CARBOXYLATION OF CROTONYL-CoA BY MAMMALIAN ENZYME SYSTEMS

1966 ◽  
Vol 44 (6) ◽  
pp. 861-878 ◽  
Author(s):  
E. Reno Tustanoff ◽  
Joseph R. Stern

In a crude dialyzed ammonium sulfate fraction (35–65% saturation) of rat liver, carbon dioxide fixation into crotonyl-CoA took place when the test system was supplemented with ATP, Mn++, glutathione, Tris–HCl buffer (pH 7.0), and KH14CO3. The products of this reaction were identified after hydrolysis as glutaconic, β-hydroxyglutaric, maionic, and 2-ethylmalonic acids. The isolation and characterization of 5-14C-glutaconyl-CoA indicated a γ-carboxylation reaction. In the presence of endogenous enoyl-CoA hydratase, crotonyl-CoA was carboxylated more readily than β-hydroxybutyryl-CoA, suggesting that the unsaturated acyl compound was the natural substrate for the enzyme system. Carboxylation of crotonyl-CoA was greatly enhanced when liver extracts were prepared from either fasted or alloxan-diabetic rats. Fixation of carbon dioxide into crotonyl-CoA was also demonstrated with an amorphous preparation of propionyl-CoA carboxylase from pig heart. The products of this reaction were identified as radioactive malonic acid and unlabeled acetaldehyde, compounds which resulted from the alkaline hydrolysis of 2-ethylidenemalonyl-CoA, formed by the α-carboxylation of crotonyl-CoA. Evidence is presented that both α- and γ-carboxylation are catalyzed by the crude liver preparation.

2019 ◽  
Vol 33 (4) ◽  
pp. 303-324 ◽  
Author(s):  
Rutairat Suttisuwan ◽  
Saranya Phunpruch ◽  
Tanatorn Saisavoey ◽  
Papassara Sangtanoo ◽  
Nuttha Thongchul ◽  
...  

1990 ◽  
Vol 269 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Y Homma ◽  
Y Emori ◽  
F Shibasaki ◽  
K Suzuki ◽  
T Takenawa

A novel bovine spleen phosphoinositide-specific phospholipase C (PLC) has been identified with respect to immunoreactivity with four independent antibodies against each of the PLC isoenzymes, and purified to near homogeneity by sequential column chromatography. Spleen contains three of the isoenzymes: two different gamma-types [gamma 1 and gamma 2, originally named as PLC-gamma [Rhee, Suh, Ryu & Lee (1989) Science 244, 546-550] and PLC-IV [Emori, Homma, Sorimachi, Kawasaki, Nakanishi, Suzuki & Takenawa (1989) J. Biol. Chem. 264, 21885-21890] respectively] and delta-type of the enzyme, but PLC-gamma 1 is separated from the PLC-gamma 2 pool by the first DEAE-cellulose column chromatography. Subsequently, PLC-delta is dissociated on the third heparin-Sepharose column chromatography. The purified enzyme has a molecular mass of 145 kDa on SDS/polyacrylamide-gel electrophoresis and a specific activity of 12.8 mumol/min per mg with phosphatidylinositol 4,5-bisphosphate as substrate. This enzyme activity is dependent on Ca2+ for hydrolysis of all these phosphoinositides. None of the other phospholipids examined could be its substrate at any concentration of Ca2+. The optimal pH of the enzyme is slightly acidic (pH 5.0-6.5).


1989 ◽  
Vol 261 (3) ◽  
pp. 811-818 ◽  
Author(s):  
N M Hooper ◽  
A J Turner

Renal dipeptidase (EC 3.4.13.11) has been solubilized from pig kidney microvillar membranes with n-octyl-beta-D-glucopyranoside and then purified by affinity chromatography on cilastatin-Sepharose. The enzyme exists as a disulphide-linked dimer of two identical subunits of Mr 45,000 each. The purified dipeptidase partitioned into the detergent-rich phase upon phase separation in Triton X-114 and reconstituted into liposomes consistent with the presence of the glycosyl-phosphatidylinositol membrane anchor. The N-terminal amino acid sequence of the amphipathic, detergent-solubilized, form of renal dipeptidase was identical with that of the hydrophilic, phospholipase-solubilized, form, locating the membrane anchor at the C-terminus of the protein. The glycosyl-phosphatidylinositol anchor of both purified and microvillar membrane renal dipeptidase was a substrate for an activity in pig plasma which displayed properties similar to those of a previously described phospholipase D. The cross-reacting determinant of the glycosyl-phosphatidylinositol anchor was generated by incubation of purified renal dipeptidase with bacterial phosphatidylinositol-specific phospholipase c, whereas the anchor-degrading activity in plasma failed to generate this determinant.


1985 ◽  
Vol 40 (1) ◽  
pp. 32-38
Author(s):  
Alois Haas ◽  
Wolfgang Wanzke ◽  
Nathan Welcman

Hydrolysis of CF3-nClnSCl with water yields the thiosulfinates CF3-nClnSS(O)CF3-nCln and thiosulfonates CF3-nClnSSO2CF3-nCln as stable intermediates. They were synthesized on a preparative scale by special reactions and characterized. A new mechanism, based upon additional reactions of the isolated products, is discussed and extended to the reaction of chlorine with water


1974 ◽  
Vol 7 (2) ◽  
pp. 239-274 ◽  
Author(s):  
Peter Leth Jørgensen

A satisfactory understanding of the functions of the sodium pump, the system responsible for the active transport of sodium and potassium, require the isolation and characterization of its protein and lipid components which are integrated in the structure of the cell membrane. The enzyme system (Na+ + K+)-ATPase, is located in membrane fragments and behaves in the test tube like the transport system in the intact cell membrane (Skou,1957) Purified preparations of this enzyme will contain some, if not all, of the components of the sodium pump.


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