scholarly journals The susceptibility towards proteolysis of intermediates during the renaturation of yeast phosphoglycerate mutase

1986 ◽  
Vol 236 (2) ◽  
pp. 617-620 ◽  
Author(s):  
C M Johnson ◽  
N C Price
Keyword(s):  
Gel Electrophoresis ◽  
Polypeptide Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Baker’S Yeast ◽  
Polyacrylamide Gel ◽  
Phosphoglycerate Mutase ◽  
Baker's Yeast ◽  
Guanidinium Chloride ◽  
Early Stages

The renaturation of the tetrameric enzyme phosphoglycerate mutase from baker's yeast after denaturation in guanidinium chloride was studied. Three proteinases (trypsin, chymotrypsin and thermolysin) cause extensive loss of activity of samples taken during the early stages of refolding. As judged by SDS/polyacrylamide-gel electrophoresis, the proteinases cause substantial degradation of the polypeptide chain with no evidence for large quantities of fragments of Mr greater than 6500. These data suggest that the early intermediates in the refolding, especially the folded monomer, possess a number of sites that are susceptible to proteolysis.

scholarly journals M BAND PROTEIN

1972 ◽  
Vol 55 (3) ◽  
pp. 681-695 ◽  
Author(s):  
Barbra L. Eaton ◽  
Frank A. Pepe
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polypeptide Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Breast Muscle ◽  
Chicken Breast ◽  
Specific Antibodies ◽  
Dodecyl Sulfate

M band protein can be specifically extracted from fresh chicken breast muscle myofibrils suspended in 5 mM Tris-HCl pH 8.0. During discontinuous polyacrylamide gel electrophoresis the isolated protein separates into three bands which can be identified as two separate components (A, B) and a complex of the two. When partially purified fractions of the separated components are combined, an increase in the intensity of the band containing the complex can be shown. The polypeptide chain weights of the two components are 100,000 (A) and 40,000 (B) daltons as estimated by sodium dodecyl sulfate- (SDS-) polyacrylamide gel electrophoresis. Antibody prepared against total M band protein stains only the M band of the myofibril and is completely absorbed by M band protein. M band protein also absorbs the M band staining specifically from antibody which stains both I and M bands. Immunodiffusion data indicate that anti-M band is a mixture of two specific antibodies, one against each component.

scholarly journals Denaturation and renaturation of the monomeric phosphoglycerate mutase from Schizosaccharomyces pombe

1987 ◽  
Vol 245 (2) ◽  
pp. 525-530 ◽  
Author(s):  
C M Johnson ◽  
N C Price
Keyword(s):  
Protein Structure ◽  
Schizosaccharomyces Pombe ◽  
Baker’S Yeast ◽  
Phosphoglycerate Mutase ◽  
Rabbit Muscle ◽  
Baker's Yeast ◽  
Guanidinium Chloride ◽  
Structure And Stability

The denaturation by guanidinium chloride of the monomeric phosphoglycerate mutase from Schizosaccharomyces pombe was studied. The loss in activity broadly parallels the changes in protein structure detected by fluorescence and c.d. Renaturation can be brought about by dilution of the denaturing agent. These processes were compared with those in the enzymes from baker's yeast and rabbit muscle, which are tetrameric and dimeric respectively. The effects of the cofactor 2,3-bisphosphoglycerate on the structure and stability of the S. pombe enzyme were also investigated.

scholarly journals S-adenosylmethionine decarboxylase from baker's yeast

1975 ◽  
Vol 151 (1) ◽  
pp. 67-73 ◽  
Author(s):  
H Pösö ◽  
R Sinervirta ◽  
J Jänne
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Baker’S Yeast ◽  
Decarboxylase Activity ◽  
Spermidine Synthase ◽  
Baker's Yeast ◽  
Purification Procedure ◽  
Adenosylmethionine Decarboxylase ◽  
Km Value

1. S-Adenosyl-L-methionine decarboxylase (S-adenosyl-L-methionine carboxy-lyase, EC 4.1.1.50) was purified more than 1100-fold from extracts of Saccharomyces cerevisiae by affinity chromatography on columns of Sepharose containing covalently bound methylglyoxal bis(guanylhydrazone) (1,1′[(methylethanediylidene)dinitrilo]diguanidine) [Pegg, (1974) Biochem J. 141, 581-583]. The final preparation appeared to be homogeneous on polyacrylamide-gel electrophoresis at pH 8.4. 2. S-Adenosylmethionine decarboxylase activity was completely separated from spermidine synthase activity [5′-deoxyadenosyl-(5′),3-aminopropyl-(1),methylsulphonium-salt-putrescine 3-aminopropyltransferase, EC 2.5.1.16] during the purification procedure. 3. Adenosylmethionine decarboxylase activity from crude extracts of baker's yeast was stimulated by putrescine, 1,3-diamino-propane, cadaverine (1,5-diaminopentane) and spermidine; however, the purified enzyme, although still stimulated by the diamines, was completely insensitive to spermidine. 4. Adenosylmethionine decarboxylase has an apparent Km value of 0.09 mM for adenosylmethionine in the presence of saturating concentrations of putrescine. The omission of putrescine resulted in a five-fold increase in the apparent Km value for adenosylmethionine. 5. The apparent Ka value for putrescine, as the activator of the reaction, was 0.012 mM. 6. Methylglyoxal bis(guanylhydrazone) and S-methyladenosylhomocysteamine (decarboxylated adenosylmethionine) were powerful inhibitors of the enzyme. 7. Adenosylmethionine decarboxylase from baker's yeast was inhibited by a number of conventional carbonyl reagents, but in no case could the inhibition be reversed with exogenous pyridoxal 5′-phosphate.

scholarly journals Purification and properties of tryptophan synthase from baker's yeast (Saccharomyces cerevisiae)

1983 ◽  
Vol 209 (1) ◽  
pp. 151-157 ◽  
Author(s):  
C J Bailey ◽  
P D Turner
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Baker’S Yeast ◽  
Tryptophan Synthase ◽  
Baker's Yeast ◽  
Terminal Amino Acid ◽  
Yeast Saccharomyces Cerevisiae ◽  
Purification And Properties ◽  
Terminal Amino

Tryptophan synthase was purified from baker's yeast. The purified enzyme exhibited one band on polyacrylamide-gel electrophoresis, had no detectable N-terminal amino acid and C-terminal alanine. The amino acid composition was close to that predicted by recent studies on the DNA sequence of the structural gene for the enzyme. Kinetic parameters for the following three activities were measured: indole-serine condensation, indolylglycerol phosphate lyase and the overall reaction of serine with 1-(indol-3-yl)glycerol 3-phosphate. The Km for indole was much lower than suggested by previous investigations, and the value of 11 microM was measured by a fluorimetric assay.

The Histones of Rainbow Trout Erythrocytes Include an Erythrocyte-Specific Histone

1975 ◽  
Vol 53 (11) ◽  
pp. 1158-1169 ◽  
Author(s):  
B. L. A. Miki ◽  
J. M. Neelin
Keyword(s):  
Rainbow Trout ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Relative Mobility ◽  
Guanidinium Chloride ◽  
Amino Acid Compositions ◽  
Exclusion Chromatography ◽  
Trout Erythrocyte

The erythrocyte histones of rainbow trout were compared with those of goose by polyacrylamide gel electrophoresis. A band analogous to goose erytbrocyte-specific histone V, but not identical in relative mobility or quantity, was found to be a component of trout erythrocyte histone. A similar component was also found in carp erythrocyte histone, but it was absent from trout liver histone. To reveal this band clearly, it was advantageous to displace the histone III monomer by oxidation.To verify the character of this protein, each of the main erythrocyte histones of trout were purified by chromatography on Amberlite CG-50, eluted with guanidinium chloride, and then further purified by exclusion chromatography on Bio-Gel P-60. Amino acid compositions of corresponding trout and goose histones, including that of the erythrocyte-specific histone, were sufficiently similar to establish their analogous identities. In general, the chromatographic and electrophoretic properties of histones I, IIb1, IIb2, and V from trout differed more from those of goose, than did their gross amino acid compositions. Comprehensive fractionation and characterization is necessary to establish identities of corresponding histone fractions.An extensive quantitative variability was found among erythrocyte-specific histones of fish. This must be reconciled with hypothetical roles for this histone in erythropoiesis.

Ultrastructural localization of specific nucleoprotein fractions

1978 ◽  
Vol 36 (2) ◽  
pp. 204-205
Author(s):  
G. L. Brown
Keyword(s):  
Gel Electrophoresis ◽  
Mouse Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ultrastructural Localization ◽  
Polyacrylamide Gel ◽  
Acid Extraction ◽  
Acid Solutions ◽  
Low Salt ◽  
Liver Nuclei ◽  
Phosphate Groups

Bismuth (Bi) stains nucleoproteins (NPs) by interacting with available amino and primary phosphate groups. These two staining mechanisms are distinguishable by glutaraldehyde crosslinking (Fig. 1,2).Isolated mouse liver nuclei, extracted with salt and acid solutions, fixed in either formaldehyde (form.) or gl utaraldehyde (glut.) and stained with Bi, were viewed to determine the effect of the extractions on Bi stainina. Solubilized NPs were analyzed by SDS-polyacrylamide gel electrophoresis.Extraction with 0.14 M salt does not change the Bi staining characteristics (Fig. 3). 0.34 M salt reduces nucleolar (Nu) staining but has no effect on interchromatinic (IC) staining (Fig. 4). Proteins responsible for Nu and glut.- insensitive IC staining are removed when nuclei are extracted with 0.6 M salt (Fig. 5, 6). Low salt and acid extraction prevents Bi-Nu staining but has no effect on IC staining (Fig. 7). When nuclei are extracted with 0.6 M salt followed by low salt and acid, all Bi-staining components are removed (Fig. 8).

Platelet Microtubule Subunit Proteins

1979 ◽  
Vol 42 (05) ◽  
pp. 1630-1633 ◽  
Author(s):  
A G Castle ◽  
N Crawford
Keyword(s):  
Epithelial Cells ◽  
Gel Electrophoresis ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Lens Epithelial Cells ◽  
Molecular Weights ◽  
Kinetics Of ◽  
Microtubular Structures

SummaryBlood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised “in vitro” to form structures which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits a and p tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerisation of cell microtubules.

Prothrombin Metz : Purification and Characterization of a Variant of Human Prothrombin

1979 ◽  
Author(s):  
M Ribieto ◽  
J Elion ◽  
D Labie ◽  
F Josso
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Polyacrylamide Gel ◽  
Cleavage Pattern ◽  
Purification And Characterization ◽  
Double Immunodiffusion ◽  
The Family

For the purification of the abnormal prothrombin (Pt Metz), advantage has been taken of the existence in the family of three siblings who, being double heterozygotes for Pt Metz and a hypoprothrombinemia, have no normal Pt. Purification procedures included barium citrate adsorption and chromatography on DEAE Sephadex as for normal Pt. As opposed to some other variants (Pt Barcelona and Madrid), Pt Metz elutes as a single symetrical peak. By SDS polyacrylamide gel electrophoresis, this material is homogeneous and appears to have the same molecular weight as normal Pt. Comigration of normal and abnormal Pt in the absence of SDS, shows a double band suggesting an abnormal charge for the variant. Pt Metz exhibits an identity reaction with the control by double immunodiffusion. Upon activation by factor Xa, Pt Metz can generate amydolytic activity on Bz-Phe-Val-Arg-pNa (S2160), but only a very low clotting activity. Clear abnormalities are observed in the cleavage pattern of Pt Metz when monitored by SDS gel electrophoresis. The main feature are the accumulation of prethrombin l (Pl) and the appearance of abnormal intermediates migrating faster than Pl.

Functional Relationship of Polymerization Sites of Vertebrate Fibrinogens

1979 ◽  
Author(s):  
C Cierniewski ◽  
T Krajewski ◽  
E Janiak
Keyword(s):  
Gel Electrophoresis ◽  
Functional Relationship ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Functional Similarity ◽  
Fractionation Method ◽  
Relationship Of ◽  
Fibrin Monomers ◽  
Fibrinogen Molecule ◽  
Cold Ethanol Fractionation

Various studies on the interaction of immobilized mammalian fibrinogen and fibrin monomers with some fibrinogen derivatives demonstrated the presence of two sets of polymerization sites in the mammalian fibrinogen molecule. We obtained the same results while investigating the fibrinogen molecules of other classes of vertebrates /Pisces. Amphibia. Aves/. Despite significant differences among their subunit structures, all of them contain polymerization sites homologous to mammalian counterparts. Moreover, due to great functional similarity, fibrinogen or fibrin monomers of the analyzed species of Pisces. Amphibia. Aves and Mammalia interacted in a specific way with immobilized pig fibrin monomers or fibrinogen, respectively. Using these pig affinity adsorbents, fibrinogen and fibrin monomers of different vertebrates were isolated directly from plasma and analyzed by SDS polyacrylamide gel electrophoresis. Polypeptide compositions of eluted proteins were identical to those obtained for corresponding fibrinogen preparations isolated by cold-ethanol fractionation method. It appears to indicate that the nature of polymerization sites in vertebrate fibrinogens is alike.

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