The photoinsertion was competed out by the presence of unlabeled UDPglucose thus suggesting that this protein contains substrate binding site for UDPglucose. Upon photolysis the 125 I-labelled probe was shown to link covalently to the 66 kDa protein. In an attempt to identify further the polypeptide chain of sterolglucoside synthase, the partially purified enzyme was treated with -5-allyl-UDPglucose, a photoactive analogue of UDPglucose, which is a substrate for this enzyme. SDS/PAGE of the purified fraction revealed the presence of two protein bands of molecular mass 66 kDa and 54 kDa. The membrane-bound sterolglucoside synthase from the yeast ''Saccharomyces cerevisiae'' has been solubilized by nonionic detergent, Nonidet P-40, Triton X-100, and partially purified by DEAE-cellulose column chromatography and ammonium sulfate fractionation. On the basis of the lack of any detectable glutamate-related enzyme activity associated with the isolated proteins and the presence of distinguishing sensitivities to analogs that inhibit glutamate transport carriers in synaptic membranes, it is proposed that the 71- kDa protein may be a component of a physiologic glutamate receptor complex in neuronal membranesĮvidence for the involvement of a 66 kDa membrane protein in the synthesis of sterolglucoside in ''Saccharomyces cerevisiae'' The neuroexcitatory analogs of L-glutamate and L-aspartate, ibotenate, quisqualate, and D-glutamate, inhibited Lglutamate binding to the isolated proteins, as did the antagonist of L-glutamate-induced neuronal excitation, L-glutamate diethylester. The glutamate-binding characteristics of these isolated protein fractions were very similar to those previously described for the 14- kDa GBP, including estimated dissociation constants for L-glutamate binding of 0.25 and 1 + M, inhibition of glutamate binding by azide and cyanide, and a selectivity of the ligand binding site for L-glutamate and L-aspartate. The major protein enriched in the most highly purified fractions was a 71- kDa glycoprotein, but a 63- kDa protein was co-purified during most steps of the isolation procedure. Since the antibodies used were raised against a 14- kDa GBP, the present studies were undertaken to explore the possibility that the 14- kDa protein may have been a proteolytic fragment of a larger M/sub r/ protein in synaptic membranes.
Immunoblot studies of synaptic membranes isolated from rat brain using antibodies raised against a previously purified glutamate-binding protein (GBP) indicated labeling of an ∼ 70- kDa protein band. International Nuclear Information System (INIS)Ĭhen, J.W. Immune labeling and purification of a 71- kDa glutamate-binding protein from brain synaptic membranes These molecules had no cytotoxic effect on macrophages and do not increase tumoricidal property of macrophages. according to our finding, 14- kDa and 47- kDa fractions of aged garlic extract are able to suppress NO production from macrophages, which can be used as a biological advantage. Cytotoxicity of macrophages' supernatant on WEHI-164 fibrosarcoma cells was not affected by garlic protein fractions (P = 0.066 for 14- kDa and P = 0.085 for 47- kDa fractions). Both 14- kDa and 47- kDa fractions significantly suppressed production of NO from macrophages (P = 0.007 and P = 0.003, respectively). MTT assay results for both 14- kDa and 47- kDa protein fractions of stimulated macrophages were not significant (P > 0.05). The amount of nitric oxide (NO) was detected in culture supernatants of macrophages by Griess reagent and furthermore, the cytotoxicity study of culture supernatants was carried out on WEHI-164 fibrosarcoma cell line as tumor necrosis factor-α bioassay. MTT assay was performed to evaluate the viability of macrophage.
Mice peritoneal macrophages were lavaged and cultured in a microtiter plate and exposed to different concentrations of garlic proteins. 14- kDa and 47- kDa protein fractions of garlic were purified. The present work aimed to assess the effect of protein fractions of garlic on peritoneal macrophages. Garlic is also an immunomodulator and its different components are responsible for different properties. Garlic (Allium sativum), traditionally being used as a spice worldwide, has different applications and is claimed to possess beneficial effects in several health ailments such as tumor and atherosclerosis. Effect of 14- kDa and 47- kDa protein molecules of age garlic extract on peritoneal macrophages.ĭaneshmandi, Saeed Hajimoradi, Monire Ahmadabad, Hasan Namdar Hassan, Zuhair Mohammad Roudbary, Maryam Ghazanfari, Tooba
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