Biomedical subjects

L Thim

Publications and source records attributed to L Thim.

At least 109 records · Page 6Linked to original sources

PubMed1990

Limited proteolysis of beta 2-microglobulin at Lys-58 by complement component C1s.

We have now demonstrated that activated complement component C1s cleaves beta 2-microglobulin at the position identical to that at which beta 2-microglobulin is cleaved in serum of patients suffering from lung cancer. The main cleavage is in the disulphide loop C-terminal to Lys-58, generating a modified form of beta 2-microglobulin with a two-chain structure. The C-terminal Lys-58 in the A chain is highly susceptible to removal by a carboxypeptidase-B-like activity causing the formation of des-Lys58-beta 2-microglobulin. This is the first demonstration of a noncomplement protein substrate for the proteolytic activity of C1s. The C1s-induced cleavage of beta 2-microglobulin can be inhibited in the presence of C1 esterase inhibitor, demonstrating a regulatory function of C1 esterase inhibitor in the C1s-induced cleavage of beta 2-microglobulin.

Amino Acid Sequence↗

PubMed1990

Purification and characterization of a low and a high molecular weight form of epidermal growth factor from rat urine.

Two forms of epidermal growth factor (EGF) have been purified to homogeneity from rat urine by immunoaffinity chromatography and gel filtration. For one of the purified peptides the molecular mass has been determined to be 5891 by mass spectrometry. This peptide consists of 51 amino acid residues. The sequence of the first 48 amino acid residues is identical to the previously published sequence for submandibular rat EGF. The C-terminal three residues (49-51) are Trp-Trp-Lys. The other purified peptide has a molecular mass of 45 kDa as determined by SDS-polyacrylamide gel electrophoresis. The N-terminal sequence is Asn-Tyr-Lys-Asp-(Cys)-Gly-Pro-Gly-Gly-(Cys)-Gly-Ser-His-Ala. Both the high and the low molecular mass form of urinary rat EGF are able to bind to the human placenta receptor for EGF.

Amino Acid Sequence↗

PubMed1990

The gamma-carboxyglutamic acid domain of human factor VIIa is essential for its interaction with cell surface tissue factor.

Previous studies indicated that both plasma-derived and recombinant human factor VIIa specifically interacted with tissue factor on the surface of a human bladder carcinoma cell line (J82). In the presence of calcium ions, factor VIIa interacted with approximately 300,000 binding sites/cell with a dissociation constant (Kd) of 3.25 nM (Sakai, T., Lund-Hansen, T., Paborsky, L., Pedersen, A. H., and Kisiel, W. (1989) J. Biol. Chem. 264, 9980-9988). In this study, we compare recombinant human factor VIIa and a preparation of recombinant human factor VIIa lacking the gamma-carboxyglutamic acid domain (GD-rVIIa) with respect to their interaction with J82 cell surface tissue factor. Interaction of GD-rVIIa with J82 monolayers at 37 degrees C was specific, saturable, and exhibited a hyperbolic profile. Scatchard plots of the binding data obtained at 37 degrees C indicated a single class of binding sites for GD-rVIIa with a Kd value of 2.5 nM. GD-rVIIa interacted with about 10,000 binding sites/cell. In contrast to the tissue factor-specific binding observed for intact factor VIIa, specific binding of GD-rVIIa to the J82 cell surface was neither influenced by calcium nor blocked by prior incubation of the cells with polyclonal anti-tissue factor apoprotein IgG. In addition, cell-bound GD-rVIIa failed to activate human factor X. These results indicate that the gamma-carboxyglutamic acid domain of factor VIIa is essential for its interaction with cell surface tissue factor.

1-Carboxyglutamic Acid↗

PubMed1990

Isolation to homogeneity and partial characterization of a histo-blood group A defined Fuc alpha 1----2Gal alpha 1----3-N-acetylgalactosaminyltransferase from human lung tissue.

The soluble histo-blood group A glycosyltransferase (Fuc alpha 1----Gal alpha 1----3-N-acetylgalactosaminyltransferase) was purified approximately 600,000-fold to homogeneity from human lung tissue. The enzyme was solubilized in 1% Triton X-100, partially purified by affinity chromatography on Sepharose 4B, and eluted with UDP. Final purification was obtained by twice repeated fast protein liquid chromatography ion exchange (Mono STM) with NaCl gradient elution and reverse-phase chromatography (proRPC) with acetonitrile gradient elution. Identity of the purified protein was established by (i) demonstration of the putative A transferase protein only in affinity-purified extracts of A but not O individuals, and (ii) specific immunoprecipitation of enzyme activity and putative protein with monoclonal antibodies. Sodium dodecyl sulfate electrophoresis revealed a single protein band with apparent Mr of approximately 40,000 under both reducing and nonreducing conditions. Digestion with N-glycanase yielded a reduction in Mr of approximately 6,000 (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), suggesting that the A transferase is a glycoprotein with N-linked carbohydrate chains. Amino acid composition and N-terminal amino acid sequence of the intact transferase, as well as of peptides released by endolysyl peptidase digest or cyanogen bromide cleavage, are presented.

ABO Blood-Group System↗

PubMed1990

Characterisation of neurotensin-immunoreactivity in porcine ileum using region-specific radioimmunoassays and chromatographic fractionation: isolation and primary structure of porcine neurotensin.

1. Neurotensin-immunoreactivity has been characterised in porcine ileum using region-specific radioimmunoassay coupled to chromatographic fractionation. 2. Two immunoreactive peptides were identified. 3. Peptide 1 was immunochemically, chromatographically and structurally identical to bovine neurotensin. 4. Peptide 2 exhibited novel immunochemical and chromatographic characteristics and represents a new neurotensin-related peptide.

Amino Acid Sequence↗

PubMed1990

Epidermal growth factor and haptocorrin in nasal secretion.

We report that nasal secretion contains the growth-promoting peptide epidermal growth factor (EGF) and a cobalamin-binding protein. Based on its amino-terminal sequence the cobalamin binding protein is classified as haptocorrin (HC). We have investigated the concentration of these components, the volume secreted, and the concentration of total protein after nasal challenge with methacholine, methacholine + ephedrine, histamine and serotonin. The EGF and HC concentrations do not differ for the four challenge agents employed. Range and median for all values obtained are 0.1-2.5 (0.7) nmol/l (n = 49) for EGF and 50-405 (190) nmol/l (n = 63) for HC. After challenge with methacholine the amount of secretion (80-630 (200) mg (n = 14) and the protein concentration (1.5-7.8 (3.5) g/l (n = 14] are lower than for any other challenge. After challenge with serotonin or histamine the amount secreted is 60-1125 (480) mg (n = 39). The concentration of protein is significantly higher (p less than 0.01) after challenge with serotonin (1.4-56.0 (17.0) g/l (n = 18] than after challenge with histamine (2.0-25.0 (5.0) g/l (n = 20].

Adult↗

PubMed1990

Pancreatic spasmolytic polypeptide, a potential growth factor for the intestine: neural control of secretion.

We studied the effect of electrical stimulation of the vagus nerves and the effect of vasoactive intestinal polypeptide (VIP) on the secretion of pancreatic spasmolytic polypeptide (PSP) from isolated perfused porcine pancreas. We measured the concentration of PSP in the pancreatic juice and in the venous effluent by radioimmunoassay. The concentration in the pancreatic juice varied between 1 and 180 micrograms/ml, and in the venous effluent between 1 and 10 ng/ml. PSP is thus mainly an exocrine product. However, the concentrations in juice and venous effluent varied in parallel. Electrical vagus stimulation increased the output in the juice of PSP approximately 30 times. Atropine (10(-6) M) prevented the increase in PSP concentration during vagus stimulation, but only partially inhibited the output. VIP (10(-8) M) increased the output of PSP but decreased the concentration. We conclude from these results that PSP secretion is controlled by neural parasympathetic mechanisms that include both cholinergic and peptidergic pathways.

Animals↗

PubMed1989

Revised amino acid sequence of pancreatic spasmolytic polypeptide exhibits greater similarity with an inducible pS2 peptide found in a human breast cancer cell line.

The published amino acid sequence of pancreatic spasmolytic polypeptide (Thim, L., Thomsen, J., Christensen, M. and Jørgensen, K.H. et al. (1985) Biochim. Biophys. Acta 827, 410-418) has been checked by a combination of mass spectroscopy and Edman degradation. The pyroglutamyl blocking group was positively identified, and residue assignments at four positions were corrected: Lys48 (not Ser), Ser63 (not Lys), Cys68 (not Ser) and Ser74 (not Cys). The revised sequence exhibits greater similarity with pS2 peptide, a 60 residue polypeptide which is induced by oestrogen in the human breast cancer cell line MCF-7 and found in malignant but not in non-malignant breast tissue.

Amino Acid Sequence↗

PubMed1989

Prosomatostatin 1-64 is a major product of somatostatin gene expression in pancreas and gut.

Prosomatostatin (pro-SS) is a peptide of 92 amino acids which contains the extensively studied somatostatin (SS) 1-28 and SS 1-14 at the C terminus. Little is known about the N-terminal part of pro-SS. In previous studies, using a radioimmunoassay against pro-SS 20-36 (sequence deduced from human cDNA sequence) we have identified a peptide with a molecular mass of approximately 8000 daltons in extracts of pancreas and intestinal mucosa. Using a variety of chromatographic procedures we have now isolated this peptide from extracts of pancreas and intestinal mucosa from pigs. The isolated peptides were sequenced on an Applied Biosystems gas phase sequenator and cleaved with the Asp-N endopeptidase for sequencing of C-terminal fragments. The peptides had an amino acid sequence identical to human pro-SS 1-64. In effluent from isolated perfused preparations of porcine small intestine and pancreas we identified upon appropriate stimulation pro-SS 20-36 immunoreactive peptides that by isocratic high pressure liquid chromatography appeared identical to pro-SS 1-64. An identical peptide was identified in pig plasma. Thus, in pancreas and gut pro-SS processing gives rise to the same pro-SS 1-64 molecule in spite of differential processing of the C terminus (SS 1-14 in pancreas and SS 1-28 in gut). The eventual hormonal role of pro-SS 1-64 may now be evaluated.

Amino Acid Sequence↗

PubMed1989

A new family of growth factor-like peptides. 'Trefoil' disulphide loop structures as a common feature in breast cancer associated peptide (pS2), pancreatic spasmolytic polypeptide (PSP), and frog skin peptides (spasmolysins).

Four peptides present in completely different biological sources have been shown to exhibit a large degree of structural similarity. The peptides include: (i) a 60 amino acid residue breast cancer associated pS2 peptide isolated from human gastric juice and the culture media of the human breast cancer cell line MCF-7; (ii) a 106 amino acid residue pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas and pancreatic juice; and (iii) a 49 and 50 amino acid residue peptide predicted from a cDNA isolated from the skin of the frog, Xenopus laevis. These peptides are characterized by having one (pS2 and the frog peptides) or two (PSP) domains of a highly conserved 38-39 amino acid residue consensus sequence not found in any other known peptides or proteins. The domain sequences contain 6 cysteine residues in nearly the same positions and it is suggested that these 6 residues are linked by 3 disulphide bonds to form a characteristic 'trefoil' disulphide loop structure common in all four peptides. From the sources of which the peptides have been isolated and from experiments showing that PSP has a growth factor stimulatory effect on MCF-7 cells, it is further suggested that these peptides may represent members of a new family of growth factors.

Amino Acid Sequence↗

PubMed1989

Growth stimulatory effect of pancreatic spasmolytic polypeptide on cultured colon and breast tumor cells.

The effects of a novel polypeptide, pancreatic spasmolytic polypeptide (PSP) on a colon carcinoma cell line (HCT 116) were examined. PSP stimulated the incorporation of [3H]thymidine into HCT 116 cells as well as cell proliferation in a dose-dependent manner. Maximal increase in [3H]thymidine incorporation of 50-60% occurred at 3-300 microM PSP. The VIP-mediated-increase in cAMP levels was reduced by PSP at greater than 1 microM concentrations. PSP is highly homologous to the estrogen-induced pS2 protein in MCF-7 breast cancer cells. We find that PSP also enhanced [3H]thymidine incorporation in MCF-7 cells. These findings indicate for the first time that PSP has growth stimulatory properties.

Animals↗

PubMed1989

Rhizomucor miehei triglyceride lipase is processed and secreted from transformed Aspergillus oryzae.

The cDNA encoding the precursor of the Rhizomucor miehei triglyceride lipase was inserted in an Aspergillus oryzae expression vector. In this vector the expression of the lipase cDNA is under control of the Aspergillus oryzae alpha-amylase gene promoter and the Aspergillus niger glucoamylase gene terminator. The recombinant plasmid was introduced into Aspergillus oryzae, and transformed colonies were selected and screened for lipase expression. Lipase-positive transformants were grown in a small fermentor, and recombinant triglyceride lipase was purified from the culture broth. The purified enzymatically active recombinant lipase (rRML) secreted from A. oryzae was shown to have the same characteristics with respect to mobility on reducing SDS-gels and amino acid composition as the native enzyme. N-terminal amino acid sequencing indicated that approximately 70% of the secreted rRML had the same N-terminal sequence as the native Rhizomucor miehei enzyme, whereas 30% of the secreted rRML was one amino acid residue shorter in the N-terminal. The recombinant lipase precursor, which has a 70 amino acid propeptide, is thus processed in and secreted from Aspergillus oryzae. We have hereby demonstrated the utility of this organism as a host for the production of recombinant triglyceride lipases.

Amino Acids↗

PubMed1989

Multiple molecular forms of insulin and glucagon-like peptide from the Pacific ratfish (Hydrolagus colliei).

The primary structure of an insulin isolated from the pancreas of the holocephalan fish, Hydrolagus colliei (Pacific ratfish), has been established as A-chain: GIVEQCCHNTCSLANLEGYCN B-chain: VPTQRLCGSHLVDALYFVCGERGFFYSPKPIRELEPLL. Three further molecular forms of insulin were also isolated and shown to have the same A-chain but truncated B-chains of 31-, 36-, and 37-amino acid residues. It is proposed that all four insulins arise from a single proinsulin by proteolytic cleavages at different sites within the C-peptide region. The insulin with 38 amino acids in the B-chain was equipotent with human insulin in inhibiting the binding of radiolabelled human insulin to rat fat cells but the maximum effect of ratfish insulin upon the transport of 3-O-methylglucose into the cells was only 65% of the maximum effect of human insulin. Two molecular forms of glucagon-like peptide were isolated from the ratfish pancreas. The primary structure of the more abundant peptide was established as HADGIYTSDVASLTDYLKSKRFVESLSNYNRKQND. The primary structure of the second peptide was the same except that it was extended from the C-terminus by the sequence RRM. It is probable, therefore, that both glucagon-like peptides also arise from a single proglucagon by different pathways of post-translational processing.

Amino Acid Sequence↗

PubMed1989

Isolation and primary structure of the C-peptide of proinsulin from the European eel (Anguilla anguilla).

1. The primary structure of the C-peptide of proinsulin from the European eel has been established as: DVEPLLGFLSPKSGQENEVDDFPYKGQGEL. The peptide was isolated from the extract of eel pancreas in a yield that was approximately equimolar with insulin. A comparison with the predicted structures of C-peptides from other teleost fishes has identified a domain in the central region of the peptide that has been more highly conserved than the rest of the molecule.

Amino Acid Sequence↗

PubMed1989

Quantitation and characterization of peptides from the C-terminal flanking region of rat and bovine preprotachykinins.

Sequence analysis of cDNAs has shown that the biosynthetic precursors of substance P (alpha-, beta-, and gamma-preprotachykinins) contain a common amino acid sequence in the C-terminal flanking region that has not been conserved between species. Antisera have been raised against the synthetic peptide Tyr-Glu-Arg-Ser-Ala-Met-Gln-Asn-Tyr-Glu, which represents rat beta-preprotachykinin-(117-126)-peptide, and used in radioimmunoassays. Antiserum R50 reacted strongly with C-flanking peptides in extracts of rat and bovine tissues whereas antiserum GP-4 reacted only with the rat peptides. The primary structure of the predominant molecular form of preprotachykinin C-flanking peptide in an extract of bovine corpus striatum was established as: Ala-Leu-Asn-Ser-Val5-Ala-Tyr-Glu-Arg-Ser10-Val-Met-Gln-Asp-Tyr1 5-Glu. This sequence represents beta-preprotachykinin-(111-126)-peptide which is equivalent to gamma-preprotachykinin-(96-111)-peptide. A C-flanking peptide with similar chromatographic properties was identified in extracts of rat brain and gut together with a second immunoreactive component that may represent a fragment or a posttranslationally modified variant. A peptide corresponding to the 37-amino-acid residue C-flanking peptide derived from alpha-preprotachykinin was not detected in the extracts as expected from the known low abundance of alpha-preprotachykinin mRNA in rat brain and gut.

Amino Acid Sequence↗

PubMed1988

Structural characterization of a high-molecular-mass form of calcitonin [procalcitonin-(60-116)-peptide] and its corresponding N-terminal flanking peptide [procalcitonin-(1-57)-peptide] in a human medullary thyroid carcinoma.

Four peptides derived from procalcitonin were isolated in high yield from an extract of a human medullary thyroid carcinoma. The peptides were identified as procalcitonin-(1-57)-peptide, procalcitonin-(60-91)-peptide (calcitonin), procalcitonin-(60-116)-peptide and procalcitonin-(96-116)-peptide (katacalcin). Determination of the amino acid sequence of procalcitonin-(1-57)-peptide has demonstrated that the Ala25-Ala26 bond in preprocalcitonin is the site of cleavage of the signal peptide. Procalcitonin-(60-116)-peptide represents calcitonin extended from its C-terminus by the sequence Gly-Lys-Lys-Arg-katacalcin, and its formation is indicative of an aberrant pathway of procalcitonin processing in the tumour cells.

Amino Acids↗

PubMed1988

Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells.

Blood coagulation factor VII is a vitamin K dependent glycoprotein which in its activated form, factor VIIa, participates in the coagulation process by activating factor X and/or factor IX in the presence of Ca2+ and tissue factor. Three types of potential posttranslational modifications exist in the human factor VIIa molecule, namely, 10 gamma-carboxylated, N-terminally located glutamic acid residues, 1 beta-hydroxylated aspartic acid residue, and 2 N-glycosylated asparagine residues. In the present study, the amino acid sequence and posttranslational modifications of recombinant factor VIIa as purified from the culture medium of a transfected baby hamster kidney cell line have been compared to human plasma factor VIIa. By use of HPLC, amino acid analysis, peptide mapping, and automated Edman degradations, the protein backbone of recombinant factor VIIa was found to be identical with human factor VIIa. Neither recombinant factor VIIa nor human plasma factor VIIa was found to contain beta-hydroxyaspartic acid. In human plasma factor VIIa, the 10 N-terminally located glutamic acid residues were found to be fully gamma-carboxylated whereas 9 full and 1 partial gamma-carboxylated residues were found in the corresponding positions of the recombinant factor VIIa molecule. Asparagine residues 145 and 322 were found to be fully N-glycosylated in human plasma factor VIIa. In the recombinant factor VIIa, asparagine residue 322 was fully glycosylated whereas asparagine residue 145 was only partially (approximately 66%) glycosylated. Besides minor differences in the sialic acid and fucose contents, the overall carbohydrate compositions were nearly identical in recombinant factor VIIa and human plasma factor VIIa.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1988

Post-translational processing of preprotachykinins. Isolation of protachykinin-(1-37)-peptide from human adrenal-medullary phaeochromocytoma tissue.

The biosynthetic precursors of the mammalian tachykinins, alpha-, beta-and gamma-preprotachykinins, contain a common N-terminal region of 74 amino acids. A polyclonal antiserum was raised against a synthetic peptide representing N-tyrosylated beta-preprotachykinin-(48-56)-peptide as predicted from the nucleotide sequence of cloned DNA complementary to human beta-preprotachykinin mRNA. By using this antiserum in radioimmunoassay, a single immunoreactive peptide was identified in an extract of a human pheochromocytoma that produced substance P and neurokinin A. Partial microsequencing and determination of the amino acid composition of the peptide indicated identity with preprotachykinin-(20-56)-peptide. Thus the data demonstrate that the Ala19-Glu20 bond in preprotachykinin is the site of cleavage of the signal peptide.

Adrenal Gland Neoplasms↗