Multiple Molecular Forms of Avian Aldolases. V. Purification and Molecular Properties of Chicken (Gallus domesticus) Liver Aldolase

1971 ◽  
Vol 49 (6) ◽  
pp. 647-657 ◽  
Author(s):  
Ronald R. Marquardt
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Diffusion Constant ◽  
Sedimentation Coefficient ◽  
Sedimentation Velocity ◽  
Gallus Domesticus ◽  
Molecular Forms ◽  
Velocity Analysis ◽  
Multiple Molecular Forms ◽  
Stokes Radius

Aldolase (fructose-1,6-diphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) was purified from chicken liver. The enzyme was shown to be homogeneous according to the following criteria: purification to a constant specific activity following sequential chromatography on carboxymethyl-Sephadex and Sephadex G-200, electrophoresis on cellulose acetate strips, sedimentation velocity analysis, absence of 10 other glycolytic enzymes, and immunodiffusion in agar.The sedimentation coefficient (s°20w 8.0), Stokes radius (47 Å), diffusion constant (D°20w 4.0 × 10−7 cm2/s), and molecular weight (160 000) are similar to those of rabbit liver aldolase and the muscle and brain enzymes from both chickens and rabbits.

Multiple molecular forms of avian aldolases. I. Crystallization and physical properties of chicken (Gallus domesticus) breast muscle aldolase

1969 ◽  
Vol 47 (5) ◽  
pp. 517-526 ◽  
Author(s):  
Ronald R. Marquardt
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Diffusion Constant ◽  
Sedimentation Coefficient ◽  
Double Diffusion ◽  
Glycolytic Enzyme ◽  
Gallus Domesticus ◽  
Molecular Forms ◽  
Rabbit Muscle ◽  
Multiple Molecular Forms

Aldolase (fructose 1,6-diphosphate-D-glyceraldehyde 3-phosphate lyase, EC 4.1.2.13) was purified and crystallized from chicken (Gallus domesticus) breast muscle.The crystalline enzyme is homogeneous according to the following criteria: purification to a constant specific activity, electrophoresis on cellulose acetate strips, absence of five other glycolytic enzyme activities, and immunodiffusion in agar.The sedimentation coefficient, diffusion constant, and molecular weight of the chicken enzyme are the same as for rabbit muscle aldolase. The ultraviolet spectra of the two proteins are the same. Electrophoretic comparison between the rabbit and chicken enzymes revealed a slightly different rate of migration.Antibodies directed against the pure chicken enzyme were prepared, and the reaction with pure chicken and rabbit aldolase was followed using the precipitin and double diffusion tests. A very pronounced reaction was observed between anti-serum and the chicken enzyme; the rabbit enzyme, in contrast, did not cross-react with the anti-serum.

Multiple molecular forms of avian aldolases. IV. Purification and properties of chicken (Gallus domestkus) brain aldolase

1970 ◽  
Vol 48 (3) ◽  
pp. 322-333 ◽  
Author(s):  
Ronald R. Marquardt
Keyword(s):  
Specific Activity ◽  
Diffusion Constant ◽  
Sedimentation Coefficient ◽  
Breast Muscle ◽  
Molecular Forms ◽  
Chicken Breast ◽  
Rabbit Brain ◽  
Muscle Enzymes ◽  
Ph Optimum ◽  
Chicken Brain

Aldolase (fructose-1,6-diphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) was purified from chicken (Gallus domesticus) brain tissue. The enzyme was shown to be homogeneous according to the following criteria: purification to a constant specific activity following sequential chromatography on DEAE and Sephadex, sedimentation velocity analysis, and electrophoresis on cellulose acetate strips.Several properties of the enzyme were determined including the Stokes radius (47 Å), diffusion constant (D020 w = 4.6 × 10−7 cm2/s), sedimentation coefficient (s020 w = 8.0), and molecular weight (155 000). The enzyme has a broad pH optimum centered around 7.2. The apparent Michaelis constants for fructose 1,6-diphosphate and fructose 1-phosphate were 7 × 10−5 M and 3 × 10−2 M, respectively. The activity ratio with the above two substrates was 30.Many of the molecular properties of this enzyme are similar to those of the rabbit brain enzyme and the muscle enzymes from both chickens and rabbits. The enzymic properties of chicken brain aldolase correspond more closely to those of the rabbit brain enzyme than they do to chicken breast muscle aldolase. The amino acid composition of chicken brain aldolase was found to be quite different from chicken breast muscle aldolase with respect to certain amino acids (methionine, cysteine, tryptophan, histidine, proline, aspartate, valine, and phenylalanine).

Multiple Forms of Phosphoglyceromutase from Chicken Breast Muscle

1972 ◽  
Vol 50 (10) ◽  
pp. 1132-1142 ◽  
Author(s):  
Eric James ◽  
R. O. Hurst ◽  
T. G. Flynn
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Diffusion Constant ◽  
Sedimentation Coefficient ◽  
Breast Muscle ◽  
Heat Inactivation ◽  
Chicken Breast ◽  
Multiple Forms ◽  
Active Components ◽  
Net Charges

Phosphoglyceromutase (2,3-diphospho-D-glycerate: 2-phospho-D-glycerate phosphotransferase, EC 2.7.5.3) has been purified from both frozen and fresh chicken breast muscle. During purification it was found that substrate, 3-phospho-D-glycerate stabilized the enzyme against heat inactivation to almost the same extent as did the cofactor 2,3-diphospho-D-glycerate.Phosphoglyceromutase prepared from frozen chicken breast muscle separated into three peaks of activity (I, II, and III) following chromatography on DEAE-Sephadex in 0.05 μ phosphate buffer, pH 8.0, using a 0.0–0.4 M NaCl gradient. Each peak of activity was shown by polyacrylamide disc gel electrophoresis at pH 9.3 to contain two enzymically active components (isoenzymes Ia Ib, IIa IIb, and IIIa IIIb). Isoenzymes in the same peak had the same specific activity. Phosphoglyceromutase prepared from fresh chicken breast muscle yielded only one peak of activity following chromatography on DEAE-Sephadex. This peak contained two enzymically active components corresponding to isoenzymes Ia and Ib. Additional peaks of activity were not produced when phosphoglyceromutase from fresh muscle was subjected to freezing and thawing.Isoenzyme Ia and mixtures of Ia and Ib, IIa and IIb, and IIIa and IIIb were homogeneous in the ultra-centrifuge sedimenting as single peaks. The sedimentation coefficient obtained for isoenzyme Ia and for Ia and Ib combined was 4.15 S, the diffusion constant 6.62 × 10−7 cm2/s, and the molecular weight calculated from both gel filtration and sedimentation data was of the order of 59 000. These results were confirmed by charge isomer studies which also showed that the isoenzymes of phosphoglyceromutase from frozen chicken breast muscle were proteins of the same size but different net charges.

scholarly journals PURIFICATION AND PHYSICAL PROPERTIES OF GROUP C STREPTOCOCCAL PHAGE-ASSOCIATED LYSIN

1971 ◽  
Vol 133 (5) ◽  
pp. 1105-1117 ◽  
Author(s):  
Vincent A. Fischetti ◽  
Emil C. Gotschlich ◽  
Alan W. Bernheimer
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Physical Properties ◽  
Total Protein ◽  
Sedimentation Coefficient ◽  
Polyacrylamide Gel ◽  
Single Band ◽  
Purification Procedure ◽  
Sh Group ◽  
Stokes Radius

A purification procedure for the Group C phage-associated lysin is described utilizing tetrathionate to protect the enzyme's -SH group(s) from thiol-inactivating agents. A 652-fold purification has been accomplished yielding a solution in which the enzyme activity corresponds to essentially a single band on polyacrylamide gel which accounts for 70% of the total protein in the preparation. A molecular weight of 101,000 and frictional ratio of 1.526 was determined for the lysin utilizing experimentally determined values for its Stokes radius and sedimentation coefficient.

scholarly journals Purification by affinity chromatography and immunological characterization of a 110kDa component of the chick oviduct progesterone receptor

1984 ◽  
Vol 217 (3) ◽  
pp. 685-692 ◽  
Author(s):  
J M Renoir ◽  
J Mester ◽  
T Buchou ◽  
M G Catelli ◽  
P Tuohimaa ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Affinity Chromatography ◽  
Specific Activity ◽  
Sedimentation Coefficient ◽  
Binding Activity ◽  
B Subunit ◽  
Stokes Radius ◽  
A Subunit ◽  
Final Yield

A 110kDa component of the chick oviduct progesterone receptor (PR) has been purified to homogeneity according to electrophoretic criteria and specific activity (assuming one progestagen-binding site/110kDa). The procedure involved affinity chromatography of 0.3 M-KCl-prepared cytosol, followed by DEAE-Sephacel chromatography (elution at 0.2 M-KCl). The final yield was about 12% in terms of binding activity. Properties of the 110kDa component indicate that it is identical with the ‘B’ subunit described previously [Stokes radius approximately 6.1 nm; sedimentation coefficient, (S20, w) approximately 4S; frictional ratio approximately 1.77]. It reacted with the IgG-G3 polyclonal antibody, but not with BF4 monoclonal antibody raised against the 8S molybdate-stabilized chick oviduct PR and reacting with its 90kDa component. Another progesterone-binding component, corresponding to the ‘A’ subunit, also previously described, was eluted from the DEAE-Sephacel column at approximately 0.08 M-KCl, and contained a peptide of molecular mass approx. 75-80kDa, which had S20, w approximately 4S in a sucrose gradient. This component was also recognized by IgG-G3, but not by BF4; it was very unstable in terms of hormone-binding activity.

Purification and partial characterization of a plasma inhibitor of tonin

1981 ◽  
Vol 59 (4) ◽  
pp. 256-261 ◽  
Author(s):  
J. Tremblay ◽  
G. Thibault ◽  
J. Gutkowska ◽  
R. Boucher ◽  
J. Genest
Keyword(s):  
Molecular Weight ◽  
Plasma Proteins ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Velocity ◽  
Exchange Chromatography ◽  
Ammonium Sulfate Precipitation ◽  
Angiotensin I ◽  
Stokes Radius

A plasma inhibitor of tonin activity in the rat, was purified by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Its purity was investigated by analytical electrophoresis on polyacrylamide gel and by ultracentrifugation sedimentation velocity. The molecular weight (360 000) of the purified inhibitor was determined by sodium dodecyl sulfate electrophoresis and its isoelectric point (4.5) by gel isoelectrofocusing. The Stokes radius (640 nm) was evaluated by gel filtration studies and a frictional ratio (f/f0) of 1.95 was calculated from the molecular weight and Stokes radius.Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by the purified inhibitor was noncompetitive and does not exceed 70%. Electrophoresis showed the same mobility for [125I]tonin bound to plasma proteins and for [125I]tonin bound to the purified inhibitor. The inhibitor may be a protein resembling half of the dimeric protease inhibitor rat α1-macroglobulin or human α2-macroglobulin

Distribution of the multiple molecular forms of glucose-6-phosphate dehydrogenase in different physiological states

1979 ◽  
Vol 57 (5) ◽  
pp. 396-401 ◽  
Author(s):  
Hsiao-Lin Chang ◽  
Darold Holten ◽  
Rom Karin
Keyword(s):  
Enzyme Activity ◽  
Mammary Gland ◽  
Rat Liver ◽  
Specific Activity ◽  
Molecular Forms ◽  
Polyacrylamide Gels ◽  
Multiple Molecular Forms ◽  
Pregnancy And Lactation ◽  
Enzyme Specific Activity ◽  
Glucose 6 Phosphate Dehydrogenase

The distribution of the multiple molecular forms of rat liver and mammary gland glucose-6-phosphate dehydrogenase was determined by electrophoresis on 5% polyacrylamide gels. In both of these organs, changes in the distribution of enzyme activity among the several forms was slight even when approximately 20- to 40-fold changes in enzyme specific activity were achieved by fasting-refeeding experiments (for liver) or during pregnancy and lactation (for mammary gland), it was concluded that the induction of glucose-6-phosphate dehydrogenase in these two organs occurs without any major redistribution among the multiple molecular forms of this enzyme.

scholarly journals Properties of smooth muscle meta-vinculin.

1987 ◽  
Vol 104 (3) ◽  
pp. 473-482 ◽  
Author(s):  
J D Siliciano ◽  
S W Craig
Keyword(s):  
Smooth Muscle ◽  
Specific Activity ◽  
Sedimentation Coefficient ◽  
High Salt ◽  
Calculated Molecular Weight ◽  
Tissue Homogenates ◽  
Stokes Radius ◽  
Triton X ◽  
Peptide Maps

Quantitative studies show that meta-vinculin is ninefold more soluble in 0.6 M salt than in the 0.01 M salt buffers used to extract vinculin. Based on this finding, we have developed a protocol for the purification of meta-vinculin in 43% yield and 98% purity from a high salt extract of gizzard smooth muscle. In contrast to our earlier extraction studies, which were done on unfixed cryostat sections (30), the present studies done on tissue homogenates show that nonionic detergents are not required for solubilization of meta-vinculin. Furthermore, neither purified nor partially purified meta-vinculin binds to Triton X-114 micelles. Purified meta-vinculin is a monomeric, asymmetric molecule with a Stokes radius of 50.9 A, a sedimentation coefficient of 6.35S, and a frictional ratio of 1.46. The calculated molecular weight of meta-vinculin is 145,000. Meta-vinculin has two isoforms of pI 5.9 and 6.2, and is phosphorylated in vivo to eightfold greater specific activity than vinculin. On immunoblots of smooth muscle proteins, [125I]meta-vinculin binds specifically to talin and also to unidentified polypeptides of 180, 150, 95, 70, 68, and 45 kD. On two-dimensional peptide maps, iodinated vinculin and meta-vinculin have at least 95% of their major chymotryptic peptides in common, but each protein also has at least one highly labeled peptide that appears to be unique. Comparative peptide maps of high salt soluble meta-vinculin and the low salt soluble 152-kD protein (described by Feramisco, J.R., J.E. Smart, K. Burridge, D. Helfman, and G.P. Thomas, 1982, J. Biol. Chem., 257:11024-11031) demonstrate extensive similarities among the vinculin-like proteins but suggest a lack of complete identity. In vivo pulse-chase experiments show that meta-vinculin and vinculin do not have a precursor-product relationship. The biochemical and structural differences found between vinculin and meta-vinculin suggest that there is a unique function for meta-vinculin in smooth muscle.

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