The lysosome periphery: biochemical and electrokinetic properties of the tritosome surface.
Normal rat liver lysosomes were isolated by the technique of loading with Triton WR-1339. Purity of the preparation was monitored with marker enzymes; a high enrichment in acid hydrolases was obtained in the tritosome fraction. In 0.0145 M NaCl, 4.5% sorbitol, 0.6 mM NaHCO(3), pH 7.2 at 25 degrees C the tritosomes had an electrophoretic mobility of -1.77 +/- 0.02 microm/s/V/cm, a zeta potential of 23.2 mV, a surface charge of 1970 esu/cm(2), and 33,000 electrons per particle surface assuming a tritosome diameter of 5 x 10(-7) m. Treatment of the tritosomes with 50 microg neuraminidase/mg tritosome protein lowered the electrophoretic mobility of the tritosome to -1.23 +/- 0.02 microm/s/V/cm under the same conditions and caused the release of 2.01 microg sialic acid/mg tritosome protein. Treatment of the tritosomes with hyaluronidase did not affect their electrophoretic mobility, while trypsin treatment elevated the net negative electrophoretic mobility of the tritosomes. Tritosome electrophoretic mobilities indicated a homogeneous tritosome population and varied greatly with ionic strength of the suspending media. pH vs. electrophoretic mobility curves indicated the tritosome periphery to contain an acid-dissociable group which likely represents the carboxyl group of N-acetylneuraminic acid; this was not conclusively proven, however, since the tritosomes lysed below a pH of 4 in the present system. Total tritosome carbohydrate (anthrone-positive material as glucose equivalents) was 0.19 mg/mg tritosome protein while total sialic acid was 3.8 microg (11.4 nmol)/mg tritosome protein. A tritosome "membrane" fraction was prepared by osmotic shock, homogenization, and sedimentation. Approximately 25% of the total tritosome protein was present in this fraction. Analysis by gas-liquid chromatography and amino acid analyzer showed the following carbohydrate composition of the tritosome membrane fraction (in microgram per milligram tritosome membrane protein): N-acetylneuraminic acid, 14.8 +/- 3; glucosamine, 24 +/- 3; galactosamine, 10 +/- 2; glucose, 21 +/- 2; galactose, 26 +/- 2; mannose, 31 +/- 5; fucose, 7 +/- 1; xylose, 0; and arabinose, 0. The results indicate that the tritosome periphery is characterized by external terminal sialic acid residues and an extensive complement of glycoconjugates. Essentially all the tritosome N-acetylneuraminic acid is located in the membrane and about 53% of it is neuraminidase susceptible.
D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D002499:Centrifugation, Density Gradient; D004586:Electrophoresis; D004912:Erythrocytes; D006801:Humans; D006821:Hyaluronoglucosaminidase; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008247:Lysosomes / Q000032:analysis / Q000378:metabolism*; D008297:Male; D008566:Membranes / Q000032:analysis / Q000378:metabolism; D009439:Neuraminidase; D009994:Osmolar Concentration; D011199:Potentiometry; D051381:Rats; D013499:Surface Properties; D013737:Testis / Q000201:enzymology; D014316:Tritium; D014357:Trypsin
D002241:Carbohydrates; D014316:Tritium; D009439:Neuraminidase; D006821:Hyaluronoglucosaminidase; D014357:Trypsin