Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In T. thermophila two forms of L-asparaginase (EC 3.5.1.1) were extracted and purified to near homogeneity which are associated with membranes. These two forms of L-asparaginase, I or II, act optimally at pH 8.6 and do not present any glutaminase or kinase activity. Both activities reach maximal values at the stationary phase of growth of T. thermophila. L-Asparaginases are solubilized by treatment of the particulates with 2% w/v Triton X-100 and then by sodium phosphate buffer pH 8.0. Both forms cross reacted with antibodies raised against T. pyriformis L-asparaginase and show isoelectric points 7.4 and 8.2. Among the metals tested, Ca2+ is the most effective in activating L-asparaginase I or II activity. Sorbitol alone up to 30% w/v in the assay mixture activates more than 10 x fold the activity of L-asparaginase II. Incubation of L-asparaginase I or II with increasing concentration of phospholipase C results in gradually loss of their activities. The relative effectiveness of a variety of phospholipids to reconstitute enzyme activity is presented as well.
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PMID:Two forms of L-asparaginase in Tetrahymena thermophila. 801 91

The intramitocondrial localization of the phosphate-activated glutaminase from Ehrlich cells has been examined by a combination of techniques, including: mitochondria subfractionation studies, chemical modification with sulfhydryl group reagents of different permeability, enzymatic digestion in both sides of the inner mitochondrial membrane, and immunological studies. Using alkaline extraction at high ionic strength, hypoosmotic shock and freezing-thawing cycle techniques, the enzyme was found in the particulate fraction. On the contrary, glutaminase activity was labile when subfractionation was carried out by digitonin/lubrol method; Western blot analysis localized the inactive enzyme in the matrix fraction. In addition, glutaminase was fully inactivated when mitoplasts were incubated with phospholipase A2 and phospholipase C. The enzyme also showed a non-linear Arrhenius plot with a break at 24 degrees C. The membrane-impermeant thiol reagents mersalyl and p-chloromercuriphenylsulfonic acid do not inhibit glutaminase activity in freeze-thawed mitochondria and mitoplasts, but N-ethylmaleimide, which is membrane permeant, strongly inhibited the enzyme. However, mersalyl and p-chloromercuriphenylsulfonic acid were effective inhibitors when the alkylation was performed on the matrix side of mitoplasts or using detergent-solubilized enzyme. Furthermore, trypsin digestion of mitoplasts was only effective inactivating glutaminase when the proteolysis was carried out on the matrix side of the vesicles. Enzyme-linked immunosorbent assay of the soluble and membrane fractions obtained in the preparation of submitochondrial particles, revealed that most of the enzyme was solubilized, but in the inactive form. Phase separation with Triton X-114 rendered most of the protein in the aqueous phase. These results taken together discard a transmembrane localization for the protein, whereas they are consistent with anchorage of glutaminase on the matrix side of the inner mitochondrial membrane, the matrix portion of the enzyme being relevant for its function.
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PMID:Submitochondrial localization and membrane topography of Ehrlich ascitic tumour cell glutaminase. 904 41