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M Record

Publications and source records attributed to M Record.

40 records · Page 3Linked to original sources
PubMed1977

Phospholipases A1 and A2 in subcellular fractions and plasma membranes of Krebs II ascites cells.

A method is described for the localization and characterization of phospholipases A1 and A2 (EC3.1.1.4) in Krebs II ascites cells, particularly in the plasma membranes. Cells were lysed with a Dounce homogenizer in an isotonic sucrose medium. Plasma membranes sediment with mitochondria and lysosomes during subcellular fractionation and are finally isolated on a continuous sucrose gradient. The membranes are localized at two levels in the gradient, at densities of 1.06 and 1.15, in which 5'-nucleotidase (EC 3.1.3.5) activity exhibits a 9- and 21-fold purification, respectively. Total contamination by endoplasmic reticulum, lysosomes, and mitochondria is 17 percent for the low-density membrane fraction and 25 percent for the high-density fraction. The phospholipases A present in Krebs II cells are active at pH 4.0 and pH 7.5. At the 2 pH values, they have A1 and A2 specificities. The intracellular distribution of acidic forms is comparable to that of acid phosphatase (EC 3.1.3.1), while neutral forms are localized like lactate dehydrogenase (EC 1.1.1.27). A small proportion of neutral phospholipase A2 has the same repartition on the sucrose gradient as nicotinamide adenine dinucleotide diaphorase (EF 1.6.4.3), an endoplasmic reticulum marker, and as 5'-nucleotidase, a plasma membrane marker.

Animals↗
PubMedDate not supplied

Characterization of exosome subpopulations from RBL-2H3 cells using fluorescent lipids.

Fluorescent lipid probes were used to track lipid trafficking between parent RBL cells and exosomes. We have checked the intracellular labeling of exosomes ("in vivo labeling") from parent cell incubated with either Bodipy-Cer, Bodipy-PC, or NBD-PC. Bodipy-PC labeled equally cells and exosomes, whereas Bodipy-Cer, a Golgi marker, was enriched in exosomes. Golgi membranes participated effectively in exosome biogenesis since cell incubation with brefeldin A leads to a modified phospholipid/protein ratio in exosomes. At the opposite, NBD-PC, a plasma membrane marker weakly labeled exosome membranes. Sorting of subpopulations indicated that the MHC-II containing exosomes were enriched in Bodipy-PC, whereas tetraspanin(CD 63 or CD81)-containing exosomes are essentially labeled with Bodipy-Cer and Bodipy-PC. These results indicated that RBL released two main subpopulations of exosomes that can be discriminated by their protein and lipid contents. When the bulk of exosomes was labeled after their purification ("in vitro labeling") with either of the above-mentioned lipid probes, the Bodipy-Cer was the only one to incorporate noticeably in all the subpopulations, indicating that the previous results obtained during "in vivo labeling" monitored real intracellular lipid trafficking between organelles and exosomes. Bodipy-Cer was further used as a tool to measure the respective amounts of each subpopulations. CD63, MHC II, and CD81-containing exosomes accounted for 47%, 32%, and 21%, respectively, of total exosomes.

Animals↗
PubMedDate not supplied

Intracellular location of acetyltransferase in the remodeling pathway of PAF biosynthesis in undifferentiated human leukemic cells (HL-60).

In order to determine the site of PAF synthesis via the remodeling pathway in undifferentiated HL-60 cells, a subcellular fractionation procedure (Ribbes et al., 1985) was adapted to separate various membrane fractions and cellular organelles of HL-60 cells across a Percoll gradient. The profile of alkyllyso-GPC:acetyl-CoA acetyltransferase activity closely paralleled the subcellular distribution of the endoplasmic reticulum marker, NADH dehydrogenase. These data indicate the acetyltransferase in the remodeling pathway of PAF synthesis is located at the same cellular site as the acyltransferase(s) that catalyze the transfer of long-chain acyl moieties from acyl-CoAs to lyso-phospholipids. Also the acetyltransferase in undifferentiated HL-60 cells appears to have a similar intracellular distribution as reported for human neutrophils.

Acetyltransferases↗