scholarly journals Purification and spectral studies on the Ca2+-binding properties of 67 kDa calcimedin

1989 ◽  
Vol 259 (3) ◽  
pp. 799-804 ◽  
Author(s):  
R S Mani ◽  
C M Kay
Keyword(s):  
Fluorescence Intensity ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Fluorescence Emission ◽  
Spectral Studies ◽  
Affinity Column ◽  
Protein Fluorescence ◽  
Hydrophobic Region ◽  
Emission Maximum

The 67 kDa calcimedin, isolated by using a phenyl-Sepharose affinity column followed by DEAE-cellulose and gel-filtration chromatographies, was homogeneous by the criterion of SDS/polyacrylamide-gel electrophoresis. In non-SDS gels, the protein moved faster in the presence of EDTA, suggesting that Ca2+ binding affects its mobility in a manner similar to other Ca2+-binding proteins such as calmodulin and S-100 proteins. The 67 kDa protein underwent a conformational change upon binding Ca2+, as revealed by u.v. difference spectroscopy and near-u.v. c.d. measurements. Tryptophan and tyrosine residues were perturbed upon Ca2+ binding, moving to a more non-polar environment in the presence of Ca2+. Upon excitation of the protein at 280 nm, the fluorescence emission maximum was centered around 325 nm, suggesting that the tryptophan residues are located in a fairly hydrophobic region. Ca2+ addition did not induce a significant change in the intrinsic protein fluorescence intensity at 325 nm. Addition of Ca2+ to the 67 kDa protein labelled with 2-p-toluidinylnaphthalene-6-sulphone (TNS) resulted in a 25% increase in fluorescence intensity, accompanied by a blue shift of the emission maximum from 442 to 432 nm. Hence, the probe in the presence of Ca2+ moves to a more non-polar microenvironment, like calmodulin and other Ca2+-binding proteins. Fluorescence titration with Ca2+ using TNS-labelled protein revealed one class of binding site, with a Kd value of 2 x 10(-5) M.

scholarly journals Spectral studies on the cadmium-ion-binding properties of bovine brain S-100b protein

1991 ◽  
Vol 276 (1) ◽  
pp. 13-18 ◽  
Author(s):  
H Donato ◽  
R S Mani ◽  
C M Kay
Keyword(s):  
Absorption Band ◽  
Fluorescence Intensity ◽  
Fluorescence Emission ◽  
Bovine Brain ◽  
Spectral Studies ◽  
Tyrosine Residue ◽  
Difference Spectroscopy ◽  
Model Complexes ◽  
Emission Maximum ◽  
Fluorescence Measurements

The effect of Cd2+ binding on bovine brain S-100b protein was studied using c.d. u.v. difference spectroscopy and fluorescence measurements. At pH 7.5, S-100b protein binds two Cd2+ ions per monomer with a Kd value of 3 x 10(-5) M. Addition of Cd2+ resulted in perturbing the single tyrosine residue (Tyr17) in the protein as indicated by u.v. difference spectroscopy and aromatic c.d. measurements. In the presence of Cd2+, the tyrosine residue moves to a more non-polar environment, since a red shift was observed in the u.v. difference spectrum. When the protein was excited at 278 nm, the tyrosine fluorescence emission maximum was centred at 306 nm. Cd2+ addition resulted in an increase in intrinsic fluorescence intensity. Fluorescence titration with Cd2+ indicated the protein binds Cd2+ with a Kd value of 3 x 10(-5) M. 2-p-Toluidinylnaphthalene-6-sulphonate-labelled protein, when excited at 345 nm, had a fluorescence emission maximum at 440 nm. Addition of Cd2+ to labelled protein resulted in a 5-fold increase in fluorescence intensity accompanied by a 5 nm blue shift in the emission maximum, suggesting that the probe, in the presence of Cd2+, moves to a hydrophobic domain. U.v. difference spectroscopic studies indicated a unique Cd2(+)-binding site on the protein, since Cd2+ addition yielded a large positive absorption band in the 240 nm region that is not found with either Ca2+ or Zn2- ions. Similar absorption bands have been observed in Cd-protein complexes such as Cd-metallothionein [Vasak, Kagi & Hill (1981) Biochemistry 20, 2852-2856] and also in model complexes of Cd2+ with 2-mercaptoethanol. This absorption band is believed to arise as a result of charge-transfer transitions between the thiolate and Cd2+. Of the two Cd2- -binding sites on the beta-chain, one must be located at the N-terminal end near the single tyrosine residue, since Cd2- and Zn2+ produced similar effects on the intrinsic protein fluorescence. The other Cd2+ site which is unique to Cd2+ must be Cys84, located at the C-terminal end.

A fluorochrome from aniline blue: structure,synthesis and fluorescence properties

1982 ◽  
Vol 35 (12) ◽  
pp. 2571 ◽  
Author(s):  
NA Evans ◽  
PA Hoyne
Keyword(s):  
Critical Micelle Concentration ◽  
Cationic Surfactant ◽  
Fluorescence Intensity ◽  
Fluorescence Emission ◽  
Aniline Blue ◽  
Fluorescence Properties ◽  
Hexadecyltrimethylammonium Bromide ◽  
Commercial Sample ◽  
Emission Maximum ◽  
Structure Synthesis

A fluorochrome has been isolated in analytically pure form from a commercial sample of the triaryl-methane dye aniline blue. Its structure has been shown to be sodium 4,4'-[carbonylbis(benzene-4,1-diyl)bis(imino)]bisbenzenesulfonate by spectroscopic means and confirmed by synthesis. Its fluorescence emission, which is markedly solvent-dependent, is 150 times greater in butan-1-ol than in water (however, the wavelength of the emission maximum is not altered significantly). In the presence of a cationic surfactant, hexadecyltrimethylammonium bromide, the fluorescence intensity reaches a maximum at approximately the critical micelle concentration of the surfactant.

scholarly journals 67 kDa calcimedin, a new Ca2+-binding protein

1986 ◽  
Vol 238 (1) ◽  
pp. 49-54 ◽  
Author(s):  
P B Moore
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
Binding Site ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Affinity Column ◽  
Binding Studies ◽  
High Affinity

A set of four proteins, termed calcimedins, are isolatable from smooth, cardiac and skeletal muscle by using a fluphenazine-Sepharose affinity column. The calcimedins show apparent Mr values of 67,000, 35,000, 33,000 and 30,000 by SDS/polyacrylamide-gel electrophoresis. The 67,000-Mr calcimedin (67 kDa calcimedin) has now been purified to homogeneity by using DEAE-cellulose chromatography followed by Ca2+-dependent binding to phenyl-Sepharose. The amino acid analysis of the 67 kDa calcimedin shows this protein does not contain trimethyl-lysine but does contain 2 mol of tryptophan/mol of protein. The 67 kDa calcimedin shows positive ellipticity in the near-u.v. range with c.d. Ca2+-binding studies indicate one high-affinity Ca2+-binding site with Kd 0.4 microM. The data show that the 67 kDa calcimedin is distinct from other Ca2+-binding proteins described to date.

scholarly journals Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei

1976 ◽  
Vol 157 (3) ◽  
pp. 559-571 ◽  
Author(s):  
J G Dann ◽  
G Ostler ◽  
R A Bjur ◽  
R W King ◽  
P Scudder ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Resistant Strain ◽  
Lactobacillus Casei ◽  
Large Scale ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Binding Constants ◽  
Affinity Column ◽  
Protein Fluorescence ◽  
Turnover Number

Dihydrofolate reductase has been purified from a methotrexate-resistant strain of Lactobacillus casei NCB 6375. By careful attention to growth conditions, up to 2.5 g of enzyme is obtained from a 400 litre culture. The purification procedure, involving poly-ethyleneimine treatment, DEAE-cellulose chromatography and affinity chromatography on methotrexate-aminohexyl-Sepharose, operates on the gram scale, with overall yields of 50-60%. Elution of the affinity column by reverse (upward) flow was used, as it led to recovery of the enzyme in a much smaller volume. The enzyme obtained appears to be more than 98% pure, as judged by gel electrophoresis, isoelectric focusing, and gel filtration. It has a mol.wt. of approx. 17900 and a turnover number of 4s-1 (50mM-triethanolamine/400mM-KCl, pH 7.2, 25 degrees C) with dihydrofolate and NADPH as substrates. The turnover number for folate is 0.02s-1. Michaelis constants for a variety of substrates have been measured by using a new fluorimetric assay (0.36 muM-dihydrofolate; 0.78 muM-NADPH), and binding constants determined by using the quenching of protein fluorescence (dihydrofolate, 2.25 X 10(6)M-1; NADPH, greater than 10(8)M-1). The pH/activity profile shows a single maximum at pH 7.3; at this pH, marked activation by 0.5M-NaCl is observed.

Modification of β–lactoglobulin by aliphatic aldehydes in aqueous solution

1994 ◽  
Vol 61 (2) ◽  
pp. 209-219 ◽  
Author(s):  
Henrik Stapelfeldt ◽  
Leif H. Skibsted
Keyword(s):  
Whey Proteins ◽  
Fluorescence Emission ◽  
Tryptophan Fluorescence ◽  
Oxidation Products ◽  
Size Exclusion ◽  
Protein Fluorescence ◽  
Two Phase ◽  
Emission Maximum ◽  
Lipid Oxidation Products ◽  
Size Exclusion Hplc

SummaryEach of the secondary lipid oxidation products pentanal, hexanal and heptanal was found to react with β–lactoglobulin (β–lg) in a two-phase model System (aqueous phosphate buffer–1-octanol) yielding fluorescent condensation products (emission maximum, 410 nm; excitation maximum, 350 nm). Protein polymers were detected by size-exclusion HPLC, and the rate of reaction paralleled the formation of fluorescent products, with the reactivity being pentanal > hexanal > heptanal. Simultaneously, the reaction also changed the intrinsic fluorescence of β–lg, and in particular pentanal reduced the intensity of tryptophan fluorescence (emission maximum, 332 nm; excitation maximum, 288 nm) by 30%. These findings are discussed with reference to the effect of peroxidizing lipids on the physical properties of whey proteins and the use of protein fluorescence (induced by the reaction with aldehydes) as marker for the oxidative status of milk and whey protein products.

scholarly journals Sponge-derived Ageladine A affects the in vivo fluorescence emission spectra of microalgae

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242464
Author(s):  
Carolin Peter ◽  
Silke Thoms ◽  
Florian Koch ◽  
Franz Josef Sartoris ◽  
Ulf Bickmeyer
Keyword(s):  
Natural Products ◽  
Fluorescence Intensity ◽  
Crucial Role ◽  
Fluorescence Emission ◽  
Emission Spectra ◽  
Fluorescence Emission Spectra ◽  
Tisochrysis Lutea ◽  
Emission Maximum ◽  
Ph Dependent

In several marine hosts of microalgae, fluorescent natural products may play an important role. While the ecological function of these compounds is not well understood, an interaction of these molecules with the photosynthesis of the symbionts has been suggested. In this study, the effect of Ageladine A (Ag A), a pH-dependent fluorophore found in sponges of the genus Agelas, on microalgal fluorescence was examined. The spectra showed an accumulation of Ag A within the cells, but with variable impacts on fluorescence. While in two Synechococcus strains, fluorescence of phycoerythrin increased significantly, the fluorescence of other Synechococcus strains was not affected. In four out of the five eukaryote species examined, chlorophyll a (Chl a) fluorescence intensity was modulated. In Tisochrysis lutea, for example, the position of the fluorescence emission maximum of Chl a was shifted. The variety of these effects of Ag A on microalgal fluorescence suggests that fluorophores derived from animals could play a crucial role in shaping the composition of marine host/symbiont systems.

scholarly journals Studies on the α-subunit of bovine brain S-100 protein

1984 ◽  
Vol 218 (3) ◽  
pp. 691-696 ◽  
Author(s):  
H R Masure ◽  
J F Head ◽  
H M Tice
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fluorescence Emission ◽  
Bovine Brain ◽  
Alpha Subunit ◽  
Tryptophan Residue ◽  
Affinity Column ◽  
Apparent Dissociation Constant ◽  
Α Subunit ◽  
S 100

A method is described for the rapid purification of both S-100 protein and calmodulin from crude bovine brain extracts by the use of a fluphenazine-Sepharose affinity column eluted stepwise with decreasing concentrations of free Ca2+. Protein containing only alpha-subunit was purified from preparations of S-100 protein by anion-exchange chromatography. This protein co-migrated with the alpha-subunit of S-100 protein on sodium dodecyl sulphate/urea/polyacrylamide-gel electrophoresis and had an amino acid composition identical with that previously reported for this subunit. The results of u.v.-absorption and fluorescence-emission spectroscopy indicate that the tryptophan residue of the purified alpha-subunit of S-100 protein undergoes a Ca2+-induced change in environment. Measurements of changes in tryptophan fluorescence with increasing Ca2+ concentrations suggest an apparent dissociation constant of the alpha-subunit for Ca2+ of 7 × 10(-5)M in the absence of K+. In the presence of 90mM-K+ this value is increased to 3.4 × 10(-4)M.

scholarly journals Purification and characterization of ribonucleases from human seminal plasma

1983 ◽  
Vol 215 (3) ◽  
pp. 605-612 ◽  
Author(s):  
C L Lee ◽  
S S L Li ◽  
C Y Li ◽  
T M Chu
Keyword(s):  
Seminal Plasma ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Affinity Column ◽  
Human Seminal Plasma ◽  
Immunological Reactions ◽  
Sepharose 4B

Four ribonucleases (RNAases I-IV) have been purified to homogeneity from human seminal plasma by precipitation with 40-75%-satd. (NH4)2SO4, followed by chromatographies on concanavalin A-Sepharose 4B, DEAE-cellulose phosphocellulose, agarose-5′-(4-aminophenylphospho)uridine 2′(3′)-phosphate (RNAase affinity column) and Sephadex G-75 or G-100. The homogeneity of these RNAases was confirmed by polyacrylamide-gel electrophoresis. Mr values for these purified RNAases were 78 000, 16 000, 13 300 and 5000 as estimated by gel filtration. Enzyme activities of RNAases I, III and IV were inhibited by Mn2+, Zn2+ and Cu2+ and activated by Na+, K+, Ba2+, Mg2+, Fe2+ and EDTA, whereas that of RNAase II was inhibited by Ba2+, Mg2+, Fe2+, Mn2+, Zn2+ and Cu2+ and activated by Na+, K+ and EDTA. RNAases I, II and IV demonstrated a higher affinity for poly(C) and poly(U) or yeast RNA, whereas RNAase III preferentially hydrolysed poly(U) over poly(C) and yeast RNA. In the presence of 5 mM-spermine, RNAase I was dissociated to a low-Mr (5000) enzyme with an increase in total RNAase enzymic activity. Xenoantiserum to each RNAase was raised and evaluated by immunoprecipitation and immunohistochemical methods. Anti-(seminal RNAase III) antiserum showed no immunological cross-reaction with RNAases of other human origin, whereas anti-(seminal RNAase I), -(RNAase II) and -(RNAase IV) antisera exhibited indistinguishable immunological reactions with serum RNAase and other human RNAases, except that anti-(seminal RNAase I) and -(RNAase antisera IV) did not react with pancreatic RNAases. Seminal RNAases I and IV were identical immunologically as shown by anti-(RNAase I) and anti-(RNAase IV) in immunodiffusion. Immunohistochemical study revealed that, among human tissues examined, only prostate expressed seminal RNAase III. These results suggested that human seminal RNAase I may be an aggregated molecule of RNAase IV and that seminal RNAases II and IV are similar to serum RNAases, whereas seminal RNAase III is a prostate-specific enzyme.

scholarly journals Affinity purification and subunit structures of β-N-acetylhexosaminidases A and B from boar epididymis

1984 ◽  
Vol 219 (3) ◽  
pp. 1009-1015 ◽  
Author(s):  
H C Parkes ◽  
J L Stirling ◽  
P Calvo
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Gradient Gel Electrophoresis ◽  
Sepharose 4B ◽  
Electrophoretic Transfer ◽  
Peptide Maps

beta-N-Acetylhexosaminidase from boar epididymis was separated into two forms, A and B, on DEAE-cellulose. Both these forms were excluded from Sepharose S-200 and had apparent Mr values of 510 000 on gradient gel electrophoresis under non-denaturing conditions. Affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylam ine coupled to CNBr-activated Sepharose 4B was used to separate and purify beta-N-acetylhexosaminidases A and B that had specific activities of 115 and 380 mumol/min per mg of protein respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of denatured beta-N-acetylhexosaminidase A gave a single major component of Mr 67 000. beta-N-Acetylhexosaminidase B also had this component, and in addition had polypeptides of Mr 29 000 and 26 000. All these polypeptides were glycosylated. Antiserum to the B form precipitated form A from solution and reacted with the 67 000-Mr component or form A after electrophoretic transfer from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose sheets. The 67 000-Mr components of forms A and B yielded identical peptide maps when digested with Staphylococcus aureus V8 proteinase, and the 29 000-Mr and 26 000-Mr components in form B may be related to the 67 000-Mr polypeptide.

Purification and characterization of a novel factor which stimulates rat ribosomal gene transcription in vitro by interacting with enhancer and core promoter elements

1990 ◽  
Vol 10 (10) ◽  
pp. 5177-5186
Author(s):  
J Zhang ◽  
S T Jacob
Keyword(s):  
Ribosomal Dna ◽  
Core Promoter ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Affinity Column ◽  
Mobility Shift ◽  
Enhancer Element ◽  
Molecular Weights ◽  
The Core ◽  
Pol I

Previous studies in our laboratory have characterized a 174-base-pair (bp) enhancer sequence in the rat ribosomal DNA spacer region that exhibits all of the characteristics of a polymerase (Pol) II enhancer. Further studies showed that at least half of the enhancer activity resides in a 37-bp motif (E1) within the 174-bp spacer sequence that is located between positions -2.183 and -2.219 kilobase pairs upstream of the initiation site. To identify the factor(s) that binds specifically to the 37-bp enhancer domain, we fractionated whole-cell extract from rat adenocarcinoma ascites cells by chromatography on a series of columns, including an oligodeoxynucleotide affinity column. The final preparation contained two polypeptides of molecular weights 79,400 and 89,100 and was completely devoid of RNA Pol I activity. Electrophoretic mobility shift analysis showed that the polypeptides in the purified preparation (designated E1BF) interacted with both the enhancer element and the core promoter. To determine whether each polypeptide can separately bind to the core promoter and the enhancer, the individual components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, renatured, and subjected to gel retardation analysis. This experiment demonstrated that both polypeptides interacted with the two cis-acting sequences. The specificity of the binding was demonstrated by competition with unlabeled 37-bp and core promoter fragments and lack of competition with nonspecific DNAs in the mobility shift assay. The 37-bp enhancer as well as the downstream sequence of the core promoter were protected by E1BF in the DNase I footprinting assay. Addition of E1BF to limiting amounts of fraction DE-B, which contains all factors essential for Pol I-directed transcription, resulted in three- to fourfold stimulation of ribosomal DNA transcription. Comparison of molecular weights and footprinting profiles did not reveal any relationship between E1BF and other Pol I trans-acting factors.

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