scholarly journals Characterization of the 70 kDa polypeptide of the Na/Ca exchanger

1999 ◽  
Vol 338 (1) ◽  
pp. 139-145 ◽  
Author(s):  
Rami I. SABA ◽  
Alex BOLLEN ◽  
André HERCHUELZ
Keyword(s):  
Wild Type ◽  
Terminal Portion ◽  
Terminal Sequence ◽  
Protein Ratio ◽  
Reducing Conditions ◽  
Sds Page ◽  
Cloned Bovine ◽  
Sarcolemmal Vesicles ◽  
Exchange Activity

The Na/Ca exchanger is associated with 160, 120 and 70 kDa polypeptides whose nature is poorly understood. We have purified and characterized the Na/Ca exchanger from bovine cardiac sarcolemmal vesicles (SLVs) by using ion-exchange and affinity chromatographies. The Na/Ca exchanger-enriched fraction was reconstituted into asolectin liposomes [lipid to protein ratio 10:1 (w/w)] that showed Na/Ca exchange activity. Under non-reducing conditions, SDS/PAGE showed a single 70 kDa polypeptide, which was further characterized by immunoblots with different antibodies: SWant, raised against the purified exchanger protein; NH2-terminus, residues 1–21; NCX1, residues 393–406; and Exon F, residues 622–644. Immunoblots under reducing conditions with SWant, NH2-terminus and NCX1 showed three bands migrating at 160, 120 and 70 kDa for SLV preparations, whereas Exon F reacted only with the 160 and 120 kDa bands. Under non-reducing conditions, immunoblots with purified reconstituted Na/Ca exchanger showed a single band at 70 kDa reacting with SWant, NH2-terminus and NCX1 but not with Exon F. We conclude that the 70 kDa protein is associated with Na/Ca exchange activity, has the same N-terminal sequence as the cloned bovine cardiac exchanger, and has its length decreased by at least 35% from its C-terminal portion as compared with that of the wild-type exchanger.

Purification and Characterization of Cytochrome c6 from the Unicellular Green Alga Scenedesmus obliquus

1997 ◽  
Vol 52 (11-12) ◽  
pp. 740-746 ◽  
Author(s):  
Röbbe Wünschiers ◽  
Thomas Zinn ◽  
Dietmar Linder ◽  
Rüdiger Schulz
Keyword(s):  
Cytochrome C ◽  
Green Alga ◽  
Scenedesmus Obliquus ◽  
Terminal Sequence ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Characterization ◽  
Unicellular Green Alga ◽  
Sds Page ◽  
Monoraphidium Braunii

Abstract Purification of a soluble cytochrome c6 from the unicellular green alga Scenedesmus obliquus by a simple and rapid method is described. The purification procedure includes ammonium sulfate precipitation and non-denaturating PAGE. The N-terminal sequence of the first 20 amino acids was determined and shows 85% similarity and 75% identity to the sequence of cytochrome c6 from the green alga Monoraphidium braunii. The ferrocyto-chrome shows typical UV/VIS absorption peaks at 552.9, 521.9 and 415.7 nm. The apparent molecular mass was estimated to be 12 kD a by SDS-PAGE. EPR-spectroscopy at 20K shows resonances indicative for two distinct low-spin heme forms.

scholarly journals Purification and partial characterization of a peroxidase from plant cell cultures of Cassia didymobotrya and biotransformation studies1

1998 ◽  
Vol 331 (2) ◽  
pp. 513-519 ◽  
Author(s):  
Alberto VITALI ◽  
Bruno BOTTA ◽  
Giuliano DELLE MONACHE ◽  
Sabrina ZAPPITELLI ◽  
Paola RICCIARDI ◽  
...  
Keyword(s):  
Cell Wall ◽  
Culture Medium ◽  
Gel Filtration ◽  
Cell Suspension Cultures ◽  
High Specificity ◽  
Coniferyl Alcohol ◽  
Terminal Sequence ◽  
Sds Page ◽  
Plant Peroxidases

An acidic peroxidase (EC 1.11.1.7) produced by cell suspension cultures of Cassia didymobotrya(wild senna) was purified from culture medium collected on the 29th day. The enzyme was shown to be a glycoprotein with a pI of 3.5, a molecular mass of approx. 43 kDa by SDS/PAGE and 50 kDa by gel filtration. The N-terminal sequence was very similar to those of other plant peroxidases. The peroxidase was characterized by a high specificity towards coniferyl alcohol and other natural phenolics such as guaiacol and ferulic and caffeic acids. These findings suggest that the enzyme is involved in lignification processes of the cell wall. Moreover, the enzyme was able to catalyse the oxidation of 4,3´,4´-trihydroxychalcone and 4,3´,4´-trihydroxy-3-methoxychalcone to the corresponding 3,3´-biflavanones, as mixtures of racemic and mesoforms.

scholarly journals Chemical Characterization of a Non-Covalently Linked Peptide from Plakalbumin

1969 ◽  
Vol 22 (1) ◽  
pp. 239 ◽  
Author(s):  
R Sleigh ◽  
R Hosken MB Smith ◽  
EOP Thompson
Keyword(s):  
Chemical Characterization ◽  
Protein Component ◽  
Terminal Portion ◽  
Terminal Sequence ◽  
Covalently Linked

A 33�residue peptide isolated from plakalbumin by dissociation with urea at pH 3 is shown to be derived from the a-terminal portion of ovalbumin. The yield of proline obtained on hydrazinolysis was the same as that from ovalbumin, whereas proline was not liberated from the protein component. The N�terminal sequence of this peptide was determined by the Edman degradative procedure to be Ser. V al.Ser .Glu.Glu.Phe.Arg.Ala.Asp.

scholarly journals Purification, Characterization of an Entomopathogenic Fungal Lectin from Purpureocillium Lilacinum, and its Involvement in Pathogenesis Leading to Mycotic Keratitis

2021 ◽  
Author(s):  
Narasimhappagari Jagadeesh ◽  
Supreeth Kulkarni ◽  
Vishwanath B Chachadi ◽  
Sanhita Roy ◽  
Shashikala Inamdar
Keyword(s):  
Proinflammatory Response ◽  
Corneal Epithelial ◽  
Purpureocillium Lilacinum ◽  
Ammonium Sulphate Precipitation ◽  
Reducing Conditions ◽  
Sds Page ◽  
Fungal Lectin ◽  
Human Corneal Epithelial Cells ◽  
Ph Range

Abstract A lectin PCL, from Purpureocillium lilacinum a saprophytic, filamentous fungus was purified from the crude extract of the mycelia using 70% ammonium sulphate precipitation followed by affinity chromatography on mucin-Sepharose 4 B column. PCL is a monomer with an apparent molecular mass of 18.5 kDa as revealed by SDS-PAGE under both reducing and non reducing conditions. PCL is a blood group non specific lectin and has highest affinity towards Chitin, Mucin, asialo mucin, Fetuin with a MIC of 0.15µg/mL and also recognizes L-fucose, galactose, lactose, N-acetly galactosamine, Hyaluronic acid. PCL is stable up to 60 ºC and within the pH range 4–8. To understand its role in pathogenesis, effect of PCL was evaluated on Human Corneal Epithelial Cells (HCECs). PCL showed strong glycan mediated binding to HCECsand PCL showed proinflammatory response at lower concentrations by stimulating secretion of IL-6, 8. In contrast PCL at higher concentrations revealed opposite effect of HCECs growth inhibition. All these results collectively support the involvement of PCL in mediating host pathogen interactions possibly leading to pathogenesis. In addition, considering the entomopathogenic effect of Purpureocillium lilacinum, PCL may be attributed for this beneficiary effect, which needs to be explored.

scholarly journals Characterization of non-covalent oligomers of proteins treated with hypochlorous acid

2003 ◽  
Vol 375 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Anna L. P. CHAPMAN ◽  
Christine C. WINTERBOURN ◽  
Stephen O. BRENNAN ◽  
T. William JORDAN ◽  
Anthony J. KETTLE
Keyword(s):  
Hypochlorous Acid ◽  
Tissue Injury ◽  
Convincing Evidence ◽  
Protein Carbonyls ◽  
Molar Ratio ◽  
Myeloperoxidase Activity ◽  
Reducing Conditions ◽  
Sds Page ◽  
Methionine Residues

Hypochlorous acid (HOCl) is a potent oxidant produced by myeloperoxidase that causes aggregation of many proteins. Treatment of apohaemoglobin and apomyoglobin with HOCl produced a regular series of oligomer bands when the proteins were separated by SDS/PAGE under reducing conditions. Aggregation was detectable at a HOCl/protein molar ratio of 0.5:1 and was maximal at ratios of 10:1–20:1. Dimers formed within 1 min of adding HOCl, and further aggregation occurred over the next 30 min. No convincing evidence for covalent cross-linking was obtained by amino acid analysis, peptide analysis or electrospray ionization-MS of HOCl-modified apomyoglobin. The latter showed an increase in mass consistent with conversion of the two methionine residues into sulphoxides. A 5-fold excess of HOCl generated approximately three chloramines on the apomyoglobin. These underwent slow decay. Protein carbonyls were formed and were almost entirely located only on the polymer bands. Conversion of positively into negatively charged groups on the protein by succinylation caused preformed aggregates to dissociate. Treatment of apomyoglobin with taurine chloramine generated methionine sulphoxides but few protein carbonyls, and did not result in aggregation. We conclude that aggregation was due to strong, non-covalent interactions between protein chains. We propose that formation of protein carbonyls and possibly chloramines, along with methionine oxidation, alters protein folding to expose hydrophobic areas on neighbouring molecules that associate to form dimers and higher-molecular-mass aggregates. This process could lead to the formation of aggregated proteins at sites of myeloperoxidase activity and contribute to inflammatory tissue injury.

scholarly journals Human synovial cells secrete a 39 kDa protein similar to a bovine mammary protein expressed during the non-lactating period

1990 ◽  
Vol 269 (1) ◽  
pp. 265-268 ◽  
Author(s):  
P Nyirkos ◽  
E E Golds
Keyword(s):  
Synovial Cell ◽  
Lung Fibroblasts ◽  
Synovial Cells ◽  
Terminal Sequence ◽  
Reverse Phase ◽  
Cell Monolayers ◽  
Human Synovial Cell ◽  
Sds Page

By SDS/PAGE analysis we have observed that human synovial cell monolayers secrete a prominent 39 kDa protein which could not be detected in skin and lung fibroblasts. This protein was purified to homogeneity by heparin-Sepharose chromatography and reverse-phase h.p.l.c. The N-terminal sequence was found to be almost identical to that of a recently described bovine protein detected in the mammary secretions during the involutionary phase of the lactational cycle. Characterization of this 39 kDa protein may provide a useful marker for classification of connective tissue cells.

CHARACTERIZATION OF RECOMBINANT HUMAN SINGLE-CHAIN LOW MOLECULAR WEIGHT UROKINASE (RE-SC-LUK)

1987 ◽  
Author(s):  
W A Günzler ◽  
B Wolf ◽  
L Flohé
Keyword(s):  
Fibrinolytic Activity ◽  
Agar Plate ◽  
Single Chain ◽  
Amidolytic Activity ◽  
Terminal Sequence ◽  
Kringle Domains ◽  
Structural Identity ◽  
Sds Page ◽  
A Chain

RE-SC-LUK obtained from recomoinant b. con Bacteria showed a molecular mass similar to that of recombinant two-chain LUK (RE-TC-LUK) as judged from SDS-PAGE. By “Western“ blot analysis immunoreactivity of RE-SC-LUK was observed with monoclonal antibodies directed against the B chain but not with those against the A1 chain of urokinase. N-terminal sequence analysis c RE-SC-LUK showed identity to the A, chain of RE-TC_LUK and provided evidence for its single-chain nature, i.e. integrity of the Lys-Ile bond which is split in TC-UK. In all other respects structural identity of RE-SC-LUK and RE-TC-LUK was demonstrated by fingerprinting of fragments. Similar to recombinant pro-urokinase (RE-SCU-PA), RE-SC-LUK exhibits only marginal amidolytic activity, which is greatly enhanced by treatment with plasmin, but considerable fibrinolytic activity in a fibrin agar plate test.Thus, RE-SC-LUK is characterized as a fragment (residues 136 -411) of RE-SCU-PA, which lacks the “growth factor” and “kringle” domains. Moreover further evidence is provided that a free N-terminus of the B chain is essential for amidolytic but not for fibrinolytic activity of urokinase in more complex systems.

Characterization of an epilepsy-associated variant of the human Cl−/HCO3−exchanger AE3

2009 ◽  
Vol 297 (3) ◽  
pp. C526-C536 ◽  
Author(s):  
Gonzalo L. Vilas ◽  
Danielle E. Johnson ◽  
Paul Freund ◽  
Joseph R. Casey
Keyword(s):  
Protein Trafficking ◽  
Specific Inhibitor ◽  
Similar Degree ◽  
Transport Activity ◽  
Wild Type ◽  
Hek 293 ◽  
293 Cells ◽  
The Brain ◽  
Exchange Activity

Anion exchanger 3 (AE3), expressed in the brain, heart, and retina, extrudes intracellular HCO3−in exchange for extracellular Cl−. The SLC4A3 gene encodes two variants of AE3, brain or full-length AE3 (AE3fl) and cardiac AE3 (cAE3). Epilepsy is a heterogeneous group of disorders characterized by recurrent unprovoked seizures that affect about 50 million people worldwide. The AE3-A867D allele in humans has been associated with the development of IGE (IGE), which accounts for ∼30% of all epilepsies. To examine the molecular basis for the association of the A867D allele with IGE, we characterized wild-type (WT) and AE3fl-A867D in transfected human embryonic kidney (HEK)-293 cells. AE3fl-A867D had significantly reduced transport activity relative to WT (54 ± 4%, P < 0.01). Differences in expression levels or the degree of protein trafficking to the plasma membrane did not account for the defect of AE3fl-A867D. Treatment with 8-bromo-cAMP (8-Br-cAMP) increased Cl−/HCO3−exchange activity of WT and AE3fl-A867D to a similar degree, which was abolished by preincubation with the protein kinase A (PKA)-specific inhibitor H89. This indicates that PKA regulates WT and AE3fl-A867D Cl−/HCO3−exchange activity. No difference in Cl−/HCO3−exchange activity was found between cultures of mixed populations of neonatal hippocampal cells from WT and slc4a3−/−mice. We conclude that the A867D allele is a functional (catalytic) mutant of AE3 and that the decreased activity of AE3fl-A867D may cause changes in cell volume and abnormal intracellular pH. In the brain, these alterations may promote neuron hyperexcitability and the generation of seizures.

scholarly journals The non-phospholipase A2 subunit of β-bungarotoxin plays an important role in the phospholipase A2-independent neurotoxic effect: characterization of three isotoxins with a common phospholipase A2 subunit

1994 ◽  
Vol 303 (1) ◽  
pp. 171-176 ◽  
Author(s):  
C C Chu ◽  
S T Chu ◽  
S W Chen ◽  
Y H Chen
Keyword(s):  
Phospholipase A2 ◽  
Terminal Sequence ◽  
Protein Fluorescence ◽  
Linear Gradient ◽  
Sds Page ◽  
Neurotoxic Effects ◽  
Beta 2 ◽  
Chick Biventer Cervicis Muscle ◽  
Derivatives Of

Three isotoxins (SP I-III) of the beta-bungarotoxin family were purified to homogeneity via a series of isolation procedures including a final step of h.p.l.c. on an SP column washed with a linear gradient of 0.2-0.6 M sodium acetate at pH 7.4. Their proportions varied greatly with the batch of venom. Each isotoxin was demonstrated by SDS/PAGE to contain a phospholipase A2 subunit and a non-phospholipase A2 subunit. The three proteins were reductively alkylated with 4-vinylpyridine and the alkylated derivatives of the two subunits of each isotoxin were separated. N-Terminal sequence analysis of the alkylated derivatives revealed that the three isotoxins probably share a common phospholipase A2 subunit but differ in their non-phospholipase A2 subunits. The non-phospholipase A2 subunits of SP II and SP III were identical with those of beta 2- and beta 1-toxin respectively, except that there was an additional valine inserted between Thr-18 and Val-19 in beta 2-toxin and Pro-18 and Val-19 in beta 1-toxin. The non-phospholipase A2 subunit of SP I differed greatly from that of SP III but was almost identical with that of SP II, except that Lys-14 and Ala-29 in SP II were replaced by Arg-14 and Glu-29 in SP I. Analysis of the effect of CaCl2 on protein fluorescence showed the existence of a low- and a high-affinity site on the different domains of each isotoxin for Ca2+ binding. The three isotoxins showed no great difference in their ability to bind Ca2+ on both the high- and low-affinity site. They had slightly different phospholipase A2 activities but differed to a great extent with respect to their neurotoxic effects. LD50 values increased in the order SP I > SP II > SP III. In contrast, the ability to inhibit the indirectly evoked contraction of chick biventer cervicis muscle was in the order SP III > SP II > SP I.

scholarly journals Construction and Initial Characterization of Escherichia coli Strains with Few or No Intact Chromosomal rRNA Operons

1999 ◽  
Vol 181 (12) ◽  
pp. 3803-3809 ◽  
Author(s):  
Tsuneaki Asai ◽  
Ciarán Condon ◽  
Justina Voulgaris ◽  
Dmitry Zaporojets ◽  
Binghua Shen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Rrna Operon ◽  
Rrna Genes ◽  
23S Rrna ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Protein Ratio ◽  
23S Rrna Genes

ABSTRACT The Escherichia coli genome carries seven rRNA (rrn) operons, each containing three rRNA genes. The presence of multiple operons has been an obstacle to many studies of rRNA because the effect of mutations in one operon is diluted by the six remaining wild-type copies. To create a tool useful for manipulating rRNA, we sequentially inactivated from one to all seven of these operons with deletions spanning the 16S and 23S rRNA genes. In the final strain, carrying no intact rRNA operon on the chromosome, rRNA molecules were expressed from a multicopy plasmid containing a single rRNA operon (prrn). Characterization of these rrndeletion strains revealed that deletion of two operons was required to observe a reduction in the growth rate and rRNA/protein ratio. When the number of deletions was extended from three to six, the decrease in the growth rate was slightly more than the decrease in the rRNA/protein ratio, suggesting that ribosome efficiency was reduced. This reduction was most pronounced in the Δ7 prrn strain, in which the growth rate, unlike the rRNA/protein ratio, was not completely restored to wild-type levels by a cloned rRNA operon. The decreases in growth rate and rRNA/protein ratio were surprisingly moderate in the rrndeletion strains; the presence of even a single operon on the chromosome was able to produce as much as 56% of wild-type levels of rRNA. We discuss possible applications of these strains in rRNA studies.

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