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.

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 Isolation and characterization of human plasma α 1-proteinase inhibitor and a conformational study of its interaction with proteinases

1976 ◽  
Vol 157 (2) ◽  
pp. 339-351 ◽  
Author(s):  
J Saklatvala ◽  
G C Wood ◽  
D D White
Keyword(s):  
Human Plasma ◽  
Isoelectric Focusing ◽  
Proteinase Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Equilibrium ◽  
Isolation And Characterization ◽  
Step Procedure ◽  
Helical Content

1. alpha 1-Proteinase inhibitor was isolated from human plasma by a five-step procedure. Isoelectric focusing showed that six components focused between pH4.85 and 4.95. 2. The mol.wt. of the inhibitor was 52000 by sedimentation equilibrium and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The amino acid and carbohydrate compositions of the inhibitor were also determined. 3. The far-u.v.c.d. (circular-dichroism) spectrum indicated that the inhibitor had about 36% alpha-helical content. 4. The loss of proteinase-inhibitory activity when the inhibitor was exposed to pH values less than 5.0 or greater than 10.5 was accompanied by small changes in the far-u.v.c.d. spectrum and large changes in the near-u.v.c.d. spectrum. The change at alkaline pH was associated with ionization of tyrosine residues. 5. Interaction of inhibitor with chymotrypsin caused perturbation of the c.d. spectrum and this was used to follow the interaction and show a 1:1 stoicheiometry. 6. C.d., electrophoresis and isoelectric focusing showed that the inhibitor-enzyme complex is degraded by free enzyme. 7. Parallel studies with trypsin indicated that it too forms a 1:1 complex with inhibitor and is degraded by excess of enzyme.

scholarly journals The microsporidian spore invasion tube. The ultrastructure, isolation, and characterization of the protein comprising the tube.

1976 ◽  
Vol 71 (1) ◽  
pp. 23-34 ◽  
Author(s):  
E Weidner
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Low Molecular Weight ◽  
Two Dimensional ◽  
Polar Tube ◽  
Isolation And Characterization ◽  
Disulfide Linkages ◽  
Microsporidian Spore ◽  
Ph Range

The extrusion apparatus of the microsporidian parasitic protozoan Nosema michaelis discharges an invasion (or polar) tube with a velocity suitalbe for piercing cells and injecting infective sporoplasm. The tube is composed of a polar tube protein (PTP) which consists of a single, low molecular weight polypeptide slightly smaller than chymotrypsinogen-A. Assembled PTP tubes resist dissociation in sodium dodecyl sulfate and brief exposures in media at extreme ends of the pH range; however, the tubes are reduced by mercaptoethanol and dithiothreitol. When acidified, mercaptoethanol-reduced PTP self-assembles into plastic, two-dimensional monolayers. Dithiothreitol-reduced PTP will not reassemble when acidified. Evidence is presented which indicates that PTP is assembled as a tube within the spore; that the ejected tube has plasticity during sporoplasm passage; and, finally, that the subunits within the tube polymer are bound together, in part, by interprotein disulfide linkages.

scholarly journals Haemoglobin from the tadpole shrimp, Lepidurus apus lubbocki Characterization of the molecule and determination of the number of polypeptide chains

1979 ◽  
Vol 183 (2) ◽  
pp. 325-330 ◽  
Author(s):  
E Ilan ◽  
E Daniel
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Sedimentation Equilibrium ◽  
Guanidinium Chloride ◽  
Tadpole Shrimp ◽  
Polypeptide Chains

Haemoglobin from the tadpole shrimp, Lepidurus apus lubbocki, was found to have a sedimentation coefficient (s020,w) of 19.3 +/- 0.2 S and a molecular weight, as determined by sedimentation equilibrium, of 798000 +/- 20000. The amino acid composition showed the lack of cysteine and cystine residues. A haem content of 3.55 +/- 0.03% was determined, corresponding to a minimal mol.wt. of 17400 +/- 200. The pH-independence in the range pH 5-11 of the sedimentation coefficient indicates a relatively high stability of the native molecule. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave one band with mobility corresponding to a mol.wt. of 34000 +/- 1500. The molecular weight of the polypeptide chain was determined to be 32800 +/- 800 by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol. The findings indicate that Lepidurus haemoglobin is composed of 24 identical polypeptide chains, carrying two haem groups each.

Isolation and characterization of myoglobin and its two major isoforms from sheep heart.

1989 ◽  
Vol 35 (5) ◽  
pp. 778-782 ◽  
Author(s):  
J T Wu ◽  
R K Pieper ◽  
L H Wu ◽  
J L Peters
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Cardiac Muscle ◽  
Ammonium Sulfate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Agarose Gel ◽  
Isolation And Characterization ◽  
Isoelectric Points

Abstract We isolated myoglobin from sheep heart by homogenizing cardiac muscle in 70%-saturated ammonium sulfate, followed by chromatography on a column containing carboxymethyl(CM)-Sephadex gel. Two major isoforms of myoglobin, designated Mb 7.9 and Mb 8.1, were separated by chromatofocusing and were distinguished by their different patterns seen on either isoelectrofocusing or on electrophoresis on polyacrylamide gel. The isoelectric points of the major bands of Mb 7.9 and Mb 8.1 were 7.4 and 7.16, respectively. Both isoforms were identical in size when examined by gel filtration chromatography but differed slightly when analyzed by polyacrylamide gradient gel in the presence of sodium dodecyl sulfate. The Mr of Mb 7.9 (15,900 Da) is slightly smaller than that of Mb 8.1 (18,400 Da). When reacted against rabbit anti-sheep myoglobin, two isoforms also appeared as two nonidentical precipitin lines on agarose gel.

scholarly journals The interaction of the calcium-binding protein (troponin C) with bivalent cations and the inhibitory protein (troponin I)

1974 ◽  
Vol 137 (2) ◽  
pp. 145-154 ◽  
Author(s):  
J. F. Head ◽  
S. V. Perry
Keyword(s):  
Skeletal Muscle ◽  
Calcium Binding ◽  
Troponin I ◽  
Binding Protein ◽  
Sodium Dodecyl ◽  
Psoas Muscle ◽  
Calcium Binding Protein ◽  
Sedimentation Equilibrium ◽  
Free Form ◽  
Inhibitory Protein

1. The molecular weight of the calcium-binding protein of rabbit white skeletal muscle was estimated to be 18500 by sedimentation equilibrium and electrophoresis in sodium dodecyl sulphate. 2. Addition of 2 Ca2+ ions per molecule produced reversible changes in the u.v.-absorption spectrum that are interpreted as arising from conformational changes in the structure of the protein. 3. Cd2+ was almost as effective as Ca2+ in producing the spectral changes. Other bivalent metal ions, particularly Mg2+, were less effective. 4. Binding of Ca2+ by the calcium-binding protein produced an increase in mobility to the anode on electrophoresis in 6m-urea at pH8.6. The Ca2+-saturated form of the protein was more retarded on gel filtration than the Ca2+-free form. 5. In the presence of Ca2+ the calcium-binding protein formed an equimolar complex with the inhibitory protein. This complex was stable in 8m-urea and in the pH range 7.0–8.6. 6. An isotope-dilution method for the measurement of the content of calcium-binding protein in whole muscle is described. In rabbit psoas muscle the ratio of actin monomers to molecules of calcium-binding protein was approx. 7:1. Similar values were obtained for red skeletal and cardiac muscle. 7. Evidence is presented indicating that in the rabbit the inhibitory protein of the troponin complex of red skeletal and cardiac muscles is different from the inhibitory protein of white skeletal muscle.

Molecular analysis of FMRFamide- and FMRFamide-related peptides (FaRPS) in the cuttlefish Sepia officinalis.

1997 ◽  
Vol 200 (10) ◽  
pp. 1483-1489 ◽  
Author(s):  
P K Loi ◽  
N Tublitz
Keyword(s):  
Basic Amino Acid ◽  
Sepia Officinalis ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Base Pairs ◽  
Reading Frame ◽  
Isolation And Characterization ◽  
The Brain

The display of complex color patterns of the cuttlefish Sepia officinalis is under the regulation of the FMRFamide-related peptide (FaRP) family, but their exact identities are unknown. We report the isolation and characterization of a full-length FaRP cDNA from the brain of S. officinalis. This cDNA is 1850 base pairs long, including an open reading frame of 996 base pairs. The cDNA encodes a precursor protein containing four FaRPs: ALSGDAFLRF, FIRF, FLRF and FMRF. Each propeptide has a C-terminal glycine residue that is presumably converted post-translationally to an amide. Every FaRP propeptide is also flanked by basic amino acid residues at the amino and carboxy termini, indicative of putative cleavage sites during post-translational processing. Each of the four FaRPs encoded by this cDNA causes chromatophore expansion when assayed in an in vitro chromatophore bioassay. Thus, it is likely that one or more of the FaRPs identified in this study are involved in controlling chromatophore activity in cuttlefish.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

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