scholarly journals Purification and partial characterization of a thiol proteinase from the thermophilic fungus Humicola lanuginosa

1982 ◽  
Vol 205 (1) ◽  
pp. 147-152 ◽  
Author(s):  
S Shenolikar ◽  
K J Stevenson
Keyword(s):  
Gel Filtration ◽  
Extracellular Fluid ◽  
Thiol Group ◽  
Thermophilic Fungus ◽  
Sedimentation Equilibrium ◽  
Amino Acid Residues ◽  
Humicola Lanuginosa ◽  
Mercuric Ions ◽  
Thiol Proteinase

An extracellular thiol proteinase was produced by the growth of a thermophilic fungus, Humicola lanuginosa, on a medium containing 2% casein, and was purified to virtual homogeneity by affinity chromatography on organomercurial columns. The essential thiol group for activity was confirmed by the inhibition of the enzyme by p-chloromercuribenzoate and mercuric ions. The enzyme, purified 27-fold from the extracellular fluid, exhibited an Mr of 23700 on gel filtration and sedimentation equilibrium. The H. lanuginosa proteinase preferentially cleaves at the C-terminal end of hydrophobic amino acid residues. This proteinase differed from the plant enzyme papain in its interaction with three affinity matrices and its substrate specificity towards synthetic substrates. This enzyme represents a unique example of a thiol proteinase obtained from a fungal source.

scholarly journals Cathepsin B, the lysosomal thiol proteinase of calf liver

1969 ◽  
Vol 114 (4) ◽  
pp. 673-678 ◽  
Author(s):  
O. Snellman
Keyword(s):  
Cathepsin B ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Chelating Agent ◽  
Thiol Group ◽  
Maximum Activity ◽  
Active Centre ◽  
Peptide Bonds ◽  
Hydrolysis Of ◽  
Thiol Proteinase

Cathepsin B from calf liver was obtained by a method involving preparation of a lysosomal–mitochondrial pellet and treatment of this pellet with acetone. The material was extracted with an acid buffer, pH4·0, and then precipitated from the extract with acetone. The precipitate was dissolved in phosphate buffer, pH7·4, and subjected to gel filtration on Sephadex G-200 and G-100. The cathepsin B emerged in a range of molecular weight much lower than 50000 as a well-defined component. The purity of this material was checked by electrophoresis. To obtain maximum activity the enzyme had to be activated with a chelating agent and a reducing agent (i.e. EDTA and cysteine). A number of different substrates were used. The enzyme was active for the hydrolysis of both peptide bonds and ester bonds and had approximately equal reactivity in the two cases. The pH-dependence of the hydrolysis was the same with both substrates. The binding of the substrates was half-maximal at pH4·5 and at pH6·8. A thiol group occurred in the active centre but this group ought to have a much higher pK than that found in this enzyme.

The Isolation and Characterization of the ATPase Inhibitory Protein (TN-I) from Bovine Cardiac Muscle

1975 ◽  
Vol 53 (11) ◽  
pp. 1207-1213 ◽  
Author(s):  
L. D. Burtnick ◽  
W. D. McCubbin ◽  
C. M. Kay
Keyword(s):  
Molecular Weight ◽  
Cardiac Muscle ◽  
Calcium Binding ◽  
Sodium Dodecyl ◽  
Basic Amino Acid ◽  
Sedimentation Equilibrium ◽  
Amino Acid Residues ◽  
Inhibitory Protein ◽  
Isolation And Characterization

The inhibitory component of the troponin complex (TN-I) was purified from bovine cardiac muscle, using a combination of ion exchange and molecular exclusion chromatographies in the presence of urea. It has the ability to inhibit the Mg2+-activated ATPase (EC 3.6.1.3) of a synthetic cardiac actomyosin preparation and this inhibition is reversed by the addition of cardiac calcium binding component of troponin (TN-C). Conventional sedimentation equilibrium experiments suggest a molecular weight for cardiac TN-I of 22 900 ± 500. However, sodium dodecyl sulfate (SDS) gels indicate a molecular weight of 27 000 ± 1000. The mobility of TN-I on SDS gels may be anomalous due to the high proportion of basic amino acid residues in the protein. Cardiac TN-I and TN-C interact to form a tight complex, even in the presence of 6 M urea. The results of this study invite direct comparison with results published for rabbit skeletal TN-I.

scholarly journals Characterization of an exocellular serine-thiol proteinase activity in Paracoccidioides brasiliensis

1995 ◽  
Vol 309 (1) ◽  
pp. 209-214 ◽  
Author(s):  
A K Carmona ◽  
R Puccia ◽  
M C F Oliveira ◽  
E G Rodrigues ◽  
L Juliano ◽  
...  
Keyword(s):  
Mercuric Acetate ◽  
Paracoccidioides Brasiliensis ◽  
Gel Filtration ◽  
Proteinase Activity ◽  
Reversible Inhibitor ◽  
Peptidase Activity ◽  
Low Protein ◽  
Optimum Ph ◽  
Thiol Proteinase

An exocellular proteinase activity has been characterized in Paracoccidioides brasiliensis culture filtrates. Chromatographic analysis showed that the activity was eluted from an anion-exchange Resource Q column at 0.08-0.1 M NaCl, and by gel filtration near ovalbumin elution, in a single peak. Purification of the proteinase, however, was hampered by the low protein yield, in contrast to the high peptidase activity. Numerous chromogenic peptidyl p-nitroanilide derivatives and internally quenched fluorescent peptides, flanked by Abz (O-aminobenzoyl) and EDDnp (ethylenediaminedinitrophenyl), were tested as substrates. Cleavage was observed with Abz-MKRLTL-EDDnp, Abz-FRLVR-EDDnp, and Abz-PLGLLGR-EDDnp at Leu-Thr, Leu-Val and Leu-Leu/Leu-Gly bonds respectively as determined by isolation of the corresponding fragments by HPLC. Leucine at P1 seemed to be restrictive for the activity of the exocellular enzyme, but threonine (P'1) and leucine (P'2) in Abz-MKRLTL-EDDnp apparently were not essential. Also, a pair of alanines could substitute for lysine (P3) and arginine (P2) in this substrate, with a decrease in the Km values. The exocellular peptidase activity of P. brasiliensis had an optimum pH of > 9.0 and was irreversibly inhibited by PMSF, mercuric acetate and p-hydroxymercuribenzoate. Inhibition of the mercuriate compounds could be partially reversed by Cys/EDTA. E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanido)butene] was a weak and reversible inhibitor, whereas EDTA and pepstatin were not inhibitory. These results suggest that P. brasiliensis exocellular enzyme belongs to the subfamily of SH-containing serine proteinases.

scholarly journals Characterization of gastric mucoproteins isolated by equilibrium density-gradient centrifugation in caesium chloride

1974 ◽  
Vol 141 (3) ◽  
pp. 633-639 ◽  
Author(s):  
Bryan J. Starkey ◽  
David Snary ◽  
Adrian Allen
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Equilibrium Density ◽  
Amino Acid Residues ◽  
Gastric Mucus ◽  
Terminal Amino Acid ◽  
Terminal Amino

1. The mucoprotein from pig gastric mucus has been purified by equilibrium centrifugation in a CsCl gradient. 2. This procedure removes the non-covalently bound protein, which is closely associated with the mucoprotein and not easily removed from it by gel filtration. 3. The purified mucoprotein is separable by gel filtration into a high-molecular-weight mucoprotein A (mol.wt. 2.3×106) and a low-molecular-weight mucoprotein B/C (mol.wt. 1.15×106). 4. These two mucoproteins have the same chemical analysis namely fucose 11.3%, galactose 26%, glucosamine 19.5%, galactosamine 8.3% and protein 13.6%. 5. Mucoprotein A contains 3.1% ester sulphate. 6. These mucoproteins are isolated without enzymic digestion and have a higher protein content than the blood-group-substance mucoproteins from proteolytic digestion of gastric mucus. Detailed amino acid analysis shows that the extra protein in the non-enzymically digested material is composed of amino acids other than serine and threonine. 7. Mucoproteins A and B/C contain respectively 130 and 9 half-cystine residues per molecule of which about 78 and 6 residues are involved in disulphide linkages. 8. Cleavage of these disulphide linkages by mercaptoethanol splits both mucoproteins into four equally sized subunits of mol.wt. 5.2×105for mucoprotein A and 2.8×104for mucoprotein B/C. 9. The sole N-terminal amino acid of mucoprotein A is aspartic acid, whereas mucoprotein B/C has several different N-terminal amino acid residues.

scholarly journals Purification of a high-molecular-weight somatoliberin (growth-hormone-releasing factor) from pig hypothalami

1983 ◽  
Vol 209 (3) ◽  
pp. 643-651 ◽  
Author(s):  
J E C Sykes ◽  
P J Lowry
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Amino Acid ◽  
Response Curve ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Amino Acid Residues ◽  
Growth Hormone Releasing Factor

Preliminary observations [Sykes & Lowry (1980) J. Endocrinol. 85, 42P-43P] had suggested that the major hypothalamic somatoliberin (growth-hormone-releasing factor) was a larger peptide than the other characterized hypothalamic factors, with an elution position on Sephadex G-50 between those of neurophysin and corticotropin. The present paper reports the isolation and preliminary characterization of pig hypothalamic somatoliberin. Acid extracts of pig stalk median eminence were purified by gel filtration and preparative and analytical high-pressure liquid chromatography to yield a preparation that was specific in the release of somatotropin (growth hormone) in vitro, giving a steep dose-response curve at doses in the range 0.20-3.0 ng. Amino acid analysis revealed a non-cysteine-containing peptide with a high number of glutamate (or glutamine) and aspartate (or asparagine) residues. The peptide had about 56-57 amino acid residues and an apparent molecular weight of 6400, in keeping with its elution position on a column of Sephadex G-50.

scholarly journals Purification and characterization of carboxylesterases from rat lung

1991 ◽  
Vol 274 (3) ◽  
pp. 693-697 ◽  
Author(s):  
R Gaustad ◽  
K Sletten ◽  
D Løvhaug ◽  
F Fonnum
Keyword(s):  
Cholinesterase Inhibitors ◽  
Organophosphorus Compounds ◽  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Amino Acid Residues ◽  
Rat Lung ◽  
Purification And Characterization ◽  
Cation Exchangers ◽  
Isoelectric Points

Carboxylesterase (EC 3.1.1.1) has played an important part in our understanding of the toxicokinetic behaviour of the organophosphorus cholinesterase inhibitors. Carboxylesterases are a heterogeneous group of enzymes that can be separated on the basis of their isoelectric points and by their substrate-specificity. We have purified the isoenzyme (pI 5.8) present in greatest activity in rat lung to near homogeneity. The enzyme was purified by (NH4)2SO4 precipitation, gel filtration, chromatofocusing, separation on anion- and cation-exchangers and hydrophobic-interaction chromatography. The purified enzyme has a molecular mass of approx. 180 kDa with subunits of 60 kDa. The substrate and inhibitor specificities of the enzyme have been characterized. Edman degradation revealed the first 19 amino acid residues of the enzyme. The N-terminus was found to be tyrosine. Inhibition of the enzyme by organophosphorus compounds differed greatly from that of cholinesterases, despite the partial analogy at the N-terminal region.

scholarly journals Purification and Characterization of a Vulnificolysin-Like Cytolysin Produced by Vibrio tubiashii

2001 ◽  
Vol 67 (8) ◽  
pp. 3707-3711 ◽  
Author(s):  
Mahendra H. Kothary ◽  
Rachel B. Delston ◽  
Sherill K. Curtis ◽  
Barbara A. McCardell ◽  
Ben D. Tall
Keyword(s):  
Vibrio Vulnificus ◽  
Chinese Hamster Ovary Cells ◽  
Gel Filtration ◽  
Chinese Hamster ◽  
Hydrophobic Interaction Chromatography ◽  
Atlantic Menhaden ◽  
Amino Acid Residues ◽  
Ovary Cells ◽  
Vibrio Tubiashii

ABSTRACT An extracellular cytolysin from Vibrio tubiashii was purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3. In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture. Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH. Twelve of the first 17 N-terminal amino acid residues (Asp-Asp-Tyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

The Immunological Characterization of Human Antibodies to Factor VIII Isolated by Immuno-Affinity Chromatography

1981 ◽  
Vol 45 (01) ◽  
pp. 060-064 ◽  
Author(s):  
M L Kavanagh ◽  
C N Wood ◽  
J F Davidson
Keyword(s):  
Factor Viii ◽  
Affinity Chromatography ◽  
Gel Filtration ◽  
Immunological Characterization ◽  
Human Antibodies ◽  
Heavy Chains ◽  
Light Chain Type ◽  
Antibody Preparations ◽  
Chain Type

SummaryNine human antibodies to factor VIII were isolated from haemophilic plasmas by affinity chromatography and gel filtration and six were subsequently subjected to immunological characterization. Three partially purified preparations were similarly characterized. Eight of the antibodies were characterized as being exclusively IgG and one preparation was found to contain IgM. Seven of the antibodies contained only a single light chain type, four being of type lambda and three of type kappa. Two antibody preparations contained both kappa and lambda light chains. In four of the preparations, only a single heavy chain sub-class could be demonstrated, three of IgG3 and one of IgG4. Of the remainder, three were a mixture of IgG3 and IgG4 sub-classes and one contained both IgG2 and IgG4. IgG sub-classification could not be achieved with the IgM-containing preparation. These results demonstrate a restricted heterogeneity of light and heavy chains in human antibodies to factor VIII.

Preparation and Characterization of NH2-Terminal Fibrinogen Bβ Fragments from N-DSK of Human Fibrinogen

1984 ◽  
Vol 51 (01) ◽  
pp. 016-021 ◽  
Author(s):  
S Birken ◽  
G Agosto ◽  
B Lahiri ◽  
R Canfield
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Human Fibrinogen ◽  
Human Volunteers ◽  
Cnbr Cleavage ◽  
Two Peaks

SummaryIn order to investigate the early release of NH2-terminal plasmic fragments from the Bβ chain of fibrinogen, substantial quantities of Bβ 1-42 and Bβ 1-21 are required as immunogens, as radioimmunoassay standards and for infusion into human volunteers to determine the half-lives of these peptides. Towards this end methods that employ selective proteolytic cleavage of these fragments from fibrinogen have been developed. Both the N-DSK fragment, produced by CNBr cleavage of fibrinogen, and Bβ 1-118 were employed as substrates for plasmin with the finding of higher yields from N-DSK. Bβ 1-42 and Bβ 1-21 were purified by gel filtration and ion-exchange chromatography on SP-Sephadex using volatile buffers. When the purified preparation of Bβ 1-42 was chromatographed on reverse-phase high performance liquid chromatography, two peaks of identical amino acid composition were separated, presumably due either to pyroglutamate or to amide differences.

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