scholarly journals Isolation of lysyl hydroxylase, an enzyme of collagen synthesis, from chick embryos as a homogeneous protein

1980 ◽  
Vol 189 (2) ◽  
pp. 247-253 ◽  
Author(s):  
T M Turpeenniemi-Hujanen ◽  
U Puistola ◽  
K I Kivirikko
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polymeric Form ◽  
Lysyl Hydroxylase ◽  
Enzyme Form

Two procedures are reported for the purification of lysyl hydroxylase, both procedures involving (NH4)2SO4 fractionation, affinity chromatography on concanavalin A-agarose and elution of the column with ethylene glycol. The additional steps in procedure A consist of gel filtration and chromatography on a hydroxyapatite column, and in procedure B of affinity chromatography on collagen linked to agarose and gel filtration. The best preparations obtained with either of the two procedures were pure when examined by sodium dodecyl sulphate-polyacrylamide-disc-gel or slab-gel electrophoresis, but about half of the preparations obtained by procedure A had minor contaminants. The specific activity of a typical preparation purified by procedure B was 13 4000 times that of the 15 000 g supernatant of the chick-embryo homogenate, with a recovery of about 4%. The molecular weight of the pure enzyme was bout 200 000 by gel filtration, and that of the enzyme subunit about 85 000 by sodium dodecyl sulphate/polyacrylamide-disc-gel or slab-gel electrophoresis. It is suggested that the active enzyme is a dimer consisting of only one type of monomer, and that a previously described enzyme form with an apparent molecular weight of about 550 000 is a polymeric form of this dimer. The catalytic-centre activity of the pure enzyme, as determined with a saturating concentration of a synthetic peptide substrate and under conditions specified, was about 3-4 mol/s per mol.

scholarly journals Molecular weight and subunit size of fatty acid synthetase from rabbit mammary gland

1976 ◽  
Vol 159 (1) ◽  
pp. 181-184 ◽  
Author(s):  
N Paskin ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Fatty Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Fatty Acid Synthetase ◽  
Subunit Size

Fatty acid synthetase purified from the mammary gland of the rabbit has a mol. wt. of 968000 as determined by gel filtration. The enzyme gave one band, corresponding to a mol.wt. of approx. 35000, on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and phenylmethanesulphonyl fluoride.

scholarly journals Rabbit Tamm–Horsfall urinary glycoprotein. Chemical composition and subunit structure

1971 ◽  
Vol 122 (5) ◽  
pp. 623-631 ◽  
Author(s):  
Anne M. S. Marr ◽  
A. Neuberger ◽  
Wendy A. Ratcliffe
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Chemical Composition ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Subunit Structure ◽  
Terminal Amino

1. Tamm–Horsfall glycoprotein from rabbit urine has been isolated and characterized. The homogeneity of the preparation has been established by a variety of procedures including disc gel electrophoresis and ultracentrifugation in aqueous solution, sodium dodecyl sulphate and formic acid. 2. The chemical composition has been determined and a carbohydrate content of approx. 31% was obtained. The relative contents of the amino acids were shown to be very similar to those in human Tamm–Horsfall glycoprotein. A trace of lipid was also detected. 3. Leucine was identified as the only N-terminal amino acid. 4. The subunit structure was investigated in the presence of sodium dodecyl sulphate by gel filtration and disc gel electrophoresis. These studies indicated that the subunit possessed a molecular weight of approx. 84000±6000. A similar value was obtained after reduction and S-alkylation of the glycoprotein indicating that the disulphide bonds were all intrachain. 5. A minimum value for the chemical molecular weight of 85000±6000 was obtained from the number of N-terminal amino acids released by cyanogen bromide cleavage of the glycoprotein. 6. The immunological properties of the glycoprotein were studied. Cross reactivity was demonstrated between human Tamm–Horsfall glycoprotein and a guinea-pig anti-rabbit Tamm–Horsfall antiserum.

scholarly journals The molecular size of N-methylglutamate dehydrogenase of Pseudomonas aminovorans

1977 ◽  
Vol 167 (2) ◽  
pp. 509-512 ◽  
Author(s):  
C W Bamforth ◽  
P J Large
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Partial Specific Volume ◽  
Triton X

N-Methylglutamate dehydrogenase, purified to a specific activity of 0.29 unit/mg of protein, gave one band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a molecular weight of 130 000. Enzyme-Triton complexes were found to have a partial specific volume of 0.73 cm3/g, suggesting that the protein binds less than 0.1 g of Triton/g of protein. A molecular weight for the intact enzyme in the presence of 1% (w/v) Triton X-100 of 550 000 suggested that the enzyme may be a tetramer.

Purification of Human Renal Renin

1978 ◽  
Vol 55 (s4) ◽  
pp. 117s-119s
Author(s):  
Eve E. Slater ◽  
Robert C. Cohn ◽  
Victor J. Dzau ◽  
Edgar Haber
Keyword(s):  
Enzymatic Activity ◽  
Affinity Chromatography ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Single Band ◽  
Kidney Cortex ◽  
Human Renin ◽  
Cadaver Kidney

1. Human renal renin has been purified 200 000-fold from cadaver kidney cortex by a method which employs affinity chromatography on aminohexyl pepstatin. 2. The product of this purification has a specific activity of 400 Goldblatt units/mg when compared with Haas human renin standard. 3. This product appears as a single band on sodium dodecyl sulphate gel and polyacrylamide-disc gel electrophoresis. Renin enzymatic activity was recovered after elution from a polyacrylamide-disc gel run at pH 7·8. 4. Yield with this method was 1%.

scholarly journals Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli

1983 ◽  
Vol 213 (1) ◽  
pp. 187-191 ◽  
Author(s):  
A Lewendon ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Purification Step ◽  
Phosphate Synthase

A procedure for the purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli is described. Homogeneous enzyme of specific activity 17.7 units/mg was obtained in 22% yield. The key purification step involves substrate elution of the enzyme from a cellulose phosphate column. The subunit Mr was estimated to be 49 000 by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The native Mr was estimated to be 55 000 by gel filtration, indicating that the enzyme is monomeric.

scholarly journals Properties of a 5′-nucleotidase purified from mouse liver plasma membranes

1973 ◽  
Vol 133 (1) ◽  
pp. 189-199 ◽  
Author(s):  
W. H. Evans ◽  
James W. Gurd
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Mouse Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Tris Buffer ◽  
Nucleotidase Activity

1. Extraction of a mouse liver plasma-membrane fraction with a detergent buffer, N-dodecylsarcosinate–Tris buffer (sarcosyl–Tris buffer), solubilized 90% of the protein and 70% of the 5′-nucleotidase activity. 2. The proteins of the sarcosyl–Tris buffer extract were fractionated by a rate-zonal centrifugation in a sucrose–detergent gradient. The major protein peak sedimented ahead of phospholipids, which mainly remained in the overlay. Glycoproteins were separated ahead of the protein peak. 3. The 5′-nucleotidase activity peak was associated with 5% of the protein applied to the gradient, and contained relatively few protein bands. 4. The 5′-nucleotidase was purified further by gel filtration on Sepharose and Sephadex columns equilibrated with sarcosyl–Tris buffer, to give a single glycoprotein band on sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. The purified enzyme was lipid-free. 5. Electrophoresis in polyacrylamide gels in sarcosyl–Tris buffers showed that the enzymic activity was coincident with the protein band. 6. The molecular weight suggested for the enzyme activity by gel filtration or centrifugation in sucrose gradients was 140000–150000. Sometimes, a minor enzyme peak of lower molecular weight was obtained. 7. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate indicated that as the polyacrylamide concentration was increased from 5 to 15%, the apparent molecular weight of the enzyme decreased from 130000 to 90000. 8. The evidence that 5′-nucleotidase is composed of two active and similar, if not identical, glycoprotein subunits and the role of detergent in effecting the separation of membrane proteins and glycoproteins are discussed. 9. Substrate requirements, pH optima and the nature of inhibition by an analogue of adenosine diphosphate are reported.

scholarly journals Biochemical Properties of Rhodanese from Almond (Prunus amygdalus) Nuts

2018 ◽  
Vol 1 (1) ◽  
pp. 17-24
Author(s):  
Adeola F. Ehigie ◽  
Mohammed A. Abdulrasak ◽  
Ona L. Ehigie
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Biochemical Properties ◽  
Prunus Amygdalus ◽  
Cyanide Detoxification ◽  
Km Value

Study of the characteristic pattern of enzymes are useful in the understanding of certain physiological and biochemical process-es. Thiosulfate: cyanide sulfurtransferase (rhodanese) is a ubiquitous multifunctional enzyme, that is believed to function in cyanide detoxification. The present study was conducted to determine the activity of rhodanese in almonds (Prunus amygdalus) that belong to the rose family, rosaceae. Rhodanese from the almond nuts was purified by ammonium sulphate precipitation, ion exchange and affinity chromatography. The molecular weight of the enzyme was determined by sodium dodecyl sulphate poly-acrylamide gel electrophoresis. The purified rhodanese from the almond nuts had a specific activity of 5.09 RU/mg with yield of 0.06%. A Km value of 11.14 mM with Vmax 0.46 RU/ml/min were obtained from KCN while a Km value of 13.95 mM with Vmax of 0.48 RU/ml/min was obtained from Na2S2O3. The substrate specificity studied indicated that Mercapto-ethanol (MCPE), Ammonium per sulfate ((NH2)2S2O8, Ammonium sulfate ((NH2)2SO4, Sodium sulfate (Na2SO4) and Sodium metabi-sulfate (Na2S2O5) cannot be substituted for sodium thiosulphate (Na2S2O3) as sulphur donor for rhodanese catalytic reaction. The optimum activity of the enzyme was observed at 50oC and an optimum pH of 8. The effect of metals on rhodanese from Almond nut showed that at 1 mM concentration of the metals used did not pronouncedly affect the activity of the enzyme metals except that of HgCl2 and MnCl2. However, the divalent metals including MnCl2 HgCl2, CaCl2, and BaCl2 inhibited the enzyme at 10 mM concentration. The molecular weight obtained from sodium dodecyl sulphate polyacrylamide gel electrophoresis was estimated to be 35 kDa. The study validates the expression of rhodanese activity in almond nut. The characteristic property of rhodanese in the plant may be exploited for bioremediation of cyanide polluted soil.

scholarly journals Rabbit β-glucuronidase. Purification and properties, and the existence of multiple forms

1974 ◽  
Vol 138 (3) ◽  
pp. 395-405 ◽  
Author(s):  
Roger T. Dean
Keyword(s):  
Molecular Weight ◽  
Isoelectric Focusing ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Multiple Forms ◽  
Hydroxyapatite Gel

1. β-Glucuronidase (EC 3.2.1.31) was purified from rabbit liver by a procedure involving autolysis, (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration, sedimentation in a sucrose gradient, and isoelectric focusing. 2. Electron microscopy revealed ferritin as the major contaminant in later stages of purification and also showed aggregates of enzyme molecules. Particular attention was paid to the removal of ferritin. 3. The purified enzyme was homogeneous in polyacrylamide-gel electrophoresis both in non-dissociating conditions and in the presence of sodium dodecyl sulphate, and in Ouchterlony gel diffusion and immunoelectrophoresis against polyspecific antisera. 4. Sedimentation in sucrose gradients gave a molecular weight of 300000, whereas gel filtration indicated 440000. 5. Subunits of 75000 molecular weight were observed in gel electrophoresis in the presence of sodium dodecyl sulphate and in gel filtration in the presence of urea. 6. The Km value for p-nitrophenyl β-d-glucuronide was 0.6mm, and the enzyme was extremely sensitive to lactone inhibitors. It was also inhibited by Hg2+ ions. 7. Multiple forms were observed in the pure enzyme by isoelectric focusing, with pI values of 4.5–5.8. Subunits showed similar heterogeneity. The origin of the multiple forms was investigated in detail, and the possibility of artifact generation largely excluded. Some of the forms of lowest pI disappeared after neuraminidase digestion. The nature of the residual heterogeneity remains to be elucidated.

The Different Forms of Antithrombin III in Serum

1977 ◽  
Vol 38 (02) ◽  
pp. 0494-0503 ◽  
Author(s):  
D. S Pepper ◽  
D Banhegyi ◽  
J. D Cash
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Human Serum ◽  
Degradation Product ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Free Form ◽  
Polymeric Form ◽  
At Iii ◽  
Human Thrombin

SummaryAntithrombin III (AT III) complexes were isolated from human serum by affinity chromatography and gel filtration. In the first step of the preparation, using heparin-agarose chromatography, we observed that the complexed form of AT III bound less strongly to the gel than the free form and that about half of the AT III was free. With further purification a 2.5 × 105 molecular weight complex was isolated. Using 125I labelled human thrombin, this complex was radioactive indicating the presence of thrombin. Only in a synthetic thrombin-AT III system was a 9 × 104 molecular weight complex detected, but not in serum. These facts suggest that in serum AT III complexes may exist in a polymeric form. Also, an AT III antigen derived from the original AT III molecule, but not complexed, was isolated which may be a degradation product.Abbreviations used: AT-III, antithrombin III. Hepes, N-2-Hydroxyethylpiperazine-N-2-Ethanesulphonic acid.

Partial purification of a boar sperm membrane protein inducing sperm agglutinating antibody

1990 ◽  
Vol 10 (2) ◽  
pp. 131-139
Author(s):  
Oyewole Adeyemo ◽  
E. O. Okegbile ◽  
O. O. Olorunsogo
Keyword(s):  
Molecular Weight ◽  
Membrane Protein ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Boar Sperm ◽  
Sperm Membrane ◽  
Triton X

For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody.

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