Effects of sulphydryl reagents on the structure of dehistonized metaphase chromosomes

1985 ◽  
Vol 73 (1) ◽  
pp. 245-260
Author(s):  
P. Jeppesen ◽  
H. Morten
Keyword(s):  
Gel Electrophoresis ◽  
Heavy Metal Ions ◽  
Core Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Linking ◽  
Polypeptide Composition ◽  
Metaphase Chromosomes ◽  
Axial Structure

Dehistonized metaphase chromosomes lose their apparent axial organization (the ‘scaffold’) and sediment more slowly following exposure to beta-mercaptoethanol (BME). We have subsequently treated BME chromosomes with reagents that oxidize protein sulphydryls to disulphides, and found that if calcium is also present during the oxidation an apparently similar axial structure is restored following dehistonization, as seen by microscopic examination. In general, however, we do not find that oxidation restores the higher sedimentation rate of dehistonized control chromosomes. Analysis of residual core protein in dehistonized chromosomes by sodium dodecyl sulphate/polyacrylamide gel electrophoresis fails to detect any differences in polypeptide composition related to the state of oxidation or to the presence or absence of visible axial organization. Combining our results with those of other workers, we conclude that the axial structure evident in dehistonized metaphase chromosomes is maintained, at least partially, by inter-protein cross-linking, although in vivo this may not be via simple disulphide bridges. Additional factors, which we have not yet characterized, but which possibly include heavy metal ions, appear to be involved in the axial organization existing in vivo.

Soluble High-Molecular-Weight E Fragments in the Plasmin-Induced Degradation Products of Cross-Linked Human Fibrin

1975 ◽  
Vol 49 (2) ◽  
pp. 149-156 ◽  
Author(s):  
P. J. Gaffney ◽  
D. A. Lane ◽  
M. Brasher
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Electrophoresis ◽  
Factor Xiii ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Linking ◽  
D Dimer

1. The factor XIII-mediated cross-linked α chains in fibrin have no effect on the nature of the fragments released during the solubilization of fibrin by plasmin. 2. Besides the known D dimer and E fragments solubilized during the lysis of cross-linked fibrin, other fragments have been observed on sodium dodecyl sulphate-polyacrylamide gel electrophoresis which have a molecular weight of about 135 000. After prolonged plasmin digestion, these fragments (U fragments) were no longer evident on the gels and the high-molecular-weight E antigen was absent. It is assumed that the E antigen was associated with the U fragments. These fragments also cross-reacted with an anti-D serum. 3. The U fragments have been tentatively presumed to be a factor XIII-mediated cross-linked D–E complex since they degrade only after prolonged degradation with plasmin. Whereas it is known that the fibrin D dimer fragment contains the cross-linked γ chain residues of the originating fibrin, the presumed covalent cross-linking of the D–E fragments has not been proved. 4. The presence of these high-molecular-weight fragments, containing the E antigen, in cross-linked human fibrin digests should be taken into account in the development of D dimer assays to monitor fibrin lysis in vivo.

scholarly journals Degradation of myofibrillar proteins by trypsin-like serine proteinases.

1982 ◽  
Vol 201 (2) ◽  
pp. 279-285 ◽  
Author(s):  
J Kay ◽  
L M Siemankowski ◽  
R F Siemankowski ◽  
J A Greweling ◽  
D E Goll
Keyword(s):  
Skeletal Muscle ◽  
Gel Electrophoresis ◽  
Cysteine Proteinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Serine Proteinase ◽  
Myofibrillar Proteins ◽  
Serine Proteinases ◽  
Bovine Trypsin

The effects of the Ca2+-activated cysteine proteinase, the rat trypsin-like serine proteinase and bovine trypsin on myofibrillar proteins from rabbit skeletal muscle are compared. 2. Myofibrils that had been treated at neutral pH with the Ca2+-dependent proteinase and with the rat enzyme were (a) analyzed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and (b) examined in the electron microscope. Treatment with each proteinase resulted in the loss of the Z-discs, but the rat enzyme caused much more extensive disruption of the ultrastructure and degraded more of the myofibrillar proteins. 3. Purified F-actin was almost totally resistant to the proteinases, whereas G-actin was degraded by the rat trypsin-like proteinase at a rate approx. 15 times faster than was obtained with bovine trypsin. 4. Similar results were obtained with alpha-actinin, whereas tropomyosin was degraded more readily by bovine trypsin than by the rat trypsin-like proteinase. 5. The implications of these findings for the non-lysosomal breakdown of myofibrillar proteins in vivo are considered.

The effects of temperature on the growth and polypeptide composition of several snow mold species

1985 ◽  
Vol 63 (12) ◽  
pp. 2311-2318 ◽  
Author(s):  
W. J. Newsted ◽  
N. P. A. Huner ◽  
J. P. Insell ◽  
M. Griffith ◽  
R. B. van Huystee
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Vegetative Growth ◽  
Gel Electrophoresis ◽  
Agar Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Relative Proportion ◽  
Sodium Dodecyl ◽  
Polypeptide Composition ◽  
Effects Of Temperature ◽  
Typhula Incarnata

Myriosclerotinia borealis (W51), Coprinus spp. (13W1 and 14W2), Typhula idahoensis (W21), and Typhula incarnata (W29) were incubated in the dark on a defined agar medium at permissive (4 °C) and nonpermissive temperatures (22 and 30 °C). Isolates of Coprinus spp. and Typhula spp. required higher temperatures than M. borealis to arrest vegetative growth completely. The effects of incubation at permissive and nonpermissive temperatures on the polypeptide compositions of M. borealis, Coprinus spp., T. idahoensis (W21), and T. incarnata (W29) were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results indicated that the number of polypeptides in the polypeptide complement of M. borealis and both Typhula species decreased significantly during incubation at nonpermissive temperatures. In contrast, Coprinus sp. (13W1) showed no significant change in the number of polypeptides observed during incubation at nonpermissive temperatures. Furthermore, there appeared to be an increase in the relative proportion of at least three polypeptides during incubation of Coprinus at nonpermissive temperatures. The significance of these species-dependent responses to nonpermissive growth temperatures is discussed.

Phosphorylation of calcineurin by glycogen synthase (casein) kinase-1

1987 ◽  
Vol 65 (10) ◽  
pp. 917-921 ◽  
Author(s):  
Toolsee J. Singh ◽  
Jerry H. Wang
Keyword(s):  
Gel Electrophoresis ◽  
Peptide Mapping ◽  
Glycogen Synthase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Casein Kinase ◽  
Casein Kinase 1 ◽  
Calcineurin Activity ◽  
Potential Substrate

A previous study demonstrated that calcineurin preparations contain variable amounts of endogenous phosphate. This observation suggests that calcineurin may be regulated by protein phosphorylation. In this study we have used calcineurin as a potential substrate for eight different protein kinases and significant phosphorylation was observed only with glycogen synthase (casein) kinase-1 (CK-1). Analysis by sodium dodecyl sulfate – polyacrylamide gel electrophoresis revealed that only subunit A of calcineurin was phosphorylated. The incorporation of 32P into calcineurin catalyzed by CK-1 ranged from 0.4 to 1.5 mol, depending on the preparation of the substrate used. Peptide mapping revealed that two major sites on calcineurin were phosphorylated. No change in calcineurin activity was observed as a result of phosphorylation. The results of this study suggest that CK-1 may be responsible for phosphorylating calcineurin in vivo.

Light microscopic and polypeptide analyses of sclerotia from mesophilic and psychrophilic pathogenic fungi

1985 ◽  
Vol 63 (12) ◽  
pp. 2305-2310 ◽  
Author(s):  
James P. Insell ◽  
N. P. A. Huner ◽  
W. Jay Newsted ◽  
R. B. van Huystee
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sclerotium Rolfsii ◽  
Pathogenic Fungi ◽  
Polypeptide Composition ◽  
Thin Walled ◽  
Hard Structures ◽  
A Minor

The structure and polypeptide composition of sclerotia of three mesophilic pathogenic fungi, Sclerotinia sclerotiorum, Sclerotium rolfsii, and Botrytis cinerea, and one psychrophilic snow mold, Myriosclerotinia borealis, were compared. The sclerotia of S. sclerotiorum and B. cinerea were black, round, hard structures which were composed of three areas: the rind, the cortex, and the medulla. Both the cortical and medullary areas of these sclerotia exhibited intensely stained inclusions. In contrast, sclerotia of M. borealis were not present as discrete entities but coalesced near the central point of inoculation. These black sclerotial masses were composed of thin-walled, pseudoparenchymal cells tightly packed together to form two distinct areas: a rind and a medullarylike region. No inclusions were evident in the medulla of cells of M. borealis sclerotia. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of sclerotial extracts of mesophilic fungi indicated the presence of major polypeptides. The polypeptide complement of B. cinerea contained a single, major polypeptide with an apparent molecular mass of 33 to 36 kDa. Similarly, sclerotia of S. sclerotiorum contained one major polypeptide of approximately 36 kDa in addition to one minor polypeptide of about 18 kDa. However, sclerotia of S. rolfsii contained a major polypeptide of about 16 kDa. Sclerotia of M. borealis contained a major polypeptide of 32 to 36 kDa and a minor polypeptide of about 16 kDa.

scholarly journals Variant specific antigens of Trypanosoma brucei exist in solution as glycoprotein dimers

1981 ◽  
Vol 193 (2) ◽  
pp. 647-650 ◽  
Author(s):  
C A Auffret ◽  
M J Turner
Keyword(s):  
Trypanosoma Brucei ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Cross Linking ◽  
Specific Antigens

Purified variant specific antigens of Trypanosoma brucei were shown to exist in solution as dimers, and occasionally as higher oligomers, as judged by gel filtration and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after treatment with bifunctional cross-linking reagents.

The αEC domain of human fibrinogen-420 is a stable and early plasmin cleavage product

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2297-2303 ◽  
Author(s):  
Dianne Applegate ◽  
Lara Stoike Steben ◽  
Kathe M. Hertzberg ◽  
Gerd Grieninger
Keyword(s):  
Gel Electrophoresis ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cleavage Product ◽  
The Other ◽  
Cross Linking ◽  
Human Fibrinogen ◽  
Western Blots ◽  
Sds Page

Abstract Human fibrinogen-420, (Eβγ)2, was isolated from plasma and evaluated for its ability to form clots and for its susceptibility to proteolysis. Clotting parameters, including cross-linking of subunit chains, of this subclass and of the more abundant fibrinogen-340 (βγ)2, were found to be similar, suggesting little impact of the unique EC domains of fibrinogen-420 on coagulation. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmic digestion patterns revealed production from fibrinogen-420 of the conventional fibrinogen degradation products, X, Y, D, and E, to be comparable to that from fibrinogen-340 in all respects except the presence of at least 2 additional cleavage products that were shown by Western blot analysis to contain the EC domain. One was a stable fragment (ECX) comigrating with a 34-kd yeast recombinant EC domain, and the other was an apparent precursor. Their release occurred early, before that of fragments D and E. Two bands of the same mobility and antibody reactivity were found in Western blots of plasma collected from patients with myocardial infarction shortly after the initiation of thrombolytic therapy.

Heterogeneity of lipoprotein Lp(a) and apolipoprotein(a).

1988 ◽  
Vol 34 (6) ◽  
pp. 1036-1040 ◽  
Author(s):  
G F Grinstead ◽  
R D Ellefson
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Core Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Single Patient ◽  
Selective Precipitation ◽  
Apolipoprotein A ◽  
Molecular Masses ◽  
Chemical Deglycosylation

Abstract We have purified Lp(a) lipoproteins from sera of four subjects by ultracentrifugation, selective precipitation, and chromatofocusing. Each subject had two forms of serum Lp(a) that were separable by chromatofocusing. We purified apolipoprotein (a) [apo(a)] from the eight isolated Lp(a)s and obtained only one form of apo(a) from each subject. The four apo(a)s seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis had different apparent molecular masses, ranging from 275 to 440 kDa. Chemical deglycosylation of the smallest apo(a) yielded a 235 kDa protein, which may be a core protein structure common to all apo(a)s. We conclude that there are many forms of serum Lp(a) and apo(a). The heterogeneity of serum Lp(a) particles can be ascribed in part to differences in size of apo(a), but other factors must account for the existence within a single patient of different Lp(a)s that contain apparently identical apo(a). One must consider the heterogeneity of Lp(a) when designing assays for this lipoprotein.

scholarly journals Characterization of an Escherichia coli O157:H7 Plasmid O157 Deletion Mutant and Its Survival and Persistence in Cattle

2007 ◽  
Vol 73 (7) ◽  
pp. 2037-2047 ◽  
Author(s):  
Ji Youn Lim ◽  
Haiqing Sheng ◽  
Keun Seok Seo ◽  
Yong Ho Park ◽  
Carolyn J. Hovde
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Differentially Expressed ◽  
Sequence Matching ◽  
Two Dimensional Gel Electrophoresis ◽  
E Coli ◽  
Cell Lysates

ABSTRACT Escherichia coli O157:H7 causes hemorrhagic colitis and hemolytic-uremic syndrome in humans, and its major reservoir is healthy cattle. An F-like 92-kb plasmid, pO157, is found in most E. coli O157:H7 clinical isolates, and pO157 shares sequence similarities with plasmids present in other enterohemorrhagic E. coli serotypes. We compared wild-type (WT) E. coli O157:H7 and an isogenic ΔpO157 mutant for (i) growth rates and antibiotic susceptibilities, (ii) survival in environments with various acidity, salt, or heat conditions, (iii) protein expression, and (iv) survival and persistence in cattle following oral challenge. Growth, metabolic reactions, and antibiotic resistance of the ΔpO157 mutant were indistinguishable from those of its complement and the WT. However, in cell competition assays, the WT was more abundant than the ΔpO157 mutant. The ΔpO157 mutant was more resistant to acidic synthetic bovine gastric fluid and bile than the WT. In vivo, the ΔpO157 mutant survived passage through the bovine gastrointestinal tract better than the WT but, interestingly, did not colonize the bovine rectoanal junction mucosa as well as the WT. Many proteins were differentially expressed between the ΔpO157 mutant and the WT. Proteins from whole-cell lysates and membrane fractions of cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Ten differentially expressed ∼50-kDa proteins were identified by quadrupole-time of flight mass spectrometry and sequence matching with the peptide fragment database. Most of these proteins, including tryptophanase and glutamate decarboxylase isozymes, were related to survival under salvage conditions, and expression was increased by the deletion of pO157. This suggested that the genes on pO157 regulate some chromosomal genes.

Sign in / Sign up

Export Citation Format

Share Document