Membrane lipids of rat liver lysosomes prepared by freeflow electrophoresis

Rat liver lysosomes were isolated by free-flow electrophoresis and were examined morphologically and enzymatically for purity. Their membrane fraction was prepared by osmotic shock and analyzed for cholesterol, phospholipids and fatty acids. The results were compared with the membrane fraction of Triton WR 1339-filled lysosomes and with mitochondria. The cholesterol content (0.269 M cholesterol per M lipid phosphorus), the sphingomyelin concentration (7.9% of total lipid phosphorus) and the degree of unsaturation of fatty acids (38–45%) were found to be intermediate between those of membranes of Triton WR 1339-filled lysosomes (“plasma membrane-like”) and mitochondria (“endoplasmic reticulum-like”). The similarity of these results with corresponding data for the Golgi apparatus support the present view concerning the formation of primary lysosomes via the Golgi apparatus. The drastic changes in the lipid composition found after overloading with Triton WR 1339 confirm that the plasma membrane participates in the formation of the secondary lysosomal membrane. The data presented here underline the significance of the analysis of membrane lipids in evaluating correlations between morphologically different but functionally closely related membrane types.     

A new method for the rapid separation of cell organelles

A rapid procedure is described for the separation of plant cell organelles from castor bean endosperm (Ricinus communis). This method is based on the reorientation of sucrose density gradients during centrifugation in a vertical rotor, thus resulting in a shorter path length and drastically reduced run times. Comparison to a separation by a standard procedure shows that, by using this method, equal resolution is possible in less than 10% spin time.     

Cardiolipin degradation by rat liver lysosomes.

The lysosomal subcellular fraction of rat liver contains acid hydrolases which can carry out the degradation of cardiolipin to yield water-soluble products and free fatty acids. The time course of appearance of the products indicates that the major catabolic route involves the sequential removal of three of the fatty acids, followed by hydrolysis to acylglycerophosphoryl glycerol (from which the fatty acid is subsequently removed) and d-glycerophosphate (which is hydrolysed to give free phosphate and glycerol). The phospholipase A activity responsible for removal of the first fatty acid is located in the lysosomal fraction.     

Neutral-sugar transport by rat liver lysosomes.

Transport of D-glucose was studied in Percoll-gradient-purified rat liver lysosomes. D-Glucose uptake had a Km of 22 mM and a t1/2 of approx. 30 s. D-Fucose, 2-deoxyglucose and methyl alpha-glucoside were the most effective competitors for uptake of D-glucose, although D-galactose, D-mannose, D-xylose and L-fucose also appeared to compete for uptake. L-Glucose was a poor competitor for uptake. No competition was observed with N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-glucuronic acid, N-acetylneuraminic acid, D-glucosamine or the amino acids L-glycine, L-lysine and L-proline. Uptake was unaffected by N-ethylmaleimide, dithiothreitol, KCl, NaCl, ATP/Mg or alteration of buffer pH. D-Glucose efflux from lysosomes was temperature-dependent, with a Q10 of 2.3, and was inhibited by cytochalasin B. Counter-transport could not be demonstrated. In contrast, L-fucose uptake had a Km of 65 mM and was largely unaffected by 5 M excess of neutral D-sugars. Both uptake and efflux of L-fucose were inhibited by cytochalasin B. It appears that lysosomes possess a facilitated transport system for D-glucose and perhaps other neutral D-sugars that is discrete from transport systems for acetylated and acidic sugars.     

A new method of DNA preparation for pulsed-field gel electrophoresis analysis

We have developed a new, labour-saving method of preparation, handling and treatment of DNA-containing agarose plugs for pulsed-field gel electrophoresis (PFGE). A plastic mould in which plugs are formed and supported during DNA purification and digestion was designed and successfully tested in a prototype device.     

Stabilization of rat liver lysosomes by new anti-inflammatory steroids in vitro

Steroid acid esters, synthesized by modifying the 17-ketol side chain of prednisolone, were tested for their in vitro ability to stabilize heavy mitochondrial lysosomes prepared from rat liver. Membrane stabilization was determined by assessing capability of steroids to decrease extrusion of the marker enzymes (acid phosphatase, beta-glucuronidase and aryl sulfatase) from lysosomes incubated in hypo-osmotic sucrose-Tris acetate buffer. Results indicated that prednisolone (1) significantly inhibited the lysosomal release of acid phosphatase as did the new anti-inflammatory steroid, methyl 20-dihydroprednisolonate. Methyl prednisolonate exhibited weak membrane stabilization capacities and 20-dihydroprednisolonic acid, a metabolic product of methyl 20-dihydroprednisolonate, showed virtually no membrane stabilization.     

A simple and rapid method for the preparation of apolipoproteins for electrophoresis.

A new method is described for the rapid preparation of apolipoproteins for polyacrylamide gel electrophoresis. It is suitable for all serum lipoproteins including chylomicrons. The procedure involves extraction with diethyl ether in the presence of trichloroacetic acid and sodium deoxycholate. The method gives an improved protein recovery, in particular with chylomicrons. In addition, samples do not require dialysis to remove salts (e.g., KBr) prior to processing; as a consequence, the procedure requires only 1 h. Due to this rapidity and the high yields, the procedure is superior to present methods utilizing ethanol-diethyl ether extraction.     

Hydrolysis of dolichyl esters by rat liver lysosomes

Dolichyl ester hydrolase activity is broadly distributed among the organs of the rat. The highest activity was found in spleen, brain, lung, and thyroid tissues, whereas this activity is very low in stomach and intestine. The esterase involved is localized to the lumen of lysosomes and, to some extent, in the plasma membranes. Hydrolysis occurs with both alpha-saturated and alpha-unsaturated polyisoprenes esterified with different fatty acids, but the rate of hydrolysis is strongly dependent on the nature of the substrate. The enzyme involved is inhibited by divalent cations, EDTA and EGTA and also by one of the products, dolichol. The esterase is activated by 3-[(3-cholamidopropyl) dimethylammonio]-1-propranesulfonic acid and taurodeoxycholate and inhibited by Triton X-100. Dolichyl esterase activity is completely inhibited by alpha- and beta-naphthyl acetate, phenylmethylsulfonyl fluoride, and beta-chloromethylmercurisulfate. These inhibitors, as well as the pH optimum for dolichyl ester hydrolysis, clearly differentiate the enzyme involved from cholesteryl esterase and triglyceride lipase. Microsomal phospholipase A hydrolyzes dolichyl esters at a slow rate only. In vivo labeling experiments with [3H]mevalonate demonstrated that newly synthesized dolichol is transported in esterified form to the lysosomes, where this lipid is slowly hydrolyzed by the esterase. The possibility is raised that the role of the fatty acyl moiety may be to target dolichol to its final location in the cell.