Immunochemical and biological characterization of calcitonin originating from transplanted medullary thyroid carcinoma in rats

1982 ◽  
Vol 99 (3) ◽  
pp. 397-403 ◽  
Author(s):  
Lisbeth Myhre ◽  
Arne Ekeland ◽  
Kaare M. Gautvik
Keyword(s):  
Biological Activities ◽  
Gel Filtration ◽  
Molecular Size ◽  
Serum Levels ◽  
Biologically Active ◽  
Subcutaneous Injections ◽  
Medullary Thyroid ◽  
Biological Potency ◽  
Medullary Thyroid Carcinomas

Abstract. The immunoreactive and biological activities of calcitonin (CT) produced by transplanted rat medullary thyroid carcinomas (MCT) have been studied. Immunoreactive CT (iCT) in serum and in MCT tissues of rats carrying tumours of generations 5–8 was characterized by gel filtration on Sephadex G-100 followed by radioimmunological measurements using region specific antiserum. The hypocalcaemic effect of sera and tumour extracts was tested in a rat bioassay. Rats with transplanted MCT of the 5th and 6th generations had mainly (70–84%) circulating iCT species of molecular size comparable to intact hormone. However, in rats with tumours of the 7th and 8th generations corresponding circulating iCT forms comprised less than 52% of total immunoreactivity while 32–38% eluted earlier. In conparison, iCT corresponding to intact hormone represented 30–50% of total immunoreactivity in the tumour extracts and no differences were observed between the generations. Subcutaneous injections of sera from MCT rats and of tumour extracts reduced the serum levels of ionized calcium in test rats. The sera containing mainly intact iCT showed the strongest biological potency. We conclude that rat MCT transplanted under the kidney capsule is able to secrete biologically active CT. However, the heterogeneity of circulating iCT increases in rats with transplanted tumours of older generations, approaching the heterogeneity of stored hormone in the gland.

Analogues of the cholecystokinin octapeptide modified in the central part of the molecule

1991 ◽  
Vol 56 (9) ◽  
pp. 1963-1970 ◽  
Author(s):  
Jan Hlaváček ◽  
Václav Čeřovský ◽  
Jana Pírková ◽  
Pavel Majer ◽  
Lenka Maletínská ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Point Of View ◽  
Biologically Active ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Cholecystokinin Octapeptide ◽  
Biological Tests ◽  
Biological Potency ◽  
Biologically Active Conformation

In a series of analogues of the cholecystokinin octapeptide (CCK-8) the amino acid residues were gradually modified by substituting Gly by Pro in position 4, Trp by His in position 5, Met by Cle in position 6, or the Gly residue was inserted between Tyr and Met in positions 2 and 3 of the peptide chain, and in the case of the cholecystokinin heptapeptide (CCK-7) the Met residues were substituted by Nle or Aib. These peptides were investigated from the point of view of their biological potency in the peripheral and central region. From the results of the biological tests it follows that the modifications carried out in these analogues and in their Nα-Boc derivatives mean a suppression of the investigated biological activities by 2-3 orders of magnitude (at a maximum dose of the tested substance of 2 . 10-2 mg per animal).This means that a disturbance of the assumed biologically active conformation of CCK-8, connected with a considerable decrease of the biological potency of the molecule, takes place not only after introduction of the side chain into its centre (substitution of Gly4), but also after the modification of the side chains of the amino acids or by extension of the backbone in further positions around this central amino acid.

Purification and characterization of tuberculin-active components from BCG

1975 ◽  
Vol 21 (12) ◽  
pp. 2019-2027
Author(s):  
M. Laguerre ◽  
R. Turcotte
Keyword(s):  
Physicochemical Properties ◽  
Guinea Pigs ◽  
Gel Filtration ◽  
Biologically Active ◽  
Polyacrylamide Gels ◽  
Active Components ◽  
Purification And Characterization ◽  
Molecular Weights ◽  
Antigenic Activity

The tuberculin activity of protoplasmic extracts isolated from living BCG was purified successively by gel filtration on Sephadex G-100 and G-75, and by electrophoresis on 7.5% and on gradient (6–18%) polyacrylamide gels. The tuberculin-active fractions, as determined in BCG-sensitized guinea pigs, were used as the starting material for each of the following fractionation steps.The physicochemical properties and the antigenic activity of the biologically active fractions have shown that a single component, or only a few ones with similar properties, possessed high tuberculin activity. These active components were proteins having relatively high molecular weights (about 72 000) and could behave as antigens.

scholarly journals Isolation and characterization of a novel haemoprotein b-559 from Bengal gram (Cicer arietinum)

1987 ◽  
Vol 243 (3) ◽  
pp. 723-728 ◽  
Author(s):  
C S Ramadoss ◽  
B C Shenoy ◽  
A Borthakur
Keyword(s):  
Gel Filtration ◽  
Molecular Size ◽  
Soret Band ◽  
Beta Band ◽  
Photosynthetic Membrane ◽  
Isolation And Characterization ◽  
Bengal Gram ◽  
Apparent Homogeneity ◽  
Reduced State

A haemoprotein was purified to apparent homogeneity from Bengal-gram seeds. The purified protein exhibited an absorption maximum at 412 nm (Soret band) that upon reduction with dithionite gave rise to a shift in the Soret band to 426 nm with concomitant appearance of an alpha-band at 559 nm and a beta-band at 530 nm. In the reduced state the Bengal-gram haemoprotein showed reactivity towards CO, nitrite and hydroxylamine. SDS/polyacrylamide-slab-gel electrophoresis showed that the haemoprotein has Mr 78,000. Gel-filtration and ultracentrifugal analyses suggest that the Bengal-gram haemoprotein is oligomeric in nature. Since it differs from photosynthetic membrane cytochrome b-559 in solubility in buffer, in reactivity towards CO and in molecular size, it appears to be a novel haemoprotein b-559.

scholarly journals Characterization of a potent human interleukin-11 agonist

2003 ◽  
Vol 375 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Dimitri HARMEGNIES ◽  
Xiao-Ming WANG ◽  
Paul VANDENBUSSCHE ◽  
Arnaud LEON ◽  
Patricia VUSIO ◽  
...  
Keyword(s):  
Biological Activities ◽  
Gel Filtration ◽  
Electrophoretic Analysis ◽  
Receptor Complex ◽  
Wild Type ◽  
Α Subunit ◽  
Receptor Chain ◽  
Interleukin 11 ◽  
New Protein

Human interleukin-11 (hIL-11) is a multi-potential cytokine that is involved in numerous biological activities, such as haematopoiesis, osteoclastogenesis, neurogenesis and female fertility, and also displays anti-inflammatory properties. IL-11 is used clinically to treat chemotherapy-induced thrombocytopenia. Because of its broad spectrum of action, improved IL-11 agonists, as well as IL-11 antagonists, could be of interest for numerous clinical applications. IL-11 signalling is dependent on the formation of a tripartite ligand–receptor complex consisting of IL-11, the IL-11R (IL-11 receptor) α subunit (responsible for the specificity of the interaction) and gp130 (glycoprotein 130) receptor β subunit (responsible for signal transduction). The interaction between IL-11 and IL-11Rα subunit occurs at its recently assigned site I. We have designed an IL-11 mutein whose hydrophobicity at site I has been increased. The mutein has been characterized in terms of structure, affinity, specificity and bioactivity. Electrophoretic analysis, gel filtration, IR spectroscopy and CD indicate that this new protein is more compact than wild-type IL-11. It binds to IL-11Rα with a three-fold-enhanced affinity, and retains the ability to recruit gp130 through site II. However, analysis of its biological activity revealed a complex pattern: although this mutein is 60–400-fold more active than wild-type IL-11 on the proliferation of 7TD1 murine hybridoma cell, it is less active than IL-11 on the proliferation of B9 cells, another murine hybridoma cell line. The results are interpreted on the basis of an IL-11 conformational change induced by the mutations, and the preferential use by the mutein of another unknown transducing receptor chain.

scholarly journals Solubilization and partial characterization of rat epididymal Δ 4-steroid 5 α-reductase (cholestenone 5 α-reductase)

1983 ◽  
Vol 211 (1) ◽  
pp. 65-74 ◽  
Author(s):  
H Scheer ◽  
B Robaire
Keyword(s):  
Reductase Activity ◽  
Sedimentation Coefficient ◽  
Molecular Size ◽  
Enzymic Activity ◽  
Subcellular Fractions ◽  
Biologically Active ◽  
Stable Form ◽  
Membrane Bound ◽  
Relative Capacity

Epididymal delta 4-steroid 5 alpha-reductase (cholestenone 5 alpha-reductase), the enzyme that catalyses the conversion of testosterone into the biologically active metabolite dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one), is a membrane-bound enzyme found in both nuclear and microsomal subcellular fractions. In order to characterize epididymal delta 4-steroid 5 alpha-reductase, it was first necessary to solubilize the enzymic activity. Of the various treatments tested, a combination of 0.5% (w/v) Lubrol WX, 0.1 M-sodium citrate and 0.1 M-KCl maintained enzymic activity at control values and solubilized 66% of total epididymal delta 4-steroid 5 alpha-reductase activity in an active and stable form. The sedimentation coefficient of solubilized delta 4-steroid 5 alpha-reductase, as determined in continuous sucrose density gradients, was greater for the microsomal than for the nuclear enzyme (11.6S compared with 10.1S). Although the apparent Km values of the enzyme for testosterone were similar in nuclear and microsomal subcellular fractions (range 1.75 × 10(-7) × 4.52 × 10(-7)M), the apparent Km of the enzyme for NADPH was about 30-fold greater for the microsomal enzyme than for the nuclear enzyme. The apparent Km of the enzyme for either substrate was not significantly altered after solubilization. The relative capacity of steroids to inhibit the enzymic activity, the pH optima and the effects of Ca2+ and Mg2+ were similar for membrane-bound and solubilized delta 4-steroid 5 alpha-reductase in both the nuclear and the microsomal fractions. The results reported demonstrate that epididymal delta 4-steroid 5 alpha-reductase can be solubilized in an active and stable form with no significant changes in the kinetic characteristics of the enzyme after solubilization; furthermore, kinetic and molecular-size differences observed for the nuclear and the microsomal forms of the enzyme suggest that there may exist at least two forms of epididymal delta 4-steroid 5 alpha-reductase.

scholarly journals Production and purification of biologically active recombinant tilapia (Oreochromis niloticus) prolactins

1991 ◽  
Vol 131 (2) ◽  
pp. 219-NP ◽  
Author(s):  
D. Swennen ◽  
F. Rentier-Delrue ◽  
B. Auperin ◽  
P. Prunet ◽  
G. Flik ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Recombinant Expression ◽  
Biological Activities ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Significant Rise ◽  
Biologically Active ◽  
Expression Vectors ◽  
Reducing Conditions ◽  
Natural Hormone

ABSTRACT Recombinant expression vectors carrying tilapia prolactin-I or -II (tiPRL-I or tiPRL-II) cDNA were constructed and the tiPRL-I and II proteins were produced in E. coli as inclusion bodies. These inclusion bodies were dissolved in 6 mol urea/1. Refolding of the proteins was followed by SDS-PAGE under non-reducing conditions so as to visualize the oxidized state of the molecules. Proteins tiPRL-I and tiPRL-II were purified by gel filtration and ion-exchange chromatography. The N-terminal sequence and bioactivities of both purified proteins were then analysed. Recombinant tiPRL-I and tiPRL-II induced a significant rise in plasma calcium levels as well as in mucocyte density in the abdominal skin epithelium. When tested on kidney membrane, both proteins exhibited potency in competing with 125I-labelled tiPRL-I for binding sites, but tiPRL-I seemed to be more potent than tiPRL-II in competing for these sites. The results obtained for the biological activities tested suggest that both recombinant prolactins were correctly refolded and had retained the full biological activity previously observed with the natural hormone preparations extracted from the animals. Journal of Endocrinology (1991) 131, 219–227

Characterization of immunoreactive dynorphin in human phaeochromocytomas

1988 ◽  
Vol 117 (1) ◽  
pp. 123-132 ◽  
Author(s):  
T. A. Howlett ◽  
G. M. Besser ◽  
L. H. Rees
Keyword(s):  
Acetic Acid ◽  
Gel Filtration ◽  
Molecular Size ◽  
Major Peak ◽  
Gel Filtration Chromatography ◽  
Post Translational Modifications ◽  
Minor Peak ◽  
Wet Weight ◽  
A Minor

ABSTRACT The prodynorphin-derived opioids, dynorphin (DYN) and α-neoendorphin (αNE) were studied in 24 human phaeochromocytomas and related tumours. Nineteen tumours, extracted in HCl (0·1 mol/l), contained concentrations of immunoreactive DYN (ir-DYN) ranging from < 0·5 to 794 pmol/g wet weight. None of the extracts in HCl contained ir-αNE (all < 2·4 pmol/g). Sephadex G-50 gel filtration chromatography of ir-DYN in HCl (0·1 mol/l) extracts of six tumours revealed three small peaks of ir-DYN of higher molecular size (approximately 12 000, 6000 and 3000 daltons), a minor peak of ir-DYN eluting just after DYN(1–17), and a broad major peak, consisting of at least three components, which was significantly retarded and eluted after the salt volume of the column. High-pressure liquid chromatography (HPLC) of these extracts revealed multiple peaks of ir-DYN, most of which did not coelute with any synthetic DYN peptides. On both gel filtration chromatography and HPLC, one of the minor peaks coeluted with DYN(1–32). None of the peaks of ir-DYN coeluted with DYN(1–17) which had been acetylated using acetic anhydride. Extracts of the same tumours in acetic acid (0·1 mol/l) yielded similar values for ir-DYN content, but parallelism in the assay was improved. Sephadex G-50 chromatography revealed a different pattern of ir-DYN with a major peak coeluting with DYN(1–17) and, in two tumours, a minor peak coeluting with DYN(1–8). Studies with HPLC revealed, however, that substantial degradation of synthetic DYN occurred during extraction in acetic acid (0·1 mol/l) in spite of the precautions taken. Phaeochromocytomas frequently contain ir-DYN in concentrations which may approach that of the mammalian pituitary. These tumours did not, however, contain ir-αNE and, with the possible exception of a small amount of DYN(1–32), the ir-DYN present did not correspond with any known sequences. Thus, whilst prodynorphin is expressed in phaeochromocytomas, it does not seem to be processed to the usual end-products, and post-translational modifications therefore seem likely. Enzymatic degradation of DYN may occur during extraction in acetic acid (0·1 mol/l), and this medium should, therefore, be avoided in studies of such labile peptides. J. Endocr. (1988) 117, 123–132

scholarly journals The human gastrin precursor. Characterization of phosphorylated forms and fragments

1988 ◽  
Vol 256 (3) ◽  
pp. 951-957 ◽  
Author(s):  
A Varro ◽  
H Desmond ◽  
S Pauwels ◽  
H Gregory ◽  
J Young ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Phosphorylation Site ◽  
Gel Filtration ◽  
Biologically Active ◽  
Antral Mucosa ◽  
Tryptic Fragment ◽  
Preferential Cleavage ◽  
Acidic Peptide ◽  
Human Gastrin

There is a potential phosphorylation site in the C-terminal region of the precursor for the acid-stimulating hormone gastrin, which is immediately adjacent to an important cleavage point. In the present study we have sought to identify, separate, quantify and characterize phosphorylated and unphosphorylated forms of human progastrin and its fragments. Identification was made by two radioimmunoassays: (a) a novel assay employing an antibody raised to intact human progastrin; and (b) an assay using antibody reacting with the C-terminal tryptic fragment of human progastrin, as well as progastrin itself. Two forms of human progastrin isolated from a gastrinoma were separated by ion-exchange h.p.l.c., and had similar elution positions on reverse-phase h.p.l.c. and on gel filtration. The more acidic peptide contained close to equimolar amounts of phosphate. On trypsinization, peptides were released that co-eluted on ion-exchange h.p.l.c. with, and had the immunochemical properties of, naturally occurring C-terminal fragments of progastrin. One of the latter was isolated and shown by Edman degradation after derivatization with ethanethiol to have the sequence Ser (P)-Ala-Glu-Asp-Glu-Asn. Similar peptides occur in antral mucosa resected from ulcer patients. The unphosphorylated forms of progastrin predominated, whereas the phosphorylated forms of the C-terminal fragments were predominant. This distribution could be explained by preferential cleavage of phosphorylated progastrin. We conclude that in human progastrin, Ser-96 can occur in the phosphorylated form; this residue immediately follows a pair of basic residues (Arg-Arg) that are cleaved during synthesis of the biologically active product.

scholarly journals Functional and biophysical characterization of recombinant human hepatocyte growth factor isoforms produced in Escherichia coli

1997 ◽  
Vol 326 (3) ◽  
pp. 763-772 ◽  
Author(s):  
Stephen J. STAHL ◽  
Paul T. WINGFIELD ◽  
Joshua D. KAUFMAN ◽  
Lewis K. PANNELL ◽  
Vittoria CIOCE ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Simple Procedure ◽  
Gel Filtration ◽  
Biologically Active ◽  
High Yield ◽  
Detailed Structural Analysis ◽  
Hepatocyte Growth

Hepatocyte growth factor (HGF) is a pluripotent secreted protein that stimulates a wide array of cellular targets, including hepatocytes and other epithelial cells, melanocytes, endothelial and haematopoietic cells. Multiple mRNA species transcribed from a single HGF gene encode at least three distinct proteins: the full-length HGF protein and two truncated HGF isoforms that encompass the N-terminal (N) domain through kringle 1 (NK1) or through kringle 2 (NK2). We report the high-level expression in Escherichia coli of NK1 and NK2, as well as the individual kringle 1 (K1) and N domains of HGF. All proteins accumulated as insoluble aggregates that were solubilized, folded and purified in high yield using a simple procedure that included two gel-filtration steps. Characterization of the purified proteins indicated chemical and physical homogeneity, and analysis by CD suggested native conformations. Although the K1 and N-terminal domains of HGF have limited biological activity, spectroscopic evidence indicated that the conformation of each matched that observed when the domains were components of biologically active NK1. Both NK1 and NK2 produced in bacteria were functionally equivalent to proteins generated by eukaryotic systems, as indicated by mitogenicity, cell scatter, and receptor binding and activation assays. These data indicate that all four bacterially produced HGF derivatives are well suited for detailed structural analysis.

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