scholarly journals Ca2+-Induced Cleavage of Human Platelet Polypeptides Mediated by the Calcium Ionophore A-23187

1977 ◽  
Author(s):  
Milica Jakábová ◽  
David R. Phillips
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Proteolytic Activity ◽  
Platelet Activation ◽  
Human Platelet ◽  
Calcium Ionophore ◽  
Lactic Dehydrogenase ◽  
Actin Binding ◽  
A 23187 ◽  
Hydrolysis Of

The effect of calcium on human platelet polypeptides was investigated. When lysed platelets were incubated with mM Ca++, two major intracellular polypeptides (Mr = 255,000 and 230,000) were found to rapidly disappear. A similar phenomenon was also observed when intact platelets were treated with the calcium ionophore A-23187 in the presence of mM Ca++. Determinations of lactic dehydrogenase activity in supernatant fractions demonstrated that these losses occurred before platelet lysis. Investigations into the identity of the high molecular weight polypeptides revealed that one (Mr = 255,000) had similar properties to actin binding protein. The loss of the high molecular weight polypeptides was accompanied by formation of lower molecular weight polypeptides (Mr = 135,000, 93,000 and 48,000), indicating that Ca++ activates a polypeptide cleavage mechanism. The Ca++-activated polypeptide cleavages were rapid, with significant changes being observed within the first 0.5 min of incubation. An obvious explanation for these effects is. that there is Ca++-activated proteolytic activity within platelets. The Ca++-activated proteolytic activity was determined by the hydrolysis of the artificial substrate azocasein. We found that more than 90% of the proteolytic activity in lysed platelets was due to Ca++-activated proteases. These studies show that Ca++-activated proteases may play an important role in platelet activation.

scholarly journals The multiple complexes formed by the interaction of platelet factor 4 with heparin

1980 ◽  
Vol 191 (3) ◽  
pp. 769-776 ◽  
Author(s):  
P E Bock ◽  
M Luscombe ◽  
S E Marshall ◽  
D S Pepper ◽  
J J Holbrook
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Chain Length ◽  
Salt Concentration ◽  
Human Platelet ◽  
Platelet Factor 4 ◽  
High Protein ◽  
Platelet Factor ◽  
The Stability ◽  
Very High

The anisotropy of the fluorescence of dansyl (5-dimethylaminonaphthalene-1- sulphonyl) groups covalently attached to human platelet factor 4 was used to detect the macromolecular compounds formed when the factor was mixed with heparin. At low heparin/protein ratios a very-high-molecular-weight compound (1) was formed that dissociated to give a smaller compound (2) when excess heparin was added. 2. A large complex was also detected as a precipitate that formed at high protein concentrations in chloride buffer. It contained 15.7% (w/w) polysaccharide, equivalent to four or five heparin tetrasaccharide units per protein tetramer. In this complex, more than one molecule of protein binds to each heparin molecule of molecular weight greater than about 6 × 10(3).3. The stability of these complexes varied with pH, salt concentration and the chain length of the heparin. The limit complexes found in excess of the larger heparins consisted of only one heparin molecule per protein tetramer, and the failure to observe complexes with four heparin molecules/protein tetramer is discussed.

scholarly journals Biodegradation of low molecular weight polyacrylamide under aerobic and anaerobic conditions: effect of the molecular weight

2020 ◽  
Vol 81 (2) ◽  
pp. 301-308 ◽  
Author(s):  
Wenzhe Song ◽  
Yu Zhang ◽  
Amir Hossein Hamidian ◽  
Min Yang
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Carbon Chain ◽  
Anaerobic Treatment ◽  
Low Molecular Weight ◽  
Amino Groups ◽  
Molecular Weights ◽  
Weight One ◽  
Hydrolysis Of ◽  
Aerobic And Anaerobic

Abstract The biodegradation of polyacrylamide (PAM) includes the hydrolysis of amino groups and cleavage of the carbon chain; however, the effect of molecular weight on the biodegradation needs further investigations. In this study, biodegradation of low molecular weight PAM (1.6 × 106 Da) was evaluated in two aerobic (25 °C and 40 °C) and two anaerobic (35 °C and 55 °C) reactors over 100 days. The removal of the low molecular weight PAM (52.0–52.6%) through the hydrolysis of amino groups by anaerobic treatment (35 °C and 55 °C) was much higher than that of the high molecular weight (2.2 × 107 Da, 11.2–17.0%) observed under the same conditions. The molecular weight was reduced from 1.6 × 106 to 6.45–7.42 × 105 Da for the low molecular weight PAM, while the high molecular weight PAM declined from 2.2 × 107 to 3.76–5.87 × 106 Da. The results showed that the amino hydrolysis of low molecular weight PAM is easier than that of the high molecular weight one, while the cleavage of its carbon chain is still difficult. The molecular weights of PAM in the effluents from the two aerobic reactors (25 °C and 40 °C) were further reduced to 4.31 × 105 and 5.68 × 105 Da by the biofilm treatment, respectively. The results would be useful for the management of wastewater containing PAM.

Calcium-Dependent Cross-Linking Processes in Platelets

1979 ◽  
Author(s):  
I. Cohen ◽  
T. Glaser
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Cross Linking ◽  
Low Molecular Weight ◽  
Tracer Concentration ◽  
Calcium Dependent ◽  
A 23187 ◽  
Polymer Formation ◽  
Total Disappearance ◽  
Low Molecular Weight Proteins

When platelet cytoplasmic Ca2+ is increased by the ionophore A 23187, there is the coincident appearance of a cross-linked polymer and the partial disappearance of five high molecular weight polypeptide bands (> 145,000). The glycoproteins show a partial disappearance of bands lb, IIb and IV and the total disappearance of hands la and Ilia. The disappearance of the protein bands, possibly contributing to the polymer formation, is prevented by histamine, aminoacetonltrile and cystamlne, which, as pseudodonor amines are known Inhibitors of factor XHIa-catalyzed cross-linking. 14C-histamine, at a tracer concentration, was incorporated into the polymer as well as into myosin, glycoproteins IIb and IIIa (α-actinln), actin and two unidentified low-molecular weight proteins. The polymer formed is also apparent in isolated membranes following the iono-phore-stimulated increase in intracellular Ca2+. These findings are unrelated to a proteolytic activity since the platelet Ca2+-dependent proteases are inhibited by leupep-tin. Ca2+-activation of a platelet cytosol transamidase would explain the data obtained. This platelet transamidase(s) may couple membrane proteins to cytoplasmic contra-tlle proteins. Thus, a new concept is proposed for the stabilization of platelet membranes and platelets as they form the hemostatic plug.

Phosphorylation Of Platelet Actin-Binding Protein During Platelet Activation

1981 ◽  
Author(s):  
Roger C Carroll ◽  
Jonathan M Gerrard
Keyword(s):  
Molecular Weight ◽  
Platelet Activation ◽  
Time Course ◽  
Binding Protein ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Platelet Protein ◽  
External Calcium

We have followed the 32P-labelling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples we found 2 to 3 fold labelling increases in actin-binding protein 30 to 60 seconds after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis of purified actin-binding protein as well as cytoskeletal cores prepared by detergent extraction of activated 32p-iabelled platelets. In addition, these cytoskeletal cores indicated that the 32P-labelled actin-binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labelling of actin-binding protein over an 8 minute time course revealed that in aggregating platelet samples rapid desphosphorylation to almost initial levels occurred between 3 and 5 minutes. A similar curve was obtained for the 20,000 apparent molecular weight peptide. This rapid dephosphorylation was shown to be dependent on platelet aggregation in the absence of external calcium or in thrombastenic platelets lacking the aggregation response to activation. These results suggest that phosphorylation of actin-binding protein initiates its association with the platelet cytoskeleton during activation.

scholarly journals Characterization of 83-kilodalton nonmuscle caldesmon from cultured rat cells: stimulation of actin binding of nonmuscle tropomyosin and periodic localization along microfilaments like tropomyosin.

1988 ◽  
Vol 106 (6) ◽  
pp. 1973-1983 ◽  
Author(s):  
S Yamashiro-Matsumura ◽  
F Matsumura
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Actin Filaments ◽  
Binding Constant ◽  
Reducing Agents ◽  
Actin Binding ◽  
Low Molecular Weight ◽  
Tropomyosin Isoforms ◽  
Apparent Binding Constant ◽  
Stimulation Of

Nonmuscle caldesmon purified from cultured rat cells shows a molecular weight of 83,000 on SDS gels, Stokes radius of 60.5 A, and sedimentation coefficient (S20,w) of 3.5 in the presence of reducing agents. These values give a native molecular weight of 87,000 and a frictional ratio of 2.04, suggesting that the molecule is a monomeric, asymmetric protein. In the absence of reducing agents, the protein is self-associated, through disulfide bonds, into oligomers with a molecular weight of 230,000 on SDS gels. These S-S oligomers appear to be responsible for the actin-bundling activity of nonmuscle caldesmon in the absence of reducing agents. Actin binding is saturated at a molar ratio of one 83-kD protein to six actins with an apparent binding constant of 5 X 10(6) M-1. Because of 83-kD nonmuscle caldesmon and tropomyosin are colocalized in stress fibers of cultured cells, we have examined effects of 83-kD protein on the actin binding of cultured cell tropomyosin. Of five isoforms of cultured rat cell tropomyosin, tropomyosin isoforms with high molecular weight values (40,000 and 36,500) show higher affinity to actin than do tropomyosin isoforms with low molecular weight values (32,400 and 32,000) (Matsumura, F., and S. Yamashiro-Matsumura. 1986. J. Biol. Chem. 260:13851-13859). At physiological concentration of KCl (100 mM), 83-kD nonmuscle caldesmon stimulates binding of low molecular weight tropomyosins to actin and increases the apparent binding constant (Ka from 4.4 X 10(5) to 1.5 X 10(6) M-1. In contrast, 83-kD protein has slight stimulation of actin binding of high molecular weight tropomyosins because high molecular weight tropomyosins bind to actin strongly in this condition. As the binding of 83-kD protein to actin is regulated by calcium/calmodulin, 83-kD protein regulates the binding of low molecular weight tropomyosins to actin in a calcium/calmodulin-dependent way. Using monoclonal antibodies to visualize nonmuscle caldesmon along microfilaments or actin filaments reconstituted with purified 83-kD protein, we demonstrate that 83-kD nonmuscle caldesmon is localized periodically along microfilaments or actin filaments with similar periodicity (36 +/- 4 nm) as tropomyosin. These results suggest that 83-kD protein plays an important role in the organization of microfilaments, as well as the control of the motility, through the regulation of the binding of tropomyosin to actin.

"Colloidal" Phosphorus Errors in Gel Chromatography

1983 ◽  
Vol 40 (10) ◽  
pp. 1614-1621 ◽  
Author(s):  
B. Kent Burnison
Keyword(s):  
Molecular Weight ◽  
Acid Hydrolysis ◽  
High Molecular Weight ◽  
Gel Chromatography ◽  
Chemical Analyses ◽  
Molybdophosphoric Acid ◽  
Molybdenum Blue ◽  
Soluble Reactive Phosphate ◽  
Colloidal Phosphorus ◽  
Hydrolysis Of

Gel chromatography has been used for the separation of 32PO4 and a high molecular weight "colloidal" 32P-labeled fraction from 32PO4-labeled lakewater. When the labeled filtrate is treated with reagents required for the molybdenum blue method for orthophosphate analysis, only a small fraction of the "colloidal" peak is hydrolyzed to orthophosphate. As the reduced molybdophosphoric acid is strongly adsorbed to the dextran gel, quantitative elution of 32PO4 can be achieved with 0.05 mol∙L−1 NaOH and 0.3% NaCl. In hardwater lakes, care must be taken to eliminate the possibility of orthophosphate precipitation at higher pH. In these lakes, it is unlikely that the discrepancy between 32PO4 bioassays and chemical analyses can be solely attributed to acid hydrolysis of "colloidal" phosphorus. Microparticulate apatite also has the potential to release soluble reactive phosphate when the acidic molybdenum blue method is used.

scholarly journals Inactivation of α2-Macroglobulin by Activated Human Polymorphonuclear Leukocytes

1994 ◽  
Vol 3 (2) ◽  
pp. 117-123 ◽  
Author(s):  
G. Deby-Dupont ◽  
J.-L. Croisier ◽  
G. Camus ◽  
D. Brumioul ◽  
M. Mathy-Hartert ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Proteolytic Activity ◽  
Polymorphonuclear Leukocytes ◽  
Cytochalasin B ◽  
Hypochlorous Acid ◽  
Myeloperoxidase Activity ◽  
Trypsin Activity ◽  
Α2 Macroglobulin ◽  
Human Polymorphonuclear Leukocytes

The proteolytic activity of trypsin releases the dye Remazol Brilliant Blue from its high molecular weight substrate, the skin powder (Hide Powder Azure, Sigma), with an increase in absorbance at 595 nm. Active α2- macroglobulin (80 μg/ml) totally inhibits the proteolytic activity of trypsin (14 μg/ml) by trapping this protease. But after a 20 min incubation of α2-macroglobulin at 37°C with 2 × 106human polymorphonuclear leukocytes activated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (10−7M) and cytochalasin B (10−8M), 100% of trypsin activity was recovered, indicating a total inactivation of α2-macroglobuHn. Incubation with granulocyte myeloperoxidase also inactivates α2-macroglobulin. Hypochlorous acid, a by-product of myeloperoxidase activity, at a concentration of 10−7M also inactivates α2-macroglobulin, which indicates that an important cause of α2-macroglobulin inactivation by activated polymorphonuclear leukocytes could be the activity of myeloperoxidase.

Effects of Thrombin, Chymotrypsin and Aggregated Gamma-Globulins on the Proteins of the Human Platelet Membrane

1977 ◽  
Vol 37 (03) ◽  
pp. 396-406 ◽  
Author(s):  
B Podolsak
Keyword(s):  
Molecular Weight ◽  
Platelet Activation ◽  
Gel Electrophoresis ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Platelet Membrane ◽  
Low Molecular Weight ◽  
Membrane Glycoproteins ◽  
Membrane Component ◽  
Before And After

SummaryAnalysis of platelet membrane proteins and glycoproteins by SDS Polyacrylamide gel electrophoresis was carried out before and after treatment with thrombin. Extended incubation with thrombin (in the presence of EDTA or adenosine, which inhibit aggregation) produced extensive changes in the bands observed. With incubation times of a few minutes however, the changes were restricted to a glycopeptide, GP IV (approx. 90,000 Daltons) and one or two polypeptides of low molecular weight, in particular polypeptide 16 (approx. 23,000 Daltons). At 0–3° C only polypeptide 16 was still hydrolyzed.Chymotrypsin, which does not activate platelets, attacked glycopeptides I, II, III but no changes were apparent in GP IV and polypeptide 16. When chymotrypsin-treated platelets were further incubated with thrombin, only GP IV and one to two low molecular weight polypeptides, especially polypeptide 16, were affected. As polypeptide 16 appears to be an integral membrane component it is possible that it, either by itself or in combination with GP IV, represents the primary thrombin substrate involved in platelet activation.Aggregated IgG, which also activates platelets, does not modify the membrane glycoproteins but does change the low molecular weight region in particular band 16.

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