Detergent Effect on Anion Exchange Perfusion Chromatography and Gel Filtration of Intact Chloroplast H+-ATP Synthase

1999 ◽  
Vol 265 (2) ◽  
pp. 520-524 ◽  
Author(s):  
Ansgar Poetsch ◽  
Holger Seelert ◽  
Jürgen Meyer zu Tittingdorf ◽  
Norbert A. Dencher
Keyword(s):  
Anion Exchange ◽  
Atp Synthase ◽  
Gel Filtration ◽  
Intact Chloroplast ◽  
Perfusion Chromatography ◽  
Detergent Effect

Bovine Factors X1And X2: Activation Peptide Based Chromatographic Differences

1979 ◽  
Author(s):  
Takashi Morita ◽  
Craig Jackson
Keyword(s):  
Amino Acid ◽  
Anion Exchange ◽  
Gel Filtration ◽  
Heart Association ◽  
Personal Communication ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Factor X ◽  
Activation Peptide ◽  
Activation Peptides

Bovine Factor X is eluted in two forms (X1and X2) from anion exchange chromatographic columns. These two forms have indistinguishable amino acid compositions, molecular weights and specific activities. The amino acid sequences containing the γ-carboxyglutamic acid residues have been shown to be identical in X1 and X2(H. Morris, personal communication). An activation peptide is released from the N-terminal region of the heavy chain of Factor X by an activator from Russell’s viper venom. This peptide can be isolated after activation by gel filtration on Sephadex G-100 under nondenaturing conditions. The activation peptides from a mixture of Factors X1 and X2 were separated into two forms by anion-exchange chromatography. The activation peptide (AP1) which eluted first was shown to be derived from Factor X1. while the activation peptiae (AP2) which eluted second was shown to be derived from X2 on the basis of chromatographic separations carried out on Factors X1 and X2 separately. Factor Xa was eluted as a symmetrical single peak. On the basis of these and other data characterizing these products, we conclude that the difference between X1 and X2 are properties of the structures of the activation peptides. (Supported by a grant HL 12820 from the National Heart, Lung and Blood Institute. C.M.J. is an Established Investigator of the American Heart Association).

scholarly journals F1F0-ATP synthase from bovine heart mitochondria: development of the purification of a monodisperse oligomycin-sensitive ATPase

1993 ◽  
Vol 295 (3) ◽  
pp. 799-806 ◽  
Author(s):  
R Lutter ◽  
M Saraste ◽  
H S van Walraven ◽  
M J Runswick ◽  
M Finel ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Gel Filtration ◽  
Primary Objective ◽  
Membrane Domain ◽  
Mitochondrial Membranes ◽  
F1 Atpase ◽  
Ammonium Sulphate Precipitation ◽  
F1f0 Atp Synthase ◽  
Hydrolysis Activity

A new procedure for the isolation of ATP synthase from bovine mitochondria has been developed, with the primary objective of producing enzyme suitable for crystallization trials. Proteins were extracted from mitochondrial membranes with dodecyl-beta-D-maltoside, and the ATP synthase was purified from the extract in the presence of the same detergent by a combination of ion-exchange and gel-filtration chromatography and ammonium sulphate precipitation. This simple and rapid procedure yields 20-30 mg of highly pure and monodisperse enzyme, evidently consisting of 14 different subunits, amongst them, in apparently stoichiometric amounts with the established subunits, subunit e, a recently discovered subunit of unknown function. The enzyme preparation has an oligomycin-sensitive ATP hydrolysis activity, and so the F1 domain is functionally associated with the membrane domain, F0. In contrast with the N-termini of some of the subunits of bovine mitochondrial F1-ATPase, those of the F1F0-ATP synthase are not degraded by proteolysis during the isolation procedure. This preparation therefore satisfies prerequisites for crystallization trials.

scholarly journals Purification of a Crenarchaeal ATP Synthase in the Light of the Unique Bioenergetics ofIgnicoccusSpecies

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Lydia J. Kreuter ◽  
Andrea Weinfurtner ◽  
Alexander Ziegler ◽  
Julia Weigl ◽  
Jan Hoffmann ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Cell Envelope ◽  
Gel Filtration ◽  
Cellular Membrane ◽  
Content Type ◽  
The Pacific ◽  
Native Electrophoresis ◽  
Molecular Masses ◽  
The Pacific Ocean

ABSTRACTIn this study, the ATP synthase ofIgnicoccus hospitaliswas purified, characterized, and structurally compared to the respective enzymes of the otherIgnicoccusspecies, to shed light on energy conservation in this unique group of archaea. The crenarchaeal genusIgnicoccuscomprises three described species, i.e.,I. hospitalisandIgnicoccus islandicusfrom hot marine sediments near Iceland andIgnicoccus pacificusfrom a hydrothermal vent system in the Pacific Ocean. This genus is unique among all archaea due to the unusual cell envelope, consisting of two membranes that enclose a large intermembrane compartment (IMC).I. hospitalisis the best studied member of this genus, mainly because it is the only known host for the potentially parasitic archaeonNanoarchaeum equitans.I. hospitalisgrows chemolithoautotrophically, and its sole energy-yielding reaction is the reduction of elemental sulfur with molecular hydrogen, forming large amounts of hydrogen sulfide. This reaction generates an electrochemical gradient, which is used by the ATP synthase, located in the outer cellular membrane, to generate ATP inside the IMC. The genome ofI. hospitalisencodes nine subunits of an A-type ATP synthase, which we could identify in the purified complex. Although the maximalin vitroactivity of theI. hospitalisenzyme was measured around pH 6, the optimal stability of the A1AOcomplex seemed to be at pH 9. Interestingly, the soluble A1subcomplexes of the differentIgnicoccusspecies exhibited significant differences in their apparent molecular masses in native electrophoresis, although their behaviors in gel filtration and chromatography-mass spectrometry were very similar.IMPORTANCETheCrenarchaeotarepresent one of the major phyla within theArchaeadomain. This study describes the successful purification of a crenarchaeal ATP synthase. To date, all information about A-type ATP synthases is from euryarchaeal enzymes. The fact that it has not been possible to purify this enzyme complex from a member of theCrenarchaeotauntil now points to significant differences in stability, possibly caused by structural alterations. Furthermore, the study subjectI. hospitalishas a particular importance among crenarchaeotes, since it is the only known host ofN. equitans. The energy metabolism in this system is still poorly understood, and our results can help elucidate the unique relationship between these two microbes.

Identification of Glucagon-Like Peptide-1(7–36) Amide in Rat Brain

1989 ◽  
Vol 26 (2) ◽  
pp. 169-171 ◽  
Author(s):  
S Yoshimoto ◽  
M Hirota ◽  
C Ohboshi ◽  
K Shima
Keyword(s):  
Rat Brain ◽  
Anion Exchange ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Experimental Conditions ◽  
Specific Radioimmunoassay ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
The Brain

Acid-urea extract of rat brain was examined by glucagon-like peptide-1 (GLP-1) specific radioimmunoassay. A single peak was observed which co-eluted with GLP-1(7–36)amide on gel filtration and anion exchange chromatography. In contrast, GLP-1(1–37) was not detected under our experimental conditions. The fact that GLP-1 (7–36)amide, but not GLP-1(1–37), was present in rat brain suggests that preproglucagon was processed in the brain in the same manner as in the intestine and not as in the pancreas.

A Potent Insect Chitinase Inhibitor of Fungal Origin

2003 ◽  
Vol 58 (11-12) ◽  
pp. 891-894 ◽  
Author(s):  
Teruhiko Nitoda ◽  
Hirokazu Usuki ◽  
Hiroshi Kanzaki
Keyword(s):  
Molecular Weight ◽  
Anion Exchange ◽  
Culture Filtrate ◽  
Inhibitory Activity ◽  
Spodoptera Litura ◽  
Gel Filtration ◽  
Fungal Strain ◽  
Water Soluble ◽  
Gel Filtration Chromatography ◽  
Soluble Polysaccharide

Abstract A water-soluble polysaccharide was isolated from the culture filtrate of a fungal strain, Sphaeropsis sp. TNPT116-Cz, as a novel insect chitinase inhibitor. It was purified to chromatographic homogeneity by ethanol precipitation, anion-exchange and gel filtration chromatography. Its molecular weight was estimated to be 16 kDa by gel filtration HPLC. Monosaccharide analysis showed that it contained glucose, galactose, N-acetylglucosamine and a deoxysugar. This polysaccharide showed potent and specific inhibitory activity against Spodoptera litura chitinase with an IC50 value of 28 nᴍ.

scholarly journals Purification and Characterization of Two Endo-β-1,4-glucanases from Mollusca, Ampullaria crossean

2005 ◽  
Vol 37 (10) ◽  
pp. 702-708 ◽  
Author(s):  
Yan-Hong Li ◽  
Rui Guo ◽  
Qiu-Yu Yin ◽  
Ming Ding ◽  
Si-Liang Zhang ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Carboxymethyl Cellulose ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Gel Filtration ◽  
Residual Activity ◽  
Hydrolytic Activity ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
The Stability

Abstract Two novel endo-β-1,4-glucanases, EG45 and EG27, were isolated from the gastric juice of mollusca, Ampullaria crossean, by anion exchange, hydrophobic interaction, gel filtration and a second round of anion exchange chromatography. The purified proteins EG45 and EG27 appeared as a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa, respectively. The optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for EG27. The optimum temperature range for EG27 was broad, between 50 °C and 60 °C; for EG45 it was 50 °C. The analysis on the stability of these two endo-β-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 even when the incubation time was prolonged to 24 h at 30 °C, whereas EG45 remained relatively stable at pH 5.0-8.0. About 85% of the activity of EG27 could be retained upon incubation at 60 °C for 24 h. However, less than 10% residual activity of EG45 was detected at 50 °C. Among different kinds of substrates, both enzymes showed a high preference for carboxymethyl cellulose. EG45, in particular, showed a carboxymethyl cellulose hydrolytic activity of 146.5 IU/mg protein. Both enzymes showed low activities to xylan (from oat spelt) and Sigmacell 101, and they were inactive to p-nitrophenyl-β-D-cellobioside, salicin and starch.

scholarly journals Measurement of vitamin B12-binding proteins of plasma. I. Technique

Blood ◽  
1975 ◽  
Vol 45 (2) ◽  
pp. 281-286
Author(s):  
JA Begley ◽  
CA Hall
Keyword(s):  
Vitamin B12 ◽  
Ammonium Sulfate ◽  
Anion Exchange ◽  
Isoelectric Focusing ◽  
Binding Proteins ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Total Plasma ◽  
Step Procedure ◽  
The Mean

The unsaturated binding capacities (UBBC) of individual vitamin B12- binding proteins in plasma were measured by a two-step procedure. Transcobalamin II (TC II) was separated by precipitation with ammonium sulfate; the “R”-type binders remaining soluble were then divided into two components by bath separation with anion exchange on DEAE- cellulose. The two R components were designated alpha1-R (TC 1) and alpha2-R (third binder, fetal binder, PV binder, TC III). Ten normal sera were studied by this technique giving a separation into TC III and total plasma R identical to that obtained simultaneously by gel filtration. The mean UBBC of TC II was 969 plus or minus 204 pg of 57 Co B12 per ml of serum. The mean contamination of the precipitated TC III with plasma R was 3%. The UBBCs of alpha 2-R and alpha 1-R were 127 plus or minus 42 and 40 plus or minus 12 pg/ml, respectively. The mean contamination of the R fraction by TC II was 14% as evaluated by gel filtration. By isoelectric focusing it was found that the alpha1-R contained principally those components isoelectric at pH isoelectric at pH of 2.9–3.2, while alpha2-R was made up of those components isoelectric at pH of 3.6 or greater.

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