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.

scholarly journals Mouse macrophage elastase. Purification and characterization as a metalloproteinase

1981 ◽  
Vol 193 (2) ◽  
pp. 589-605 ◽  
Author(s):  
M J Banda ◽  
Z Werb
Keyword(s):  
Culture Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Serine Proteinase ◽  
Peritoneal Macrophages ◽  
Serine Proteinases ◽  
Endogenous Inhibitor ◽  
Purification And Characterization ◽  
Predominant Form ◽  
Mouse Peritoneal Macrophages

Macrophage elastase was purified from tissue-culture medium conditioned by inflammatory mouse peritoneal macrophages. Characterized as a secreted neutral metalloproteinase, this enzyme was shown to be catalytically and immunochemically distinct from the mouse pancreatic and mouse granulocyte elastases, both of which are serine proteinases. Inhibition profiles, production of nascent N-terminal leucine residues and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of degraded elastin indicated that macrophage elastase is an endopeptidase, with properties of a metalloproteinase, rather than a serine proteinase. Macrophage elastase was inhibited by alpha 2-macroglobulin, but not by alpha 1-proteinase inhibitor. Macrophage elastase was resolved into three chromatographically distinct forms. The predominant form had mol.wt. 22 000 and was purified 4100-fold. Purification of biosynthetically radiolabelled elastase indicated that this form represented less than 0.5% of the secreted protein of macrophages. Approx. 800% of the starting activity was recovered after purification. Evidence was obtained for an excess of an endogenous inhibitor masking more than 80% of the secreted activity.

scholarly journals Purification of rabbit bone inhibitor of collagenase

1981 ◽  
Vol 195 (1) ◽  
pp. 159-165 ◽  
Author(s):  
T E Cawston ◽  
W A Galloway ◽  
E Mercer ◽  
G Murphy ◽  
J J Reynolds
Keyword(s):  
Tissue Culture ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Serine Proteinases ◽  
Bacterial Collagenase ◽  
Rabbit Bone

1. Rabbit bones in tissue culture synthesize an inhibitor of collagenase during the first 4 days of culture. 2. The inhibitor was purified by a combination of gel filtration, concanavalin A--Sepharose chromatography, ion-exchange chromatography and zinc-chelate affinity chromatography. 3. The purified inhibitor migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had a mol.wt. of 28000. 4. The inhibitor blocked the activity of the metalloproteinases collagenase, gelatinase, neutral proteinase III (proteoglycanase), human leucocyte collagenase and gelatinase, but not thermolysin or bacterial collagenase. The serine proteinases plasmin and trypsin were not inhibited. 5. The inhibitor interacted with purified rabbit bone collagenase with 1:1 stoichiometry. 6. The inhibitory activity was lost after incubation for 1 h at 90 degrees C, after treatment with trypsin (250 micrograms/ml) at 37 degrees C for 30 min and after reduction and alkylation.

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.

scholarly journals Purification and some physico-chemical and enzymic properties of a calcium ion-activated neutral proteinase from rabbit skeletal muscle

1979 ◽  
Vol 183 (2) ◽  
pp. 339-347 ◽  
Author(s):  
Jean-Louis Azanza ◽  
Jacques Raymond ◽  
Jean-Michel Robin ◽  
Patrick Cottin ◽  
André Ducastaing
Keyword(s):  
Skeletal Muscle ◽  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Troponin I ◽  
Proteinase Inhibitors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Rabbit Skeletal Muscle ◽  
Neutral Proteinase

Ca2+-activated neutral proteinase was purified from rabbit skeletal muscle by a method involving DEAE-Sephacel chromatography, affinity chromatography on organomercurial–Sepharose and gel filtration on Sephacryl S-200 and Sephadex G-150. The SDS (sodium dodecyl sulphate)/polyacrylamide-gel-electrophoresis data show that the purified enzyme contains only one polypeptide chain of mol.wt. 73000. The purification procedure used allowed us to eliminate a contaminant containing two components of mol.wt. about 30000 each. Whole casein or α1-casein were hydrolysed with a maximum rate at 30°C, pH7.5, and with 5mm-CaCl2, but myofibrils were found to be a very susceptible substrate for this proteinase. This activity is associated with the destruction of the Z-discs, which is caused by the solubilization of the Z-line proteins. The activity of the proteinase in vitro is not limited to the removal of Z-line. SDS/polyacrylamide-gel electrophoresis on larger plates showed the ability of the proteinase to degrade myofibrils more extensively than previously supposed. This proteolysis resulted in the production of a 30000-dalton component as well as in various other higher- and lower-molecular-weight peptide fragments. Troponin T, troponin I, α-tropomyosin, some high-molecular-weight proteins (M protein, heavy chain of myosin) and three unidentified proteins are degraded. Thus the number of proteinase-sensitive regions in the myofibrils is greater than as previously reported by Dayton, Goll, Zeece, Robson & Reville [(1976) Biochemistry15, 2150–2158]. The Ca2+-activated neutral proteinase is not a chymotrypsin- or trypsin-like enzyme, but it reacted with all the classic thiol-proteinase inhibitors for cathepsin B, papain, bromelain and ficin. Thus the proteinase was proved to have an essential thiol group. Antipain and leupeptin are also inhibitors of the Ca2+-activated neutral proteinase.

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.

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.

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.

scholarly journals Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4223-4234 ◽  
Author(s):  
M Furlan ◽  
R Robles ◽  
B Lamie
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Von Willebrand Factor ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Peptide Bond ◽  
Von Willebrand ◽  
Willebrand Factor

Proteolytic cleavage of von Willebrand factor (vWF) takes place in the circulating blood of healthy subjects and is increased in some patients with von Willebrand disease type 2A. The hemostatically active large vWF multimers are degraded to smaller less active forms. It has been suggested that the polypeptide subunit of vWF is cleaved at the peptide bond 842Tyr-843Met. We purified (approximately 10,000-fold) from human plasma a vWF-degrading protease, using chelating Sepharose, hydrophobic interaction chromatography, and gel filtration. The enzyme was found to be virtually absent in the platelet lysates obtained by repeated freezing and thawing. The proteolytic activity was associated with a high molecular weight protein (approximately 300 kD) as judged by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. vWF was resistant against the protease in a neutral buffer at physiological ionic strength but became degraded at low salt concentration or in the presence of 1 mol/L urea. No degradation of human fibrinogen, bovine serum albumin, of calf skin collagen by the purified protease was noted under the same experimental conditions. Proteolytic activity showed a pH optimum at 8 to 9 and was strongly inhibited by chelating agents, whereas only slow inhibition was observed with N-ethylmaleimide. There was no inhibition by iodoacetamide, leupeptin, or serine protease inhibitors. The best peptidyl diazomethyl ketone inhibitor was Z-Phe-Phe-CHN2. Activation by divalent metal ions was found to increase in the following order: Zn2+ approximately Cu2+ approximately CD2+ approximately Ni2+ approximately Co2+ <Mn2+ <Mg2+ <Ca2+ <Sr2+ <Ba2+. The observed properties of the vWF- degrading enzyme differ from those of all other hitherto described proteases. Purified vWF was incubated with the protease, and the degraded material subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis after disulfide reduction. The size, amino acid composition, and amino terminal sequence of the reduced fragments confirmed that the peptide bond 842Tyr-843Met had been cleaved, ie, the same bond that has been proposed to be cleaved in vivo.

Formation of Stable Complexes Between Urokinase and a Human Urinary Component

1985 ◽  
Vol 53 (01) ◽  
pp. 036-041 ◽  
Author(s):  
Witold Cieplak ◽  
David A Hart
Keyword(s):  
Complex Formation ◽  
Gel Electrophoresis ◽  
Proteinase Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Serine Proteinase ◽  
Serine Proteinase Inhibitor ◽  
Diisopropyl Fluorophosphate ◽  
Sds Page ◽  
Multiple Species

SummaryHuman urine was found to contain multiple species of urokinase (UK)-like plasminogen activator (PA) activity when subjected to concentration and/or dialysis and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. Untreated, freshly voided urine contained only Mr = 52,000 and 35,000 UK while dialyzed or undialyzed urine concentrates contained additional PA activity at Mr = 80,000 and 95.000. SDS-PAGE of incubation mixtures of radioiodinated UK (Mr = 52,000 or 35,000) and urine concentrates revealed the presence of radiolabeled complexes with MrS of 80,000 and 95.000. The bond(s) involved in complex formation was also relatively resistant to heat and reduction. Treatment of radioiodinated UK with the serine proteinase inhibitor, p-nitrophenyl guanidinobenzoate, prior to incubation with dialyzed, concentrated urine prevented formation of the complexes. In addition, the enzymatic activity of the Mr = 80,000 and 95,000 species was unaffected by diisopropyl fluorophosphate. These results indicate that UK forms SDS-stable complexes with a urinary component that has a Mr of approximately 40,000. The results further suggest that these complexes express PA activity when analyzed by SDS- PAGE and zymography.

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