Analytical subcellular fractionation of cultivated mouse resident peritoneal macrophages

Abstract

Resident peritoneal macrophages of the mouse, cultivated for 3 d, have been studied by quantitative subcellular fractionation using differential centrifugation and density equilibration in linear gradients of sucrose. Density equilibration experiments were carried out on untreated cytoplasmic extracts, on cytoplasmic extracts treated with digitonin or sodium pyrophosphate, and on cytoplasmic extracts derived from cells cultivated for 24 h in the presence of Triton WR-1339. The enzyme distributions obtained distinguished six typical behaviors characteristic of distinct subcellular entities. Acid α-galactosidase and other acid hydrolases displayed the highest average velocity of sedimentation and equilibrium density. Culturing in a medium that contained Triton WR-1339 markedly decreased their density, most likely as a result of Triton WR-1339 accumulation within lysosomes. Cytochrome c oxidase and the sedimentable activity of malate dehydrogenase showed a narrow density distribution centered around 1.17, very similar under all the experimental situations; their rate of sedimentation fell within the range expected for mitochondria. Catalase was particle-bound and exhibited structure-linked latency (80 percent); it was released in soluble and fully active form by digitonin, but this required a much higher concentration than in the case of lysosomal enzymes. Differences relative to all the other enzymes studied suggest the existence of a particular species of organelles, distinctly smaller than mitochondria, and possibly related to peroxisomes. Many enzymes were microsomal in the sense that the specific activities, but not the yields, were greater in microsomes than in other fractions obtained by differential centrifugation. These enzymes were distinguished in three groups by their properties in density equilibration experiments. NAD glycohydrolase, alkaline phosphodiesterase I, and 5'-nucleotidase had low equilibrium densities but became noticeably more dense after addition of digitonin. The other microsomal enzymes were not shifted by digitonin, in particular N-acetylglucosaminyltransferase and galactosyltransferase, which otherwise equilibrated at the same position in the gradient. We assign the digitonin-sensitive enzymes to plasma membranes and possibly to related endomembranes of the cells, and the two glycosyltransferases to elements derived from the Golgi apparatus. Finally, α-glucosidase, sulphatase C, NADH cytochrome c reductase, NADPH cytochrome c reductase, and mannosyltransferase, equilibrated at a relatively high density but were shifted to lower density values after addition of sodium pyrophosphate. These properties support their association with elements derived from the endoplasmic reticulum.

Paraoxonase 2 (PON2) in the mouse central nervous system: A neuroprotective role?

“…Briefly, cells were collected and homogenized as described by Darte and Beaufay, (1983) in ice-cold lysis buffer containing 250 mM sucrose, 50 mM Tris–HCl (pH 7.4, 18°C), 5 mM MgCl 2 , 1 mM DDT and 1 mM PMSF, using a tight fitting Teflon pestle, and then disrupted by ten slow passages through a syringe needle (16G × 0.5 cm). After centrifugation at 800 × g at 4°C for 15 min, the supernatant was removed and saved, and the pellet was resuspended for another series of five passages through the needle.…”

Isolation of plasma membrane from human blood monocytes

“…A very small percentage of the enzyme is present in the Golgi [see 7, 611. b) The detection of NAD' nucleosidase activity in a distribution profile paralleling that of 5'-nucleotidase. The "AD-splitting enzyme' is a plasma membrane ectoenzyme of mouse splenic and peritoneal macrophages [8,20,62]. The present report establishes NAD' nucleosidase as a plasma membrane enzyme of human monocytes.…”

“…cm-3 was obtained for rabbit alveolar macrophage plasma membrane [6] whilst lesser shifts (up to + 0.035 g . cm-3 , were obtained for mouse peritoneal macrophage [8], guinea pig peritoneal macrophage [11] and rat liver [72, 731 plasma membranes. These differences may reflect species variations in the Cholesterol concentration of the membrane.…”

“…Detailed analytical subcellular fractionation, in which relatively weI1-defined plasma membranes were obtained, has been carried out on rabbit alveolar macrophages [3 -61, mouse peritoneal macrophages [7,8] and guinea pig peritoneal macrophages 19 -1 I]. Other investigations involving subcellular fractionation included studies on rabbit alveolar macrophages [12-141, guinea pig peritoneal macrophages [15], mouse peritoneal macrophages [16 -211, mouse bonemarrow-derived macrophages [22] and on the mouse macrophage-like cell line J774 [23].…”

The cytotoxicity of chrysotile asbestos fibers to pulmonary alveolar macrophages I. Effects of inhibitors of ADP-ribosyl transferase

“…However, the failure by 5 mM 3-AB to protect PAM exposed to asbestos fibers casts doubt on a major contribution of the mono-ADPRT system in the cytotoxic reaction (Rankin et al, 1987). As for NAD glycohydrolases, a characteristic marker of peritoneal and alveolar macrophages (Artman et al, 1978;Darte et al, 1983), low concentrations of 3-AB (2 mM) do not inhibit this enzyme (Williams et al, 1983). Comparatively membrane-bound NADase can be inhibited by high concentrations of NAM (Ricci et al, 1986).…”