across the membrane can be uncoupled from the translocation process proper.

Amino Acid Sequence↗

PubMed1991

Dual effects of vasoactive intestinal peptide (VIP) on leucocyte migration.

Vasoactive intestinal peptide, at different concentrations, was tested on the migration of leucocytes by using the sealed capillary migration test. Vasoactive intestinal peptide, at 10(-7)-10(-9)M, inhibited, while at 10(-12)-10(-14)M, stimulated mononuclear leucocyte migration. The migration of polymorphonuclear leucocytes was inhibited by vasoactive intestinal peptide at 10(-6)-10(-9)M, a stimulation was found at 10(-13)-10(-14)M. The inhibiting effect of vasoactive intestinal peptide on leucocyte migration was abolished when vasoactive intestinal peptide was split into C- and N-terminal fragments, while a stimulating effect was retained in the N-terminal fragment, at 10(-14)M, for mononuclear cells. Helodermin and peptide T, as well as two other members of the secretin-glucagon family, secretin and gastric inhibitory peptide, had no effect on the migration. When VIP antiserum was tested, it had an inhibiting effect, which was not seen with control serum, supporting a physiological effect of the lower vasoactive intestinal peptide concentrations. Vasoactive intestinal peptide seems to have dual effects on mononuclear and polymorphonuclear leucocyte migration and, generally, intact vasoactive intestinal peptide seems to be needed for these effects.

Cell Migration Inhibition↗

PubMed1991

A fragment of triosephosphate isomerase competes with the vasoactive intestinal polypeptide (VIP) for binding to the VIP receptor.

A 28-residue N-terminal fragment of triosephosphate isomerase, TIM(1-28), has been purified from porcine upper intestine. It competes with VIP for binding to the VIP receptor on rat liver plasma membranes with an IC50 value of 2.8 mM, about 1000 times higher than that for VIP binding to the membranes. Except for a single positional identity and the number of amino acid residues, the amino acid sequences of TIM(1-28) and VIP are unrelated as regards primary structure. However, the ability to bind to the same receptor site may indicate common three-dimensional structural properties.

Amino Acid Sequence↗

PubMed1990

Purification of thymocyte growth peptide (TGP) from sheep thymus. Relationship to FTS/thymulin.

Thymocyte growth peptide (TGP) initiates DNA synthesis in immature thymocytes and has previously been characterized as an acidic peptide isolated from calf thymus. We now report the isolation of TGP from sheep thymus and show it to be a nonapeptide with a large N-terminal blocking moiety characterized by high UV absorbance. The amino acid composition is identical to FTS, consisting of 2 Gly, 2 Ser, 2 Glx, 1 Ala, 1 Lys, 1 Asx. In contrast to FTS, TGP is acidic with an apparent isoelectric point of 4.2 and a high UV absorbance at 270-280 nm. Reverse phase chromatography of TGP at an acidic pH results in a change of the molecule and the appearance of two new compounds TGP-A and TGP-B, both with less than 50% of the original TGP activity. Full activity could be restored by the addition of ZnCl2 to TGP-A. Both TGP-A and B have some amino acid composition and high UV absorbance as native TGP. We propose that TGP consists of a non-peptide moiety bound to the N-terminal of the nonapeptide Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn and that the active molecule is stabilized by Zn2+.

Amino Acid Sequence↗

PubMed1990

Processing of prosecretin: isolation of a secretin precursor from porcine intestine.

A precursor to the gastrointestinal hormone secretin has been isolated. The starting material for the purification of the precursor was a peptide fraction purified from pig intestinal extracts, containing peptides with a molecular weight higher than that of secretin. The purification could be followed by measurement of secretin bioactivity (alkali secreted in the pancreatic juice of anesthetized cat). Sequence analysis of the isolated secretin precursor revealed a 71-amino acid residue polypeptide that contained the sequence of secretin N terminally, followed by a Gly-Lys-Arg sequence and a C-terminal extension of 41-amino acid residues. With the exception of an arginine residue, which occurs directly after the Gly-Lys-Arg sequence, the remainder of the C-terminal residues in this precursor are identical to the 40 C-terminal residues predicted by the recently described cDNA sequence for porcine preprosecretin. Compared to the deduced preprosecretin sequence, a stretch of 32 amino acid residues directly following the Gly-Lys-Arg sequence is missing in the now purified secretin precursor. This implies that differential splicing may occur when the secretin gene transcript is processed to mRNA.

Amino Acid Sequence↗

PubMed1988

Synthesis, cloning and expression in Escherichia coli of artificial genes coding for biologically active elongated precursors of the vasoactive intestinal polypeptide.

Synthetic genes coding for elongated precursors of the vasoactive intestinal polypeptide (VIP) were synthesized and cloned in a highly efficient Escherichia coli expression vector. The synthetic genes code for VIP with its methionine (at position 17) replaced by leucine and elongated at the C-terminus by Gly (vasoactive intestinal polypeptide-Gly, i.e. VIPa) or by Gly-Lys-Arg (vasoactive intestinal polypeptide-Gly-Lys-Arg, i.e. VIPb). The synthetic genes fused to the N-terminal part of the E. coli beta-galactosidase gene were expressed to yield high amounts of fusion proteins reaching upon induction at least 60% of the total cellular protein. The fusion proteins of 314 and 316 amino acids carrying in their C-terminal portion either the 29 or 31 amino acids long VIP precursor polypeptide were shown to be immunoreactive with VIP antisera and were further purified and cleaved by CNBr. The resulting purified peptide precursors (VIPa and VIPb) were recognized by VIP receptors in rat liver plasma membranes and by antibodies to porcine VIP in a radioimmunoassay. Both precursors activated adenylate cyclase in rat liver membranes and stimulated pancreatic secretion in the cat. The affinity and potency of the cloned precursors is close to that of VIP purified from porcine intestine, suggesting that the elongated VIP precursors may act even without being converted into the C-terminal amide form of the peptide. The elongated VIP precursors expressed in E. coli may provide a cheap, large-scale source of experimental material for studies on VIP actions.

Amino Acid Sequence↗

PubMed1986

Characterization of cat insulin.

Cat insulin was isolated and both chains were characterized by determination of the primary structures. The molecule was found to differ from human insulin at four positions, A8 (Ala), A10 (Val), A18 (His), and B30 (Ala). A comparison with other known insulin structures suggests that cat insulin has an uncommon property: it appears to be the only insulin found so far with His at position A18. The difference is compatible with a conserved overall conformation but this histidine occupies a position close to the suggested receptor interacting area and may influence some binding properties.

Amino Acid Sequence↗

PubMed1985

A proform of secretin with high secretin-like bioactivity.

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.

Amino Acid Sequence↗

PubMedDate not supplied

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.

Amino Acids↗

PubMedDate not supplied

Isolation and primary structure of VIP from sheep brain.

The amino acid sequence of the vasoactive intestinal polypeptide (VIP) is well conserved between species. Thus, all mammalian VIPs isolated so far, except that of the guinea pig, have the same amino acid sequence. This study describes the isolation and primary structure of sheep brain VIP. The purification was followed with a bioassay and a VIP receptor assay. The amino acid sequence of the isolated sheep VIP is identical to that of the pig, human, ox, rat, rabbit, goat and dog VIP.

Amino Acid Sequence↗

PubMedDate not supplied

SecA-dependence of the translocation of a large periplasmic loop in the Escherichia coli MalF inner membrane protein is a function of sequence context.

We have analysed the translocation of a large periplasmic loop in the Escherichia coli MalF inner membrane protein when placed in different sequence contexts and under conditions when the function of the SecA protein is inhibited. The results show that the degree of SecA-dependence varies with sequence context: while translocation of the large loop in its normal context is only minimally affected by SecA inhibition, translocation is much more sensitive to SecA inhibition when the loop is placed in the context of other inner membrane proteins. Conversely, when the large MalF loop is replaced by segments from other proteins, translocation of those segments is again very sensitive to SecA inhibition. Thus, SecA-dependence is not an all-or-none phenomenon and is not only a simple function of, e.g. the length of a translocated segment or the hydrophobicity of the flanking transmembrane segments.

ATP-Binding Cassette Transporters↗