Ultracentrifugation of Acetylated Thrombin

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.

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FURTHER STUDIES ON THE PURIFICATION OF THROMBIN

Canadian Journal of Biochemistry and Physiology ◽

10.1139/o58-067 ◽

1958 ◽

Vol 36 (6) ◽

pp. 603-611 ◽

Cited By ~ 45

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.

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Peptides Associated with Bovine Thrombin Preparations

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654574 ◽

1961 ◽

Vol 06 (03) ◽

pp. 424-434 ◽

Cited By ~ 1

Author(s):

Ricardo H. Landaburu ◽

Charles R. Harmison ◽

Walter H. Seegers

Keyword(s):

Amino Acid ◽

Specific Activity ◽

Rate Of Change ◽

Urea Solution ◽

Terminal Amino Acid ◽

Bovine Thrombin ◽

Sedimentation Constant ◽

Main Protein ◽

Terminal Amino ◽

Polypeptide Chains

SummaryWhen bovine thrombin was prepared by one method the N-terminal amino acid was glutamic acid while with another method it was threonine. In urea solution these N-terminal amino acids were removed in association with peptides and the N-terminal amino acid of the main protein was leucine. Urea treated thrombin had the same specific activity as the original from which it was prepared, and also had the same carbohydrate content. It was, however, less soluble in water and had a higher viscosity. The sedimentation constant was concentration dependent. Before treatment with urea the rate of change of the sedimentation constant with concentration was positive. After urea treatment it was negative. Extrapolation to zero concentration gave the same value for thrombin as for urea treated thrombin. The peptides removed from thrombin function to alter the properties of the protein. They are attached by bonds that are broken in urea solution. Very likely the original prothrombin molecule is, in part at least, composed of sub-units consisting of polypeptide chains held together by bonds broken by reagents such as sodium citrate. In addition to alanine, bovine prothrombin has arginine as N-terminal amino acid, but the latter was uncovered only in urea solution.

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METHODS OF PROTEIN EXTRACTION FROM NEUROSPORA CRASSA

Canadian Journal of Microbiology ◽

10.1139/m64-005 ◽

1964 ◽

Vol 10 (1) ◽

pp. 29-35 ◽

Cited By ~ 4

Author(s):

G. J. Stine ◽

W. N. Strickland ◽

R. W. Barratt

Keyword(s):

Glutamic Acid ◽

Neurospora Crassa ◽

Total Protein ◽

Extraction Method ◽

Protein Extraction ◽

Specific Activity ◽

Dry Weight ◽

Acid Dehydrogenase ◽

Total Enzyme

Nine methods for disrupting the mycelium of Neurospora crassa have been compared. Protein percentages are calculated per gram dry weight of mycelium. A TPN-specific glutamic acid dehydrogenase was extracted and the efficiency of each extraction method is given as total enzyme extracted and specific activity. In terms of total protein, total enzyme, and practicality of the method, the Hughes Press, the French Press and the Raper–Hyatt Press were found to be the most efficient. The advantages and limitations of each method are considered.

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CONCENTRATION AND PURIFICATION OF BACTERIOPHAGE

The Journal of General Physiology ◽

10.1085/jgp.21.3.335 ◽

1938 ◽

Vol 21 (3) ◽

pp. 335-366 ◽

Cited By ~ 40

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.

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THE PHYSIOLOGY OF HOST-PARASITE RELATIONS: IX. FURTHER OBSERVATIONS ON THE ACCUMULATION OF RADIOACTIVE SUBSTANCES AT RUST INFECTIONS

Canadian Journal of Botany ◽

10.1139/b61-121 ◽

1961 ◽

Vol 39 (6) ◽

pp. 1393-1407 ◽

Cited By ~ 18

Author(s):

Michael Shaw

Keyword(s):

Specific Activity ◽

Dry Weight ◽

Insoluble Fraction ◽

Pentose Phosphate ◽

Accumulation Ratio ◽

X Ray ◽

Specific Activities ◽

Leaf Segments ◽

Wheat Leaves ◽

Host Parasite

Wang (Can. J. Botany, 38, 635–642 (1960)) concluded that the accumulation of radioactivity observed on radioautographs at infection sites on rusted leaves fed with C14-labelled substances was 'apparent' rather than real. The ‘accumulation ratio’ is defined as the ratio of the specific activities (c.p.m./mg dry weight of intact tissue) of rust-infected to uninfected areas of infected leaves. Theoretical considerations relating to the radioautography of leaves labelled with C14 and to the measurement of ‘accumulation ratios’ by extraction of C14-labelled substances from rusted and uninfected segments of infected leaves, as well as experimental data, show that Wang's conclusion is not generally applicable.Experimentally, it was shown using polymethacrylate C14 sources that differences in distance between sources and X-ray film of the order of 100 μ had no effect on the intensity of autoradiographs. Rust-infected leaves, fed with radioactive glucose, were radiographed between X-ray plates. Localization of radioactivity at infection sites was observed on both ‘dorsal’ and ‘ventral’ radiographs, indicating a real accumulation per unit area. Ventral were more radioactive than dorsal surfaces. The main development of the fungus occurred on the former. Radioautography revealed that C14 from glucose-1-C14, glucose-6-C14, and uniformly labelled glucose fed to excised wheat leaves became localized at 10-day-old rust infections in 2 hours. ‘Accumulation ratios’ calculated from the specific activity of leaf segments remained close to 1.0 for at least 6 hours after introduction of the tracer, but increased to more than 2 after 24 hours. When ‘accumulation ratios’ were calculated from the specific activities of individual pustules (excised with a punch 1 mm in diameter) and interpustular disks, values greater than 1 were observed in 2 hours, thus confirming the results of autoradiography. Differences between the ‘accumulation ratios’ observed with glucose-6-C14 and glucose-1-C14 were consistent with an increased role of the pentose phosphate pathway at infection sites. Incorporation of C14 from uniformly labelled glucose into the alcohol-insoluble fraction of rusted leaf segments was 2.5-fold that in uninfected segments in 6 hours and 3.65-fold in 24 hours. The humin formed during hydrochloric acid hydrolysis accounted for approximately 50% of the activity of the alcohol-insoluble material. The ‘accumulation ratio’ for the alcohol-soluble material was only 1.56 after 24 hours.All the results support the view (Shaw and Samborski, Can. J. Botany, 34, 389–405 (1956)) that there is a quantitative, metabolically dependent accumulation of C14 from radioactive glucose at vigorous rust infections. The relative roles of fungus and host in this process are discussed briefly.

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PROPERTIES OF BOVINE Ac-GLOBULIN CONCENTRATES AND METHODS OF PREPARATION

Canadian Journal of Biochemistry and Physiology ◽

10.1139/y63-271 ◽

1963 ◽

Vol 41 (1) ◽

pp. 2409-2421 ◽

Cited By ~ 3

Author(s):

Nobuo Aoki ◽

Charles R. Harmison ◽

Walter H. Seegers

Keyword(s):

Human Plasma ◽

Ammonium Sulphate ◽

Protein Concentration ◽

Room Temperature ◽

Specific Activity ◽

Barium Carbonate ◽

Dry Weight ◽

Physical Chemical ◽

Bovine Plasma ◽

Refrigerator Temperature

A procedure is described for retaining bovine plasma Ac-globulin activity as one part of the protein from plasma for every 1000 parts removed. The yields averaged 15%. The procedure involves removal of prothrombin with barium carbonate, isoelectric fractionation, fractionation with ammonium sulphate, chromatography on Amberlite IRC-50, and a second fractionation with ammonium sulphate. The procedure requires 2 days; however, the first day completes up to chromatography and the concentrate at that time is quite useful for many purposes. It is more stable than the product obtained after chromatography and the yields are higher. In absence of salts Ac-globulin is quite insoluble at pH 5.0. The final product usually contained some impurity. With the analytical ultra-centrifuge the S20in 0.1 M potassium chloride solution was found to be 4.2 at a protein concentration of 12.4 mg/ml. The specific activity was 1500 U./mg dry weight. Bovine plasma contains 120 U./ml or about 9 mg/100 ml. Assuming the same specific activity for human plasma the concentration is most likely near 1 mg/100 ml. The best stability conditions found were: 50% glycerol, pH 7.0, and 0.1 M calcium chloride. Under those conditions at room temperature all activity was retained 6 to 7 hours, at refrigerator temperature 24 hours, and at −60 °C for 1 month. In rabbits, antibodies were readily produced. Oxidizing agents destroyed the activity, while reducing agents did not, nor did they tend to stabilize. SH blocking agents destroyed the activity. The loss of activity in the presence of 0.0025 M parachloromercuribenzoate was recovered with 0.04 M cysteine. The molecule deteriorated while attempts were made to obtain physical chemical data; consequently, the molecular weight was calculated from an amino acid analysis and found to be 98,800. The reliability of this value is problematical. Human plasma was analyzed and found to contain 13 U./ml Ac-globulin. After 4 days storage, at room temperature, the prolonged prothrombin time of that plasma was completely restored with 13 units of Ac-globulin, which is equivalent to 8 μg.

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Characterization of Multiple forms of Human Thrombin

10.1055/s-0039-1682720 ◽

1977 ◽

Author(s):

J.J. Gorman ◽

P.A. Castaldi

Keyword(s):

Specific Activity ◽

Peptide Mapping ◽

Multiple Forms ◽

Bovine Thrombin ◽

Molecular Weights ◽

Human Thrombin ◽

Affinity Resin ◽

C 50 ◽

High Degree

Human thrombin was obtained by activation of partially purified human prothrombin with venom of the Australian Taipan (oxyuranus scutellatus scutellatus).The crude thrombin was precipitated with ammonium sulphate and subsequently purified by chromatography on Sephadex G-75 CM-Sephadex C-50 and the affinity resin am inobenzamidine-CH-Sepharose. The final preparation had a specific activity of 1700 units per absorbance unit (A| cm 280n m Was herterogenous as shown by urea-acrylamide gel electrophoresis at acid pH and by isoelectric focusing. SDS-acrylamide electrophoresis revealed molecular weights of 39,000, 28,000, 25-23,000 and 15-12,000 for these proteins. The 39,000 dalton species predominated (greater than 90%) when the enzyme was inhibited with phenyImethanesuI phony I fluoride prior to dialysis against 0.02M sod i urn phosphate (pH 8.0) containing 0.1% SDS. Lack of such inhibition reduced the amount of the 39,000 dalton species to less than 60% with concomitant increase in the smaller species. Increase in the smaller species also occurred during incubation in 0.IM NaCI-0.I M Tris buffer (pH 8.0).Peptide mapping studies indicated that the smaller species were structurally related to the 39,000 dalton species. Amino acid compositions of tryptic peptides indicated a high degree of homology with bovine thrombin.It has been established that human thrombin can exist in at least two secondary structural forms of different molecular weights, probably due to autolytic degradation of the largest (39,000 dalton) protein species.

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A Fatty Acyl Coenzyme A Reductase Promotes Wax Ester Accumulation in Rhodococcus jostii RHA1

Applied and Environmental Microbiology ◽

10.1128/aem.00902-17 ◽

2017 ◽

Vol 83 (20) ◽

Cited By ~ 6

Author(s):

James Round ◽

Raphael Roccor ◽

Shu-Nan Li ◽

Lindsay D. Eltis

Keyword(s):

Specific Activity ◽

Dry Weight ◽

Fatty Acyl ◽

Wax Esters ◽

Wax Ester ◽

Wild Type ◽

Considerable Potential ◽

Commodity Chemicals ◽

Acyl Coenzyme A ◽

Rhodococcus Jostii

ABSTRACT Many rhodococci are oleaginous and, as such, have considerable potential for the sustainable production of lipid-based commodity chemicals. Herein, we demonstrated that Rhodococcus jostii RHA1, a soil bacterium that catabolizes a wide range of organic compounds, produced wax esters (WEs) up to 0.0002% of its cellular dry weight during exponential growth on glucose. These WEs were fully saturated and contained primarily 31 to 34 carbon atoms. Moreover, they were present at higher levels during exponential growth than under lipid-accumulating conditions. Bioinformatics analyses revealed that RHA1 contains a gene encoding a putative fatty acyl coenzyme A (acyl-CoA) reductase (FcrA). The purified enzyme catalyzed the NADPH-dependent transformation of stearoyl-CoA to stearyl alcohol with a specific activity of 45 ± 3 nmol/mg · min and dodecanal to dodecanol with a specific activity of 5,300 ± 300 nmol/mg · min. Deletion of fcrA did not affect WE accumulation when grown in either carbon- or nitrogen-limited medium. However, the ΔfcrA mutant accumulated less than 20% of the amount of WEs as the wild-type strain under conditions of nitric oxide stress. A strain of RHA1 overproducing FcrA accumulated WEs to ∼13% cellular dry weight under lipid-accumulating conditions, and their acyl moieties had longer average chain lengths than those in wild-type cells (C17 versus C16). The results provide insight into the biosynthesis of WEs in rhodococci and facilitate the development of this genus for the production of high-value neutral lipids. IMPORTANCE Among the best-studied oleaginous bacteria, rhodococci have considerable potential for the sustainable production of lipid-based commodity chemicals, such as wax esters. However, many aspects of lipid synthesis in these bacteria are poorly understood. The current study identifies a key enzyme in wax ester synthesis in rhodococci and exploits it to significantly improve the yield of wax esters in bacteria. In so doing, this work contributes to the development of novel bioprocesses for an important class of oleochemicals that may ultimately allow us to phase out their unsustainable production from sources such as petroleum and palm oil.

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[3H]Fucose incorporation by healing skin wounds and the effect of transglutaminase inhibitors

Canadian Journal of Biochemistry ◽

10.1139/o82-096 ◽

1982 ◽

Vol 60 (8) ◽

pp. 777-781 ◽

Cited By ~ 4

Author(s):

J. Michael Bowness

Keyword(s):

Sequential Extraction ◽

Specific Activity ◽

Dry Weight ◽

Insoluble Residue ◽

Skin Wounds ◽

Salt Solutions ◽

Healing Wounds ◽

Made In

Punch wounds (3 mm) were made in the skin of rats and the animals were killed after 1 or 3 days. Plugs (4 mm) of wounded and unwounded skin were incubated in vitro with [3H]fucose. The labelled plugs were homogenized and subjected to sequential extraction with buffered salt solutions, ethanol–ether, and 8 M urea – 50 mM dithiothreitol (DTT). Nondialysable counts in the extracts and insoluble residue were determined and the incorporation of label by wounded and unwounded skin plugs was compared. Wound plugs showed a greater total incorporation of [3H]fucose. In addition, a greater proportion of [3H]fucose was found in the urea–DTT extracts. The highest specific activity (disintegrations per minute t [3H]fucose per milligram dry weight) was found in a finely dispersed precipitate, sedimenting at 10 000 × g but not at 1000 × g. The transglutaminase inhibitors aminoacetonitrile and dansyl cadaverine were found to increase the extractability of a portion of the material which incorporated [3H]fucose without affecting the total incorporation. These results show that healing wounds have an increased biosynthetic capacity for an insoluble fucosylated glycoprotein fraction and they suggest that transglutaminase is necessary to make this fraction fully insoluble.

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