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.

Modulation of posttranslational processing of gastrin precursor in dogs

1990 ◽  
Vol 258 (6) ◽  
pp. G904-G909 ◽  
Author(s):  
A. Varro ◽  
J. Nemeth ◽  
J. Bridson ◽  
J. Lonovics ◽  
G. J. Dockray
Keyword(s):  
Gel Filtration ◽  
Exchange Chromatography ◽  
Biologically Active ◽  
Metabolic Clearance ◽  
Posttranslational Processing ◽  
Antral Mucosa ◽  
Gastrin Cells ◽  
Tryptic Fragment ◽  
G Cells ◽  
Basal Plasma

The precursor of the acid-stimulating hormone gastrin is processed in pyloric antral gastrin cells by steps involving sulfation, phosphorylation, cleavage, and amidation. We describe here changes in posttranslational processing in dogs with a surgically excluded antrum; in the preparation we used there was an intact pylorus but antral mucosa was excluded from the normal influence of the luminal contents. Three to five months after the operation, basal plasma gastrin increased from 30.1 +/- 4.0 to 66.1 +/- 16.1 pmol/l, and concentrations of gastrin in the excluded mucosa were 9.23 +/- 1.75 compared with 3.2 +/- 0.56 nmol/g in control antral mucosa. Calculations based on the metabolic clearance rate and plasma and tissue gastrin concentrations suggest two-fold lower fractional release rates from the excluded G-cells compared with normal G-cells. Radioimmunoassay of tissue extracts using antisera specific for the extreme COOH-terminus of progastrin, for glycine-extended G-17, and for the COOH-terminus of G-17, combined with gel filtration and ion exchange chromatography, indicated normal endopeptidase cleavage of progastrin. However there was significantly reduced phosphorylation of the COOH-terminal tryptic fragment of progastrin, and there was also decreased conversion of Gly-extended intermediates to the biologically active COOH-terminally amidated forms of gastrin. Thus, in spite of hypergastrinaemia, the excluded antral mucosa showed evidence of decreased secretory rates associated with decreased progastrin phosphorylation and amidating enzyme activity. The results suggest that contact of antral mucosa with the luminal contents is able to modulate the posttranslational processing of progastrin and so determine the production of biologically active hormone.

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 Purification and characterization of human neuropeptide Y from adrenal-medullary phaeochromocytoma tissue

1984 ◽  
Vol 219 (3) ◽  
pp. 699-706 ◽  
Author(s):  
R Corder ◽  
P C Emson ◽  
P J Lowry
Keyword(s):  
Amino Acid ◽  
Neuropeptide Y ◽  
Amino Acid Analysis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Carboxypeptidase Y ◽  
Purification And Characterization ◽  
Reverse Phase ◽  
Tryptic Fragment

Human neuropeptide Y was isolated from acid extracts of adrenal-medullary phaeochromocytoma tissue. After (NH4)2SO4 fractionation, the neuropeptide Y-like immunoreactivity was purified from the resolubilized 80%-saturation-(NH4)2SO4 peptide-rich precipitate, by gel filtration, cation-exchange chromatography and reverse-phase high-pressure liquid chromatography. Amino acid analysis of the peptide revealed a composition almost identical with that of the pig peptide, the exception being the loss of one leucine residue and its replacement with methionine. Tryptic digestion of the peptide and subsequent amino acid analysis of the fragments further confirmed the identity of the peptide. Carboxypeptidase Y digestion of the (1-19)-peptide tryptic fragment has shown the methionine to be located at position 17 in human neuropeptide Y.

Enolase from Lobster (Homarus americanus)

1971 ◽  
Vol 28 (6) ◽  
pp. 879-882 ◽  
Author(s):  
M. John Chapman ◽  
Christopher Chin ◽  
Finn Wold
Keyword(s):  
Heat Treatment ◽  
Ion Exchange ◽  
Ammonium Sulfate ◽  
Catalytic Properties ◽  
Gel Filtration ◽  
Homarus Americanus ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Acetone Fractionation

Enolase has been isolated from lobster muscle by acetone fractionation, heat treatment, ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Preliminary characterization of the pure enzyme shows that the catalytic properties are very similar to those of the enolases from rabbit and fish.

scholarly journals Characterization of the antibodies detected by the microscopic agglutination test for bovine leptospirosis

1974 ◽  
Vol 73 (3) ◽  
pp. 425-432 ◽  
Author(s):  
J. A. Morris ◽  
S. N. Hussaini
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Agglutination Test ◽  
Microscopic Agglutination Test ◽  
Density Gradient Ultracentrifugation ◽  
Selective Precipitation ◽  
Bovine Leptospirosis

SummaryThe nature of the antibodies detectable by the microscopic agglutination test for bovine leptospirosis was examined. Density gradient ultracentrifugation, gel filtration and disulphide-bond-reduction experiments indicated that antileptospiral agglutinating activity was present in both IgM and IgG immunoglobulin fractions. This was confirmed by selective precipitation of specific antibody classes and ion-exchange chromatography.

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.

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.

scholarly journals A Boophilus microplus vitellin-degrading cysteine endopeptidase

Parasitology ◽  
2003 ◽  
Vol 126 (2) ◽  
pp. 155-163 ◽  
Author(s):  
A. SEIXAS ◽  
P. C. DOS SANTOS ◽  
F. F. VELLOSO ◽  
I. DA SILVA VAZ ◽  
A. MASUDA ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Boophilus Microplus ◽  
Hard Tick ◽  
Purification And Characterization ◽  
Basic Residue ◽  
Cysteine Endopeptidase ◽  
Sds Page

Here we describe the purification and characterization of a vitellin (VT) degrading cysteine endopeptidase (VTDCE) from eggs of the hard tick Boophilus microplus. A homogeneous enzyme preparation was obtained by chromatographic fractionation on ion-exchange and gel filtration columns and an autolysis step. This step consisted of incubation of a semipurified enzyme (after the first ion-exchange chromatography) at pH 4·0 that dissociated the enzyme from VT, to which VTDCE is naturally tightly associated. The enzyme purity was confirmed by capillary and native gel electrophoresis, and SDS–PAGE suggested the enzyme is a dimer of 17 and 22 kDa. VTDCE was active upon several synthetic substrates, with a preference for a hydrophobic or a basic residue in P1, and a hydrophobic residue in P2. VTDCE also hydrolysed haemoglobin, albumin, gelatin and vitellin. VTDCE is inactive in the absence of DTT and was totally inhibited by E-64, indicating it is a cysteine endopeptidase. Our results suggest that VTDCE is a major enzyme involved in yolk processing during B. microplus embryogenesis.

scholarly journals The purification and characterization of subcomponent C1s of the first component of bovine complement

1979 ◽  
Vol 183 (3) ◽  
pp. 579-588 ◽  
Author(s):  
R D Campbell ◽  
N A Booth ◽  
J E Fothergill
Keyword(s):  
Ion Exchange ◽  
Gel Electrophoresis ◽  
Molecular Sieve ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Equimolar Amount ◽  
Terminal Part ◽  
Purification And Characterization ◽  
Human Counterpart

Bovine C1s, a subcomponent of the first component of complement, was purified in good yield by a combination of euglobulin precipitation and ion-exchange and molecular-sieve chromatography. Approx. 10 mg can be obtained from 3 litres of serum, representing a yield of 11%. The C1s is obtained in zymogen form, with a mol.wt. of 85000-88000, determined by gel filtration and SDS/polyacrylamide-gel electrophoresis. It is haemolytically active when tested with human C1q and C1r. Activation can be achieved by incubation with human C1r, resulting in cleavage of the C1s chain into two chains of 65000 and 27000 mol.wt. and the generation of an isoleucine N-terminal residue on the smaller chain. Active C1s binds an equimolar amount of di-isopropyl phosphorfluoridate to the smaller chain, which is the C-terminal part in the zymogen. The chains can be separated by ion-exchange in 8 M-urea. All of these characteristics show that bovine C1s is very similar to its human counterpart.

scholarly journals PURIFICATION AND CHARACTERIZATION OF POLYGALACTURONASE OF `FUJI' APPLES.

HortScience ◽  
1992 ◽  
Vol 27 (6) ◽  
pp. 653e-653
Author(s):  
Seung-Ryeul Shin ◽  
Jae-Kyun Byun ◽  
Kyung-Ho Chang
Keyword(s):  
Ion Exchange ◽  
Column Chromatography ◽  
Malus Domestica ◽  
Gel Filtration ◽  
Reducing Sugar ◽  
Purification And Characterization ◽  
Stable Temperature ◽  
Ion Exchange Column ◽  
Optimum Ph

Polygalacturonase (PG) was purified from apple, Malus domestica Borkh, cv. Fuji by gel filtration, CM cellulose ion exchange column chromatography and characterized by means of several biochemical methods. Two forms of isozymes, PG-I and PG-II, were detected and the activities of PG-I were found to be higher than PG-II. The Km and Vmax values were calculated to be 1.54 mg/ml and 0.25 μM with reducing sugar 1 ml/30min., respectively. The PG was active between pH 3 and 8 with the optimum pH of about 4-5. The stable temperature for the PG was below 55°C with 30°C optimum. The PG activities were increased by Na* and Cu**, but were inhibited by Ag*, EDTA and SDS.

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