scholarly journals Post-translational processing of progastrin: inhibition of cleavage, phosphorylation and sulphation by brefeldin A

1993 ◽  
Vol 295 (3) ◽  
pp. 813-819 ◽  
Author(s):  
A Varro ◽  
G J Dockray
Keyword(s):  
Ion Exchange ◽  
Secretory Granule ◽  
Secretory Pathway ◽  
Specific Activity ◽  
Brefeldin A ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
Gastrin Cells ◽  
Tryptic Fragment ◽  
C Terminus

The precursor for the acid-stimulating hormone gastrin provides a useful model for studies of post-translational processing because defined sites of cleavage, amidation, sulphation and phosphorylation occur within a dodecapeptide sequence. The factors determining these post-translational processing events are still poorly understood. We have used brefeldin A, which disrupts transport from rough endoplasmic reticulum to the Golgi complex, to examine the mechanisms of cleavage, phosphorylation and sulphation of rat progastrin-derived peptides. Biosynthetic products were detected after immunoprecipitation using antibodies specific for the extreme C-terminus of progastrin, followed by reversed-phase and ion-exchange h.p.l.c. Gastrin cells incorporated [3H]tyrosine, [32P]phosphate and [35S]sulphate into both progastrin and its extreme C-terminal tryptic (nona-) peptide. Ion-exchange chromatography resolved four forms of the C-terminal tryptic fragment of progastrin which differed in whether they were phosphorylated at Ser96, sulphated at Tyr103, both or neither. The specific activity of [3H]tyrosine in the peak that was both phosphorylated and sulphated was higher than in the others. Brefeldin A inhibited the appearance of [3H]tyrosine-labelled C-terminal tryptic fragment but there was an accumulation of labelled progastrin and a peptide corresponding to the C-terminal 46 residues of progastrin. Brefeldin A also inhibited incorporation of 32P and 35S into both progastrin and its C-terminal fragment. Thus phosphorylation of Ser96, sulphation of Tyr103 and cleavage at Arg94-Arg95 depend on passage of newly synthesized progastrin along the secretory pathway; as brefeldin A is thought to act proximal to the trans-Golgi, these processing steps would appear to occur distal to this point. The data also indicate that the stores of unphosphorylated C-terminal tryptic fragment are not available for phosphorylation, implying that this modification occurs proximal to the secretory granule; cleavage is known to occur in the secretory granule which suggests that it occurs after phosphorylation.

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

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.

scholarly journals Isolation, in the intact state, of the pterin molybdenum cofactor from xanthine oxidase

1989 ◽  
Vol 263 (2) ◽  
pp. 477-483 ◽  
Author(s):  
J Deistung ◽  
R C Bray
Keyword(s):  
Aqueous Solution ◽  
Ion Exchange ◽  
Xanthine Oxidase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Molybdenum Cofactor ◽  
Anaerobic Conditions ◽  
Intact State

A procedure is described for isolation of the pterin molybdenum cofactor, in the active molybdenum-containing state, starting from purified milk xanthine oxidase. The method depends on the use of anaerobic-glove-cabinet techniques and on working in aqueous solution, in the presence of 1 mM-Na2S2O4. SDS was used to denature the protein, followed by ion-exchange chromatography and gel filtration. The cofactor, obtained at concentrations up to 0.5-1.0 mM, was fully active in the nit-1 assay [Hawkes & Bray (1984) Biochem. J. 214, 481-493], with a specific activity of 22 nmol of NO2-/min per pg-atom of Mo (with 15% molybdate-dependence). The Mr, determined by gel filtration, was about 610, consistent with the structure proposed by Kramer, Johnson, Ribeiro, Millington & Rajagopalan [(1987) J. Biol. Chem. 262, 16357-16363]. At pH 5.9, under anaerobic conditions, the cofactor was stable for at least 300 h at 20-25 degrees C.

scholarly journals Purification and Characterization of an Arginine Aminopeptidase from Lactobacillus sakei

2002 ◽  
Vol 68 (4) ◽  
pp. 1980-1987 ◽  
Author(s):  
Yolanda Sanz ◽  
Fidel Toldrá
Keyword(s):  
Specific Activity ◽  
Divalent Cations ◽  
Gel Filtration ◽  
Sulfhydryl Group ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Lactobacillus Sakei ◽  
C Terminus ◽  
Arginine Aminopeptidase

ABSTRACT An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from Lactobacillus sakei. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37°C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu2+, Hg2+, and Zn2+) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The Km values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 μM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for L. sakei in the meat environment.

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