Rabbit Cardiac Myosin. I. Physical and Chemical Characterization of the Native Molecule

1975 ◽  
Vol 53 (2) ◽  
pp. 164-174 ◽  
Author(s):  
William T. Wolodko ◽  
Cyril M. Kay
Keyword(s):  
Molecular Weight ◽  
Atpase Activity ◽  
High Speed ◽  
Deae Cellulose ◽  
Sedimentation Equilibrium ◽  
Experimental Program ◽  
Activity Measurement ◽  
Cardiac Myosin ◽  
Skeletal Myosin ◽  
Physical And Chemical

Rabbit cardiac myosin, isolated from frozen tissue, was effectively purified by batchwise treatment with DEAE-cellulose in addition to using dilution–precipitation techniques. An extensive experimental program was subsequently carried out with respect to the enzymic, amino acid, optical, and physicochemical properties of native cardiac myosin. This program has included the following: examination of the effects of pH and varying concentrations of ATP, CaCl2, MgCl2, and PCMB on its ATPase activity; measurement of its circular dichroic spectrum in solvent buffers, at different pH or containing ATP in the absence or presence of Ca2+ or Mg2+ ions; study of the concentration dependence of its viscosity and sedimentation velocity at low temperatures; and investigation of its molecular weight by the Archibald method and low- and high-speed sedimentation equilibrium. The results of these studies were consistent with the interpretation that cardiac myosin is comprised of highly asymmetric, semi-rigid molecules with a molecular weight in the order of 4.7 × 105, which display non-ideality even in solvent buffers of high ionic strength at neutral pH. In addition, computer analysis of the high-speed sedimentation equilibrium data has provided evidence for the presence of a self-association reaction at low protein concentration. Even though the specific ATPase activity of cardiac myosin was found to be approximately one-third that reported for skeletal myosin in all cases, it was concluded, on the basis of the essentially analogous physical and chemical properties of rabbit cardiac and skeletal myosin, that the two proteins are very similar in terms of molecular size, shape, and secondary structure.

scholarly journals Physicochemical properties of Planorbis corneus erythrocruorin

1975 ◽  
Vol 149 (2) ◽  
pp. 437-445 ◽  
Author(s):  
E J Wood ◽  
L J Mosby
Keyword(s):  
Molecular Weight ◽  
High Speed ◽  
Sedimentation Coefficient ◽  
Sedimentation Equilibrium ◽  
Alkaline Conditions ◽  
Planorbis Corneus ◽  
A Subunit ◽  
Partial Dissociation ◽  
Equilibrium Ultracentrifugation ◽  
Intermediate Value

The erythrocruorin from the snail Planorbis corneus had a sedimentation coefficient, s020,w, of 33.5 ± 0.31S, and a molecular weight of 1.65 × 10(6) ± 0.04 × 10(6) by high-speed sedimentation-equilibrium ultracentrifugation. The amino acid composition and absorption spectrum of the protein are reported. A very low number of half-cystine residues was found, corresponding to 0.4 residue per haem group. The haem content was 2.76 ± 0.22%, corresponding to a protein molecular weight of about 22300. Under both acid and alkaline conditions partial dissociation took place to yield mixtures of products that could not be identified. A subunit corresponding to that containing one haem group was not obtained under any of the dossociating conditions tried. Electron microscopy revealed a ring-shaped molecule about 12.2 ± 0.5 nm in diameter. The native erythrocruoerin bound O2 co-operatively, the intermediate value of h in Hill plots having values between 1.7 and 3.4 depending on the conditions.

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.

Partial amino acid sequence of the wheat germ Ec protein. Comparison with another protein very rich in half-cystine and glycine: wheat germ agglutinin

1984 ◽  
Vol 62 (9) ◽  
pp. 908-913 ◽  
Author(s):  
Theo Hofmann ◽  
David I. C. Kells ◽  
Byron G. Lane
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Wheat Germ Agglutinin ◽  
High Speed ◽  
Wheat Germ ◽  
Sodium Dodecyl ◽  
Sedimentation Equilibrium ◽  
Partial Sequence ◽  
Edman Degradation

A wheat germ protein (Ec), the dominant site of cysteine incorporation during early (E) germination of isolated wheat embryos, has been partially sequenced by automated Edman degradation. The sequence of residues 1–59 is not significantly similar to the amino acid sequence known for any other protein, including wheat germ agglutinin which, like Ec, is very rich in half-cystine and glycine. The partial sequence for Ec contains an almost identical pattern of half-cystine residues in segments 6–20 and 35–48, a duplication which includes 10 of the 12 half-cystine residues in the sequence. The partial sequence of Ec may include a large part of the complete sequence, but this remains uncertain because it has not been possible to arrive at a definitve estimate of molecular weight using different physical techniques. Protein Ec can be prepared from a reticulocyte lysate in which cell-free synthesis is programmed by bulk wheat germ mRNA. Determination of the distribution of half-cystine moieties between residues 1 and 20 by Edman degradation of the [35S]cysteine-labeled product of cell-free synthesis shows that it is devoid of an N-terminal extension. Unlike wheat germ agglutinin, Ec does not seem to arise by processing of a conspicuously larger precursor protein. Unlike Ec, another wheat germ protein, Em, the most conspicuous methionine-labeled protein when cell-free protein synthesis is directed by wheat germ mRNA, is refractory to direct sequence analysis by Edman degradation. However, again unlike Ec, uncertainty about the molecular weight of Em, based on its mobility in different sodium dodecyl sulphate – polyacrylamide gel systems, has been resolved by virtue of Em being ideally suited to study by the Yphantis high-speed sedimentation equilibrium method.

scholarly journals Isolation and characterization of dermatan sulphate proteoglycan from human uterine cervix

1983 ◽  
Vol 209 (2) ◽  
pp. 497-503 ◽  
Author(s):  
N Uldbjerg ◽  
A Malmström ◽  
G Ekman ◽  
J Sheehan ◽  
U Ulmsten ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Uterine Cervix ◽  
Proteinase Inhibitors ◽  
Gradient Centrifugation ◽  
Average Molecular Weight ◽  
Deae Cellulose ◽  
Sedimentation Equilibrium ◽  
Dermatan Sulphate ◽  
Guanidinium Chloride ◽  
Isolation And Characterization

Proteoglycans were extracted from human uterine cervix with 4 M-guanidinium chloride in the presence of proteinase inhibitors. They were purified by density-gradient centrifugation in 4 M-guanidinium chloride/CsCl (starting density 1.32 g/ml) followed by DEAE-cellulose and Sepharose chromatography. Only one polydisperse proteoglycan was found. s020,w was 2.1S and the weight-average molecular weight was 73 000 (sedimentation-equilibrium centrifugation) to 110 500 (light-scattering). The core protein was monodisperse, with an apparent molecular weight of 47 000. The proteoglycan contained about 30% protein and probably two or three glycosaminoglycan side chains per molecule. High contents of aspartate, glutamate and leucine were found. The glycan moiety of the proteoglycan was exclusively dermatan sulphate, with a co-polymeric structure with approximately equal quantities of iduronic acid- and glucuronic acid-containing disaccharides.

scholarly journals Studies on the subunit structure of 4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating) from Pseudomonas acidovorans

1975 ◽  
Vol 145 (2) ◽  
pp. 305-309 ◽  
Author(s):  
R Jeffcoat
Keyword(s):  
Molecular Weight ◽  
High Speed ◽  
Guanidine Hydrochloride ◽  
Sodium Dodecyl ◽  
Potassium Phosphate ◽  
Sedimentation Equilibrium ◽  
Subunit Structure ◽  
Before And After ◽  
Lysine Residues ◽  
Peptide Map

1. Homogeneous preparations of D-4-deoxy-5-oxoglutarate hydro lyase (decarboxylating)(EC4.2.1.41) were analysed in the ultracentrifuge by the high-speed sedimentation-equilibrium method of Yphantis (1964). The molecular weight in 0.1 M-potassium phosphate buffer, pH 7.2, in 6M-guanidine hydrochloride and in 0.1 M-beta-mercaptoethanol in 6M-guanidine hydrochloride was 113,000, 56,000 and 30,400 respectively. Polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate indicated a minimum molecular weight of 30,500. 2. Measurement of the thiol content of the enzyme, before and after reduction with NaBH4 or dithiothreitol under denaturing conditions, indicated the presence of eight thiol residues and two interchain disulphide bridges/enzyme molecule. 3. Amino acid analysis showed that the intact enzyme contains a total of approximately 100 arginine and lysine residues, but digestion of the enzyme with trypsin yielded about 49 peptides staining with ninhydrin in a peptide “map”. 4. With the knowledge that the enzyme contains only two substrate-binding sites, it is suggested that the enzyme probably consists of four polypeptide chains arranged in an alpha2beta2 confirmation.

Preparation and Properties of Human Prothrombin Complex

1970 ◽  
Vol 24 (03/04) ◽  
pp. 325-333 ◽  
Author(s):  
G. H Tishkoff ◽  
L. C Williams ◽  
D. M Brown
Keyword(s):  
Molecular Weight ◽  
Proteolytic Enzyme ◽  
Enzyme Inhibitor ◽  
Specific Activity ◽  
Crystalline Form ◽  
Sedimentation Equilibrium ◽  
Crystalline Complex ◽  
Interaction Product ◽  
Proteolytic Enzyme Inhibitor ◽  
Purification Scheme

SummaryAs a corollary to our previous studies with bovine prothrombin, we have initiated a study of human prothrombin complex. This product has been isolated in crystalline form as a barium glycoprotein interaction product. Product yields were reduced compared to bovine product due to the increased solubility of the barium glycoprotein interaction product. On occasion the crystalline complex exhibited good yields. The specific activity of the crystalline complex was 1851 Iowa u/mg. Further purification of human prothrombin complex was made by removal of barium and by chromatography on Sephadex G-100 gels. The final product evidenced multiple procoagulant activities (II, VII, IX and X). The monomeric molecular weight determined by sedimentation equilibrium in a solvent of 6 M guanidine-HCl and 0.5% mercaptoethanol was 70,191 ± 3,057 and was homogeneous with respect to molecular weight. This product was characterized in regard to physical constants and chemical composition. In general, the molecular properties of human prothrombin complex are very similar to the comparable bovine product. In some preparations a reversible proteolytic enzyme inhibitor (p-aminophenylarsonic acid) was employed in the ultrafiltration step of the purification scheme to inhibit protein degradation.

Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver

1984 ◽  
Vol 62 (5) ◽  
pp. 276-279 ◽  
Author(s):  
C. H. Lin ◽  
W. Chung ◽  
K. P. Strickland ◽  
A. J. Hudson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Enzymatic Synthesis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Aromatic Amino Acids ◽  
Adenosylmethionine Synthetase ◽  
Cellulose Column

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.

scholarly journals Identity and Origin of the ATPase activity associated with neuronal microtubules. I. The ATPase activity is associated with membrane vesicles.

1983 ◽  
Vol 96 (5) ◽  
pp. 1298-1305 ◽  
Author(s):  
D B Murphy ◽  
R R Hiebsch ◽  
K T Wallis
Keyword(s):  
Molecular Weight ◽  
Atpase Activity ◽  
High Molecular Weight ◽  
Brain Tissue ◽  
Membrane Vesicles ◽  
Microtubule Associated Proteins ◽  
In Vitro Assembly ◽  
Microtubule Protein ◽  
Associated Proteins

Microtubule protein purified from brain tissue by cycles of in vitro assembly-disassembly contains ATPase activity that has been postulated to be associated with microtubule-associated proteins (MAPs) and therefore significant for studies of microtubule-dependent motility. In this paper we demonstrate that greater than 90% of the ATPase activity is particulate in nature and may be derived from contaminating membrane vesicles. We also show that the MAPs (MAP-1, MAP-2, and tau factors) and other high molecular weight polypeptides do not contain significant amounts of ATPase activity. These findings do not support the concept of "brain dynein" or of MAPs with ATPase activity.

scholarly journals A chromatographic preparation of ox growth hormone

1966 ◽  
Vol 100 (3) ◽  
pp. 593-600 ◽  
Author(s):  
M Wallis ◽  
HBF Dixon
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Gel Electrophoresis ◽  
Anterior Pituitary ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Starch Gel Electrophoresis ◽  
Highly Active ◽  
Cross Linkage ◽  
Starch Gel

1. A method is described for the chromatographic preparation of ox growth hormone. It involves chromatography of an extract of anterior pituitary lobes on DEAE-cellulose, followed by rechromatography on a dextran gel of low cross-linkage (Sephadex G-100). 2. The product is highly active in growth-hormone assays, and is obtained in good yield. It was homogeneous by several criteria, but showed some heterogeneity on starch-gel electrophoresis. 3. The molecular weight of the hormone was estimated from its behaviour on gel-filtration columns under various conditions. Evidence that the hormone may dissociate into sub-units under some conditions is presented.

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