Platelet-derived growth factor. Isolation by a large-scale procedure and analysis of subunit composition

Platelet-derived growth factor was purified from fresh platelets by a large-scale procedure not involving the use of SDS (sodium dodecyl sulphate). The product, 0.5 mg of platelet-derived growth factor, obtained from about 3 × 10(13) platelets migrated as a single component in analytical gel electrophoresis in the presence of SDS and showed no inhomogeneity on sedimentation-equilibrium analysis in the ultracentrifuge. It had a high specific activity, 2 ng of platelet-derived growth factor/ml (70pM) being equivalent to 1% (v/v) human serum in an assay for multiplication-stimulating activity. Amino acid analysis revealed that platelet-derived growth factor contains all the common amino acids, except tryptophan, but no hexosamine. The molecular weight of platelet-derived growth factor, as determined by sedimentation-equilibrium analysis, was about 33 000. A similar value was obtained by gel electrophoresis in SDS under non-reducing conditions. In the presence of reducing agents the factor molecule was converted into two distinct components of lower molecular weight (17 000 and 14 000 respectively), as demonstrated by protein staining. The molecular model implicated by these findings is that platelet-derived growth factor consists of two different polypeptides chains, linked by disulphide bridges.

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

Canadian Journal of Biochemistry ◽

10.1139/o73-208 ◽

1973 ◽

Vol 51 (11) ◽

pp. 1551-1555 ◽

Cited By ~ 35

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.

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

Biochemical Journal ◽

10.1042/bj1950389 ◽

1981 ◽

Vol 195 (2) ◽

pp. 389-397 ◽

Cited By ~ 30

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.

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The molecular size of N-methylglutamate dehydrogenase of Pseudomonas aminovorans

Biochemical Journal ◽

10.1042/bj1670509 ◽

1977 ◽

Vol 167 (2) ◽

pp. 509-512 ◽

Cited By ~ 8

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.

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The purification and molecular properties of the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Neurospora crassa

Biochemical Journal ◽

10.1042/bj1970427 ◽

1981 ◽

Vol 197 (2) ◽

pp. 427-436 ◽

Cited By ~ 9

Author(s):

G A Nimmo ◽

J R Coggins

Keyword(s):

Molecular Weight ◽

Neurospora Crassa ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Deae Cellulose ◽

Polyacrylamide Gel ◽

Sedimentation Equilibrium ◽

Phosphate Synthase

Neurospora crassa contains three isoenzymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, which are inhibited by tyrosine, tryptophan and phenylalanine respectively, and it was estimated that the relative proportions of the total activity were 54%, 14% and 32% respectively. The tryptophan-sensitive isoenzyme was purified to homogeneity as judged by polyacrylamide-gel electrophoresis and ultracentrifugation. The tyrosine-sensitive and phenylalanine-sensitive isoenzymes were only partially purified. The three isoenzymes were completely separated from each other, however, and can be distinguished by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and Ultrogel AcA-34 and polyacrylamide-gel electrophoresis. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the tryptophan-sensitive isoenzyme contained one type of subunit of molecular weight 52000. The molecular weight of the native enzyme was found to be 200000 by sedimentation-equilibrium centrifugation, indicating that the enzyme is a tetramer, and the results of cross-linking and gel-filtration studies were in agreement with this conclusion.

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Isolation and Partial Characterization of Adenylate Cyclase-Enriched Membranes from Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661517 ◽

1986 ◽

Vol 55 (02) ◽

pp. 178-183

Author(s):

N Shimada ◽

M Tsubokura ◽

N Kimura

Keyword(s):

Molecular Weight ◽

Adenylate Cyclase ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

High Specific Activity ◽

Total Activity ◽

Human Platelets ◽

Protein Profiles

SummaryIsolation of adenylate cyclase-enriched membranes from human platelets was attempted using glycerol lysis technique followed by ultracentrifugation on discontinuous sucrose gradients composed of 24, 30, 34, 37, and 41% (w/w). Adenylate cyclase activity was enriched 4-fold in sample/24% sucrose interface, 7-fold in 24%/30% sucrose interface, and 4-fold in 30%/ 34% sucrose interface fractions with the recovery of 15-20% of the total activity. The enrichment and subcellular distribution of adenylate cyclase resembled in general those of phosphodiesterase and acid phosphatase with slight differences in each other. Protein profiles from SDS-polyacrylamide gel electrophoresis showed that the heavy chain of myosin (Mr = 200,000) was enriched in sample/24% sucrose interface and lower molecular weight proteins in 34%/37% sucrose interface and pellet. The interface fractions between 24 and 34% sucrose were, therefore, collected as adenylate cyclase-enriched membranes.Adenylate cyclase associated with the membranes displayed high specific activity (0.1 and 1-2 nmol/min/mg protein in the absence and presence of stimulants, respectively), and possessed sensitivities to prostaglandins (E1, I2, and D2) as well as cholera toxin. Activation of adenylate cyclase by these compounds required added GTP, indicating that the contamination of the membrane preparations with GTP-like substance (s) was minimal, if at all present.

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The purification and properties of urocanase from Pseudomonas testosteroni

Biochemical Journal ◽

10.1042/bj1710041 ◽

1978 ◽

Vol 171 (1) ◽

pp. 41-50 ◽

Cited By ~ 5

Author(s):

A J Hacking ◽

M V Bell ◽

H Hassall

Keyword(s):

Gel Electrophoresis ◽

Guanidine Hydrochloride ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Equilibrium Analysis ◽

Sedimentation Equilibrium ◽

Carbonyl Groups ◽

Prosthetic Group ◽

Purification And Properties ◽

Pseudomonas Testosteroni

Urocanase (urocanate hydratase, EC 4.2.1.49) purified from Pseudomonas testosteroni has a mol.wt. of 118000 determined by sedimentation-equilibrium analysis. Ultracentrifugation in 6M-guanidine hydrochloride and polyacrylamide-gel electrophoresis in sodium dodecyl sulphate show that the enzyme consists of two identical or very similar subunits. It is, like urocanase isolated from other sources, inhibited by reagents that react with carbonyl groups. Although urocanase from Ps. testosteroni is strongly inhibited by NaBH4, no evidence could be obtained for the presence of covalently bound 2-oxobutyrate as a prosthetic group; this is in contrast with findings elsewhere for urocanase from Pseudomonas putida. Urocanase from Ps. testosteroni does not contain pyridoxal 5′-phosphate as a coenzyme and in this respect is similar to all urocanases studied in purified form.

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

Biochemical Journal ◽

10.1042/bj1890247 ◽

1980 ◽

Vol 189 (2) ◽

pp. 247-253 ◽

Cited By ~ 30

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.

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An improved procedure for the isolation of 3-phosphoglycerate kinase from yeast

Biochemical Journal ◽

10.1042/bj1220089 ◽

1971 ◽

Vol 122 (1) ◽

pp. 89-92 ◽

Cited By ~ 51

Author(s):

R. K. Scopes

Keyword(s):

Molecular Weight ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Phosphoglycerate Kinase ◽

X Ray Diffraction ◽

X Ray ◽

Starch Gel ◽

Crystalline Forms

3-Phosphoglycerate kinase has been isolated from yeast by a new procedure. Over 1g was obtained from 450g of granulated baker's yeast; it had a specific activity of up to 940units/mg at 30°C. Six distinct crystalline forms have been grown, at least one of these being suitable for X-ray diffraction studies. The crystalline preparation is pure, judged by starch-gel or sodium dodecyl sulphate–polyacrylamide-gel electrophoresis; the latter method indicating that the enzyme is monomeric, with a molecular weight near to 50000.

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Biochemical Properties of Rhodanese from Almond (Prunus amygdalus) Nuts

Pan African Journal of Life Sciences ◽

10.36108/pajols/8102/10(0140) ◽

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.

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The subunit structure of jack-bean urease

Biochemical Journal ◽

10.1042/bj1130669 ◽

1969 ◽

Vol 113 (4) ◽

pp. 669-677 ◽

Cited By ~ 25

Author(s):

C. J. Bailey ◽

D. Boulter

Keyword(s):

Molecular Weight ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Polyacrylamide Gel ◽

Phenyl Isothiocyanate ◽

Subunit Structure ◽

Jack Bean ◽

Subunit Molecular Weight

1. Urease of specific activity 160–180 Sumner units/g. (Sumner, 1951) was purified from jack-bean meal. The preparation was pure on the basis of polyacryl-amide-gel electrophoresis and N-terminal studies. 2. By using both the 1-fluoro-2,4-dinitrobenzene method and the phenyl isothiocyanate method a single N-terminal methionine residue was found. 3. A single C-terminal sequence -Tyr-Leu-Phe was found by studies with carboxypeptidase A, carboxypeptidase B and hydrazinolysis. 4. N-Bromosuccinimide cleavage showed that five unique tryptophan sequences were present: Trp-Ala, Trp-Glu, Trp-Gly, Trp-Met and Trp-Arg. 5. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate showed that urease had a subunit molecular weight of 76000. 6. The yield of N- and C-terminal amino acids, the number of tryptic peptides and tryptophan sequences and the above polyacrylamide-gel electrophoretic measurement all suggest that urease contains a single structural subunit of molecular weight 75000.

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