Reinigung und Charakterisierung des an T2-Phagen gebundenen Lysozyms

1961 ◽  
Vol 16 (6) ◽  
pp. 363-368 ◽  
Author(s):  
W. Katz ◽  
W. Weidel
Keyword(s):  
Molecular Weight ◽  
Specific Activity

Procedures for the isolation, and some properties of the particle-bound T2 phage lysozyme are described. The enzyme appears to have a higher molecular weight (21 000) and a correspondingly lower specific activity than the homologous free lysozyme, which otherwise it resembles most closely.

Preparation and Properties of Human Prothrombin Complex

1970 ◽  
Vol 24 (03/04) ◽  
pp. 325-333 ◽  
Author(s):  
G. H Tishkoff ◽  
L. C Williams ◽  
D. M Brown
Keyword(s):  
Molecular Weight ◽  
Proteolytic Enzyme ◽  
Enzyme Inhibitor ◽  
Specific Activity ◽  
Crystalline Form ◽  
Sedimentation Equilibrium ◽  
Crystalline Complex ◽  
Interaction Product ◽  
Proteolytic Enzyme Inhibitor ◽  
Purification Scheme

SummaryAs a corollary to our previous studies with bovine prothrombin, we have initiated a study of human prothrombin complex. This product has been isolated in crystalline form as a barium glycoprotein interaction product. Product yields were reduced compared to bovine product due to the increased solubility of the barium glycoprotein interaction product. On occasion the crystalline complex exhibited good yields. The specific activity of the crystalline complex was 1851 Iowa u/mg. Further purification of human prothrombin complex was made by removal of barium and by chromatography on Sephadex G-100 gels. The final product evidenced multiple procoagulant activities (II, VII, IX and X). The monomeric molecular weight determined by sedimentation equilibrium in a solvent of 6 M guanidine-HCl and 0.5% mercaptoethanol was 70,191 ± 3,057 and was homogeneous with respect to molecular weight. This product was characterized in regard to physical constants and chemical composition. In general, the molecular properties of human prothrombin complex are very similar to the comparable bovine product. In some preparations a reversible proteolytic enzyme inhibitor (p-aminophenylarsonic acid) was employed in the ultrafiltration step of the purification scheme to inhibit protein degradation.

Existence of lipid vesicles containing platelet-activating factor in endothelial cell lysate

1992 ◽  
Vol 12 (1) ◽  
pp. 15-21
Author(s):  
S. Kojima ◽  
K. Nara ◽  
Y. Inada ◽  
S. Hirose ◽  
Y. Saito
Keyword(s):  
Molecular Weight ◽  
Endothelial Cell ◽  
Specific Activity ◽  
Gel Filtration ◽  
Platelet Activating Factor ◽  
Lipid Vesicles ◽  
Specific Factor ◽  
Cell Lysate ◽  
Aggregation Activity ◽  
Molecular Weight Fractions

Platelet aggregation activity due to platelet-activating factor (PAF) was detected at high molecular weight (HMW) and low molecular weight fractions after gel-filtration chromatography of cell lysate of endothelial cells. [3H]PAF added to the cell lysate was similarly distributed after chromatography. The radioactivity associated with HMW fraction was not reduced by digesting the lysate with trypsin, suggesting that PAF was not making complexes with proteins but was included in lipid vesicles in cell lysate. Further evidence showed that an unknown specific factor(s) was needed to form these PAF-containing lipid vesicles. Radioactivity was not found in HMW fraction when [3H]PAF was mixed with cell lysate of vascular smooth muscle cells. When monomeric PAF was added to endothelial cell lysate, the specific activity of aggregation decreased to the level exerted by endogenous PAF-containing lipid vesicles due to incorporation into lipid vesicles. PAF in the form of lipid vesicles was more stable in plasma than monomeric form.

scholarly journals CONCENTRATION AND PURIFICATION OF BACTERIOPHAGE

1938 ◽  
Vol 21 (3) ◽  
pp. 335-366 ◽  
Author(s):  
John H. Northrop
Keyword(s):  
Molecular Weight ◽  
Small Molecules ◽  
Specific Activity ◽  
Active Agent ◽  
Pure Substance ◽  
Solubility Curve ◽  
Denatured Protein ◽  
Sedimentation Constant ◽  
Rate Of Sedimentation ◽  
Living Organisms

1. A method for isolating a nucleoprotein from lysed staphylococci culture is described. 2. It is homogeneous in the ultracentrifuge and has a sedimentation constant of 650 x 10–13 cm. dyne–1 sec.–1, corresponding to a molecular weight of about 300,000,000. 3. The diffusion coefficient varies from about 0.001 cm.2/day in solutions containing more than 0.1 mg. protein/ml. to 0.02 in solutions containing less than 0.001 mg. protein/ml. The rate of sedimentation also decreases as the concentration decreases. It is suggested, therefore, that this protein exists in various sized molecules of from 500,000–300,000,000 molecular weight, the proportion of small molecules increasing as the concentration decreases. 4. This protein is very unstable and is denatured by acidity greater than pH 5.0, by temperature over 50°C. for 5 minutes. It is digested by chymo-trypsin but not by trypsin. 5. The loss in activity by heat, acid, and chymo-trypsin digestion is roughly proportional to the amount of denatured protein formed under these conditions. 6. The rate of diffusion of the protein is the same as that of the active agent. 7. The rate of sedimentation of the protein is the same as that of the active agent. 8. The loss in activity when susceptible living or dead bacteria are added to a solution of the protein is proportional to the loss in protein from the solution. Non-susceptible bacteria remove neither protein nor activity. 9. The relative ultraviolet light absorption, as determined directly, agrees with that calculated from Gates' inactivation experiments in the range of 2500–3000 Å. u. but is somewhat greater in the range of 2000–2500 Å. u. 10. Solubility determinations showed that most of the preparations contained at least two proteins, one being probably the denatured form of the other. Two preparations were obtained, however, which had about twice the specific activity of the earlier ones and which gave a solubility curve approximating that of a pure substance. 11. It is suggested that the formation of phage may be more simply explained by analogy with the autocatalytic formation of pepsin and trypsin than by analogy with the far more complicated system of living organisms.

Novel Catalatic Proteins of Bakers' Yeast. I. An Atypical Catalase

1973 ◽  
Vol 51 (11) ◽  
pp. 1551-1555 ◽  
Author(s):  
Tony C. M. Seah ◽  
A. R. Bhatti ◽  
J. G. Kaplan
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Native Protein ◽  
Wild Type ◽  
Major Band ◽  
Subunit Molecular Weight ◽  
Purification And Properties

At any stage of growth of a wild-type bakers' yeast, some 20% of the catalatic activity of crude extracts is not precipitable by means of antibody prepared against the typical catalase (catalase T), whose purification and properties have been previously described. Some of this catalatic activity is due to the presence of an atypical catalase (catalase A), a heme protein, with a molecular weight estimated as 170 000 – 190 000, considerably lower than that of the usual catalases (225 000 – 250 000). Preparations of catalase A were found to be homogeneous in the analytical ultracentrifuge and in polyacrylamide gel electrophoresis. Its subunit molecular weight, determined from its iron content, was 46 500, virtually the same as that of the major band obtained in gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native protein is tetrameric. Its specific activity is in the range of those reported for other typical catalases.

scholarly journals The interrelations between high- and low-molecular-weight forms of normal and mutant (Krabbe-disease) galactocerebrosidase

1980 ◽  
Vol 189 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Yoav Ben-Yoseph ◽  
Melinda Hungerford ◽  
Henry L. Nadler
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Krabbe Disease ◽  
Low Molecular Weight ◽  
Molecular Weight Form

Galactocerebrosidase (β-d-galactosyl-N-acylsphingosine galactohydrolase; EC 3.2.1.46) activity of brain and liver preparations from normal individuals and patients with Krabbe disease (globoid-cell leukodystrophy) have been separated by gel filtration into four different molecular-weight forms. The apparent mol.wts. were 760000±34000 and 121000±10000 for the high- and low-molecular-weight forms (peaks I and IV respectively) and 499000±22000 (mean±s.d.) and 256000±12000 for the intermediate forms (peaks II and III respectively). On examination by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the high- and low-molecular-weight forms revealed a single protein band with a similar mobility corresponding to a mol.wt. of about 125000. Antigenic identity was demonstrated between the various molecular-weight forms of the normal and the mutant galactocerebrosidases by using antisera against either the high- or the low-molecular-weight enzymes. The high-molecular-weight form of galactocerebrosidase was found to possess higher specific activity toward natural substrates when compared with the low-molecular-weight form. It is suggested that the high-molecular-weight enzyme is the active form in vivo and an aggregation process that proceeds from a monomer (mol.wt. approx. 125000) to a dimer (mol.wt. approx. 250000) and from the dimer to either a tetramer (mol.wt. approx. 500000) or a hexamer (mol.wt. approx. 750000) takes place in normal as well as in Krabbe-disease tissues.

scholarly journals Analysis of Anticoagulation Activities of Heparins - Is usp Heparin Assay Reliable?

1977 ◽  
Author(s):  
Linus L. Shen ◽  
Grant H. Barlow
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
Specific Activity ◽  
Half Time ◽  
Clotting System ◽  
Plasma Clot ◽  
Heparin Fractions ◽  
Time Determination ◽  
True Activity

The in vitro “over-all” activity of heparins was analyzed by the technique of rigidity measurements with plasmas upon recalcification. This technique allows the measurement of the entire clotting process of a heparinized plasma, and is thus more sensitive and reliable than clotting time determination. Contrary to its action to the clotting of whole blood, heparin reduces the final rigidity of a plasma clot only when the degree of heparinization exceeds certain limit. A log-log plot of clotting half-time vs. amount of heparin may be used to compare the anticoagulation activity of various heparins in human plasma and in sheep plasma. Results show that heparin with molecular weight around 20,000 possesses highest specific activity, the activity drops sharply when molecular weight increases or decreases. The anticoagulation activity of heparin in human plasma, expressed by the increase in clotting half-time is up to 100 times more effective than that in sheep plasma but responds less sensitively to the change in heparin concentration. Using the same heparin standard, the specific activity of certain heparin fractions assayed in human plasma differs from that assayed in sheep plasma. The discrepancy increases with the decrease in heparin molecular weight. The discrepancy was also observed with some heparins of different tissues and sources. The USP heparin assay, which uses sheep plasma as the assay medium, therefore does not necessarily reflect the true activity in human blood clotting system.

scholarly journals Purification, characterization and radioimmunoassay of adenosine deaminase from human leukaemic granulocytes

1981 ◽  
Vol 195 (2) ◽  
pp. 389-397 ◽  
Author(s):  
D A Wiginton ◽  
M S Coleman ◽  
J J Hutton
Keyword(s):  
Molecular Weight ◽  
Adenosine Deaminase ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Periodic Acid Schiff ◽  
Apparent Homogeneity

Adenosine deaminase was purified 3038-fold to apparent homogeneity from human leukaemic granulocytes by adenosine affinity chromatography. The purified enzyme has a specific activity of 486 mumol/min per mg of protein at 35 degrees C. It exhibits a single band when subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, non-denaturing polyacrylamide-gel electrophoresis and isoelectric focusing. The pI is 4.4. The enzyme is a monomeric protein of molecular weight 44000. Both electrophoretic behaviour and molecular weight differ from those of the low-molecular-weight adenosine deaminase purified from human erythrocytes. Its amino acid composition is reported. Tests with periodic acid-Schiff reagent for associated carbohydrate are negative. Of the large group of physiological compounds tested as potential effectors, none has a significant effect. The enzyme is specific for adenosine and deoxyadenosine, with Km values of 48 microM and 34 microM respectively. There are no significant differences in enzyme function on the two substrates. erythro-9-(2-Hydroxy non-3-yl) adenine is a competitive inhibitor, with Ki 15 nM. Deoxycoformycin inhibits deamination of both adenosine and deoxyadenosine, with an apparent Ki of 60-90 pM. A specific antibody was developed against the purified enzyme, and a sensitive radioimmunoassay for adenosine deaminase protein is described.

scholarly journals ISOLATION, CRYSTALLIZATION, AND PROPERTIES OF PEPSIN INHIBITOR

1941 ◽  
Vol 24 (3) ◽  
pp. 325-338 ◽  
Author(s):  
Roger M. Herriott
Keyword(s):  
Molecular Weight ◽  
Isoelectric Point ◽  
Specific Activity ◽  
Milk Clotting ◽  
Pepsin Inhibitor

A method has been described for the isolation and crystallization of swine pepsin inhibitor from swine pepsinogen. Solubility experiments and fractional recrystallization show no drift in specific activity. The reversible combination of pepsin with the inhibitor was found to obey the mass law. The inhibitor is quite specific, failing to act on other proteolytic and milk clotting enzymes. The inhibitor is destroyed by pepsin at pH 3.5. Chemical and physical studies indicate that the inhibitor is a polypeptide of approximately 5,000 molecular weight with an isoelectric point at pH 3.7. It contains arginine, tyrosine, but no tryptophane and has basic groups in its structure.

FURTHER STUDIES ON THE PURIFICATION OF THROMBIN

1958 ◽  
Vol 36 (1) ◽  
pp. 603-611 ◽  
Author(s):  
Walter H. Seegers ◽  
Walter G. Levine ◽  
Robert S. Shepard
Keyword(s):  
Molecular Weight ◽  
Phosphate Buffer ◽  
Esterase Activity ◽  
Room Temperature ◽  
Specific Activity ◽  
Dry Weight ◽  
The Other ◽  
Resin Column ◽  
Sedimentation Constant ◽  
Single Symmetrical Peak

Purified biothrombin (bovine) was fractionated with the use of amberlite IRC-50 columns to obtain resin thrombin with an activity of 4100 units/mg. dry weight or 45,000 units/mg. tyrosine. As obtained from a resin column in 0.3 M phosphate buffer, pH 8.0, the thrombin is stable for 5 days at room temperature. At 4 °C. about 70% of the activity remains after 20 weeks. The maximum molecular weight is estimated by comparing with the specific activity (2000 units/mg.) and molecular weight (62,700) of purified prothrombin as follows: 2000/4100 × 62,700 or 30,600 as the probable molecular weight. Resin thrombin can lose its fibrinogen-clotting power while esterase activity is retained. On the other hand the esterase activity can be depressed without diminishing the clotting activity. Resin thrombin lyses fibrin. When examined in an ultracentrifuge a single symmetrical peak was found with a sedimentation constant of S = 3.9 (20 °C., 0.1 M KCl, 5.5 mg./ml.) Citrate thrombin was also fractionated with the use of IRC-50 to obtain material with a specific activity of 47,000 units/mg. tyrosine.

Concentration and purification of rubella virus hemagglutinin by hollow fiber ultrafiltration and sucrose density centrifugation

1980 ◽  
Vol 26 (11) ◽  
pp. 1334-1339 ◽  
Author(s):  
M. Trudel ◽  
P. Payment
Keyword(s):  
Molecular Weight ◽  
Tissue Culture ◽  
Vero Cell ◽  
Hollow Fiber ◽  
Rubella Virus ◽  
Specific Activity ◽  
Sucrose Density Gradient ◽  
Hollow Fibers ◽  
Cell Monolayers ◽  
Molecular Weight Cutoff

Large volumes of rubella virus were produced in Vero cell monolayers which were grown in the Corbeil-Bellco TM system. Infectious tissue culture fluids were concentrated at least 600 times in less than 4 h by ultrafiltration on hollow fibers with a molecular weight cutoff of 100 000. Recovery of the hemagglutinating activity was 75%. Rubella virus was purified by three successive sucrose density gradient centrifugations using a combination of discontinuous and linear gradients. Specific activity was increased 1000-fold.

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