Kinetic and topographical studies of the phosphatidylcholine: ceramide choline phosphotransferase in plasma membrane particles from mouse ascites cells.

The content of endogenous phospholipids in plasma membrane preparations from Ehrlich ascites cells was depleted by exposure to phospholipase C. The enzyme catalyzing the phosphatidylcholine: ceramide choline phosphotransferase reaction was inactivated by this treatment. However, the activity could be restored with exogenous phosphatidylcholines, demonstrating the dependence of the reaction upon the presence of this substrate. Phosphatidylcholines containing unsaturated fatty acids were 10-fold more effective substrates than the saturated molecular species. The activation energy of the reaction was determined to be 17.2 kcal/mol. Selective trypsin treatment of the plasma membranes suggests that the cholinephosphotransferase may have an asymmetric orientation. The reaction kinetics followed a rate equation similar to that of the ping-pong reaction mechanism, which suggests the formation of an enzyme-bound intermediate of the phosphocholine group being transferred. These results are discussed in terms of possible biological functions of the enzyme.

The phosphorylcholine acceptor in the phosphatidylcholine:ceramide cholinephosphotransferase reaction. Is the enzyme a transferase or a hydrolase?

The cholinephosphotransferase reaction is shown to be catalyzed by an enzyme which has no hydrolytic activity and which is different from a phospholipase C type activity also present in these plasma membrane preparations. Diacylglycerols and sphingosine, at a concentration above 0.4 mM, are effective inhibitors of sphingomyelin formation in the presence of 0.3 mM free ceramide, the true acceptor in this reaction. Free sphingosine is not an acceptor for the cholinephosphate group, as the anticipated reaction product, sphingosylphosphocholine , could not be detected. Sphingosine inhibition may result from its structural similarity to the natural substrates of the reaction, ceramide and diacylglycerols. From the data obtained with cholesterol, triacylglycerols, acetylated ( triacetyl ) sphingosine and acetylated ceramides used as potential inhibitors of the reaction it is concluded that the free hydroxyl group at C1 of the sphingosine backbone or of the glycerol moiety of diacylglycerols and a non-polar residue consisting of an aliphatic chain were prerequisites for inhibitory activity. These results are discussed in terms of substrate specificity of the enzyme catalyzing the transfer reaction. Some of the factors influencing the regulation of the phosphatidylcholine/sphingomyelin ratio in the plasma membrane were related to the topography of sphingomyelin in the outer half-layer of the plasma membrane.

[Surgical interventions on the gallbladder and the biliary tract in the aged]. / Chirurgische Eingriffe an der Gallenblase und dem Gallengangssystem bei alten Menschen.

The surgery of the gall bladder by gallstones, bile duct and alterations of the duodenal papilla are conducted with an increase of lethality. This increased lethality is caused by accompanying ill effects, especially chronical pancreatitis, cholangitis, disturbances of the liver. The indications to operate old patients are occlusions of duodenal papilla relapsing colics with or less occlusing icterus, emphysema of gall bladder or perforation. A careful narcosis is necessary by disease of circulation, diabetes, bronchitis and emphysema of the lung. Simple cholestectomies in old patients have a mortality of 0-1%. Interventions on bile ducts, transduodenal papillotomia, choledochoduodenotomy have an mortality of 4-8%. The intraoperative cholangiography is always necessary. Operations on bile duct are finished by inserting a T-drain. The distal portion of the T-drain are not emissed through the duodenal papilla, because a pancreatitis can be released. The principal postoperative complication is the pancreatitis, consumption coagulopathy, bronchopneumonia, cholangitis with intermitting fever and injuries of the liver parenchym. The persisting pancreatitis can be treated with infusions.

The role of endogenous phosphatidylcholine and ceramide in the biosynthesis of sphingomyelin in mouse fibroblasts.

The intracellular location of sphingomyelin formation via the cholinephosphotransferase reaction from both endogenous an exogenous phosphatidylcholine and ceramide substrates has been studied in the subcellular membrane fractions prepared from mouse fibroblasts. The enzyme was found to be located in both the plasma membrane and the Golgi fractions. Activity in the Golgi fraction was stimulated to a greater extent by the addition of exogenous ceramide than was the activity in the plasma membrane fraction. It is concluded that endogenous phosphatidylcholine is available to the cholinephosphotransferase at saturating concentration and, therefore, is not rate-limiting. In contrast, the very low concentration of endogenous ceramide seems to limit the reaction rate, necessitating supplementation with exogenous material Both endogenous substrates are shown to be utilized in an intramembranous rather than an intermembranous reaction. The capacity of the plasma membrane fraction to synthesize sphingomyelin from endogenous phosphatidylcholine and ceramide was found to be sufficiently high to account for the rate of net synthesis of plasma membrane-bound sphingomyelin observed in the logarithmically multiplying cell culture. In contrast, the Golgi fraction displayed only 26% of the expected capacity, but it was stimulated 6-fold by the addition of exogenous ceramide. These results demonstrate that the total cellular sphingomyelin of the mouse fibroblasts can be provided via the cholinephosphotransferase pathways and that the plasma membrane and the Golgi fraction are most probably the intracellular sites of sphingomyelin biosynthesis.

The formation of sphingomyelin from phosphatidylcholine in plasma membrane preparations from mouse fibroblasts.

The enzymatic formation of radioactive sphingomyelin from [14C]choline-labeled phosphatidylcholine was demonstrated to reside exclusively in the plasma membrane fraction of mouse fibroblasts. This activity has several properties in common with the phosphatidylcholine ceramide phosphocholine transferase of mouse liver microsomes. The enzyme has little if any phospholipase C activity and isotope dilution experiments suggest that phosphatidylcholine is the substrate rather than it is converted to CDP choline, phosphocholine, free choline or glycerophosphocholine prior to the transfer reaction. The activity is stimulated by the addition of bovine serum albumin and MnCl2 to the incubation mixtures. The plasma membrane localization of the enzyme suggests that it may have a central role in the biosynthetic pathways for sphingomyelin in mouse fibroblasts.

A biosynthetic method for the preparation of high specific activity 32P-labeled phospholipids.

32P-Labeled phospholipids with specific activities up to 400 mCi/mmole as well as [32P]CDP-choline were prepared by cultivation of mouse fibroblasts or mouse Ehrlich ascites cells in the presence of [32P]orthophosphate. The method was also used to prepare [methyl-3H]choline-labeled glycerophospholipids from [3H]choline. The yields and the specific activities of the phospholipids were significantly lower when preparations of ox white blood cells were used.

[Spontaneous peripheral proteolysis/2nd communication: Effect of peripheral proteolysis (author's transl)]. / Spontane periphere Proteolyse. 2. Wirkung der peripheren Proteolyse

With the modified Astrup-plate method a spontaneous peripheral proteolysis was often found in chronical infections of the skin and mucous membranes. The origin of this proteolysis and its effects on the bacterial growth and wound healing were discussed.

[Spontaneous Peripheral Proteolysis/1st Communication: method and results of clinical examinations (author's transl)]. / Spontane periphere Proteolyse. 1. Mitteilung: Methodik und klinische Untersuchungsergebnisse

With a modificated Astrup-fibrin plate-method also an inhibition of proteolysis can be registrated. In various medical areas a spontaneous peripheral proteolysis had been found, especially so in chronical bacterial infections.

[Spontaneous peripheral proteolysis as a disturbing factor (author's transl)]. / Die spontane periphere Proteolyse als Störfaktor

With a modification of the Astrup - fibrin - plate method about 300 patients were investigated. In chronic infections of the skin and mucous membranes a spontaneous peripheral proteolysis was found. There is a connection between infections, proteolysis and chronic continuance.