scholarly journals Purification of human urinary prokallikrein. Identification of the site of activation by the metalloproteinase thermolysin

1985 ◽  
Vol 232 (3) ◽  
pp. 851-858 ◽  
Author(s):  
Y Takada ◽  
R A Skidgel ◽  
E G Erdös
Keyword(s):  
Amino Acids ◽  
Human Plasma ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Plasma Kallikrein ◽  
Double Immunodiffusion ◽  
C Terminus ◽  
Hydrophobic Amino Acids

Human urinary active kallikrein and prokallikrein were separated on DEAE-cellulose and octyl-Sepharose columns and both purified to homogeneity by affinity chromatography, gel filtration and hydrophobic h.p.l.c. Prokallikrein was monitored during purification by trypsin activation followed by determination of both amidase and kininogenase activity. After trypsin activation, purified prokallikrein had a specific kininogenase activity of 39.4 micrograms of bradykinin equivalent/min per mg and amidase activity of 16.5 mumol/min per mg with D-Val-Leu-Arg-7-amino-4-trifluoromethylcoumarin. Purified active kallikrein had a specific activity of 47 micrograms of bradykinin/min per mg. The molecular mass of prokallikrein was 48 kDa on electrophoresis and 53 kDa on gel filtration whereas active kallikrein gave values of 46 kDa and 53 kDa respectively. Antisera to active and prokallikrein were obtained. In double immunodiffusion and immunoelectrophoresis, antiserum to active kallikrein reacted with active and pro-kallikrein. Antiserum to prokallikrein contained antibodies to determinants not found in active kallikrein, presumably due to the presence of the activation peptide in the proenzyme. Human prokallikrein can be activated by thermolysin, trypsin and human plasma kallikrein. Activation of 50% of the prokallikrein (1.35 microM) was achieved in 30 min with 25 nM-thermolysin, 78 nM-trypsin or 180 nM-human plasma kallikrein. Thus thermolysin was the most effective activator. Thermolysin activated prokallikrein by releasing active kallikrein with N-terminal Ile1-Val2. Thus human tissue (glandular) prokallikrein can be activated by two types of enzymes: serine proteinases, which cleave at the C-terminus of basic amino acids, and by a metalloproteinase that cleaves at the N-terminus of hydrophobic amino acids.

scholarly journals The Isolation of a Potent Plasma Kallikrein with Weak Plasminogen Activator Activity

1977 ◽  
Author(s):  
M.J. Gallimore ◽  
E. Fareid ◽  
H. Stormorken
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Specific Activity ◽  
Gel Filtration ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Plasma Kallikrein ◽  
Chromogenic Substrate ◽  
Plasminogen Activator Activity ◽  
Sepharose 4B

Kallikrein was isolated from human plasma by the following procedures: removal of euglobulins at pH 5.3; QAE Sephadex chromatography: gel filtration on Sephadex G-150 and G-100. The partially purified preparation was then freed of contaminants by running it down a column of Sepharose-4B to which had been linked antibodies to IgG and pre-PTA. Pre-kallikrein and kallikrein activities were monitored during the fractionation procedures using a new synthetic chromogenic substrate for plasma kallikrein (Chromozyme-PK, Penta-pharm, Basle, Switzerland). 5 mg of enzyme was obtained with a specific activity of 3.75 Chromozyme PK (CPK) units/mg at 22° (yield = 9.5%: purification factor = 6250) and the yield of kinin from heated plasma was 1.79 μg/CPK unit/min. The kallikrein exhibited very weak plasminogen activator activity when tested on fibrin plates and in fibrin clot lysis assays(1 CPK unit =0.133 CTA units urokinase). Some other properties of the enzyme are discussed.

Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver

1984 ◽  
Vol 62 (5) ◽  
pp. 276-279 ◽  
Author(s):  
C. H. Lin ◽  
W. Chung ◽  
K. P. Strickland ◽  
A. J. Hudson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Enzymatic Synthesis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Aromatic Amino Acids ◽  
Adenosylmethionine Synthetase ◽  
Cellulose Column

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.

scholarly journals The determination of oestradiol and oestrone in the plasma of the domestic fowl by a method involving the use of labelled derivatives

1968 ◽  
Vol 106 (1) ◽  
pp. 77-86 ◽  
Author(s):  
J. E. O'Grady
Keyword(s):  
Human Plasma ◽  
Paper Chromatography ◽  
Domestic Fowl ◽  
Specific Activity ◽  
High Specific Activity ◽  
Double Labelling ◽  
Plasma Samples ◽  
Labelling Technique ◽  
Preliminary Purification

1. A method involving the use of triple-labelled derivatives has been developed for the determination of total oestrone and oestradiol in the plasma of the domestic fowl. The double-labelling technique devised by Svendsen (1960) for the determination of free oestrogens in human plasma was modified to enable the total oestrogen recovery to be determined for each sample. 2. [6,7−3H2]Oestradiol-17β is added to the plasma samples (1–10ml.), which are hydrolysed with acid and the phenolic steroids then extracted and partially purified. The extract is esterified with iodobenzene-p[35S]-sulphonyl chloride of high specific activity. After addition of standard oestrogen [131I]iodobenzene-p-sulphonates the esters are finally purified by paper chromatography. 3. The oestrogens are determined by comparing the 3H/35S and 131I/35S ratios in the purified esters with similar ratios of appropriate standards. 4. With this procedure the recoveries of oestrone and oestradiol after hydrolysis were 70–85% and 72–84% respectively, and after hydrolysis and preliminary purification 38–53% and 39–51% respectively. With this procedure up to 500ng. of oestradiol can be determined. The sensitivity of the technique for oestrone is 3·0ng. and for oestradiol 2·1ng. 5. The ranges of oestradiol and oestrone concentrations found in six plasma samples were 8·3–21·4ng./ml. and 15·2–31·6ng./ml. respectively.

scholarly journals A New Procedure for Purification of Crotalase From Crotalus Adamanteus Venom

1977 ◽  
Author(s):  
F.S. Markland ◽  
J. Chou ◽  
Y. Shih ◽  
H. Pirkle
Keyword(s):  
Sodium Chloride ◽  
Large Scale ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fold Increase ◽  
Tris Buffer ◽  
High Yield ◽  
Crude Venom ◽  
Diamondback Rattlesnake

A new procedure has been developed for large scale, rapid purification of crotalase, the thrombin-1ike enzyme from the venom of the eastern diamondback rattlesnake (Crotalus adamanteus). The three step procedure involves: (1) molecular sieve chromatography on Sephadex G-100 in 0.04 M Tris buffer containing 0.10 M sodium chloride, pH 7.1; (2) gradient elution from DEAE-cellulose with sodium acetate buffer, pH 7.0; and (3) affinity chromatography on p-aminobenzamidine Sepharose using a spacer of 6-aminohexanoic acid. Crotalase was eluted from the affinity resin by 0.05 M Tris buffer containing 0.10 M sodium chloride and 0.15 M benzamidine-hydrochloride, pH 9.0, after first washing with the Tris buffer containing 0.40 M sodium chloride. From the crude venom, pure enzyme was obtained with an overall recovery of 40-60% of clotting activity and a 90-100 fold increase in specific activity. Crotalase was shown to be pure by Polyacrylamide disk gel electrophoresis which gave one band. The molecular weight was estimated to be approximately 31,000 by gel filtration on a calibrated Sephadex G-100 column. Amino acid analysis was performed and the composition was shown to be very similar to that reported earlier (F.S. Markland and P.S. Damus, J. Biol. Chem. 246: 6460, 1971). Clotting activity of the enzyme was not inhibited by heparin, either with or without plasma, whereas, thrombin was rapidly inactivated by heparin in the presence of plasma. In conclusion, we have developed a rapid and reproducible procedure for isolation in high yield of large quantities of the thrombin-like enzyme from the venom of the eastern diamondback rattlesnake. Studies are continuing on the primary structure and possible clinical applications of this enzyme.

scholarly journals AnkB, a Periplasmic Ankyrin-Like Protein inPseudomonas aeruginosa, Is Required for Optimal Catalase B (KatB) Activity and Resistance to Hydrogen Peroxide

2000 ◽  
Vol 182 (16) ◽  
pp. 4545-4556 ◽  
Author(s):  
Michael L. Howell ◽  
Eyad Alsabbagh ◽  
Ju-Fang Ma ◽  
Urs A. Ochsner ◽  
Martin G. Klotz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hydrogen Peroxide ◽  
Cytoplasmic Membrane ◽  
Membrane Vesicles ◽  
Gene Encoding ◽  
Rnase Protection ◽  
C Terminus ◽  
Hydrophobic Amino Acids ◽  
Increased Sensitivity ◽  
Membrane Spanning

ABSTRACT In this study, we have cloned the ankB gene, encoding an ankyrin-like protein in Pseudomonas aeruginosa. TheankB gene is composed of 549 bp encoding a protein of 183 amino acids that possesses four 33-amino-acid ankyrin repeats that are a hallmark of erythrocyte and brain ankyrins. The location ofankB is 57 bp downstream of katB, encoding a hydrogen peroxide-inducible catalase, KatB. Monomeric AnkB is a 19.4-kDa protein with a pI of 5.5 that possesses 22 primarily hydrophobic amino acids at residues 3 to 25, predicting an inner-membrane-spanning motif with the N terminus in the cytoplasm and the C terminus in the periplasm. Such an orientation in the cytoplasmic membrane and, ultimately, periplasmic space was confirmed using AnkB-BlaM and AnkB-PhoA protein fusions. Circular dichroism analysis of recombinant AnkB minus its signal peptide revealed a secondary structure that is ∼65% α-helical. RNase protection and KatB- and AnkB-LacZ translational fusion analyses indicated that katBand ankB are part of a small operon whose transcription is induced dramatically by H2O2, and controlled by the global transactivator OxyR. Interestingly, unlike the spherical nature of ankyrin-deficient erythrocytes, the cellular morphology of anankB mutant was identical to that of wild-type bacteria, yet the mutant produced more membrane vesicles. The mutant also exhibited a fourfold reduction in KatB activity and increased sensitivity to H2O2, phenotypes that could be complemented in trans by a plasmid constitutively expressing ankB. Our results suggest that AnkB may form an antioxidant scaffolding with KatB in the periplasm at the cytoplasmic membrane, thus providing a protective lattice work for optimal H2O2 detoxification.

Purification and characterization of rat epididymal neutral β-galactosidase and its changes during in vivo development

1993 ◽  
Vol 71 (1-2) ◽  
pp. 22-26 ◽  
Author(s):  
Pratima Dutta ◽  
Gopal C. Majumder
Keyword(s):  
Concanavalin A ◽  
Sexual Maturity ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Tissue Homogenate ◽  
Affinity Column ◽  
Ph Optimum

A neutral β-D-galactosidase has been partially purified from rat epididymis and characterized. The enzyme having molecular mass of approximately 50 kilodaltons has been purified 400-fold by using calcium phosphate gel adsorption, DEAE-cellulose chromatography, Sephadex G-100 gel filtration, and concanavalin A - agarose affinity chromatography. Although the neutral enzyme binds to the concanavalin A affinity column, the activity could be eluted with α-methyl mannoside only if the buffer contained salt (NaCl) at a concentration as high as 0.3 M. The enzyme was of cytosolic origin, since 90% of the total enzymic activity of the tissue homogenate was recovered in the soluble fraction of these cells. The neutral β-galactosidase was not dependent on metal ions for its activity and it had a pH optimum of 7.0. Zn2+, p-chloromercuribenzoate, Hg2+, and Pb2+ served as potent inhibitors of the enzyme. There was a marked increase (approximately fourfold) in the specific activity of the neutral β-galactosidase during sexual maturity of epididymis in vivo.Key words: neutral β-galactosidase, rat epididymal, cytosolic, developmental, sexual maturity.

scholarly journals Allelic Diversity of the Two Transferrin Binding Protein B Gene Isotypes among a Collection of Neisseria meningitidisStrains Representative of Serogroup B Disease: Implication for the Composition of a Recombinant TbpB-Based Vaccine

2000 ◽  
Vol 68 (9) ◽  
pp. 4938-4947 ◽  
Author(s):  
Bachra Rokbi ◽  
Geneviève Renauld-Mongenie ◽  
Michèle Mignon ◽  
B. Danve ◽  
David Poncet ◽  
...  
Keyword(s):  
Amino Acids ◽  
Bactericidal Activity ◽  
Immunoglobulin G ◽  
Binding Protein ◽  
Restriction Analysis ◽  
Allelic Diversity ◽  
Gene Variants ◽  
C Terminus ◽  
Specific Antisera

ABSTRACT The distribution of the two isotypes of tbpB in a collection of 108 serogroup B meningococcal strains belonging to the four major clonal groups associated with epidemic and hyperendemic disease (the ET-37 complex, the ET-5 complex, lineage III, and cluster A4) was determined. Isotype I strains (with a 1.8-kbtbpB gene) was less represented than isotype II strains (19.4 versus 80.6%). Isotype I was restricted to the ET-37 complex strains, while isotype II was found in all four clonal complexes. The extent of the allelic diversity of tbpB in these two groups was studied by PCR restriction analysis and sequencing of 10 newtbpB genes. Four major tbpB gene variants were characterized: B16B6 (representative of isotype I) and M982, BZ83, and 8680 (representative of isotype II). The relevance of these variants was assessed at the antigenic level by the determination of cross-bactericidal activity of purified immunoglobulin G preparations raised to the corresponding recombinant TbpB (rTbpB) protein against a panel of 27 strains (5 of isotype I and 22 of isotype II). The results indicated that rTbpB corresponding to each variant was able to induce cross-bactericidal antibodies. However, the number of strains killed with an anti-rTbpB serum was slightly lower than that obtained with an anti-TbpA+B complex. None of the sera tested raised against an isotype I strain was able to kill an isotype II strain and vice versa. None of the specific antisera tested (anti-rTbpB or anti-TbpA+B complex) was able to kill all of the 22 isotype II strains tested. Moreover, using sera raised against the C-terminus domain of TbpB M982 (amino acids 352 to 691) or BZ83 (amino acids 329 to 669) fused to the maltose-binding protein, cross-bactericidal activity was detected against 12 and 7 isotype II strains, respectively, of the 22 tested. These results suggest surface accessibility of the C-terminal end of TbpB. Altogether, these results show that although more than one rTbpB will be required in the composition of a TbpB-based vaccine to achieve a fully cross-bactericidal activity, rTbpB and its C terminus were able by themselves to induce cross-bactericidal antibodies.

scholarly journals Some properties of cytochrome b5 from liver microsomes of man, monkey, pig and chicken

1969 ◽  
Vol 115 (4) ◽  
pp. 849-856 ◽  
Author(s):  
F. G. Nóbrega ◽  
P. S. Araujo ◽  
M. Pasetto ◽  
I. Raw
Keyword(s):  
Amino Acids ◽  
Spectral Properties ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Gradient Elution ◽  
Closely Related Species ◽  
Ammonium Sulphate Fractionation ◽  
Terminal Amino

1. Cytochrome b5 was released from liver microsomes of man, monkey, pig and chicken by incubation with a crude lipase preparation. 2. By using DEAE-cellulose chromatography, ammonium sulphate fractionation, Sephadex-gel filtration and a final gradient elution on DEAE-Sephadex A-50, cytochromes b5 were obtained from the four species studied, all possessing similar spectral properties. 3. Stokes radii of the cytochromes were measured by gel filtration. 4. N-Terminal amino acids for the different cytochromes were serine for man and monkey, alanine for pig and glycine for chicken. 5. Amino acid analyses of the cytochromes are presented. 6. Peptide ‘fingerprint’ patterns of tryptic digests of the different cytochromes are discussed and clearly show increasing similarity for more closely related species.

RADIOIMMUNOASSAY OF GROWTH HORMONE IN HUMAN PLASMA

1972 ◽  
Vol 71 (4) ◽  
pp. 649-664 ◽  
Author(s):  
Kristian F. Hanssen
Keyword(s):  
Growth Hormone ◽  
Human Plasma ◽  
Coefficient Of Variation ◽  
Degradation Products ◽  
Peak Plasma ◽  
Gel Filtration ◽  
Tolerance Test ◽  
Precipitation Reaction ◽  
Insulin Tolerance

ABSTRACT The double antibody radio-immunoassay (pre-precipitation technique) for immunoreactive growth hormone (IRHGH) in human plasma has been evaluated on the basis of dilution and recovery experiments as well as by the investigation of accuracy and reproducibility. Given optimal conditions for the precipitation reaction, the method seems suitable for determination of plasma IRHGH. No cross-reaction between the precipitating antiserum and human gammaglobulin was demonstrated. Furthermore, by using an incubation period of 6 days for the reaction between 125I-HGH and the precipitated HGH antibodies, no serum factor influencing this reaction could be demonstrated. The results of the plasma IRHGH determinations were shown to be independent of the percentage of the radioactive degradation products present in the tracer HGH. The lower detection limit is better than 0.39 ng/ml. The cumulated within-assay coefficient of variation was between 4–7% and the cumulated between-assay coefficient of variation 10%. By using gel filtration, IRHGH in the plasma was shown to be nonhomogeneous when considering molecular weight. The normal fasting values in the ambulatory state was (Mean ± sd) ng/ml: Men 1.48 ± 0.33; women 3.83 ± 3.47. During the insulin tolerance test the mean peak plasma IRHGH was lower in children than in adults (0.02 > P > 0.01). It is suggested that this is due to the influence of sex steroids in adult subjects.

scholarly journals The Nα-acetylenkephalin carboxypeptidase activity of N-acetyltyrosine deacetylase from monkey kidney. Purification, characterization and substrate specificity

1983 ◽  
Vol 209 (2) ◽  
pp. 471-479 ◽  
Author(s):  
S T George ◽  
A S Balasubramanian
Keyword(s):  
Amino Acids ◽  
Gel Electrophoresis ◽  
High Speed ◽  
Metal Chelate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Protein Band ◽  
Monkey Kidney ◽  
Hydrophobic Amino Acids

N alpha-Acetylenkephalin carboxypeptidase was co-purified with N-acetyltyrosine deacetylase from monkey kidney. Almost 90% of the activity from the homogenate was recovered in a high-speed supernatant without the use of detergents. The crucial steps in the purification were Cibacron Blue F3GA-Sepharose chromatography (involving negative and positive binding sequentially) and metal chelate affinity chromatography. The purified enzyme showed three bands on gel electrophoresis under non-denaturing conditions. All the three bands exhibited both N-acetyltyrosine deacetylase and N-acetylenkephalin carboxypeptidase activity, indicating their co-migration, Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence and absence of 2-mercaptoethanol gave a single protein band of mol.wt. 34 000. The native enzyme was a dimer of mol.wt. 66 000 as observed on Bio-Gel P-300 gel filtration. The carboxypeptidase removed two amino acids from the C-terminal end of either N-acetyl[Met5]- or N-acetyl[Leu5]-enkephalin. Non-acetylated enkephalins were less active as substrates. Peptides with their carboxy end blocked were inactive as substrates. Models suggested for carboxypeptidase A [Hartsuck & Lipscomb (1971) Enzymes 3, 1-56] support the idea that the kidney N-acetylated aromatic amino acid deacetylase or acylase III [Endo (1978) Biochim. Biophys. Acta 523, 207-217] can act as a carboxypeptidase on peptides having hydrophobic amino acids at the C-terminal end.

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