scholarly journals Phospholipase A2 enzyme from the venom of Egyptian honey bee Apis mellifera lamarckii with anti-platelet aggregation and anti-coagulation activities

Author(s):  
Doaa A. Darwish ◽  
Hassan M. M. Masoud ◽  
Mohamed M. Abdel-Monsef ◽  
Mohamed S. Helmy ◽  
Hind A. Zidan ◽  
...  

Abstract Background Honey bee venom contains various enzymes with wide medical and pharmaceutical applications. Results The phospholipase A2 (PLA2) has been apparently purified from the venom of Egyptian honey bee (Apis mellifera lamarckii) 8.9-fold to a very high specific activity of 6033 U/mg protein using DEAE–cellulose and Sephacryl S-300 columns. The purified bee venom PLA2 is monomeric 16 kDa protein and has isoelectric point (pI) of 5.9. The optimal activity of bee venom PLA2 was attained at pH 8 and 45 °C. Cu2+, Ni2+, Fe2+, Ca2+, and Co2+ exhibited a complete activating effect on it, while Zn2+, Mn2+, NaN3, PMSF, N-Methylmaleimide, and EDTA have inhibitory effect. Conclusions The purified bee venom PLA2 exhibited anti-platelet aggregation and anti-coagulation activities which makes it promising agent for developing novel anti-clot formation drugs in future.

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.


1985 ◽  
Vol 249 (3) ◽  
pp. H439-H449 ◽  
Author(s):  
R. Bunger

Pyruvate compartmentation and lactate dehydrogenase (LDH) were studied in isolated perfused working guinea pig hearts. The mean intracellular pyruvate (Pyr) contents increased with perfusate Pyr (0-2 mM) but varied only slightly with glucose (0-10 mM) and additional insulin (0.04-5 U/l), respectively. With 5-10 mM glucose plus 5 U/l insulin, but not with Pyr or lactate (Lac) as substrates, a near equilibrium between the LDH and the glycerol-3-phosphate dehydrogenase seemed to exist. Evidence for an inhibitory effect of Pyr on the activity of the LDH system of the perfused hearts was not obtained. With [U-14C]glucose as sole substrate, the specific activity of coronary venous Lac was near half that of precursor glucose. 14CO2 production was thus in quantitative agreement with rates of pyruvate oxidation that were determined as glucose uptake minus (Pyr + Lac) release. In contrast, with 0.2 mM [1-14C]Pyr plus 5 mM glucose, the ratio of 14CO2 production to specific activity of Lac overestimated Pyr oxidation judged from myocardial substrate balances and O2 uptake, respectively; here, at least three pools of [14C]HCO-3 and [14C]lac, respectively, were kinetically demonstrable during washout of trace amounts of 14C-labeled Pyr. Evidently, the specific activity of Lac was equivalent to that of mitochondrial oxidized Pyr provided [14C]glucose was the sole or major precursor of cellular pyruvate. However, exogenously applied [1-14C]Pyr of high specific activity seemed to induce intracellular formation of both a highly and lowly labeled Pyr; the latter Pyr compartment did not seem in ready equilibrium with the cell physiologically prevailing highly labeled Pyr pool.


RSC Advances ◽  
2015 ◽  
Vol 5 (80) ◽  
pp. 65214-65220 ◽  
Author(s):  
Aleš Marek ◽  
Mahadeo R. Patil ◽  
Tomáš Elbert

A convenient method for the synthesis of tritium-labeled brassinosteroids with very high specific activity is reported.


2021 ◽  
Vol 8 ◽  
Author(s):  
Michiel Van de Voorde ◽  
Charlotte Duchemin ◽  
Reinhard Heinke ◽  
Laura Lambert ◽  
Eric Chevallay ◽  
...  

Samarium-153 (153Sm) is a highly interesting radionuclide within the field of targeted radionuclide therapy because of its favorable decay characteristics. 153Sm has a half-life of 1.93 d and decays into a stable daughter nuclide (153Eu) whereupon β− particles [E = 705 keV (30%), 635 keV (50%)] are emitted which are suitable for therapy. 153Sm also emits γ photons [103 keV (28%)] allowing for SPECT imaging, which is of value in theranostics. However, the full potential of 153Sm in nuclear medicine is currently not being exploited because of the radionuclide's limited specific activity due to its carrier added production route. In this work a new production method was developed to produce 153Sm with higher specific activity, allowing for its potential use in targeted radionuclide therapy. 153Sm was efficiently produced via neutron irradiation of a highly enriched 152Sm target (98.7% enriched, σth = 206 b) in the BR2 reactor at SCK CEN. Irradiated target materials were shipped to CERN-MEDICIS, where 153Sm was isolated from the 152Sm target via mass separation (MS) in combination with laser resonance enhanced ionization to drastically increase the specific activity. The specific activity obtained was 1.87 TBq/mg (≈ 265 times higher after the end of irradiation in BR2 + cooling). An overall mass separation efficiency of 4.5% was reached on average for all mass separations. Further radiochemical purification steps were developed at SCK CEN to recover the 153Sm from the MS target to yield a solution ready for radiolabeling. Each step of the radiochemical process was fully analyzed and characterized for further optimization resulting in a high efficiency (overall recovery: 84%). The obtained high specific activity (HSA) 153Sm was then used in radiolabeling experiments with different concentrations of 4-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA). Even at low concentrations of p-SCN-Bn-DOTA, radiolabeling of 0.5 MBq of HSA 153Sm was found to be efficient. In this proof-of-concept study, we demonstrated the potential to combine neutron irradiation with mass separation to supply high specific activity 153Sm. Using this process, 153SmCl3 suitable for radiolabeling, was produced with a very high specific activity allowing application of 153Sm in targeted radionuclide therapy. Further studies to incorporate 153Sm in radiopharmaceuticals for targeted radionuclide therapy are ongoing.


1977 ◽  
Vol 55 (8) ◽  
pp. 865-868 ◽  
Author(s):  
K. K. Batra ◽  
J. R. Wagner ◽  
E. L. R. Stokstad

The composition of folate coenzymes in romaine lettuce was studied. Lettuce extract was purified on QAE-Sephadex A-25 and folate compounds were separated into a monoglutamate fraction and a polyglutamate fraction by chromatography on Sephadex G-15. Both the mono- and poly-glutamate fractions were resolved on DEAE-cellulose. Positive identification of DEAE peaks was made by further cochromatography with high specific activity radioactive marker folate compounds and with differential microbiological assay. The distribution of folate compounds in lettuce is as follows: 32% 5-CH3-H4PteGlu; 1% 5-CHO-H4PteGlu; 3% 5-CHO-H4PteGlu4; 9% 5-CH3-H4PteGlu4; 13% 5-CHO-H4PteGlu5; and 31% 5-CH3-H4PteGlu5.


1987 ◽  
Author(s):  
A D Purdon ◽  
J B Smith

Previously, we have shown that 1-acyl-2-arachidonoyl glycero-phosphocholine (GPC) is the main source of arachidonic acid in thrombin-stimulated (5 U/ml) human platelets. Thus 1-acyl-2-3H-arachidonoyl GPC was dispersed in Tris buffer, 0.01 M, pH 7.5, 0.01 M CaCl2 for use a substrate for the assay of phospholipase A2 activity in human platelets. The released 3H-arachidonate(AA) was isolated by thin layer chromatography following Bligh and Dyer extraction of the enzyme-substrate incubate. Phospholipase A2 (PLA2) specific for this phospholipid was thought to be membrane bound and of low activity when solubilized, however, we have found, that provided resting platelets are gently sonicated while suspended in tyrode's buffer in the presence of suitable concentrations of protease inhibitors and metal chelators (EGTA, EDTA), a large amount of soluble PLA2 activity can be isolated following centrifugation to remove membranes. The enzyme required calcium for activity and was inactive in the presence of EGTA. No activity was found in the secretate from thrombin-stimulated cells, indicating that the PLA2 assayed at pH 7.5 was not lysosomal. PLA2 was further purified by DEAE cellulose chromatography where a 5 times increase in specific activity was achieved. It is known that OAG (1-oleoyl-2-acetyle-glycerol) augments deacylation of 1,2 diradyl GPC in platelets stimulated with suboptimal levels of ionophore A23187. Thus the effect of OAG stimulation of platelets on the distribution of soluble PLA2 was studied. Platelets (109 cells/ml) suspended in tyrode's buffer and stimulated with 100 ug/ml OAG or 5 U/ml thrombin (10 min, 37°C., 10 min, without stirring), showed a considerable decrease in soluble PLA2 activity suggesting a partitioning of soluble PLA2 into the membrane bilayer. Thus a model for PLA2 action is suggested in which binding of the cytosolic enzyme to its site of hydrolysis is induced by diglyceride-perturbation of the membrane, phospholipid, bilayer phase.


1974 ◽  
Vol 29 (3-4) ◽  
pp. 161-168 ◽  
Author(s):  
K. H. Trautmann ◽  
A. Schuler ◽  
M. Suchý ◽  
H.-K. Wipf

Abstract A method is presented permitting the qualitative and quantitative determination of all three presently known hormones (JH1-3). The determination is based on the method of radioactive isotope dilution, whereby a very small known amount of tritium-labelled JH-1 is added to the ether extract of the particular species. The addition of radioactive JH-1 permits the isolation of all three hormones, because of their similar behaviour during the chosen work up. The quantitative determination was carried out by gas chromatography and the identification was confirmed with the help of retention-times and GC-MS combination. The method was checked by using an extract of Hyalophora cecropia. For the first time methyl 10,11-epoxy-3,7,11-trimethyl-2-trans-6-trans-dodecadienoate (JH-3) could also be identified as the juvenile hormone of Melo­lontha melolontha. In Vanessa io larvae, Tenebrio molitor larvae and adults and in Musca domestica larvae none of the three known hormones could be detected. The preparation of JH-1 labelled with tritium in the methyl group of the ester was accomplished with very high specific activity (4.34 Ci/mmol) of the tritiated acid with diazomethane.


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