scholarly journals Purification and properties of a ribosomal casein kinase from rabbit reticulocytes

1977 ◽  
Vol 165 (3) ◽  
pp. 511-518 ◽  
Author(s):  
O G Issinger
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Gradient Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Casein Kinase ◽  
Purification And Properties ◽  
Casein Kinases ◽  
Acidic Proteins ◽  
Rabbit Reticulocytes

A casein kinase was isolated and purifed from rabbit reticulocytes. About 90% of the enzyme activity co-sedimented with the ribosomal fraction, whereas about 10% of the enzyme activity was found in the ribosome-free supernatant. Both casein kinases (the ribosome-bound enzyme as well as the free enzyme) showed identical activity and the same molecular weight. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis a single band of about 70 000 mol.wt. was observed. Sucrose-gradient analysis, however, showed that the enzyme activity sedimented with a s20,w of approx. 7.5S. This observation suggested that the casein kinase is a dimer composed of subunits of identical molecular weight. The enzyme utilizes GTP as well as ATP as a phosphoryl donor. It preferentially phosphorylates acidic proteins, in particular the model substrates casein and phosvitin. Casein kinase is cyclic AMP-indepenoent. The Km values for ATP and GTP with phosvitin as a substrate were determined as 1.2 and 8.8 micrometer respectively.

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.

Isoelectric points and molecular weights of salt-extractable ribosomal proteins

1976 ◽  
Vol 54 (12) ◽  
pp. 1029-1033 ◽  
Author(s):  
M Saleem ◽  
Burr Atkinson
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Molecular Weights ◽  
Isoelectric Points ◽  
Acidic Proteins ◽  
Gel Isoelectric Focusing ◽  
Liver Ribosomes

Rat liver ribosomes prepared in low salt buffer contain basic and acidic proteins not found on ribosomes washed in high salt buffer. Proteins extracted from liver ribosomes by 500 mM KCl were characterized by acid urea–polyacrylamide gel electrophoresis, sodium dodecyl sulfate – polyacrylamide gel electrophoresis and gel isoelectric focusing. The salt-solubilized proteins contain 12 polypeptides with a molecular weight over 67 000, several polypeptides with molecular weights less than 67 000, and three polypeptides whose molecular weight exceeded 130 000. Ten to 12 of the proteins were basic, and about 24 acidic proteins were partially or wholly extracted from the ribosomes. Four of the acidic proteins have isoelectric points less than 4.5.

scholarly journals Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland

1975 ◽  
Vol 151 (2) ◽  
pp. 263-270 ◽  
Author(s):  
S A Betts ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Phosphogluconate Dehydrogenase ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Final Purification Step

1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.

scholarly journals A Thermostable and Alkalitolerant Arabinofuranosidase by Streptomyces lividus

2020 ◽  
pp. 35-47
Author(s):  
Abimbola Olajide ◽  
Felicia C. Adesina ◽  
Abiodun A. Onilude
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Solid State ◽  
Solid State Fermentation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Palm Kernel ◽  
Residual Activity ◽  
Apparent Homogeneity ◽  
Production Temperature

Aim: The study aimed at producing and purifying thermostable and alkalitolerant microbial arabinofuranosidase using local Palm Kernel Cake (PKC) as substrate. Study Design: This is an experimental design in which samples were collected thrice and  subjected to laboratory analyses from which quantitative data were obtained and analysed. Place and Duration of Study: Ibadan, Nigeria, Five months. Methodology: Bacterial strains were isolated from degrading PKC by serial dilution and pour plate technique on formulated Modified Basal Salt Agar Medium and incubated at 50°C for enzyme activity screening. Plates were afterwards flooded with 1% congo red solution for visualization of hydrolysis zone. Its arabinofuranosidase activity was optimized in solid state fermentation in PKC. Production temperature, pH, moisture content, inoculum size and agitation were studied for optimization test. Optimal production temperature and pH for arabinofuranosidase by isolate was 45°C and pH 9. Produced arabinofuranosidase was purified to apparent homogeneity with ammonium sulphate precipitation, dialysis and column chromatography techniques. Stability of arabinofuranofuranosidase obtained to temperature, pH, substrate concentration and some ions was determined as well as its molecular weight using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: Isolate with highest arabinofuranosidase activity was selected and identified as Streptomyces lividus. Purity level attained was 16.36 fold. Enzyme had a specific activity of 25.4 U/mg, and total enzyme activity of 13.2 U.  Molecular weight of enzyme appeared as a band of 30 kDa. Purified arabinofuranosidase enzyme revealed optimum temperature and pH as 60oC and 9 respectively. Enzyme was stable over a broad pH range of 3-11, and temperature of 30-80oC. Residual activity after incubating for 1 hour at 70oC was 64%. Enzyme kinetics studies showed Km and Vmax values for P-nitrophenyl arabinofuranoside were 2.3mM and 0.7U/min respectively. Conclusion: Apart from Solid State Fermentation (SSF) of PKC being a potential fermentation technique for production of arabinofuranosidase by Streptomyces lividus, the enzyme was highly stable.

scholarly journals Purification and properties of pantohenase from Pseudomonas fluorescens

1976 ◽  
Vol 157 (2) ◽  
pp. 409-413 ◽  
Author(s):  
R K Airas ◽  
E A Hietanen ◽  
V T Nurmikko
Keyword(s):  
Molecular Weight ◽  
Pseudomonas Fluorescens ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Purification Procedure ◽  
Subunit Molecular Weight ◽  
Purification And Properties

Pantothenase (EC 3.5.1.22) from Pseudomonas fluorescens UK-1 was purified to homogeneity as judged by disc-gel electrophoresis and isoelectric focusing. The purification procedure consisted of four steps: DEAE-Sephadex chromatography, (NH4)2SO4 precipitation, hydroxyapatite chromatography and preparative polyacrylamide-gel electrophoresis. Gel filtration on Ultrogel AcA 34 was used to determine the molecular weight, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to study the subunit molecular weight. The enzyme appeared to be composed of two subunits with mol.wts. of approx. 50000 each. The total mol.wt. of the enzyme was thus about 100000. The isoelectric point was 4.7 at 10 degrees C.

scholarly journals Microbial metabolism of amino alcohols. Purification and properties of coenzyme B12-dependent ethanolamine ammonia-lysae of Escherichia coli

1978 ◽  
Vol 175 (2) ◽  
pp. 555-563 ◽  
Author(s):  
Carol M. Blackwell ◽  
John M. Turner
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Coenzyme B12 ◽  
Apparent Homogeneity ◽  
Purification And Properties ◽  
Protein Components ◽  
Inhibited Enzyme

1. The 120-fold purification of ethanolamine ammonia-lyase from Escherichia coli extracts, to apparent homogeneity, is described. Ethanolamine, dithiothreitol, glycerol and KCl protected the apoenzyme from inactivation. 2. At the optimum pH7.5, Km values for ethanolamine and coenzyme B12 were 44μm and 0.42μm respectively. The Km for ethanolamine was markedly affected by pH, transitions occurring at pH7.0 and 8.35. 3. The enzyme was specific for ethanolamine as substrate, none of the 18 analogues tested being active. l-2-Aminopropan-l-ol (Ki 0.86μm), dl-1-aminopropan-2-ol (Ki 2.2μm) and dl-1,3-diaminopropan-2-ol (Ki 88.0μm) inhibited competitively. 4. Enzyme activity was inhibited, irreversibly and non-competitively, by the coenzyme analogues methylcobalamin (Ki 1.4nm), hydroxocobalamin (Ki 2.1nm) and cyanocobalamin (Ki 4.8nm). 5. Iodoacetamide inhibited in the absence of ethanolamine, but only slightly in its presence. p-Hydroxymercuribenzoate inhibited markedly even in the presence of ethanolamine. Dithiothreitol and 2-mercaptoethanol (less effectively) restored activity to the enzyme dialysed against buffer containing ethanolamine. 6. Although K+ ions stabilized the enzyme during dialysis or storage, they were not necessary for activity. 7. Gel filtration showed the enzyme to be of high molecular weight, ultracentrifugal studies giving s20,w of 16.4 and an estimated mol.wt. 560400. The isoelectric point for the apoenzyme was approx. pH5.0. inhibited enzyme activity at concentrations above 1m (95% inhibition at 3m) and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated protein subunits of mol.wt. 61400. 8. Immunological studies showed that the E.coli enzyme was closely related to those of other enterobacteria, but only distantly to that of Clostridium sp. A double precipitin band suggested that the apoenzyme may be made up of two protein components.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

Purification and partial characterization of cysteine-glutamate transaminase from rat liver

1977 ◽  
Vol 55 (9) ◽  
pp. 958-964 ◽  
Author(s):  
M. P. C. Ip ◽  
R. J. Thibert ◽  
D. E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Labile Hydrogen ◽  
Apparent Homogeneity

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.

Extracellular alkaline phosphatase from marine bacteria: purification and properties of extracellular phosphatase from a marine Pseudomonas sp.

1980 ◽  
Vol 26 (7) ◽  
pp. 833-838 ◽  
Author(s):  
Hiromi Kobori ◽  
Nobuo Taga
Keyword(s):  
Molecular Weight ◽  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Marine Bacteria ◽  
Divalent Cations ◽  
Maximal Activity ◽  
Pseudomonas Sp ◽  
Purification And Properties ◽  
Extracellular Alkaline Phosphatase ◽  
Increased Pressure

Extracellular alkaline phosphatase produced by a marine Pseudomonas was purified to electrophoretic homogeneity. The molecular weight of the enzyme was estimated to be 100 000. The enzyme had maximal activity at pH 11.5. The enzyme was completely inhibited by 1 mM EDTA. However, divalent cations reversed the enzyme inhibition and their order of effectiveness on the reaction was Zn2+ > Ca2+ > Mn2+ > Mg2+ > Sr2+ > Co2+. The enzyme activity was affected by the species of anion whose order of effectiveness was demonstrated to follow the lyotrophic series, Cl− > Br− > NO3−> ClO4− > SCN−. The activity of phosphatase was accelerated linearly by increased pressure until up to 1000 atm (1 atm = 101.325 kPa), and the enzyme activity at 1000 atm was 3.2 times higher than that at 1 atm.

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