scholarly journals Amelogenins. Purification and partial characterization of proteins from developing bovine dental enamel

1973 ◽  
Vol 131 (3) ◽  
pp. 471-484 ◽  
Author(s):  
F. Michael Eggert ◽  
Grania A. Allen ◽  
Ralph C. Burgess
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Dental Enamel ◽  
Polyacrylamide Gel ◽  
Sedimentation Equilibrium ◽  
Amino Acid Compositions ◽  
Small Proteins ◽  
Equilibrium Ultracentrifugation

1. Procedures are described for the purification of amelogenin electrophoretic components and their analysis for homogeneity by polyacrylamide-gel electrophoresis at both acidic and alkaline pH values. 2. Most of these components belonged to two main groups, termed the J group and the C group after their major electrophoretic components. Sodium dodecyl sulphate-polyacrylamide-gel electrophoresis indicated that, within each group, proteins were of similar size, but the C-group proteins were larger than those of the J group. 3. By sedimentation-equilibrium ultracentrifugation and amino acid analysis, the four J-group components were found to be very small proteins (mol. wt. 5500–3000) and, except for one, similar in amino acid composition. The components of the C group were found to be proteins of moderate size (mol. wt. 16800–16100) with very similar amino acid compositions. A third minor amelogenin group of intermediate size was also found, but not further analysed. Details of the results of the ultracentrifuge studies are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50014 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5. 4. Two of the J-group components were similar to amelogenins isolated by other workers. 5. All amelogenins analysed were rich in proline, glutamic acid, histidine and methionine, and contained no half-cystine. Their amino acid compositions, combined with their molecular weights, serve to distinguish the amelogenins from both collagens and keratins.

scholarly journals Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure

1983 ◽  
Vol 213 (1) ◽  
pp. 225-234 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Bovine Liver ◽  
Polyacrylamide Gel ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.

Plant NAD-Dependent Glutamate Dehydrogenase. Purification, Molecular Properties and Metal Ion Activation of the Enzymes from Lemna minor and Pisum sativum

1980 ◽  
Vol 35 (3-4) ◽  
pp. 213-221 ◽  
Author(s):  
Heinz-Walter Scheid ◽  
Adelheid Ehmke ◽  
Thomas Hartmann
Keyword(s):  
Amino Acid ◽  
Pisum Sativum ◽  
Glutamate Dehydrogenase ◽  
Gel Electrophoresis ◽  
Metal Ion ◽  
Lemna Minor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Equilibrium ◽  
Electron Microscopic

Abstract Glutamate dehydrogenase (ʟ-glutamate: NAD+ oxidoreductase (deaminating) EC 1.4.1.2) has been purified to homogeneity from Lemna minor and seeds of Pisum sativum. As established by polyacrylamide gel electrophoresis the Pisum-enzyme constitutes a multiple pattern of seven char­ge isoenzymes whereas the Lemna enzyme shows one single protein band. Molecular weights of 230 000 were calculated for both enzymes by sedimentation equilibrium measurements (Pisum-enzyme) and comparative gel filtration (Lemna-enzyme). Sodium dodecyl sulfate gel electrophoresis and electron microscopic observations revealed that both enzymes are composed of four identical subunits (molecular weight 58 500) arranged in a tetraedric structure. The amino acid compositions of both enzymes are similar to those of various hexameric glutamate dehydrogenases. The N-terminal amino acid of the Pisum-enzyme is alanine. Both enzymes require Ca2+ for maximal catalytic activity. For the Lemna-enzyme the K0.5 values for Ca2+ are 22 µᴍ (NADH-dependent reaction) and 4 µᴍ (NAD+ -dependent reaction), respectively. Ca2+ which to some extent can be replaced by Zn2+ does not affect the enzyme aggregation but seems to govern a reversible equilibrium between catalytically active and inactive enzyme forms.

scholarly journals Purification and properties of a cellulase from Aspergillus niger

1977 ◽  
Vol 165 (1) ◽  
pp. 33-41 ◽  
Author(s):  
P L Hurst ◽  
J Nielsen ◽  
P A Sullivan ◽  
M G Shepherd
Keyword(s):  
Amino Acid ◽  
Aspergillus Niger ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Cellulolytic Enzyme ◽  
Protein Amino Acid ◽  
Ph Optimum

A cellulolytic enzyme was isolated from a commercial cellulase preparation form Aspergillus niger. A yield of about 50mg of enzyme was obtained per 100g of commerial cellulase. The isolated enzyme was homogeneous in the ultracentrifuge at pH 4.0 and 8.0, and in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but showed one major and two minor bands in disc gel electrophoresis. No carbohydrate was associated with the protein. Amino acid analysis revealed that the enzyme was rich in acidic and aromatic amino acids. Data from the amino acid composition and dodecyl sulphate/polyacrylamide-gel electrophoresis indicated a molecular weight of 26000. The purified enzyme was active towards CM-cellulose, but no activity towards either cellobiose or p-nitrophenyl beta-D-glucoside was detected under the assay conditions used. The pH optimum for the enzyme was pH 3.8-4.0, and it was stable at 25 degrees C over the range pH 1-9; maximum activity (at pH 4.0) was obtained at 45 degrees C. The cellulase was more stable to heat treatment at pH 8.0 than at 4.0. Kinetic studies gave pK values between 4.2 and 5.3 for groups involved in the enzyme-substrate complex.

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

1981 ◽  
Vol 197 (2) ◽  
pp. 427-436 ◽  
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.

scholarly journals Two low-molecular-weight Ca2+-binding proteins isolated from squid optic lobe by phenothiazine-Sepharose affinity chromatography

1983 ◽  
Vol 209 (3) ◽  
pp. 797-802 ◽  
Author(s):  
J F Head ◽  
S Spielberg ◽  
B Kaminer
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Content ◽  
Bovine Brain ◽  
Polyacrylamide Gel ◽  
Dependent Manner

We have isolated two Ca2+-binding proteins from squid optic lobes, each of which is also able to bind phenothiazines in a Ca2+-dependent manner. These proteins have each been purified and partly characterized. One of the proteins corresponds to calmodulin, in that it has a similar amino acid content to bovine brain calmodulin, including a single residue of trimethyl-lysine, it co-migrates with bovine calmodulin both on alkaline-urea- and on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis, and will activate calmodulin-dependent phosphodiesterase. The second protein has the same subunit molecular weight as calmodulin, as determined by SDS/polyacrylamide-gel electrophoresis, Mr 17 000, but migrates more slowly than this protein on alkaline-urea-gel electrophoresis. It has an amino acid composition distinct from calmodulin, containing no trimethyl-lysine, its CNBr fragments migrate on alkaline gels in a pattern distinct from those of calmodulin and it shows little ability to activate phosphodiesterase. The u.v.-absorption spectra of the proteins indicate the absence of tryptophan and the presence of a high phenylalanine/tyrosine ratio in each. Both proteins also bind 3-4 calcium ions/mol at 0.1 mM-free Ca2+ and each binds chlorpromazine in a Ca2+-dependent manner.

scholarly journals Purification of rat fibrinogen and its constituent chains

1978 ◽  
Vol 169 (3) ◽  
pp. 653-658 ◽  
Author(s):  
Irina A. M. Van Ruijven-Vermeer ◽  
Willem Nieuwenhuizen
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rat Plasma ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Gel Chromatography

Rat fibrinogen was purified from rat plasma by using lysine–Sepharose chromatography, repeated precipitation with 25%-satd. (NH4)2SO4 and gel chromatography on Sepharose 6B. To minimize proteolytic activity, rats were injected intravenously with Trasylol before bleeding and the collected blood was treated with Trasylol and di-isopropyl phosphorofluoridate. A 95%-clottable preparation was obtained in 70–75% yield; it proved to be free of factor XIII and plasminogen. It showed a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and on disc electrophoresis in 8m-urea. Alanine was the only detectable N-terminal amino acid. After reduction and modification of the thiol groups, the material could be separated into three distinct chains (Aα, Bβ and γ) by pore-limit polyacrylamide slab-gel electrophoresis in the presence of sodium dodecyl sulphate. The amino acid compositions of the whole fibrinogen and of the separated modified chains were determined. The molecular weights were 61000, 58000 and 51000 for Aα-, Bβ- and γ-chains respectively. Our results for the chains are in contrast with previous reports on rat fibrinogen [Bouma & Fuller (1975) J. Biol. Chem.250, 4678–4683; Stemberger & Jilek (1976) Thromb. Res.9, 657–660], in which no separation between Aα- and Bβ-chains was achieved on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis for 3h. Evidence is presented that this is probably due to Aα-chain degradation as a result of incomplete inhibition of proteolytic enzymes during the purification. Complete inhibition of proteolytic activities is essential in all steps of the present purification procedure.

scholarly journals Purification and characterization of a phospholipase A2 from the venom of the coral snake, Micrurus fulvius microgalbineus (Brown and Smith)

1979 ◽  
Vol 179 (3) ◽  
pp. 603-606 ◽  
Author(s):  
L D Possani ◽  
A C Alagòn ◽  
P L Fletcher ◽  
M J Varela ◽  
J Z Juliá
Keyword(s):  
Amino Acid ◽  
Phospholipase A2 ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Coral Snake ◽  
Phospholipase Activity ◽  
Crude Venom

A phospholipase A2 was purified from the Mexican coral snake Micrurus fulvius microgalbieus (Brown and Smith). Gel filtration of the soluble crude venom on Sephadex g-50 resolved five fractions, of which fraction II had 98% of the total phospholipase activity. This fraction was rechromatographed on a CM-cellulose column that resolved eight fractions, four of which had an important phospholipase activity. The first fraction (II-1) was homogeneous by polyacrylamide-gel electrophoresis and displayed a phospholipase specific activity of 920 units/mg of protein. The apparent molecular weight as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 14000. The amino acid analysis revealed the presence of 119 amino acid residues, with 12 half-cystines. the N-terminal sequence was shown to be Ser-Leu-Leu-Asx-Phe-Lys-Asx-Met-Ile-Glu-Ser-Thr..., which is homologous with that of phospholipases from other snake venoms.

scholarly journals New proline-rich proteins in isolated insect Z-discs

1980 ◽  
Vol 191 (2) ◽  
pp. 333-339 ◽  
Author(s):  
G M Sainsbury ◽  
B Bullard
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Flight Muscle ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Ionic Strength ◽  
Polyacrylamide Gel ◽  
Protein Rich

Z-discs were isolated from Lethocerus (waterbug) flight muscle by removing the contractile proteins from myofibrils with a solution of high ionic strength. Sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis confirmed a previous report that major Z-disc proteins had subunit mol.wts of 200 000, 180 000, 105 000, 95 000, 42 000 and 35 000. A protein of subunit mol.wt 25 000 was found in once-washed Z-discs but was degraded or was removed by successive washes. In addition, a protein of high molecular weight (less than 300 000) was found in Z-discs. Proteins of subunit mol.wts. 42 000, 35 000 and 25 000 were individually sliced from SDS/polyacrylamide gels and eluted. Amino acid analysis showed that the 35 000-subunit-mol.wt. protein was not, as was previously suggested, tropomyosin, but was a distinct Z-disc protein rich in proline. Calculations based on the amino acid analysis showed that this protein contained substantial hydrophobic regions. Preliminary investigations into the isoelectric point and a method of isolation of the 35 000-subunit-mol.wt. Z-disc protein are described. This protein was found in slices cut from SDS/polyacrylamide-gel electrophoretograms of whole myofibrils. The protein of 42 000 subunit mol.wt. was shown by amino acid analysis to be actin and the 25 000-subunit-mol.wt. Z-disc protein was proline-rich.

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