scholarly journals Limited digestion of α-actinin in the presence of F-actin

BIOPHYSICS ◽  
2011 ◽  
Vol 7 ◽  
pp. 29-34
Author(s):  
You Jia ◽  
Masaaki Kuroda
Keyword(s):  
Limited Digestion

scholarly journals Structural defects in rat liver deoxyribonucleic acid. Endogenous single-strained regions in comparison with damage induced in vivo by a carcinogen

1979 ◽  
Vol 179 (2) ◽  
pp. 341-352 ◽  
Author(s):  
B W Stewart ◽  
P H Huang ◽  
M J Brian
Keyword(s):  
Rat Liver ◽  
Structural Damage ◽  
Deae Cellulose ◽  
Minor Component ◽  
Structural Defects ◽  
Structural Abnormality ◽  
Denaturation Kinetics ◽  
Limited Digestion ◽  
A Minor

Rat liver DNA may be separated into two fractions by stepwise elution from benzoylated-DEAE-cellulose with NaCl and caffeine solutions respectively. Other studies using bacterical and yeast DNA suggested that the first fraction contains native DNA, whereas the second may exhibit some degree of single-stranded character. In the present experiments, chromatography of DNA was monitored by labelling in vivo with [methyl-3H]thymidine in rats previously subjected to partial hepatectomy. In animals killed up to 1 h after thymidine injection, radioactivity eluted in the second fraction was inversely related to the incorporation time, being greatest when animals were killed 10 min after radioisotope injection. However, for most experiments, animals were allowed to survive 2-4 weeks after surgery before use, analysis being made on non-dividing DNA. Under these conditions, the proportion of caffeine-eluted DNA was decreased by subjecting the preparation to shear, before chromatography. A procedure that resulted in 12% of the recovered radioactivity being eluted with caffeine was adopted for experiments involving comparisons of the two DNA fractions. Under these conditions, cross-contamination could be detected by rechromatography, but this did not preclude distinction being made between the two fractions in terms of DNA structure. NaCl-eluted DNA did not bind to nitrocellulose filters. Caffeine-eluted DNA was retained by the filters and released by washing with 3mM-Tris/HCl, pH9.4. The fractions did not differ in terms of isopycnic centrifugation in CsCl. The NaCl-eluted fraction migrated as a single band in polyacrylamide gels, and this pattern was not modified by prior digestion with Neurospora crassa endonuclease. In contrast, caffeine-eluted DNA contained a minor component having a wide molecular-weight distribution and was subject to limited digestion by the endonuclease. The kinetics of denaturation of NaCi-eluted DNA in the presence of formaldehyde, in common with unfractionated DNA, were consistent with double-stranded structure. The same analysis of caffeine-eluted DNA revealed structural abnormality equivalent to two defects per 10000 base-pairs. The data are consistent with the minor fraction of rat liver DNA, separated by using benzoylated-DEAE-cellulose, containing regions of local denaturation. We previously showed that administration of the hepatocarcinogen dimethylnitrosamine is associated with an increase in the proportion of caffeine-eluted DNA. In terms of most analysis, differences between DNA fraction from nitrosamine-treated rats were similar to differences exhibited by preparations from control animals. However, structural analysis using denaturation kinetics indicated defects in both the NaCl- and caffeine-eluted DNA isolated from nitrosamine-treated rats. The two fractions differed from each other in that caffeine-eluted DNA exhibited a degree of structural damage far greater than that detected in any preparation from control animals...

scholarly journals Neutrophil elastase produces 52-kD and 30-kD glucocorticoid receptor fragments in the cytosol of human leukemia cells

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 860-868
Author(s):  
CW Distelhorst ◽  
KE Janiga ◽  
KJ Howard ◽  
SE Strandjord ◽  
EJ Campbell
Keyword(s):  
Glucocorticoid Receptor ◽  
Neutrophil Elastase ◽  
Glucocorticoid Receptors ◽  
Leukemia Cell ◽  
Specific Inhibitor ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Receptor Fragments ◽  
Limited Digestion

Characterization of glucocorticoid receptors in leukemia cells is important to understand mechanisms of glucocorticoid resistance but has been impeded by receptor fragmentation in cytosol extracts. We recently found that formation of 52- and 30-kilodalton (kD) glucocorticoid receptor fragments in cytosol of leukemia cells is due to proteolysis and is blocked by diisopropylfluorophosphate (DFP). In the present study, we identify a 28-kD serine protease in cytosol of leukemia cells that binds [3H]DFP and correlates with the formation of 52- and 30-kD receptor fragments. This protease is immunoprecipitated by antiserum to neutrophil elastase. Limited digestion of [3H]dexamethasone-21-mesylate- labeled receptors by purified neutrophil elastase produces 52- and 30- kD receptor fragments. Receptor fragmentation in the cytosol of leukemia cells in inhibited by methoxysuccinyl-alanyl-alanyl-prolyl- valyl-chloromethylketone, a highly specific inhibitor of neutrophil elastase. The addition of as few as 5% neutrophils to a lymphoid cell suspension provides sufficient elastase to produce receptor fragmentation. Our findings indicate that neutrophil elastase is responsible for receptor fragmentation in the cytosol of leukemia cells. The neutrophil elastase may be endogenous to the leukemia cells or may come from neutrophils that contaminate leukemia cell suspensions.

Re-Investigation of the Protein Structure of Coenzyme B12-Dependent Diol Dehydrase

1987 ◽  
Vol 42 (4) ◽  
pp. 353-359 ◽  
Author(s):  
Katsuyuki Tanizawa ◽  
Nobuyoshi Nakajima ◽  
Tetsuo Toraya ◽  
Hidehiko Tanaka ◽  
Kenji Soda
Keyword(s):  
Protein Structure ◽  
Membrane Fraction ◽  
Enzyme Purification ◽  
Soluble Fraction ◽  
Proteolytic Cleavage ◽  
Subunit Structure ◽  
Coenzyme B12 ◽  
Enzyme Preparations ◽  
Protein Components ◽  
Limited Digestion

We have purified diol dehydrase, an adenosylcobalamin-dependent enzyme, from Klebsiella pneumoniae by two different procedures to re-investigate its protein structure; one including its extraction with detergent from the membrane fraction, and the other consisting of only chromato­graphic separations of the soluble fraction. The enzyme preparations obtained by these two methods were different in the subunit structure, but both are identical in molecular weight, and in enzymological and immunochemical properties. In addition, the enzyme preparation obtained from the membrane fraction dissociated reversibly into two dissimilar protein components (F and S) in the absence of substrate, as did the preparation from the soluble fraction. Although the subunit multiplicity of component S might be partly due to proteolytic cleavage during the enzyme purification as revealed by limited digestion with trypsin, component F is not a product of proteolytic cleavage of component S, but a primordial and essential constituent of the enzyme.

CHARACTERIZATION OF THE VON WILLEBRAND FACTOR-BINDING DOMAIN OF PLATELET MEMBRANE GLYCOPROTEIN Ib

1987 ◽  
Author(s):  
M Handa ◽  
K Titani ◽  
K Takio ◽  
Z M Ruggeri
Keyword(s):  
Von Willebrand Factor ◽  
Binding Domain ◽  
Platelet Membrane ◽  
Membrane Glycoprotein ◽  
Tryptic Fragment ◽  
Amino Terminal ◽  
Platelet Membrane Glycoprotein ◽  
Limited Digestion ◽  
Von Willebrand ◽  
Willebrand Factor

We have previously obtained immunochemical evidence that the von Willebrand factor (vWF)-binding domain of the platelet membrane glycoprotein (GP) Ib is located near the amino terminus of the a subunit (Journal of Biological Chemistry 261: 12579-12585, 1986). We have now determined the complete amino acid sequence of the 45 kDa tryptic fragment of glycocalicin that contains this domain. Purified glycocalicin was subjected to limited digestion with trypsin and the proteolytic fragments were separated by size-exclusion high-pressure liquid chromatography. Two fragments of 45 kDa and 84 kDa, respectively, were obtained under nonreducing conditions. After reduction and S-carboxymethylation, the 84 kDa fragment was unchanged, while the 45 kDa fragment yielded two new fragments, one of 35 kDa and the other of 7 kDa. This finding proves the existence of a trypsin cleavage site within a disulfide loop. Two primary sets of overlapping fragments were obtained by cleavage of the carboxymethylated protein at methionyl and lysyl bonds following treatment with cyanogen bromide and Achromobacter protease I, respectively. Additional fragments were obtained by treatment of glycocalicin with Staphylococcus aureus V8 protease and Serratia marcescens protease. Analysis of all these fragments provided data that allowed determination of the sequence of the amino terminal 299 residues of the GP Ib a-chain. This includes the 45 kDa tryptic fragment containing the vWF-binding domain. This 299-residue sequence, corresponding approximately to two thirds of the α-chain polypeptide, is largely hydrophobic and contains only two N-linked and one O-linked carbohydrate chains. A hydrophilic region exists between residues 215-299, with a cluster of ten negatively charged residues at 269-287. This area is likely to attract positively charged molecules. The hydrophilic, highly glycosylated (at Ser/Thr residues) region corresponding to the previously described "macroglycopeptide" begins at residue 292. The determined sequence of glycocalicin contains a region with seven repeats, indicative of gene duplication, and is highly homologous to human leucine-rich α2-glycoprotein.

Effect of Structural Changes of Collagen on Collagen-platelet Reaction

1975 ◽  
Author(s):  
M. Yamanaka ◽  
Y. Nagai ◽  
M. Kodama
Keyword(s):  
Platelet Aggregation ◽  
Structural Changes ◽  
Tertiary Structure ◽  
Shape Change ◽  
Critical Length ◽  
Sequential Reaction ◽  
Initial Shape ◽  
Soluble Collagen ◽  
Skin Collagen ◽  
Limited Digestion

Removal of telopeptides from the acid soluble calf skin collagen with proctase did not affect the platelet aggregation. Limited digestion with tadpole collagenase at 20° C maintained the ability of collagen to aggregate platelets, when it was measured at 20°C., although its ability was completely abolished at 37° C. Neither α1 and α2-components of collagen did show the ability to aggregate platelets. Denaturation of the soluble collagen by heating at 45° C for 10 min destroyed its ability to aggregate platelets, although an initial shape change of platelets was observed.These results suggest that there may be different mechanisms in relation to the structure of collagen in the adhesion of platelets and the aggregation of platelets as a sequential reaction and that the tertiary structure of the soluble collagen composed of three polypeptides chains with a some critical length may be necessary for the platelet aggregation.

Isolation and preliminary characterization of histone H1.b allelic variants from quail erythrocytes

Genome ◽  
1998 ◽  
Vol 41 (5) ◽  
pp. 709-719 ◽  
Author(s):  
Jan Palyga ◽  
James M Neelin
Keyword(s):  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cation Exchange Resin ◽  
Gel Permeation Chromatography ◽  
Histone H1 ◽  
Linker Histone ◽  
Allelic Variants ◽  
Limited Digestion

Our goal was to purify and characterize the allelic variants H1b1 and H1b2 of histone H1.b, one of the seven subtypes of this linker histone extracted from Japanese quail erythrocyte nuclei. These variants are revealed phenotypically as band H1.3 or part of band H1.4 by polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS). All H1 subtypes together were separated from H5 by gel-permeation chromatography through Bio-Gel P-150. H1 was then fractionated on a column of the cation-exchange resin Amberlite CG-50 by using a shallow guanidine hydrochloride gradient, which enriched subtype H1.b together with H1.z and overlapping with subtypes H1.a and H1.b. Alternatively purification of subtypes was achieved electrophoretically: total H1 fractions from quail with different H1 phenotypes were first resolved into sub-types by PAGE in acetic acid - urea; after staining, the appropriate H1.b bands from several parallel gel pieces were excised and the histone was concentrated by PAGE in SDS. After fragmentation of H1.b in the gel pieces with N-bromosuccinimide (NBS), PAGE in SDS indicated no difference between H1b1 and H1b2 in the C-terminal "half" of the polypeptides. In contrast, limited digestion with endoprotease V8 from Staphylococcus aureus has shown that differences, probably by a few residues in length, reside in the N-terminal part of the molecule, close to the amino-terminus.Key words: quail histone H1.b, electrophoretic fractionation, linker histone variant.

scholarly journals The structure of Artemia sp. (brine shrimp) haemoglobins. Purification of a structural unit to homogeneity

1985 ◽  
Vol 227 (3) ◽  
pp. 917-924 ◽  
Author(s):  
L Moens ◽  
M L Van Hauwaert ◽  
G Wolf
Keyword(s):  
Brine Shrimp ◽  
Structural Unit ◽  
Single Type ◽  
Structural Units ◽  
Limited Digestion

The extracellular haemoglobins (Mr 260 000) of the brine shrimp Artemia sp. were cleaved by limited digestion with subtilisin. Structural units of Mr 16 000, which can bind dioxygen reversibly, were isolated. Analysis of the 16 000-Mr fraction (E) reveals the presence of a limited number of structural units. A single type of structural unit, E1 (Mr 15 800; pI4.8), was purified to homogeneity and characterized.

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