Proteolytic processing of secretory proteins in Paramecium: immunological and biochemical characterization of the precursors of trichocyst matrix proteins

1992 ◽  
Vol 103 (2) ◽  
pp. 349-361 ◽  
Author(s):  
S.J. Shih ◽  
D.L. Nelson
Keyword(s):  
Cross Sections ◽  
Disulfide Bonds ◽  
Biochemical Characterization ◽  
Rabbit Antiserum ◽  
Anion Exchange Chromatography ◽  
Cell Extract ◽  
Exchange Chromatography ◽  
Proteolytic Processing ◽  
Matrix Proteins ◽  
Secretory Proteins

We used polyclonal serum raised against mature trichocyst matrix proteins to detect their unprocessed precursors, a group of proteins (45-55 kDa) present in the whole-cell extract. These precursor proteins were partially purified from the soluble fraction of wild-type cells by ammonium sulfate precipitation and anion-exchange chromatography. Using monoclonal antibodies against each of four families of mature (processed) matrix proteins, we showed that each family was derived from a separate group of precursors. Our results also suggest that in three of four precursors, those in which the mature proteins consist of disulfide-linked heterodimers, intrachain disulfide bonds form before proteolytic processing. Purified precursors eluted from preparative SDS-gels were used to raise rabbit antiserum, which after preadsorption with mature processed proteins specifically recognized precursors, as judged by ELISA and immunoblots. In cross-sections of developing trichocysts, the anti-precursor serum after preadsorption no longer stained the central, paracrystalline region, but still stained the peripheral as well as the structureless region of the secretory granule. In trichocyst-developing mutants tl (trichless) and ftA (football A), the precursors for all four groups of mature proteins were present but their processing was affected: severely blocked in tl (which has no recognizable crystalline trichocyst matrix), and partially blocked in ftA (which has some abnormal trichocyst matrices with crystalline centers). These observations constitute further evidence that proteolytic processing of precursors occurs in parallel with crystallization.

scholarly journals Expression of TRH and TRH-like peptides in a human glioblastoma-astrocytoma cell line (U-373-MG)

2000 ◽  
Vol 166 (3) ◽  
pp. 697-703 ◽  
Author(s):  
SI Garcia ◽  
PI Porto ◽  
VN Martinez ◽  
AL Alvarez ◽  
S Finkielman ◽  
...  
Keyword(s):  
Cell Line ◽  
Anion Exchange Chromatography ◽  
Cell Extract ◽  
Exchange Chromatography ◽  
Human Glioblastoma ◽  
Cholinergic Agonist ◽  
Astrocytoma Cell Line ◽  
Astrocytoma Cell ◽  
A Cell ◽  
Positive Immunostaining

The human glioblastoma-astrocytoma cell line U-373-MG shows morphological features typical of its neuroectodermal origin. Cells showed positive immunostaining for the glial fibrillary acidic protein. We used this cell culture for studying the putative production of TRH and TRH-related peptides. In a cell extract and conditioned medium, cation and anion exchange chromatography and HPLC revealed the presence of TRH and acidic TRH-like peptides which were identified, at least in part, as pGlu-Glu-ProNH(2). These findings demonstrated that U-373-MG cells are able to produce and release these peptides. Further evidence of TRH synthesis was obtained by amplification using RT-PCR of a 396 bp fragment that corresponds to the TRH precursor mRNA. Our results therefore suggest that the U-373-MG cell line may be a useful model for studying the regulation of TRH and TRH-related peptide production and the interaction of these peptides with other classical neurotransmitter systems. In fact, pilocarpine (a muscarinic cholinergic agonist) enhanced and nicotine (a nicotinic cholinergic agonist) decreased TRH and TRH-related compound production by this cell line. These data also point out that glia may produce substances with neuromodulatory action.

Genetic and biochemical characterization of an exopolygalacturonase and a pectate lyase fromYersinia enterocolitica

1999 ◽  
Vol 45 (5) ◽  
pp. 396-403 ◽  
Author(s):  
Ching-Hsing Liao ◽  
Larry Revear ◽  
Arland Hotchkiss ◽  
Brett Savary
Keyword(s):  
High Performance ◽  
Biochemical Characterization ◽  
Amperometric Detection ◽  
Pectate Lyase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Human Pathogen ◽  
Gene Encoding ◽  
Reading Frame ◽  
Chromatography Analysis

Yersinia enterocolitica, an invasive foodborne human pathogen, degrades polypectate by producing two depolymerizing enzymes, pectate lyase (PL) and polygalacturonase (PG). The gene encoding the PG activity, designated pehY, was located in a 3-kb genomic fragment of Y. enterocolitica ATCC 49397. The complete nucleotide sequence of this 3-kb fragment was determined and an open reading frame consisting of 1803 bp was predicted to encode a PG protein with an estimated Mrof 66 kDa and pI of 6.3. The amino acid sequence of prePG showed 59 and 43% identity to that of the exopolygalacturonase (exoPG) of Erwinia chrysanthemi and Ralstonia solanacearum, respectively. The Y. enterocolitica PG overproduced in Escherichia coli was purified to near homogeneity using perfusion cation exchange chromatography. Analysis of the PG depolymerization products by high performance anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD) revealed the exolytic nature of this enzyme. The Y. enterocolitica PL overproduced in E. coli was also partially purified and the Mrand pI were estimated to be 55 kDa and 5.2, respectively. HPAEC-PAD analysis of the PL depolymerization products indicated the endolytic nature of this enzyme. Southern hybridization analyses revealed that pehY and pel genes of Y. enterocolitica are possibly encoded in the chromosome rather than in the plasmid. Purified exopolygalacturonase (over 10 activity units) was unable to macerate plant tissues.Key words: pectinase activities, human pathogen, HPLC analysis, pehY gene.

scholarly journals Purification and Characterization of a Prolyl Aminopeptidase from Debaryomyces hansenii

2003 ◽  
Vol 69 (1) ◽  
pp. 227-232 ◽  
Author(s):  
Tomás Bolumar ◽  
Yolanda Sanz ◽  
M.-Concepción Aristoy ◽  
Fidel Toldrá
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Debaryomyces Hansenii ◽  
Cysteine Proteases ◽  
Anion Exchange Chromatography ◽  
Cell Extract ◽  
Exchange Chromatography ◽  
Prolyl Aminopeptidase ◽  
Ph Range ◽  
Meat Fermentation

ABSTRACT A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of Debaryomyces hansenii CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45°C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate, p-chloromercuribenzoic acid, and Hg2+, which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The Km for proline-7-amido-4-methylcoumarin was 40 μM. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about D. hansenii proteases and their possible roles in meat fermentation.

scholarly journals Evidence for defects in membrane traffic in Paramecium secretory mutants unable to produce functional storage granules

1994 ◽  
Vol 124 (6) ◽  
pp. 893-902 ◽  
Author(s):  
MC Gautier ◽  
N Garreau de Loubresse ◽  
L Madeddu ◽  
L Sperling
Keyword(s):  
Intracellular Transport ◽  
Proteolytic Processing ◽  
Matrix Proteins ◽  
Morphological Data ◽  
Secretory Proteins ◽  
Wild Type ◽  
Intracellular Traffic ◽  
Storage Granules ◽  
Precursor Molecules ◽  
Mutant Cells

The ciliated protozoan Paramecium has a regulated secretory system amenable to genetic analysis. The secretory storage granules, known as trichocysts, enclose a crystalline matrix with a genetically determined shape whose biogenesis involves proteolytic maturation of a family of precursor molecules into a heterogeneous set of small acidic polypeptides that crystallize within the maturing vesicles. We have developed an original pulse-chase protocol for monoxenic Paramecium cultures using radiolabeled bacteria to study the processing of trichocyst matrix proteins in wild-type and mutant cells. In wild-type cells, proteolytic processing is blocked in the presence of monensin and otherwise rapidly completed after approximately 20 min of chase, suggesting that the conversion occurs in the trans-Golgi and/or in small vesicles soon after sorting to the regulated pathway, probably before crystallization begins. In trichless mutant cells, which contain no visible trichocysts, secretory proteins are synthesized but not processed and we report constitutive secretion of the uncleaved precursor molecules. The mutation thus appears to affect sorting to the regulated pathway and should prove useful for analysis of the sorting machinery and of the relationship between sorting and proteolytic processing of secretory proteins. In mutants bearing misshapen trichocysts with poorly crystallized contents (tam33, tam38, stubbyA), the proteolytic processing of the trichocyst matrix proteins appears to be normal, while both pulse-chase and morphological data indicate that intracellular transport is perturbed, probably between ER and Golgi. Precursor molecules are present in the mutant trichocysts but not in wild-type trichocysts and may account for the defective crystallization. Our analysis of these mutants suggests that the temporal coordination of intracellular traffic plays a regulatory role in granule maturation.

scholarly journals Growth and form of secretory granules involves stepwise assembly but not differential sorting of a family of secretory proteins in Paramecium

2001 ◽  
Vol 114 (5) ◽  
pp. 875-886 ◽  
Author(s):  
L. Vayssie ◽  
N. Garreau de Loubresse ◽  
L. Sperling
Keyword(s):  
Multigene Family ◽  
Secretory Granules ◽  
Three Dimensional ◽  
Proteolytic Processing ◽  
Matrix Proteins ◽  
Secretory Proteins ◽  
Synthetic Genes ◽  
Double Label ◽  
Stepwise Assembly ◽  
Crystalline Array

Paramecium trichocysts are voluminous secretory vesicles consisting of a spindle-shaped body surmounted by a tip that serves to anchor them at exocytotic sites in the plasma membrane. This constrained shape is conferred by the proteins stored in the vesicles, which form an insoluble three-dimensional crystalline array. The constituent polypeptides (Trichocyst Matrix Proteins, TMPs), which assemble during trichocyst biogenesis, are produced by proteolytic processing of soluble proproteins encoded by a large multigene family. In order to investigate the functional significance of the TMP multigene family, which assures the synthesis of a mixture of related polypeptides, we have designed synthetic genes for heterologous expression of three different mature polypeptides, which were used to obtain sequence-specific rabbit antisera. We used these antisera to carry out immunolocalization experiments with wild-type trichocysts at different stages of development and found that the trichocyst matrix consists of two concentric layers containing different TMPs, and that the assembly of each layer corresponds to a distinct phase of trichocyst growth. Examination of mutant trichocysts created by targeted gene silencing of different TMP genes showed that the layer containing the products of the silenced genes is specifically affected, as are all subsequently assembled parts of the structure, consistent with an ordered assembly pathway. This stepwise assembly is not controlled by differential sorting of the TMPs, as single and double label experiments provided evidence that the different TMPs are delivered together to post-Golgi vesicles and developing trichocysts. We present a model for trichocyst biogenesis in which TMP assembly is controlled by protein processing.

scholarly journals PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF A T/TN SPECIFIC LECTIN FROM LEPECHINIA BULLATA SEEDS (LAMIACEAE)

2017 ◽  
Vol 9 (10) ◽  
pp. 165
Author(s):  
Wilches Torres A. ◽  
Rojas Caraballo J. ◽  
Sanabria E. ◽  
Reyes MontaÑo E ◽  
FernÁndez Alonso Jl ◽  
...  
Keyword(s):  
Isoelectric Focusing ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reducing Conditions ◽  
Sds Page ◽  
Unique Band

Objective: This study focused on purifying and characterizing a lectin from Lepechinia bullata (L. bullata) seeds, and determining its specificity towards tumour-associated carbohydrate-antigens.Methods: Pigments were removed by washing the seeds with NH4OH 0.1 M pH 9.4 and treating the crude extracts with Pectinex®. The purification procedure consisted of anion exchange chromatography on diethylaminoethyl (DEAE)-Sephadex followed by affinity chromatography. For the characterization, the phase was used polyacrylamide gel electrophoresis-sodium dodecyl sulphate (SDS-PAGE), isoelectric focusing, hemagglutination assays, enzyme-linked lectinosorbent assay (ELLA) and thermal shift assay (TSA).Results: 6.2 mg of lectin were obtained from 100 g of seeds. It was able to agglutinate enzymatically treated erythrocytes with a minimal required lectin concentration of 7 μg. ml-1. Strong binding to asialo bovine submaxillary mucine (aBSM) was determined, corroborating Tn recognition.The isoelectric focusing showed a unique band at pH 8.5. Lectin pure shown bands at 28, 48 and 93 kDa by SDS-PAGE, with an incomplete dissociation of the last species despite trying several reduction conditions. By preparative electrophoresis under different conditions, three species were observed too, in all fractions one band at 28 kDa on Tricine-PAGE in reducing and no reducing conditions were found.Amino acid composition, carbohydrate content, thermal stability and Ca2+and Mn2+requirements were determined. N-acetylgalactosamine (GalNAc) and desialylated mucins inhibited the agglutinant activity on human cells. Fetuin inhibited hemagglutination of rabbit erythrocytes.Conclusion: A new lectin was isolated and characterized from L. bullata seeds, it recognizes T/Tn antigen and shows some similarities with other Lamiaceae lectins.

Purification, Biochemical Characterization and Decolorization Efficiency of Laccases from Peach and Cherry Cultures of Pleutorus eryngii: A Comparative Study

2020 ◽  
Vol 27 (7) ◽  
pp. 623-634 ◽  
Author(s):  
Merve Akpinar ◽  
Raziye Ozturk Urek
Keyword(s):  
Biochemical Characterization ◽  
Gel Filtration ◽  
Industrial Effluents ◽  
Anion Exchange Chromatography ◽  
Biochemical Properties ◽  
Exchange Chromatography ◽  
Kinetic Characteristics ◽  
Chemical Agents ◽  
Decolorization Efficiency

Background:: Laccases (Lacs) are used potentially in industrial and biotechnological applications such as decolorization of dyes, degradation of industrial effluents, delignification, etc. thanks to their large varieties of substrate specificities and excellent catalytic efficiencies. The efficient utilizations of Lacs in these applications mostly depend on the identifying their biochemical properties. Objective: The goal of this research is to investigate the purification, biochemical characterization and decolorization efficiencies of Lacs. Methods: Pleurotus eryngii was incubated on peach (PC) and cherry (CC) wastes under optimized solid state fermentation conditions. Then, the enzymes extracts were purified by ammonium sulfate precipitation, anion exchange chromatography, gel filtration, respectively. Lacs fractions were subjected to electrophoretic analyses as well as their structural and kinetic characteristics. Also, the effects of selected chemical agents on purified Lacs activities and determination of decolorization efficiencies were studied. Results: As the results of purification processes of Lacs from both cultures, 3.94-fold purification was obtained for PC, while it was 5.34 for CC. The electrophoretic results of purified Lacs illustrated the single bands of protein (30±1 kDa) in accordance with the results after gel filtration. The Km values of Lacs from PC and CC were respectively detected as 1.1381 and 0.329 mM for ABTS. The selected agents partially/completely inhibited Lac activities. The highest decolorization efficiencies of purified Lacs from PC and CC were separately obtained as 53 and 11.8%. Conclusion: The results clearly indicated that the performances of Lacs from both cultures in decolorization application are different from each other depending their activities, biochemical and kinetic characteristics.

Functional reconstitution of an eicosanoid-modulated Cl− channel from bovine tracheal smooth muscle

2002 ◽  
Vol 282 (3) ◽  
pp. C567-C577 ◽  
Author(s):  
Dany Salvail ◽  
Martin Cloutier ◽  
Eric Rousseau
Keyword(s):  
Inhibitory Concentration ◽  
Biochemical Characterization ◽  
Anion Exchange Chromatography ◽  
Biochemical Properties ◽  
Exchange Chromatography ◽  
Dependent Manner ◽  
Open Probability ◽  
Concentration Dependent Manner ◽  
Lipid Environment ◽  
Channel Control

We describe the biochemical properties of an eicosanoid-modulated Cl−channel and assess the mechanisms by which the epoxyeicosatrienoic acids (EETs) alter both its unitary conductance and its open probability ( P o). After a purification protocol involving wheat-germ agglutinin affinity and anion-exchange chromatography, the proteins were sequentially inserted into liposomes, which were then fused into PLBs. Functional and biochemical characterization tests confirm that the Cl− channel is a 55-kDa glycosylated monomer with voltage- and Ca2+concentration-independent activity. 5,6- and 8,9-EET decreased the conductance of the native channel (control conductance: 70 ± 5 pS in asymmetrical 50 mM trans/250 mM cis CsCl) in a concentration-dependent manner, with respective 50% inhibitory concentration values of 0.31 and 0.42 μM. These regioisomers similarly decreased the conductance of the purified channel (control conductance value: 75 ± 5 pS in asymmetrical 50 mM trans/250 mM cis CsCl), which had been stripped of its native proteic and lipidic environment. On the other hand, 5,6- and 8,9-EETs decreased the P o of the native channel with respective 50% inhibitory concentration values of 0.27 and 0.30 μM but failed to alter the P o of the purified protein. Thus we suggest that the effects of these EETs on channel conductance likely result from direct interactions of EET− anions with the channel pore, whereas the alteration of P o requires a lipid environment of specific composition that is lost on solubilization and purification of the protein.

Sign in / Sign up

Export Citation Format

Share Document