scholarly journals Isolation and characterization of the major oligosaccharide of human platelet membrane glycoprotein GPIb

1982 ◽  
Vol 205 (1) ◽  
pp. 81-90 ◽  
Author(s):  
P A Judson ◽  
D J Anstee ◽  
J R Clamp
Keyword(s):  
Human Platelet ◽  
Human Platelets ◽  
Membrane Glycoprotein ◽  
Molar Ratios ◽  
Isolation And Characterization ◽  
Molar Basis ◽  
Membrane Bound ◽  
Almost All

Treatment of intact human platelets with chymotrypsin released a glycopolypeptide that was shown to be derived from the major membrane glycoprotein, GPIb. The glycopolypeptide contained 59% carbohydrate on a molar basis and was rich in serine, threonine and proline. Almost all the carbohydrate could be released from the glycopolypeptide by treatment with alkali in the presence of NaBH4. The major component (comprising 80% of the released sugar) was purified and shown to be a hexasaccharide containing sialic acid, galactose, N-acetylglucosamine and N-acetylgalactosaminitol in the molar ratios 2:2:1:1. Two possible structures for this hexasaccharide are proposed on the basis of the known biosynthetic pathways of mucus-type glycoproteins. Our data is consistent with the occurrence of an O-glycosidically linked oligosaccharide on one amino acid in four of the glycopolypeptide. These results suggest that glycoprotein Ib can best be described as a membrane-bound mucus-type glycoproteins. Our data are consistent with the occurrence of an O- in the process by which platelets adhere to the exposed subendothelium of damaged blood-vessel walls. The possible role of the glycopolypeptide portion of GPIb in this process was investigated. Neither the major oligosaccharide nor the glycopolypeptide itself inhibited ristocetin-induced platelet agglutination at the concentrations tested. It is suggested that the carbohydrate moieties of GPIb molecules at the cell surface interact to form a barrier to macromolecules. Such a barrier could play a major role in modulating platelet function.

Isolation and Characterization of Human Platelet β-Thromboglobulin

1975 ◽  
Author(s):  
D. S. Pepper ◽  
S. Moore ◽  
J. D. Cash
Keyword(s):  
Molecular Weight ◽  
Plasma Proteins ◽  
Human Platelet ◽  
Weight Fraction ◽  
Human Platelets ◽  
Low Molecular Weight ◽  
Purification Procedure ◽  
Isolation And Characterization

The thrombin released products from washed human platelets were separated by filtration on 4% agarose in 0.15 M NaCl. The high molecular weight PF4 complex was dissociated and re-chromatographed in 0.75 M NaCl. The low molecular weight fraction, including β thromboglobulin and a low MW anti-heparin was freed of plasminogen anti-activator by dissociation and chromatography in pH 3.5 pyridine acetic acid. The anti-activator was irreversibly denatured and albumin was removed in the void volume of the column. A more suitable purification procedure for recovery of all activities was affinity chromatography on heparin-agarose. The anti-activator was excluded and could be obtained free of plasma proteins by Sephadex G-200 chromatography. The βTG eluted at 0.3 M NaCl and the low MW anti-heparin at 1.5 M NaCl. The pure βTG (MW 36,000) was injected into rabbits and the resulting antiserum used to produce a radioimmunoassay for the release reaction in vivo.

ISOLATION AND CHARACTERIZATION OF HUMAN PLATELET MEMBRANE GLYCOPROTEINS Ia AND IIa

1987 ◽  
Author(s):  
D Bienz ◽  
T Wager ◽  
K J Clemetson
Keyword(s):  
Lens Culinaris ◽  
Human Platelet ◽  
Protein Solutions ◽  
Membrane Glycoproteins ◽  
Platelet Surface ◽  
Isolation And Characterization ◽  
Sds Page ◽  
Triton X

Glycoproteins (GP) Ia and IIa are relatively minor components of the platelet surface with similar molecular properties. Nieuwenhuis et al. (Nature 319, 470-72, 1985) described a patient whose platelets show no response to collagen. The correlating lack of GPIa in the platelets of this patient suggests this glycoprotein being the receptor for collagen. Santoro (Cell, 46, 913-20, 1986) described a 160 kDa glycoprotein that binds to collagen in the presence of MG2 + and is possibly identical with GPIa. The role of GPIIa is still unknown but a similar molecule has also been found on endothelial cells. It has been suggested that GPIa and GPIIa are complexed with a further membrane component GPIc. The two glycoproteins show only slight difference in molecular weight, isoelectric point and in their affinity for various lectins. As a result they coisolate using most separation techniques.GPIa is usually associated with the cytoskeleton while GPIIa is mostly found in the soluble phase. GPIa is dissociated from the cytoskeleton by addition of 2% SDS (final conc.) and sonication. Performing Triton X-114 phase partition, GPIIa is found in the detergent phase. After the detergents of the GPIa and GPIIa enriched protein solutions are exchanged with the non-ionic octanoyl-N-methyl glucamide, the glycoproteins are further purified by affinity chromatography on wheat germ agglutinin-Sepharose followed by Lens culinaris lectin-Sepharose both of which bind GPIa and GPIIa. A major contaminant during the purification is GPIb. Final purification of GPIa and GPIIa was obtained by preparative SDS-PAGE using electroelution into a membrane trap. Latest results show an enrichment of GPIa and a lack of GPIb in pseudopodes, isolated by the method of Rotman et al. (Proc. Natl. Acad. Sci. USA, 4357-61, 1982).

Differential Ability of Agonists to Express Distinct Pools of Fibrinogen (Gpllb/llla) Receptors which Can Mediate the Aggregation of Human Platelets

1989 ◽  
Vol 62 (03) ◽  
pp. 955-961 ◽  
Author(s):  
Ian S Watts ◽  
Rebecca J Keery ◽  
Philip Lumley
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Surface Membrane ◽  
Blood Platelet ◽  
Human Platelets ◽  
Membrane Glycoprotein ◽  
Platelet Surface ◽  
Human Platelet Aggregation ◽  
Differential Ability ◽  
Blood Platelet Aggregation

SummaryWe have investigated the effect of two procedures that modify human platelet surface membrane glycoprotein (Gp) IIb and IIIa complexes upon whole blood platelet aggregation to a range of agonists. (A) Irreversible disruption of complexes by temporary (30 min) Ca2+-deprivation with EGTA at 37° C. (B) Binding of a monoclonal antibody M148 to the complex. EGTA exposure abolished aggregation to ADP, adrenaline and PAF. In contrast, full aggregation curves to collagen and U-46619 could still be established. EGTA exposure reduced M148 binding to platelets by 80%. Excess M148 abolished aggregation to ADP, PAF, collagen and U-46619. However, upon removal of unbound antibody from platelets full aggregation curves to collagen and U-46619 but not to ADP and PAF could be re-established. Thus human platelet aggregation to ADP, PAF and adrenaline appears absolutely dependent upon surface membrane GpIIb/IIIa complexes. In contrast, collagen and U-46619 cause expression of an additional distinct pool of Gp complexes inaccessible to EGTA and M148 in unstimulated platelets which is intimately involved in aggregation to these agonists.

scholarly journals Isolation and characterization of a membrane protein from rat erythrocytes which inhibits lysis by the membrane attack complex of rat complement

1992 ◽  
Vol 284 (1) ◽  
pp. 169-176 ◽  
Author(s):  
T R Hughes ◽  
S J Piddlesden ◽  
J D Williams ◽  
R A Harrison ◽  
B P Morgan
Keyword(s):  
Affinity Purification ◽  
Membrane Attack Complex ◽  
Erythrocyte Membranes ◽  
Dependent Manner ◽  
Inhibitory Protein ◽  
Rat Erythrocytes ◽  
Butanol Extract ◽  
Isolation And Characterization ◽  
Membrane Bound

The membrane attack complex (MAC) of complement in humans is regulated by several membrane-bound proteins; however, no such proteins have so far been described in other species. Here we report the isolation and characterization of a rat erythrocyte membrane glycoprotein of molecular mass 21 kDa which inserts into cell membranes and is a potent inhibitor of the rat MAC. This protein, here called rat inhibitory protein (RIP), was first partially purified by column chromatography from a butanol extract of rat erythrocyte membranes. Monoclonal antibodies (Mabs) were raised against RIP and used for its affinity purification. Affinity-purified RIP was shown to inhibit in a dose-dependent manner the cobra venom factor (CVF)-mediated ‘reactive’ lysis of guinea pig erythrocytes by rat complement. Conversely, the anti-RIP MAbs 6D1 and TH9 were shown to markedly enhance the CVF-mediated lysis of rat erythrocytes by rat complement. RIP acted late in the assembly of the MAC (at or after the C5b-8 stage) and was releasable from the membranes of rat erythrocytes by phosphatidylinositol-specific phospholipase C. These features, together with its size, deglycosylation pattern and N-terminal amino acid sequence, lead us to conclude that RIP is the rat homologue of the human MAC-inhibitory protein CD59 antigen.

scholarly journals Isolation and Characterization of Bacteriophage ZCSE6 against Salmonella spp.: Phage Application in Milk

Biologics ◽  
2021 ◽  
Vol 1 (2) ◽  
pp. 164-176
Author(s):  
Abdallah S. Abdelsattar ◽  
Anan Safwat ◽  
Rana Nofal ◽  
Amera Elsayed ◽  
Salsabil Makky ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Raw Milk ◽  
Salmonella Spp ◽  
Isolation And Characterization ◽  
Food Borne ◽  
Wide Range ◽  
Almost All ◽  
And Control ◽  
Milk And Dairy Products

Food safety is very important in the food industry as most pathogenic bacteria can cause food-borne diseases and negatively affect public health. In the milk industry, contamination with Salmonella has always been a challenge, but the risks have dramatically increased as almost all bacteria now show resistance to a wide range of commercial antibiotics. This study aimed to isolate a bacteriophage to be used as a bactericidal agent against Salmonella in milk and dairy products. Here, phage ZCSE6 has been isolated from raw milk sample sand molecularly and chemically characterized. At different multiplicities of infection (MOIs) of 0.1, 0.01, and 0.001, the phage–Salmonella interaction was studied for 6 h at 37 °C and 24 h at 8 °C. In addition, ZCSE6 was tested against Salmonella contamination in milk to examine its lytic activity for 3 h at 37 °C. The results showed that ZCSE6 has a small genome size (<48.5 kbp) and belongs to the Siphovirus family. Phage ZCSE6 revealed a high thermal and pH stability at various conditions that mimic milk manufacturing and supply chain conditions. It also demonstrated a significant reduction in Salmonella concentration in media at various MOIs, with higher bacterial eradication at higher MOI. Moreover, it significantly reduced Salmonella growth (MOI 1) in milk, manifesting a 1000-fold decrease in bacteria concentration following 3 h incubation at 37 °C. The results highlighted the strong ability of ZCSE6 to kill Salmonella and control its growth in milk. Thus, ZCSE6 is recommended as a biocontrol agent in milk to limit bacterial growth and increase the milk shelf-life.

Isolation and characterization of SRF accessory proteins

1993 ◽  
Vol 340 (1293) ◽  
pp. 325-332 ◽  
Keyword(s):  
Ternary Complex ◽  
Map Kinases ◽  
Serum Response Factor ◽  
Regulatory Element ◽  
Isolation And Characterization ◽  
Activation Of Transcription ◽  
Serum Response ◽  
Dna Binding Properties

Many genes which are regulated by growth factors contain a common regulatory element, the serum response element (SRE). Activation of transcription by the SRE involves a ternary complex formed between a ubiquitous factor, serum response factor (SRF), and a second protein, p62/TCF. We used a yeast genetic screen to isolate cDNAs encoding a protein, SAP-1, with the DNA binding properties of p62/TCF. The SAP-1 sequence contains three regions of homology to the previously uncharacterized Elk-1 protein, which also acts as an SRF accessory protein. Only two of these regions are required for cooperative interactions with SRF in the ternary complex. The third contains several conserved sites for the MAP kinases, whose activity is regulated in response to growth factor stimulation. We discuss the potential role of these proteins in regulation of the c-fos SRE.

scholarly journals Cover Feature: Isolation and Characterization of Different Homometallic and Heterobimetallic Complexes of Nickel and Zinc Ions by Controlling  Molar Ratios and Solvents (Eur. J. Inorg. Chem. 24/2019)

2019 ◽  
Vol 2019 (24) ◽  
pp. 2862-2862
Author(s):  
Saroj Kumar Kushvaha ◽  
Selvakumar Arumugam ◽  
Bhaskaran Shankar ◽  
Rabi Shankar Sarkar ◽  
Venkatachalam Ramkumar ◽  
...  
Keyword(s):  
Zinc Ions ◽  
Molar Ratios ◽  
Heterobimetallic Complexes ◽  
Isolation And Characterization
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