Mixed micelles of sphingomyelin and phosphatidylcholine with nonionic surfactants. Effect of temperature and surfactant polydispersity.

Mixed micelle formation of the polydisperse nonionic surfactant Triton X-100 as well as its homogeneous analogue, p-(1,1,3,3-tetramethylbutyl)-phenoxynonaoxyethylene glycol (OPE-9), with bovine brain sphingomyelin or dipalmitoyl phosphatidylcholine has been characterized by column chromatography on 6% agarose. At 40 degrees C, mixtures of OPE-9 and either sphingomyelin or dipalmitoyl phosphatidylcholine give a narrow size distribution for mixed micelles. A this temperature the size distribution of Triton X-100-containing mixed micelles is complicated because of the polydispersity of the oxyethylene chains. At 20 degrees C narrow size distributions are observed for mixed micelles of sphingomyelin/Triton X-100 and sphingomyelin/OPE-9 up to at least 0.06 mol fraction of lipid. For dipalmitoyl phosphatidylcholine this is observed only with OPE-9. At intermediate mol fractions of lipid (around 0.25), two populations of mixed micelles exist for sphingomyelin/Trition X-100, sphingomyelin/OPE-9, and dipalmitoyl phosphatidylcholine/OPE-9. At high mol fractions of lipid only one population of mixed micelles again exists. At 20 degrees C, sphingoymelin forms a clear solution with Triton X-100 and OPE-9 to a lipid mol fraction of at least 0.46 and 0.67, respectively. Dipalmitoyl phosphatidylcholine forms a clear solution with OPE-9 to a lipid mol fraction of at least 0.57 at the same temperature. Triton X-100 and dipalmitoyl phosphatidylcholine do not form stable, clear solutions at 20 degrees C unless the lipid mol fraction is extremely low. These results show that surfactant polydispersity and temperature are important determinants in the solubilization of lipids by nonionic surfactants. It is also shown that pure surfactant micelles and lipid/surfactant mixed micelles do not co-exist in the same solution.     

Action of cobra venom phospholipase A2 on the gel and liquid crystalline states of dimyristoyl and dipalmitoyl phosphatidylcholine vesicles

The activity of phospholipase A2 from cobra venom toward phospholipid in single-walled, sonicated vesicles was analyzed, particularly with respect to its activity toward the saturated phosphatidylcholines in the gel and liquid crystalline states. When egg phosphatidylcholine vesicles are used as substrate, the phospholipase has an apparent Km of 4.4 mM, an apparent Vmax of 100 mumol min-1 mg-1 of protein, and a pH optimum of 5.0 at 40 degrees C. The phospholipase hydrolyzed the gel state of dimyristoyl phosphatidylcholine vesicles and dipalmitoyl phosphatidylcholine vesicles at a rate 2 to 3 times greater than the liquid crystalline state, taking into account temperature effects on the enzymatic reaction itself. The results suggest that, toward sonicated vesicles, there is no specific enhancement of the rate when the both liquid crystalline and gel states are present together, as has been suggested to occur for multibilayers studied with other phospholipases. An apparent stimulation of activity as the reaction proceeded was observed above the phase transition temperature. This might be attributed to an increase in the phase transition temperature caused by free fatty acids so that, in the presence of reaction products, the enzyme is actually hydrolyzing gel state phospholipid which was found to be the preferred lipid state for phospholipase activity.     

A rapid and sensitive method for the detection of histone peptides

Fluorescamine has been used to obtain a peptide map of a mixture of histones (H₃, H₂A, H₂B, and H₄) prepared from oocytes of Xenopus laevis. Fluorescamine was found to be more sensitive than o-phthalaldehyde or ninhydrin-Cd for the detection of peptide fragments obtained from tryptic digestion of oocyte histones of X. laevis and the peptic digestion of the β chain of insulin. Using the β chain of insulin for a comparison, the 8 major peptide fragments could be separated by electrophoresis within 30 min and were detectable at the picomols level. Some 70 peptide spots of X. laevis oocyte histones were resolved, thus permitting the analysis of this complex mixture of polypeptides without the need for prior separation.     

Analysis of phospholipase C (Bacillus cereus) action toward mixed micelles of phospholipid and surfactant.

The action of phospholipase C (Bacillus cereus) toward mixed micelles of phosphatidylcholine and the nonionic surfactant Triton X-100 is analyzed according to the “surfaceas-cofactor” kinetic scheme recently proposed for characterizing the action of lipolytic enzymes [Deems, R. A., Eaton, B. R., and Dennis, E. A. (1975) J. Biol. Chem.250, 9013–9020]. According to this scheme, the enzyme first associates with the surface or mixed micelles, where the dissociation constant is K_s^A. The enzyme, now part of the mixed micelle surface, then binds the substrate phospholipid molecule in its active site and this binding is related to the Michaelis constant, K_m^B. The surface, or mixed micelles in this scheme, behaves kinetically as a cofactor in that, under initial rate conditions, the surface properties of the mixed micelles are virtually unchanged after catalysis. For phospholipase C with egg phosphatidylcholine as substrate, it was found that at pH 6.4 (the pH optimum for the enzyme) and 40 °C, V is about 2 × 10³ μmol min^?1 (mg of protein)^?1. K_s^A is about 2 mm and K_m^B is 1 to 2 × 10^?10 mol cm^?2. The kinetic constants for phospholipase C are compared with those previously reported for phospholipase A₂ and the membrane-bound enzyme phosphatidylserine decarboxylase determined under similar conditions.     

Molecular changes in cell surface membranes resulting from trypsinization of sarcoma 180 tumor cells

Sarcoma-180 tumor cells in culture or grown as an ascites form in the CD-1 mouse have been subjected to mild trypsinization procedures in order to study morphological and molecular changes resulting from proteolysis. The cells attached to a substratum become rounded within 20 min and most undergo cell division, but they do not detach from the substratum. Removal of trypsin permits the cells to go back to their original spindle shape over an 8–20 h period.Surface membranes were isolated from trypsinized ascites and cultured cells and subjected to dodecyl sulfate-acrylamide gel electrophoresis. Both cell types showed the same two kinds of changes in electrophoretic patterns. First, there was a loss of glycoproteins from both cell types, even though they show different complements of cell surface glycoproteins. Second, there is a loss of high molecular weight polypeptides, which have previously been suggested to play a role in membrane stabilization and cell shape. These results further implicate these polypeptides in the control of cell morphology and offer circumstantial evidence for transmembrane interactions of surface glycoproteins with the high molecular weight polypeptides as a factor in controlling cell morphology.     

Improved histological localization of GABA-transaminase activity in rat cerebellar cortex after aldehyde fixation

Summary A method for the chemical fixation of the enzyme GABA-transaminase in nervous tissue is described. It is shown that after perfusion with a formaldehyde/glutaraldehyde fixative, activity of the enzyme in cerebellar cortex is demonstrable whilst cellular morphology is preserved. Results from the improved technique have shown new sites of GABA-transaminase activity in cerebellar cortex. In view of these results a special function for glial cells in this area of brain has been suggested.