Purification Of A New High Mw Single Chain Form Of Urokinase (UK) From Urine

1981 ◽  
Author(s):  
S S Husain ◽  
V Gurewich ◽  
B Lipinski
Keyword(s):  
Disulfide Bonds ◽  
Specific Activity ◽  
Gel Filtration ◽  
Chain Structure ◽  
Isolation Procedure ◽  
Single Chain ◽  
Native Form ◽  
The Uk ◽  
Chain Form ◽  
Fibrin Thrombi

Purified UK exists in 2 forms, a high MW (55,000 daltons) and a low MW (33,000 daltons) enzyme. The former is composed of 2 chains held together by disulfide bonds and is believed to be a precursor of the latter. Little affinity for fibrin has been ascribed to either form. We have purified a third form of UK using affinity chromatography on fibrin-celite, a method which we developed to purify the major plasminogen activator from blood. When freshly voided urine was exposed to fibrin-celite, approximately 20% of the UK present was tightly bound to the fibrin. This high affinity UK (HAUK) was eluted in a sharp peak with arginine (0.2 M). Purification was achieved by gel filtration (Sephadex G-200) of the activator peak. SDS gel electrophoresis showed a single band (56,000 daltons) which remained intact after exposure to reducing agents, indicating that HAUK has a single chain structure and may be the native form of UK. The specific activity of HAUK is relatively low (40,000-50,000 CTA u/mg) suggesting that it may be a proactivator. Freshly voided urine and a rapid isolation procedure are necessary if degradation of HAUK is to be avoided. The unique high fibrin affinity of HAUK, which is not shared by the other 2 forms of UK, may make it a more specific and efficient fibrinolytic agent by confining and concentrating the enzymatic activity to the fibrin surface. The attachment of a suitable radiolabel to HAUK may also provide a useful tool for the detection of intravascular fibrin thrombi.

Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

1982 ◽  
Vol 47 (03) ◽  
pp. 197-202 ◽  
Author(s):  
Kurt Huber ◽  
Johannes Kirchheimer ◽  
Bernd R Binder
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Urine ◽  
Gel Filtration ◽  
Chain Structure ◽  
The Other ◽  
Single Chain ◽  
Apparent Homogeneity ◽  
Sequential Adsorption

SummaryUrokinase (UK) could be purified to apparent homogeneity starting from crude urine by sequential adsorption and elution of the enzyme to gelatine-Sepharose and agmatine-Sepharose followed by gel filtration on Sephadex G-150. The purified product exhibited characteristics of the high molecular weight urokinase (HMW-UK) but did contain two distinct entities, one of which exhibited a two chain structure as reported for the HMW-UK while the other one exhibited an apparent single chain structure. The purification described is rapid and simple and results in an enzyme with probably no major alterations. Yields are high enough to obtain purified enzymes for characterization of UK from individual donors.

PURIFICATION AND CHARACTERIZATION OF PAI-1 FROM HUMAN ENDOTHELIAL CELLS

1987 ◽  
Author(s):  
N A Booth ◽  
I R MacGregor ◽  
N R Hunter ◽  
B Bennett
Keyword(s):  
Endothelial Cells ◽  
Specific Activity ◽  
Umbilical Vein ◽  
Gel Filtration ◽  
Rabbit Antiserum ◽  
Protein Band ◽  
Single Chain ◽  
Step Procedure ◽  
Umbilical Vein Endothelial Cells ◽  
Pai 1

Platelet α-granules and endothelial cells contain an inhibitor of plasminogen activator, which inhibits both t-PA and u-PA. The inhibitor (PAI-1) is detectable after SDS-PAGE and zymography on fibrin/plasmin-ogen/u-PA detector gels. We have purified endothelial PAI-1 by a simple two-step procedure. Serum-free conditioned medium from human umbilical vein endothelial cells, grown in microcarrier culture, was fractionated on Sephadex CM-50, a cation exchanger, followed by gel filtration on Sephacryl S-200. Aprotinin was included throughout the procedure to maintain the activity of the inhibitor. The PAI-1 was purified 2000-fold with a recovery of about 7%. The purified protein had a specific activity of 8500 U/mg protein and the activity could be stimulated 14-fold by 4M guanidine. The purified PAI-1, of M 48000, was a single-chain glycoprotein.The ptoduct wasapparently homogeneous on a silver-stained SDS-polyacrylamide gel, the protein band co-migrating with PAI activity. Further, a rabbit antiserum raised against the purified PAI-1 revealed only a single band on immunoblots of material from each stage of the purification. The immunoglobulin fraction ofthe antiserum, incorporated into the detector gel for zymographic analysis, neutralised the inhibitor from plasma, platelets and endothelial cells, confirming their identity. Preincubation of PAI-1 from these sources with the immunoglobulin prevented formation of a complex with t-PA or u-PA. This purification procedure, in which no denaturants are employed, provides a homogeneous preparation of PAI-1 that is useful for studies on the stimulatory effects of denaturants. The antiserum raised has allowed the development of a sensitive ELISA, specific for PAI-1.

INVOLVEMENT OF FACTOR XII (F XII) AND PREKALLIKREIN (PKK) IN THE ACTIVATION OF UROKINASE (UK)-RELATED PROTEINS IN HUMAN PLASMA.

1987 ◽  
Author(s):  
D J Binnema ◽  
G Dooijewaard
Keyword(s):  
Human Plasma ◽  
Specific Activity ◽  
Antigenic Determinants ◽  
Gel Chromatography ◽  
Type I ◽  
Factor Xii ◽  
Single Chain ◽  
Sds Page ◽  
The Uk ◽  
Related Proteins

Recently it has been shown that in human plasma two types of UK-related proteins occur: Type I, plasma UK, with UK-related antigenic determinants directly accessible to anti-UK antibodies and Type II with UK-related antigenic determinants which become accessible only after SDS treatment and separation of polypeptides on PAGE. In this study we compared the molecular and enzymic properties of the two types in: 1. plasma activated by dextran sulphate (DXS) euglobulin precipitation, 2. plasma that was not activated and 3. plasma deficient in F XII, depleted in PKK and subsequently activated by DXS. ACA 34 gel chromatography, SDS PAGE, fibrin underlay zymography and immunoblotting were used. Results:Conclusions: 1. The UK-related subunits of T1 and TII are active when cleaved, but relatively inactive in the single-chain form. 2. The presence of F XII and PKK is indispensable for activation of TII, but not for that of TI; TII contributes to the F Xll-de-pendent plasminogen activator activity reported earlier, TI to the F Xll-independent part. 3. Activation of TI by DXS with no F XII and PKK present impairs the formation of the 150,000 form. 4. The specific activity of TII is rather low, but its concentration in plasma (not shown) is at least ten times that of TI.

scholarly journals Conformation, structure and activation of bovine cathepsin D. Unfolding and refolding studies

1984 ◽  
Vol 218 (2) ◽  
pp. 601-608 ◽  
Author(s):  
T Lah ◽  
M Drobniĉ-Koŝorok ◽  
V Turk ◽  
R H Pain
Keyword(s):  
Cathepsin D ◽  
Specific Activity ◽  
Single Chain ◽  
Guanidinium Chloride ◽  
Single Polypeptide Chain ◽  
Acid Environment ◽  
Single Polypeptide ◽  
Kinetics Of ◽  
Chain Form ◽  
Covalent Complex

Cathepsin D is found in the cell in two forms, one a single polypeptide chain (Mr 44 000) and the other a non-covalent complex of two peptides of Mr 14 000 and 30 000. These correspond to the N-terminal and C-terminal regions of the single chain from which they originate. It has been shown that the two forms of the enzyme are closely similar in secondary-structure content, in aromatic amino acid environment and in denaturation behaviour. The two-chain enzyme has half the specific activity of the single-chain form. The denaturation and renaturation of the single-chain cathepsin D has now been studied by c.d., fluorescence and enzyme activity. Activity is lost irreversibly on unfolding, but the loss of backbone ellipticity and of folded aromatic environment is 75% reversible. The enzyme unfolds in two main stages, and the kinetics of these transitions indicate the existence of at least two intermediate forms between the native and the fully unfolded states. A further form of the enzyme exists in 0.5 M-guanidinium chloride. It is characterized by having an activity 40% greater than that of the native state. This increase is not reversed on removing the denaturant. The similarities between cathepsin D and pepsin are discussed.

Studies on the Biochemistry of Urokinase

1977 ◽  
Vol 38 (04) ◽  
pp. 0801-0808 ◽  
Author(s):  
Eng Bee Ong ◽  
Mercedes E. Soberano ◽  
Alan J. Johnson ◽  
Guenther Schoellmann
Keyword(s):  
Affinity Chromatography ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Blue Staining ◽  
Single Chain ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Sds Page ◽  
Excess Substrate

SummaryDirect evidence for an active center histidine residue in urokinase (UK) was obtained with use of newly synthesized peptide chloroketones Ac-Gly-Lys-CH2C1 and Nle-Gly-Lys-CH2C1. Stoichiometric inactivation by DFP provided further evidence that UK is a serine protease. Essential histidine and serine residues were both located in the heavy chain of the 47,0 M. W.UK. The high M.W. form can be converted (catalytically) to the low M. W. form.9 partially purified human urinary UK preparations (5 with predominantly high M. W. UK), varying in purity and proportion of high and low M. W. forms, were found to be heterogeneous by a number of acrylamide electrophoretic procedures. 7 preparations had strikingly similar molar activities at excess substrate, except for the lower values found in 2 predominantly high M. W. UK preparations from the same supplier. 2 high M. W. UK preparations from another supplier showed a definite increase in activity when assayed at low plasminogen concentration, but this effect was abolished after gel filtration (Sephadex G-25), by further purification with affinity chromatography, or when assayed with excess plasminogen.The high and low M. W. forms of UK (47,000 and 33,400 M. W.), isolated and purified by Sepharose-EACA-agmatine affinity chromatography were shown to be homogeneous by Coomassie Blue staining after SDS-polyacrylamide gel electrophoresis (PAGE) and by 14C-DFP and 14C-NPGB incorporation before SDS-PAGE. Comparative properties of the high M. W. vs low M. W. forms were as follows: specific activity (104,000 IU/mg vs 226,000) ; 2 chains (33,100 and 18,600 M.W.) linked by disulfide(s) vs a single chain; pi 8.60 (major subform) and pi 8.90 (minor subform) vs pi 8.35, 8.60, 8.70 (major subform) and pi 8.05 (minor subform); and second order kinetics for DFP inactivation (400 vs 770 M−1 min−1). The molar activities were similar (9.6 × 109 and 10.2 × 109IUm/mole) for each form.

scholarly journals Evolution of phosphagen kinase1: Isolation, characterization and cDNA-derived amino acid sequence of two-domain arginine kinase from the sea anemone Anthopleura japonicus

1997 ◽  
Vol 328 (1) ◽  
pp. 301-306 ◽  
Author(s):  
Tomohiko SUZUKI ◽  
Yoshitada KAWASAKI ◽  
Takahiro FURUKOHRI
Keyword(s):  
Creatine Kinase ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Arginine Kinase ◽  
Gel Filtration ◽  
Sea Anemone ◽  
The Body ◽  
Native Form ◽  
Significant Similarity

Arginine kinase (AK) was isolated from the body wall muscle of the primitive sea anemone Anthopleura japonicus by Ultrogel AcA34 gel filtration, DEAE-32 chromatography and elution on a Cosmogel-SP column. The denatured molecular mass as determined with SDS/PAGE was 80 kDa, twice that of the usual AK subunit, indicating that this AK has an unusual two-domain structure. The native form was eluted on a Superose 12 column with the same retention time as that of rabbit homodimeric creatine kinase, indicating that Anthopleura AK is a monomer of 80 kDa. The isolated enzyme gave a specific activity of 100-120 μmol of Pi/min per mg of protein in the pH range 7.9-9.1 for the forward reaction. The enzyme is fully activated by Ca2+, as it is with Mg2+. The cDNA-derived amino acid sequence of 715 residues of Anthopleura AK was determined. The validity of the sequence was supported by chemical sequencing of internal tryptic peptides. A bridge intron of 686 bp, which separates the two domains of Anthopleura AK, is present between the second and third nucleotide in the codon of Ala-364. This is the first two-domain AK to be sequenced. Anthopleura AK shows 48-54% amino acid sequence identity with known invertebrate AKs, and also shows a lower, but significant, similarity (39-46%) to marine worm glycocyamine kinase and rabbit creatine kinase.

Human Plasminogen Activator From Heart And Vascular Endothelium

1981 ◽  
Author(s):  
N A Booth ◽  
B Bennett
Keyword(s):  
Plasminogen Activator ◽  
Vascular Endothelium ◽  
Limited Proteolysis ◽  
Detailed Comparison ◽  
Plasminogen Activators ◽  
Chain Structure ◽  
Regulatory Function ◽  
Single Chain ◽  
Proteolytic Activation ◽  
Chain Form

Plasminogen activators play a central role in fibrinolysis. At present little is known about the interrelationships and molecular properties of these protein(s). There have been conflicting reports on the number of polypeptide chains present in the active molecules; plasminogen activators, purified from human uterus and human plasma after venous occlusion, are reported to consist of two chains, each of approximately 30,000 MW, linked by disulphide bonds. In contrast, the protein purified from human vascular endothelium consists of a single chain of 67,000 MW. Evidence for proteolytic activation of a single chain precursor to a two-chain form has been obtained by Wallen, in that the protein could be isolated in the single chain form only if inhibitors of proteolysis were present throughout purification. The two-chain form was found to possess significantly greater activity. By analogy with other plasma systems, generation of an active two-chain form from a single-chain precursor may serve a regulatory function, but the role of limited proteolysis of plasminogen activator remains to be elucidated.Using modifications of reported methods, we have obtained improved purification of plasminogen activator from human heart and vascular endothelium by similar procedures. This has allowed detailed comparison of the enzyme from these two sources, in terms of chain structure, active site labelling and carbohydrate content. In particular, the effect of variation of conditions during purification on the chain structure of the isolated molecule has been studied.

scholarly journals Human tissue-type plasminogen activator. Production in continuous serum-free cell culture and rapid purification

1985 ◽  
Vol 226 (3) ◽  
pp. 631-636 ◽  
Author(s):  
E K O Kruithof ◽  
W D Schleuning ◽  
F Bachmann
Keyword(s):  
Plasminogen Activator ◽  
Human Tissue ◽  
Specific Activity ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Tissue Type ◽  
Single Chain ◽  
Serum Free ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

A simplified procedure for the production and purification of human tissue-type plasminogen activator (t-PA) is described. Bowes-melanoma cells were maintained in continuous serum-free culture. The cell nutrient consisted of Dulbecco's modified Eagle's medium (DMEM) supplemented with insulin (5 mg/litre), transferrin (5 mg/litre), progesterone (1 nM), cortisol (10 nM), aprotinin (2 X 10(4) units/litre) and a mixture of trace elements. t-PA accumulated in the culture medium at a rate of 40 units/day per ml and was harvested every third day. Cell losses during each harvest, leading to a steady decline of enzyme yields, were compensated for by treating the cells with 5% (v/v) fetal-bovine serum in DMEM every 6-8 weeks. t-PA was rapidly purified by a combination of cation-exchange chromatography and gel filtration. The procedure yielded mainly single-chain t-PA of a specific activity of 80 000 to 100 000 units/mg.

scholarly journals Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Camel Liver

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Mahmoud A. Ibrahim ◽  
Abdel-Hady M. Ghazy ◽  
Ahmed M. H. Salem ◽  
Mohamed A. Ghazy ◽  
Mohamed M. Abdel-Monsef
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Divalent Cations ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Native Form ◽  
Native Page ◽  
Sds Page ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Sepharose 4B

Glucose-6-phosphate dehydrogenase from camel liver was purified to homogeneity by ammonium sulfate precipitation and a combination of DEAE-cellulose, Sephacryl S-300 gel filtration, and 2′, 5′ ADP Sepharose 4B affinity chromatography columns. The specific activity of camel liver G6PD is increased to 1.80438 units/mg proteins with 63-fold purification. It turned out to be homogenous on both native PAGE and 12% SDS PAGE, with a molecular weight of 64 kDa. The molecular weight of the native form of camel liver G6PD was determined to be 194 kDa by gel filtration indicating a trimeric protein. The Km value was found to be 0.081 mM of NADP+. Camel liver G6PD displayed its optimum activity at pH 7.8 with an isoelectric point (pI) of pH 6.6–6.8. The divalent cations MgCl2, MnCl2, and CoCl2 act as activators; on the other hand, CaCl2 and NiCl2 act as moderate inhibitors, while FeCl2, CuCl2, and ZnCl2 are potent inhibitors of camel liver G6PD activity. NADPH inhibited camel liver G6PD competitively with Ki value of 0.035 mM. One binding site was deduced for NADPH on the enzyme molecule. This study presents a simple and reproducible purification procedure of G6PD from the camel liver.

Cryo-electron microscopy of two-dimensional crystals of reduced type B botulinum neurotoxin

1992 ◽  
Vol 50 (1) ◽  
pp. 530-531
Author(s):  
P. F. Flicker ◽  
V.S. Kulkarni ◽  
J. P. Robinson ◽  
G. Stubbs ◽  
B. R. DasGupta
Keyword(s):  
Disulfide Bonds ◽  
Clostridium Botulinum ◽  
Structural Changes ◽  
Two Dimensional ◽  
Type B ◽  
Single Chain ◽  
Muscle Paralysis ◽  
Cryo Electron Microscopy ◽  
Disulfide Linkages ◽  
Intracellular Action

Botulinum toxin is a potent neurotoxin produced by Clostridium botulinum. The toxin inhibits release of neurotransmitter, causing muscle paralysis. There are several serotypes, A to G, all of molecular weight about 150,000. The protein exists as a single chain or or as two chains, with two disulfide linkages. In a recent investigation on intracellular action of neurotoxins it was reported that type B neurotoxin can inhibit the release of Ca++-activated [3H] norepinephrine only if the disulfide bonds are reduced. In order to investigate possible structural changes in the toxin upon reduction of the disulfide bonds, we have prepared two-dimensional crystals of reduced type B neurotoxin. These two-dimensional crystals will be compared with those of the native (unreduced) type B toxin.

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