Plasminogen-Activator in Human Early Milk: Its Partial Purification and Characterization

1981 ◽  
Vol 45 (02) ◽  
pp. 121-126 ◽  
Author(s):  
Utako Okamoto ◽  
Noboru Horie ◽  
Yoko Nagamatsu ◽  
Jun-Ichiro Yamamoto
Keyword(s):  
Plasminogen Activator ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Partial Purification ◽  
Order Kinetic ◽  
Diisopropyl Fluorophosphate ◽  
Step Procedure ◽  
Activity Band ◽  
C 50 ◽  
Gel Isoelectric Focusing

SummaryMilk plasminogen-activator was partially purified from human transitional milk collected at about 10 days after delivery, by a five-step procedure involving chloroform treatment, ammonium sulfate precipitation, and column chromatography on Sephadex G-150, CM Sephadex C-50 and DEAE Sephadex A-50. This gave milk-activator with a maximum purification factor of about 2,400-fold with respect to the skimmed milk. The CM Sephadex-step preparation showed, on polyacrylamide gel electrophoresis, a single plasminogen-activator activity band located between the bands of albumin and prealbumin of human serum. This preparation exhibited no kinin forming activity. The activator hydrolyzed acetyl-glycyl-L-lysine methyl ester with similar order kinetic constants to urokinase, and was inhibited strongly by diisopropyl-fluorophosphate. The molecular weight of the activator as estimated by gel filtration was approximately 86,000, the isoelectric points as estimated by gel isoelectric focusing were pH 7.2, 6.9 and 6.6, and the activator activity was not quenched by antiurokinase globulin, indicating that the milk-activator is a different entity from urokinase.

Secretion of Plasminogen Activators by Cultured Bovine Endothelial Cells: Partial Purification, Characterization and Evidence for Multiple Forms

1981 ◽  
Vol 45 (03) ◽  
pp. 219-224 ◽  
Author(s):  
W E Laug
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Plasminogen Activators ◽  
Maximum Activity ◽  
Partial Purification ◽  
Diisopropyl Fluorophosphate

SummaryEndothelial cells were obtained from the aortae of newborn calves and cloned. High plasminogen activator (PA) activity was detected in the supernatant medium and the cell lysates of confluent cultures. The PA activity in the growth medium increased steadily during 12 hrs of incubation, indicating active enzyme secretion by these cells. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of the concentrated medium demonstrated the presence of four plasminogen activators with apparent molecular weights of 77,000 (±3000), 43,000 (±2000), 26,000 (±1500) and 14,500 (±1500) respectively. The 43,000, 26,000 and 14,500 molecular weight forms could be converted to radioactive derivates by active site labeling with 3H diisopropyl fluorophosphate (3H DFP) while the 77,000 Dalton form took up only traces of this radioactively labeled compound. The 43,000 molecular weight form was partially purified by means of salt precipitation and gel filtration. This enzyme preparation activated plasminogen by proteolytic cleavage with maximum activity at pH 7.5-8.5 and demonstrated a specific activity of 80,000 CTA (Committee on Thrombolytic Agents) units/mg protein when tested on 125I-fibrin in the presence of plasminogen. This PA was rapidly and irreversibly inhibited by diisopropyl fluorophosphate (DFP), suggesting that it was a serine protease. The partially purified enzyme was extremely labile at temperatures from 0-60° C, but could be stabilized by lowering the pH to 3 or by the addition of albumin.

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

Partial purification and characterization of isopenicillin N epimerase activity from Streptomyces clavuligerus

1983 ◽  
Vol 29 (11) ◽  
pp. 1526-1531 ◽  
Author(s):  
Susan E. Jensen ◽  
Donald W. S. Westlake ◽  
Saul Wolfe
Keyword(s):  
Pyridoxal Phosphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Streptomyces Clavuligerus ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Major Protein ◽  
Major Protein Band ◽  
Isopenicillin N

Epimerase activity, which converts isopenicillin N to penicillin N, has been partially purified from cell-free extracts of Streptomyces clavuligerus. No stimulating cofactors of this activity were found, and neither EDTA nor anaerobic incubation caused significant inhibition of activity. Although pyridoxal phosphate did not stimulate epimerase activity, the presence of this cofactor was necessary for the stabilization of enzymic activity during the purification process. Epimerase activity was purified 35.5-fold by a combination of salt precipitation, gel filtration, and ion exchange chromatography. Gel filtration indicated that the epimerase has a molecular weight of 60 000 and sodium dodecyl sulphate – polyacrylamide gel electrophoresis of the 35.5-fold purified epimerase showed a major protein band running near that location. Pyridoxal phosphate antagonists did not uniformly inhibit epimerase activity, but the inhibitory effect of hydroxylamine could be partially reversed by pyridoxal phosphate.

scholarly journals Partial purification and properties of the external NADH dehydrogenase from cuckoo-pint (Arum maculatum) mitochondria

1984 ◽  
Vol 224 (1) ◽  
pp. 171-179 ◽  
Author(s):  
I R Cottingham ◽  
A L Moore
Keyword(s):  
Nadh Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Purification And Properties ◽  
Nadh Dehydrogenases ◽  
Arum Maculatum ◽  
A Minor ◽  
Considerable Loss

The external NADH dehydrogenase has been purified from Arum maculatum (cuckoo-pint) mitochondria by phosphate washing, extraction with deoxycholate, ion-exchange and gel-filtration chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows, when the gel is silver-stained, that the purified enzyme contains two major bands of Mr 78 000 and 65 000 and a minor one of Mr about 76 000. It is not possible at present to determine which of these, or which combination, constitutes the dehydrogenase. The enzyme contains non-covalently bound FAD and a small amount of FMN. Since the conditions of purification lead to considerable loss of flavin and possibly iron-sulphur centres, it is not possible to decide with certainty whether the enzyme is a flavo- or ferroflavo-protein. The enzyme has been distinguished from the other NADH dehydrogenases on the basis of its substrate specificity, its capability of reducing electron acceptors such as ubiquinone-1 and 2,6-dichlorophenol-indophenol and its sensitivity towards Ca2+, EGTA and dicoumarol.

Comparison of LHRH-peptidase and plasminogen activator activity in rat testis extracts

1997 ◽  
Vol 9 (7) ◽  
pp. 659 ◽  
Author(s):  
Mark P. Hedger ◽  
Michael D. Culler
Keyword(s):  
Plasminogen Activator ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Plasmin Activity ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Gonadotrophin Releasing Hormone ◽  
Secreted Proteases

Testicular LHRH-peptidase and testicular urokinase-type plasminogen activator are Sertoli cell-secreted proteases which display similar molecular properties. However, there is relatively little information regarding the substrate specificity and potential cross-reactivity of these enzymes. Testicular extracts were prepared from homogenates of whole rat testes and assessed by LHRH-peptidase assay, and by radial caseinolysis assays for plasminogen activator and plasmin-like activity. Following partial purification of the protease activities in testicular extracts by gel filtration and ion-exchange chromatography, it was conrmed that testicular LHRH-peptidase and plasminogen activator are clearly separable. There was no detectable plasmin-like activity in the testicular extracts; however, the extracts were found to contain an inhibitor, or inhibitors, of both plasminogen activator and plasmin activity. In addition to LHRH and Gly 6 -substituted LHRH analogues, the partially purified LHRH-peptidase degraded both angiotensins I and II, but not the gonadotrophin-releasing-hormone-associated peptide derived from the LHRH precursor molecule. These properties of the LHRH-peptidase provide further evidence that it is a testis-specific prolyl endopeptidase, involved in regulating and/or limiting peptide activity in the testis.

scholarly journals Innate Immunity in Lobsters: Partial Purification and Characterization of a Panulirus cygnus Anti-A Lectin

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Robert L. P. Flower
Keyword(s):  
Innate Immunity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Spiny Lobster ◽  
Gel Filtration ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Rat Erythrocytes ◽  
Group A ◽  
Panulirus Cygnus

A lectin detected in haemolymph from the Australian spiny lobster Panulirus cygnus agglutinated human ABO Group A cells to a higher titre than Group O or B. The lectin also agglutinated rat and sheep erythrocytes, with reactivity with rat erythrocytes strongly enhanced by treatment with the proteolytic enzyme papain, an observation consistent with reactivity via a glycolipid. The lectin, purified by affinity chromatography on fixed rat-erythrocyte stroma, was inhibited equally by N-acetylglucosamine and N-acetylgalactosamine. Comparison of data from gel filtration of haemolymph (behaving as a 1,800,000 Da macromolecule), and polyacrylamide gel electrophoresis of purified lectin (a single 67,000 Da band), suggested that in haemolymph the lecin was a multimer. The purified anti-A lectin autoprecipitated unless the storage solution contained chaotropic inhibitors (125 mmol/L sucrose: 500 mmol/L urea). The properties of this anti-A lectin and other similar lectins are consistent with a role in innate immunity in these invertebrates.

Rat lingual lipase: partial purification, hydrolytic properties, and comparison with pancreatic lipase

1984 ◽  
Vol 247 (4) ◽  
pp. G385-G393 ◽  
Author(s):  
I. M. Roberts ◽  
R. K. Montgomery ◽  
M. C. Carey
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Pancreatic Lipase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Partial Purification ◽  
Ph Optimum ◽  
Dodecyl Sulfate ◽  
Triton X

We have partially purified lingual lipase from the serous glands of rat tongue. With a combination of Triton X-100 extraction or Triton X-114 phase-separation techniques, Bio-Bead SM-2 treatment, dialysis, and gel filtration on Sephadex G-200 or Sephacryl S-300, we obtained a sparingly soluble lipid-free protein demonstrating hydrolytic activity against triglycerides and negligible phospholipase or cholesteryl esterase activities. Compared with homogenate, specific activities of the enzyme were enriched 3- to 5-fold prior to gel filtration and 10-fold after gel filtration. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration under denaturing conditions (6 M guanidine X HCl or 0.1% sodium dodecyl sulfate) revealed one major glycoprotein band with Mr approximately 50,000. Gel filtration of the active enzyme in 0.1% Triton X-100 gave an Mr approximately 270,000-300,000, suggesting extensive self-aggregation. With both tributyrin and triolein, the pH optimum of the purified enzyme was 4.0 and activity extended from pH 2.0 to 8.0. In contrast to purified human pancreatic lipase, lingual lipase hydrolyzed triglyceride emulsions and mixed micelles stabilized with both short-chain (dihexanoyl) and long-chain (egg) lecithin and were inhibited only slightly (18-25%) by micellar concentrations of two common bile salts, taurodeoxycholate and taurocholate. Our results suggest that the hydrolysis of dietary fat by lingual lipase may extend from the pharynx through the esophagus and stomach and into the upper small intestine.

Isolation And Characterization Of A Plasminogen Activator (PA) From Human Myocardial Tissue

1981 ◽  
Author(s):  
B R Binder ◽  
G Reissert ◽  
R Beckmann
Keyword(s):  
Plasminogen Activator ◽  
Human Heart ◽  
Strong Dependence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Myocardial Tissue ◽  
System A ◽  
Isolation And Characterization ◽  
Apparent Homogeneity

Pig heart is one of the major sources for isolation of PAs. However, no attempt has been made to isolate PA from human heart. It was the aim of the study to isolate a PA from human myocardial tissue and to compare it with the vascular plasminogen activator (VPA) derived from cadaver vessel eluates.Human heart was obtained from cadavers and myocardial tissue was prepared, homogenized, and after extraction with a neutral buffer, a pH=4.2 extract was obtained containing about 60-70% of PA activity originally present in the homogenate. The latter extract was made 2 M with (NH4)2SO4 at pH=7.0 precipitating all of the activator activity. PA activity could be purified to apparent homogeneity by reverse ammonium sulfate gradient solubilization followed by hydrophobic interaction chromatography on octly- Sepharose, affinity chromatography on arginin-Sepharose and gel filtration on Sephadex G-150. The material obtained showed a single band in SDS polyacrylamide gel electrophoresis corresponding to Mr=70.000 and the region of the gel where PA activities could be eluted.Kinetic analysis of the purified activator with synthetic paranitroanilide substrates revealed a KM of 0.8 mM,4 mM, and 0.4 mM for H-D-Ile-Pro-Arg-paranitroanilide, H-D-Val- Gly-Arg-paranitroanilide, and H-D-Phe-Aze-Arg-paranitro- anilide, respectively, which were almost identic to those obtained with the VPA using the same subtrates. Plasminogen activation with the myocardial PA showed in a purified system a strong dependence on the presence of fibrin as it could be observed with VPA.From the similar isolation characteristics, the molecular weight, the amidolytic activities, and the fibrin effect on plasmin formation it can be concluded that the PA isolated from human myocardial tissue is similar or identic to the VPA.

Purification of PRL receptors from toad kidney: comparisons with rabbit mammary PRL receptors

1988 ◽  
Vol 254 (3) ◽  
pp. C372-C382 ◽  
Author(s):  
M. Dunand ◽  
J. P. Kraehenbuhl ◽  
B. C. Rossier ◽  
M. L. Aubert
Keyword(s):  
Mammalian Species ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Structural Features ◽  
Polyclonal Antiserum ◽  
Partial Purification ◽  
Molecular Characteristics ◽  
Binding Characteristics ◽  
Sds Page

The binding characteristics of the prolactin (PRL) receptors present in toad (Bufo marinus) kidneys were investigated and compared to those of PRL receptors present in rabbit mammary glands. The molecular characteristics of the Triton X-100 solubilized renal and mammary PRL receptors were assessed by gel filtration and by migration analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after affinity labeling of the binding sites with 125I-human growth hormone. Similar results were obtained for both receptors. Partial purification of the toad PRL receptor could be achieved by affinity chromatography. The molecular weight of this purified receptor could be determined by analysis on SDS-PAGE. With the use of a polyclonal antiserum raised against a purified preparation of rabbit mammary PRL receptor, one or several antigenic epitope(s) could be identified on the core of the toad renal PRL receptor. In conclusion, although the structure and the biological role(s) of PRL have substantially changed during evolution, the receptor for this hormone has retained many of its structural features as could be assessed between an amphibian and a mammalian species on functionally different target tissues.

scholarly journals Denaturant-induced stimulation of the beta-migrating plasminogen activator inhibitor in endothelial cells and serum

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1298-1305 ◽  
Author(s):  
LA Erickson ◽  
CM Hekman ◽  
DJ Loskutoff
Keyword(s):  
Endothelial Cells ◽  
Sodium Dodecyl Sulfate ◽  
Human Serum ◽  
Plasminogen Activator ◽  
Serum Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Zone Electrophoresis ◽  
Dodecyl Sulfate

Cultured bovine aortic endothelial cells and human serum contain plasminogen activator inhibitors (PAIs) that are immunologically related. In the present study, the electrophoretic mobilities, molecular weights (mol wt), and activities of these PAIs were compared. When fractionated by agarose zone electrophoresis, both PAIs migrated with beta mobility as compared with the mobilities of human plasma/serum proteins. Two-dimensional electrophoretic analysis, employing agarose zone electrophoresis in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension, indicated that these beta-PAIs comigrated, both having a mol wt of approximately 50,000. The activity of the PAI in endothelial cell- conditioned medium is enhanced severalfold by treatment with either sodium dodecyl sulfate or guanidine. In preliminary experiments, we were unable to stimulate the PAI activity of undiluted serum by similar treatments. However, the PAI activities in both diluted serum and gel- filtered or electrophoretically fractionated serum were enhanced by treatment with these denaturants. The gel filtration studies also revealed that serum contains multiple forms of the beta-PAI. These forms may represent polymeric PAI and/or complexes between the PAI and other serum components. These findings indicate that the primary PAIs in bovine endothelial cells and human serum are not only immunologically related but are also biochemically similar.

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