Nuclear magnetic resonance studies of blood platelets

1980 ◽  
Vol 289 (1037) ◽  
pp. 413-423 ◽  
Keyword(s):  
Molecular Weight ◽  
Metal Ions ◽  
High Molecular Weight ◽  
Human Platelet ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Biological Significance ◽  
Divalent Metal Ions ◽  
Motional State ◽  
High Concentrations

Blood platelets contain membrane-enclosed granules which have inside them high concentrations of 5-hydroxytryptamine (serotonin) along with adenine nucleotides and divalent metal ions. 19 F n.m.r. of fluorinated serotonin incorporated into the granules of both human and pig intact platelets has shown that the motional state of the serotonin is restricted. Comparison with 31 P n.m.r. experiments indicates that this restriction of motion is a consequence of high molecular weight aggregates formed by the adenine nucleotides and metal ions, and that it varies with the species from which the platelets are obtained. In the case of human platelet granules, at least, these high molecular weight aggregates are present in the absence as well as in the presence of serotonin. The biological significance of these data is briefly discussed.

scholarly journals Identification of high-affinity (Kd 0.35 mumol/L) and low-affinity (Kd 7.9 mumol/L) platelet binding sites for ADP and competition by ADP analogues

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 110-116 ◽  
Author(s):  
JR Jefferson ◽  
JT Harmon ◽  
GA Jamieson
Keyword(s):  
Platelet Activation ◽  
Dissociation Constants ◽  
Partial Agonist ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Ionophore A23187 ◽  
High Affinity ◽  
Binding Isotherms ◽  
High Affinity Site ◽  
High Concentrations

Steady-state binding of ADP to blood platelets and isolated membranes has not previously been obtained because of complications arising from metabolism of the ligand and dilution due to its secretion from storage granules. In the present studies, competition binding isotherms (n = 9) using paraformaldehyde-fixed platelets showed that [2–3 H]ADP bound to two sites with a small amount (approximately 5% of total) of nonspecific binding: 410,000 +/- 40,000 sites of low affinity (Kd 7.9 +/- 2.0 mumol/L) and 160,000 +/- 20,000 sites of high affinity (Kd 0.35 +/- 0.04 mumol/L) corresponding to the ADP concentration required for activation in fresh platelets (0.1–0.5 mumol/L). All agonists and antagonists examined were able to compete with ADP at the high-affinity site. The strong platelet agonists 2-methylthio ADP and 2-(3- aminopropylthio)ADP competed with ADP at the high-affinity site with dissociation constant values of 7 mumol/L and 200 mumol/L, respectively. The partial agonist 2′,3′-dialdehyde ADP and the weak agonist GDP also competed at the high-affinity site with Kd values of 5 mumol/L and 49 mumol/L, respectively. The sequence of binding affinities of other adenine nucleotides at the high-affinity site corresponded to their relative activities as known antagonists of platelet activation by ADP; namely, ADP(Kd 0.35 mumol/L) approximately equal to ATP (Kd 0.45 mumol/L) much greater than AMP (Kd 360 mumol/L). Adenosine and 2-chloroadenosine did not compete with ADP. ADP binding to the high-affinity site was inhibited by p-mercuribenzene sulfonate (Ki 250 mumol/L) but only very weakly by 5′-p- fluorosulfonylbenzoyladenosine (Ki 1 mmol/L). All the above nucleotides also competed with ADP at the low-affinity sites but, because of the high concentrations of competing nucleotide required, dissociation constants at this site were obtained only for ATP (21 mumol/L), 2-MeS ADP (200 mumol/L) and 2′,3′-dialdehyde ADP (270 mumol/L). 8-Bromo ADP competed strongly with ADP at the high-affinity site (Kd 0.40 mumol/L) but weakly if at all at the low-affinity site. 8-Bromo ADP inhibited platelet activation induced by ADP (EC50 approximately 100 mumol/L) but not by collagen, thrombin, or ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS).

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 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.

Determination of Free and Bound Cd, Zn, Cu, and Ag Ions in Lobster (Homarus americanus) Digestive Gland Extracts by Gel Chromatography Followed by Atomic Absorption Spectrophotometry and Polarography

1993 ◽  
Vol 76 (4) ◽  
pp. 794-798 ◽  
Author(s):  
Chiu L Chou ◽  
John F Uthe ◽  
Robert D Guy
Keyword(s):  
Molecular Weight ◽  
Metal Ions ◽  
Atomic Absorption ◽  
Gel Permeation Chromatography ◽  
Weight Fraction ◽  
Atomic Absorption Spectrophotometry ◽  
Divalent Metal ◽  
Divalent Metal Ions ◽  
Absorption Spectrophotometry ◽  
Gel Permeation

Abstract Gel-permeation chromatography followed by atomic absorption spectrophotometric and polarographic analyses were used to measure free and bound divalent metal ions in lobster digestive gland extracts prepared with or without oc-toluenesulfonyl fluoride, a protease inhibitor. Chromatography on Sephadex G-50 or G-100 yielded 3 UV-absorbing peaks, which corresponded, respectively, to the void volume, a medium molecular weight fraction, and a low molecular weight fraction that contained free divalent metal ions. In protease-inhibited extracts, only Zn+2 was found, whereas Cd, Cu, and Ag were bound in high and medium molecular weight fractions. Cd+2 and Zn+2 were rapidly released from their bound forms in the absence of a protease inhibitor, and their presence was confirmed by polarography with EDTA. Gel-permeation chromatography coupled with atomic absorption spectrophotometry offers a rapid method for following changes in the concentrations of bound and free metal ions during processing of shellfishbased foodstuffs.

Inhibition of the Platelet Reaction by a High Molecular Weight Phosphoglycoprotein Isolated from Human Platelet Plasma Membranes

1981 ◽  
Vol 45 (02) ◽  
pp. 130-135 ◽  
Author(s):  
R Apitz-Castro ◽  
G Fonseca ◽  
V Michelena ◽  
M R Cruz
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Plasma Membranes ◽  
Human Platelet ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Secondary Wave ◽  
Platelet Reaction ◽  
Induced Aggregation ◽  
External Calcium

SummaryThe effect of a phospho-glycoprotein (HMW-GP), obtained from human platelet plasma membranes, on the aggregation and secretion of human platelets was studied. Incubation of PRP with 4 to 16 μg/ml of HMW-GP results in inhibition of ADP-, Epinephrine-, Collagen-, and Thrombin-induced platelet aggregation. The effect is mainly reflected on the secondary wave of aggregation. The inhibitory effect is partially overcome by higher concentration of the inducers, however, even under these conditions, a clear tendency towards disaggregation is observed. 5HT release (Col-induced) is strongly decreased from 50% to 4.5. The inhibitory effect on Thrombin-induced aggregation is markedly dependent on external calcium, being maximal at 5 mM calcium. The HMW-GP does not bind ADP or Thrombin. Membrane conformation is markedly affected, as evidenced by the effect of HMW-GP on the iodination of surface polypeptides of intact platelets. It is suggested that interaction of HMW-GP with the platelet membrane blocks the signal(s) transmission that links stimulus to activation. The inhibition observed might just represent an experimental amplification of the endogenous modulatory function that has been proposed for this high molecular weight phosphoglycoprotein.

scholarly journals Molecular Basis for Ser/Thr Specificity in PKA Signaling

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1548 ◽  
Author(s):  
Matthias J. Knape ◽  
Maximilian Wallbott ◽  
Nicole C. G. Burghardt ◽  
Daniela Bertinetti ◽  
Jan Hornung ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Substrate Specificity ◽  
Metal Ions ◽  
Protein Kinases ◽  
Molecular Basis ◽  
Kinase Inhibitor ◽  
Adenine Nucleotides ◽  
Catalytic Efficiency ◽  
Specific Protein ◽  
Divalent Metal Ions

cAMP-dependent protein kinase (PKA) is the major receptor of the second messenger cAMP and a prototype for Ser/Thr-specific protein kinases. Although PKA strongly prefers serine over threonine substrates, little is known about the molecular basis of this substrate specificity. We employ classical enzyme kinetics and a surface plasmon resonance (SPR)-based method to analyze each step of the kinase reaction. In the absence of divalent metal ions and nucleotides, PKA binds serine (PKS) and threonine (PKT) substrates, derived from the heat-stable protein kinase inhibitor (PKI), with similar affinities. However, in the presence of metal ions and adenine nucleotides, the Michaelis complex for PKT is unstable. PKA phosphorylates PKT with a higher turnover due to a faster dissociation of the product complex. Thus, threonine substrates are not necessarily poor substrates of PKA. Mutation of the DFG+1 phenylalanine to β-branched amino acids increases the catalytic efficiency of PKA for a threonine peptide substrate up to 200-fold. The PKA Cα mutant F187V forms a stable Michaelis complex with PKT and shows no preference for serine versus threonine substrates. Disease-associated mutations of the DFG+1 position in other protein kinases underline the importance of substrate specificity for keeping signaling pathways segregated and precisely regulated.

scholarly journals Ein makroporöses Polyacrylamid für immunologische Diffusionstechniken / Macroporous Polyacrylamide, its Use for Immunological Diffusion Techniques

1972 ◽  
Vol 27 (6) ◽  
pp. 683-688 ◽  
Author(s):  
Helmut Determann ◽  
Axel Walch
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Monomer Concentration ◽  
Agarose Gels ◽  
Γ Globulins ◽  
Sample Application ◽  
Total Monomer Concentration ◽  
High Molecular Weight Proteins ◽  
High Concentrations

Gel layers of crosslinked polyacrylamide with different porosities were synthesized and their properties compared. At high concentrations of bifunctional monomers macroporous gels were formed, one of which (total monomer concentration 14%, methylene-bis-acrylamide 40%) permitted even high molecular weight proteins (e. g. γ-globulins) to diffuse more readily than in 2 per cent agar or agarose gels. Different immunological techniques were modified for application with this polymer. It offers advantages especially with respect to porosity, storability and sample application for quantitative diffusion techniques.

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