scholarly journals A cystatin-like cysteine proteinase inhibitor from venom of the African puff adder (Bitis arietans)

1987 ◽  
Vol 246 (3) ◽  
pp. 795-797 ◽  
Author(s):  
H J Evans ◽  
A J Barrett
Keyword(s):  
Gel Electrophoresis ◽  
Cathepsin B ◽  
Proteinase Inhibitor ◽  
Cysteine Proteinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Minor Component ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Cysteine Proteinase Inhibitor ◽  
Strong Activity

Venoms from eight snakes have been screened for inhibitory activity against papain, strong activity being found in that of the African puff adder, Bitis arietans. The inhibitor from B. arietans venom has been purified by affinity chromatography on carboxymethyl-papain-Sepharose and ion-exchange chromatography. The inhibitor had an apparent Mr of 13,000 in SDS/polyacrylamide gel electrophoresis, and pI value of 6.5 (major component) or 6.3 (minor component). Values of Ki for the inhibition of papain, cathepsin B and dipeptidyl peptidase I were 0.10, 2.7 and 0.23 nM, respectively; chicken calpain was not inhibited.

scholarly journals Human plasma α-cysteine proteinase inhibitor. Purification by affinity chromatography, characterization and isolation of an active fragment

1984 ◽  
Vol 221 (2) ◽  
pp. 445-452 ◽  
Author(s):  
A D Gounaris ◽  
M A Brown ◽  
A J Barrett
Keyword(s):  
Affinity Chromatography ◽  
Human Plasma ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Proteinase Inhibitor ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cysteine Proteinase Inhibitor

Human plasma alpha-cysteine proteinase inhibitor (alpha CPI) was purified by a two-stage method: affinity chromatography on S-carboxymethyl-papain-Sepharose, and high-resolution anion-exchange chromatography. The protein was obtained as a form of Mr about 64 000 and material of higher Mr (about 100 000). In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with reduction, both forms showed a major component of Mr 64 000. An antiserum was raised against alpha CPI, and ‘rocket’ immunoassays showed the mean concentration in sera from 19 individuals to be 35.9 mg/dl. Both low-Mr and high-Mr forms of alpha CPI were confirmed to be sialoglycoproteins by the decrease in electrophoretic mobility after treatment with neuraminidase. alpha CPI was shown immunologically to be distinct from antithrombin III and alpha 1-antichymotrypsin, two serine proteinase inhibitors from plasma with somewhat similar Mr values. alpha CPI was also distinct from cystatins A and B, the two intracellular low-Mr cysteine proteinase inhibitors from human liver. Complexes of alpha CPI with papain were detectable in immunoelectrophoresis, but dissociated to free enzyme and intact inhibitor in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The stoichiometry of binding of papain was close to 1:1 for both low-Mr and high-Mr forms. alpha CPI was found to be a tight-binding inhibitor of papain and human cathepsins H and L (Ki 34 pM, 1.1 nM and 62 pM respectively). By contrast, inhibition of cathepsin B was much weaker, Ki being about 35 microM. Dipeptidyl peptidase I also was weakly inhibited. Digestion of alpha CPI with bromelain gave rise to an inhibitory fragment of Mr about 22 000, which was isolated.

Rapid separation of bovine caseins by mass ion exchange chromatography

1989 ◽  
Vol 56 (3) ◽  
pp. 391-397 ◽  
Author(s):  
K. F. Ng-Kwai-Hang ◽  
J. P. Pélissier
Keyword(s):  
Liquid Chromatography ◽  
Ion Exchange ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Rapid Separation ◽  
Rapid Isolation ◽  
Time Required

SummaryThe rapid isolation of major bovine caseins in gram quantities was investigated. Whole casein was precipitated from individual cow's milk by adjusting the pH to 4·6 and the precipitated casein was suspended in 4·5 M urea (pH 8·0) containing 0·02 M imidazole and 0·03 M β-mercaptoethanol, and bound on a QAE Zeta Prep 250 cartridge. Stepwise elution with the urea/imidazole β-mercaptoethanol buffer and varying amounts of NaCl gave five well resolved peaks, which were identified by polyacrylamide gel electrophoresis and fast protein liquid chromatography to be pure γ-casein, κ-casein. β-casein, β-casein and αs-casein, respectively. The ion exchange cartridge was regenerated by flushing with buffer containing 0·50 Μ-NaCl followed by equilibration with starting buffer before separation of next sample. The time required to run each sample including cartridge regeneration and equilibration was 4 hours.

scholarly journals Purification of rabbit bone inhibitor of collagenase

1981 ◽  
Vol 195 (1) ◽  
pp. 159-165 ◽  
Author(s):  
T E Cawston ◽  
W A Galloway ◽  
E Mercer ◽  
G Murphy ◽  
J J Reynolds
Keyword(s):  
Tissue Culture ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Serine Proteinases ◽  
Bacterial Collagenase ◽  
Rabbit Bone

1. Rabbit bones in tissue culture synthesize an inhibitor of collagenase during the first 4 days of culture. 2. The inhibitor was purified by a combination of gel filtration, concanavalin A--Sepharose chromatography, ion-exchange chromatography and zinc-chelate affinity chromatography. 3. The purified inhibitor migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had a mol.wt. of 28000. 4. The inhibitor blocked the activity of the metalloproteinases collagenase, gelatinase, neutral proteinase III (proteoglycanase), human leucocyte collagenase and gelatinase, but not thermolysin or bacterial collagenase. The serine proteinases plasmin and trypsin were not inhibited. 5. The inhibitor interacted with purified rabbit bone collagenase with 1:1 stoichiometry. 6. The inhibitory activity was lost after incubation for 1 h at 90 degrees C, after treatment with trypsin (250 micrograms/ml) at 37 degrees C for 30 min and after reduction and alkylation.

Charge Properties of Mitochondrial Matrix Proteins

1975 ◽  
Vol 53 (11) ◽  
pp. 1150-1157 ◽  
Author(s):  
P. M. Strasberg ◽  
K. B. Freeman
Keyword(s):  
Gel Electrophoresis ◽  
Matrix Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Matrix Proteins ◽  
Mitochondrial Matrix ◽  
Total Fraction ◽  
Acidic Nature

Proteins of the rat liver mitochondrial matrix have been separated into anionic (acidic), cationic (basic), and neutral groups by electrophoresis. These groups represent 69, 8, and 23% of the total matrix protein, respectively, compared to 69, 21, and 10% for the cytosol protein. The acidic nature of the mitochondrial matrix proteins has been confirmed by cellulose ion-exchange chromatography, isoelectric focusing in sucrose gradients, and amino acid analysis. The anionic, cationic, and neutral matrix proteins were separated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis into 18, 6, and 5 bands, respectively, compared to 22 bands for the total fraction. The significance of the charge properties of these proteins in terms of mitochondrial biogenesis is discussed.

Purification, characterization, and antifungal activity of chitinase from Fusarium chlamydosporum, a mycoparasiteto groundnut rust, Puccinia arachidis

1998 ◽  
Vol 44 (7) ◽  
pp. 646-651 ◽  
Author(s):  
N Mathivanan ◽  
V Kabilan ◽  
K Murugesan
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Puccinia Arachidis ◽  
Fusarium Chlamydosporum ◽  
Groundnut Rust ◽  
Germ Tubes

Chitinase (EC 3.2.1.14) was isolated from the culture filtrate of Fusarium chlamydosporum and purified by ion-exchange chromatography and gel filtration. The molecular mass of purified chitinase was 40 kDa as estimated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Chitinase was optimally active at a pH of 5 and stable from pH 4 to 6 and up to 40°C. Among the metals and inhibitors tested, mercuric chloride completely inhibited the enzyme activity. The activity of chitinase was high on colloidal and pure chitin. The purified chitinase inhibited the germination of uredospores of Puccinia arachidis and also lysed the walls of uredospores and germ tubes. The results from these experiments indicated that chitinase of F. chlamydosporum plays an important role in the biocontrol of groundnut rust. Key words: Fusarium chlamydosporum, chitinase, purification, Puccinia arachidis, uredospores.

Effective pretreatment by cysteine proteinase inhibitor for improved analysis of protein components of trematodes on SDS-PAGE

1990 ◽  
Vol 64 (3) ◽  
pp. 175-179 ◽  
Author(s):  
M. Itoh ◽  
S. Sato ◽  
A. Moriyama ◽  
M. Sasaki
Keyword(s):  
Proteinase Inhibitor ◽  
Cysteine Proteinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Clonorchis Sinensis ◽  
Sodium Dodecyl ◽  
Heating Process ◽  
Cysteine Proteinase Inhibitor ◽  
Electrophoretic Patterns ◽  
Sds Page ◽  
Protein Components

ABSTRACTSince Fasciola sp. contained proteolytic enzyme(s), it was confirmed that degradation took place in protein components in extracts of the liver flukes, which resulted in lack of clarity of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Degradation was shown to occur mostly during a heating process of the extract samples. The proteolytic activity in the extracts was completely blocked and electrophoretic patterns were improved only by the use of cysteine proteinase inhibitor N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). Great improvement was also noted in electrophoretic patterns of the extracts of other trematodes, such as Paragonimus westermani, P. miyazakii and Clonorchis sinensis, when their extracts were treated with E-64.

Biosynthèse "In Vitro" de l'Hormone Béta-Lipotropique de Boeuf

1974 ◽  
Vol 52 (5) ◽  
pp. 349-358 ◽  
Author(s):  
X. Bertagna ◽  
M. Lis ◽  
C. Gilardeau ◽  
M. Chrétien
Keyword(s):  
Amino Acids ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Usual Method ◽  
Pituitary Hormone ◽  
Melanocyte Stimulating Hormone

Sheep beta-lipotropic hormone (β-LPH) is a pituitary hormone made of 90 amino acids and having a portion of its sequence (41–58) identical with the structure of beta-melanocyte-stimulating hormone (β-MSH). We hypothetized that β-LPH could be the biological precursor of β-MSH. We studied the biosynthesis of these two molecules by monitoring the incorporation of radioactive amino acids in beef pituitary slices. We separated β-LPH from the other radioactive proteins with the usual method of purification described previously and we characterized the proteins by ion-exchange chromatography, gel filtration, and polyacrylamide gel electrophoresis. Our results show that the pituitary slices synthesized a radioactive β-LPH which has all the characteristics of non-radioactive β-LPH. However, in the conditions used, we could not demonstrate any biosynthesis of β-MSH after 4 h incubation. These results suggest that the conversion of β-LPH into β-MSH, if it exists, is a slow process and should be studied in more prolonged incubations.

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