The isolation and characterization of the tropomyosin binding component (TN-T) of bovine cardiac troponin

1976 ◽  
Vol 54 (6) ◽  
pp. 546-552 ◽  
Author(s):  
L. D. Burtnick ◽  
W. D. McCubbin ◽  
C. M. Kay
Keyword(s):  
Cardiac Muscle ◽  
Gel Electrophoresis ◽  
Calcium Binding ◽  
Cardiac Troponin ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chemical Properties ◽  
Polyacrylamide Gel ◽  
Sedimentation Equilibrium ◽  
Isolation And Characterization ◽  
Binding Component

The tropomyosin binding component (TN-T) of troponin was purified from bovine cardiac muscle using a combination of ion exchange chromatographies in the presence of urea. Sedimentation equilibrium experiments suggest a molecular weight for cardiac TN-T of 36 300 ± 2 000, consistent with a value of 37 000 ± 1 000 determined by polyacrylamide gel electrophoresis. Calculations based upon circular dichroism spectra indicate an apparent α-helical content of 43 ± 3% for TN-T. Polyacrylamide gel electrophoresis and the effects of the calcium binding component (TN-C) upon the solubility of TN-T suggest that the two cardiac troponin components can interact with each other. Cosedimentation analysis of solutions containing cardiac tropomyosin and TN-T provide evidence for complex formation involving these two proteins. The data presented on the physical and chemical properties of TN-T, as well as the interaction studies indicate that the cardiac muscle regulatory system operates in a manner similar to that proposed for skeletal muscle.

scholarly journals Physical and chemical properties of human plasma β2-macroglobulin

1978 ◽  
Vol 173 (1) ◽  
pp. 27-38 ◽  
Author(s):  
P K Hall ◽  
R C Roberts
Keyword(s):  
Human Plasma ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chemical Properties ◽  
Sedimentation Coefficient ◽  
Binding Activity ◽  
Polyacrylamide Gel ◽  
Sedimentation Equilibrium

Alpha2-M (alpha2-macroglobulin) was purified from human plasma by two different procedures. As well as having no detectable impurities by the usual criteria for testing the homogeneity of protein preparations, these alpha2M preparations showed a single component, after reduction in urea, of 185000 daltons by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weight of the alpha2M was found to be 718000 by sedimentation equilibrium experiments using the gravimetrically determined -v of 0.731 ml/g. The interaction of several proteinases with alpha2M was studied by using a novel discontinuous polyacrylamide-gel system, which showed clear separation of the enzyme-complexed alpha2M from the free alpha2M. These studies indicated that urokinase, as well as trypsin, chymotrypsin, plasmin and thrombin forms complexes with alphaM. The cleavage of the 185000-dalton subunit to a 85000-dalton species on interaction of trypsin with alpha2M was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after reduction of the alpha2M-trypsin complex in urea. The amino acid composition, carbohydrate content, absorption coefficient at 280 nm, the specific refractive increment and the sedimentation coefficient for these alpha2M preparations were measured. The stability of the trypsin-binding activity of the alpha2M preparations was also studied under several storage situations.

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.

The Isolation and Characterization of the ATPase Inhibitory Protein (TN-I) from Bovine Cardiac Muscle

1975 ◽  
Vol 53 (11) ◽  
pp. 1207-1213 ◽  
Author(s):  
L. D. Burtnick ◽  
W. D. McCubbin ◽  
C. M. Kay
Keyword(s):  
Molecular Weight ◽  
Cardiac Muscle ◽  
Calcium Binding ◽  
Sodium Dodecyl ◽  
Basic Amino Acid ◽  
Sedimentation Equilibrium ◽  
Amino Acid Residues ◽  
Inhibitory Protein ◽  
Isolation And Characterization

The inhibitory component of the troponin complex (TN-I) was purified from bovine cardiac muscle, using a combination of ion exchange and molecular exclusion chromatographies in the presence of urea. It has the ability to inhibit the Mg2+-activated ATPase (EC 3.6.1.3) of a synthetic cardiac actomyosin preparation and this inhibition is reversed by the addition of cardiac calcium binding component of troponin (TN-C). Conventional sedimentation equilibrium experiments suggest a molecular weight for cardiac TN-I of 22 900 ± 500. However, sodium dodecyl sulfate (SDS) gels indicate a molecular weight of 27 000 ± 1000. The mobility of TN-I on SDS gels may be anomalous due to the high proportion of basic amino acid residues in the protein. Cardiac TN-I and TN-C interact to form a tight complex, even in the presence of 6 M urea. The results of this study invite direct comparison with results published for rabbit skeletal TN-I.

Isolation and Characterization of J-chain from Bovine Colostral Immunoglobulin M

1975 ◽  
Vol 53 (9) ◽  
pp. 943-949 ◽  
Author(s):  
R. Komar ◽  
T. K. S. Mukkur
Keyword(s):  
Gel Electrophoresis ◽  
Fast Moving ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Immunoglobulin M ◽  
J Chain ◽  
Isolation And Characterization ◽  
Moving Band ◽  
Covalently Bound

Purified bovine colostral intact immunoglobulin M (IgM) exhibited the presence of an anodal, single, fast moving band (noncovalently bound form) when subjected to analytical polyacrylamide gel electrophoresis at an alkaline pH in urea. Reduced and alkylated or sulfitolysed bovine colostral IgM (devoid of the noncovalently bound form) also showed the presence of a similar band (covalently bound form). The molecular weight of both the covalently bound and noncovalently bound forms of the fast component was determined to be 16 500 by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. In addition, the non-covalently bound form of the fast-moving component was found to be antigenically identical to the covalently bound form. The noncovalently bound form sedimented as a single peak at 1.56 S. Antiserum against the fast-moving component precipitated neither bovine colostral IgG nor μ-chains and bovine serum albumin, but precipitated native or denatured intact IgM (devoid of the non-covalently bound form) and human J-chains and vice versa, thus permitting the fast-moving components to be classified as J-chains. Radioalkylation experiments revealed the presence of 9.7 sulfhydryl groups per mole, for both the covalently and non-covalently bound forms of bovine J-chain. The stoichiometry of J-chain, determined from the densitometric tracing of the reduced and alkylated bovine colostral IgM (devoid of the noncovalently bound J-chain) in stained analytical polyacrylamide gels, revealed the presence of one J-chain per IgM molecule. On the other hand the amount of non-covalently bound form of J-chain was determined to be 1.2 per molecule of IgM.

scholarly journals The exocellular dd-carboxypeptidase-endopeptidase from Streptomyces albus G. Purification and chemical properties

1978 ◽  
Vol 175 (3) ◽  
pp. 793-800 ◽  
Author(s):  
C Duez ◽  
J M Frère ◽  
F Geurts ◽  
J M Ghuysen ◽  
L Dierickx ◽  
...  
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Molecular Sieve ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chemical Properties ◽  
Polyacrylamide Gel ◽  
Streptomyces Albus ◽  
Enzyme I ◽  
And Chemical Properties

The exocellular DD-carboxypeptidase-endopeptidase of Streptomyces albus G was purified to protein homogeneity and compared with the exocellular DD-carboxypeptidases-transpeptidases of Streptomyces R61 and Actinomadura R39. The S. albus G enzyme, as it is isolated, occurs in two forms. Enzyme I (30% of the total amount) and enzyme II (70% of the total amount) are identical in all respects, except that, by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, enzyme I has an apparent mol. wt. (9000) that is half of that found by molecular-sieve filtration under non-denaturing conditions. Irrespective of the technique used, enzyme II has an apparent mol. wt. of about 18500.

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.

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