Purification and characterization of human phosphatidylserine synthases 1 and 2

2009 ◽  
Vol 418 (2) ◽  
pp. 421-429 ◽  
Author(s):  
Shiho Tomohiro ◽  
Ayako Kawaguti ◽  
Yukiyo Kawabe ◽  
Sakae Kitada ◽  
Osamu Kuge
Keyword(s):  
Feedback Control ◽  
Substrate Specificity ◽  
Mammalian Cells ◽  
Enzyme Assay ◽  
The Other ◽  
Significant Extent ◽  
Purification And Characterization ◽  
Intact Cells ◽  
Base Exchange

PS (phosphatidylserine) in mammalian cells is synthesized by two distinct base-exchange enzymes, PSS1 (PS synthase 1) and PSS2, which are responsible for the conversion of PC (phosphatidylcholine) and PE (phosphatidylethanolamine) respectively into PS in intact cells. The PS synthesis in cultured mammalian cells is inhibited by exogenous PS, and this feedback control occurs through inhibition of PSSs by PS. In the present study, we purified epitope-tagged forms of human PSS1 and PSS2. The purified PSS2 was shown to catalyse the conversion of PE, but not PC, into PS, this being consistent with the substrate specificity observed in intact cells. On the other hand, the purified PSS1 was shown to catalyse the conversion of both PC and PE into PS, although PSS1 in intact cells had been shown not to contribute to the conversion of PE into PS to a significant extent. Furthermore, we found that the purified PSS2, but not the purified PSS1, was inhibited on the addition of PS to the enzyme assay mixture, raising the possibility that there was some difference between the mechanisms of the inhibitory actions of PS towards PSS1 and PSS2.

scholarly journals Lysosomal degradation of glycoproteins and glycosaminoglycans. Efflux and recycling of sulphate and N-acetylhexosamines

1986 ◽  
Vol 235 (3) ◽  
pp. 707-713 ◽  
Author(s):  
L H Rome ◽  
D F Hill
Keyword(s):  
Cultured Cells ◽  
The Other ◽  
Biosynthetic Pathways ◽  
Significant Extent ◽  
Lysosomal Degradation ◽  
Intact Cells ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Glycoprotein Degradation ◽  
Macromolecule Degradation

Lysosomal degradation of the carbohydrate portion of glycoproteins and glycosaminoglycans produces monosaccharides and sulphate, which must efflux from the lysosomes before re-entering biosynthetic pathways. We examined the degradation of glycoproteins and glycosaminoglycans by lysosomes isolated from cultured human diploid fibroblasts. Cells were grown for 24 h in medium containing [3H]glucosamine and [35S]sulphate. When lysosomes are isolated from these cells, they contain label primarily in macromolecules (glycoproteins and glycosaminoglycans). Glycoprotein degradation by isolated lysosomes was followed by measuring the release of tritiated sugars from macromolecules and efflux of these sugars from the organelles. Glycosaminoglycan degradation was monitored by the release of both tritiated sugars and [35S]sulphate. During macromolecule degradation, the total amounts of free [35S]sulphate, N-acetyl[3H]glucosamine and N-acetyl[3H]galactosamine found outside the lysosome parallels the amounts of these products released by degradation. The total degradation of glycoproteins and glycosaminoglycans by intact cultured cells was also examined. The lysosomal contribution to degradation was assessed by measuring inhibition by the lysosomotropic amine NH4Cl. After 48 h incubation, inhibition by NH4Cl exceeded 55% of glycoprotein and 72% of glycosaminoglycan degradation. Recycling of [3H]hexosamines and [35S]sulphate by intact cells was estimated by measuring the appearance of ‘newly synthesized’ radioactively labelled macromolecules in the medium. Sulphate does not appear to be appreciably recycled. N-Acetylglucosamine and N-acetylgalactosamine, on the other hand, are reutilized to a significant extent.

scholarly journals Characterization of Hybrid Toluate and Benzoate Dioxygenases

2003 ◽  
Vol 185 (18) ◽  
pp. 5333-5341 ◽  
Author(s):  
Yong Ge ◽  
Lindsay D. Eltis
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Acinetobacter Calcoaceticus ◽  
The Other ◽  
Β Subunit ◽  
Structural Determinants ◽  
Α Subunit ◽  
Benzoate Dioxygenase

ABSTRACT Toluate dioxygenase of Pseudomonas putida mt-2 (TADOmt2) and benzoate dioxygenase of Acinetobacter calcoaceticus ADP1 (BADOADP1) catalyze the 1,2-dihydroxylation of different ranges of benzoates. The catalytic component of these enzymes is an oxygenase consisting of two subunits. To investigate the structural determinants of substrate specificity in these ring-hydroxylating dioxygenases, hybrid oxygenases consisting of the α subunit of one enzyme and the β subunit of the other were prepared, and their respective specificities were compared to those of the parent enzymes. Reconstituted BADOADP1 utilized four of the seven tested benzoates in the following order of apparent specificity: benzoate > 3-methylbenzoate > 3-chlorobenzoate > 2-methylbenzoate. This is a significantly narrower apparent specificity than for TADOmt2 (3-methylbenzoate > benzoate ∼ 3-chlorobenzoate > 4-methylbenzoate ∼ 4-chlorobenzoate ≫ 2-methylbenzoate ∼ 2-chlorobenzoate [Y. Ge, F. H. Vaillancourt, N. Y. Agar, and L. D. Eltis, J. Bacteriol. 184:4096-4103, 2002]). The apparent substrate specificity of the αBβT hybrid oxygenase for these benzoates corresponded to that of BADOADP1, the parent from which the α subunit originated. In contrast, the apparent substrate specificity of the αTβB hybrid oxygenase differed slightly from that of TADOmt2 (3-chlorobenzoate > 3-methylbenzoate > benzoate ∼ 4-methylbenzoate > 4-chlorobenzoate > 2-methylbenzoate > 2-chlorobenzoate). Moreover, the αTβB hybrid catalyzed the 1,6-dihydroxylation of 2-methylbenzoate, not the 1,2-dihydroxylation catalyzed by the TADOmt2 parent. Finally, the turnover of this ortho-substituted benzoate was much better coupled to O2 utilization in the hybrid than in the parent. Overall, these results support the notion that the α subunit harbors the principal determinants of specificity in ring-hydroxylating dioxygenases. However, they also demonstrate that the β subunit contributes significantly to the enzyme's function.

Purification and Characterization of a Cysteine Endopeptidase fromVasconcellea quercifoliaA. St.-Hil. Latex Displaying High Substrate Specificity

2010 ◽  
Vol 58 (20) ◽  
pp. 11027-11035 ◽  
Author(s):  
M. José Torres ◽  
Sebastián A. Trejo ◽  
M. Inés Martin ◽  
Claudia L. Natalucci ◽  
Francesc X. Avilés ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Purification And Characterization ◽  
High Substrate ◽  
Cysteine Endopeptidase
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