Crude Lysosomal Fraction Research Articles

Possible heterogeneity of the distribution of lysosomal marker enzymes among “lysosomal particles” of rat liver

The postnuclear supernatant and the crude lysosomal fraction were layered onto Ficoll gradients and centrifuged. After centrifugation ten fractions were ontained and each fraction was analyzed for protein, cytochrome oxidase, D-alanine oxidase and three to six acid hydrolases known as lysosomal enzymes. Latent and total activity were measured in the case of the acid hydrolases. Six marker lysosomal enzymes were distributed in broad patterns from the bottom to the top of the gradient on centrifugation of the postnuclear supernatant, though the markers of other particles were recovered in one or two fractions. After similar centrifugation of the crude lysosome fraction the ratios of the activity of three acid hydrolases differed, depending on their position on the gradient. These results indicate the presence of lysosomes heterogeneous in their content of acid hydrolases.

Activity of -amylase and -1,4-glucosidase in subfractions of human liver homogenates.

Activity of α-amylase and α-1,4-glucosidase was studied in subfractions of human liver homogenates obtained from operative biopsies in 22 patients operated on for uncomplicated gallstone disease. No activity of α-amylase was found in subfractions of homogenate. The activity of α-1,4-glucosidase was distributed in the same way as acid phosphatase and β-glucuronidase activity upon centrifugal fractionation of homogenate. The highest activity of α-1,4-glucosidase per unit of protein was found in the crude lysosomal fraction (L-fraction). The activity was easily solubilized from the L-fraction pellet. The lysosomal origin of the enzyme appears unambiguous considering its subcellular distribution and its acid hydrolytic properties.

Isolation and properties of lysosomes from dark-grown potato shoots

A method is described for the isolation of lysosomal fractions from dark-grown potato shoots using a single stage separation on a Ficoll gradient. Peaks of acid hydrolase activity consisting of acid phosphatase, phosphodiesterase, ribonuclease, carboxylic esterase and β-glycerophosphatase were well separated from peaks of mitochondrial and glyoxysomal enzymes. A heavy lysosomal fraction with particle diameters from 0.1 to 1.6 μ and density of 1.10 g cm(-3) containing relatively low hydrolase activity was distinguishable from a light fraction with diameters 0.025 to 0.6 μ and density of 1.07 g cm(-3) with a higher level of hydrolase activity. Both fractions appeared heterogeneous by electron microscopy, but the fine structure of the membranes of both heavy and light lysosomes was similar. The heavy lysosomal fraction was rich in autophagic vacuoles (secondary lysosomes) containing organelles and amorphous cytoplasmic material. Both fractions were rich in ribonucleic acid.Freezing and thawing, high speed blending and ultrasonication either singly or in combination solubilised a maximum of ca. 30% of the acid phosphatase from crude lysosomal fractions derived from dark-grown potato shoots. Treatment with Triton X-100 and deoxycholate released appreciably more enzyme activity but acetone and carbon tetrachloride failed to solubilise any acid phosphatase. Only detergent treatments gave marked overrecovery of enzyme and indicated structure-linked latency. Liberation of enzyme from lysosomes varied with pH and was almost complete at both extremes of pH. Crude snake venom was rapid and effective in solubilising acid phosphatase from lysosomal preparations, purified phospholipase A was less effective and phospholipases C and D had negligible effects. Phospholipase and venom mediated release of acid phosphatase was accompanied by the coincident release of an acid end-product. Gel filtration of acid phosphatase liberated from heavy and light lysosomal fractions by snake venom digestion revealed that each of these fractions was characterised by the presence of distinct molecular forms of the enzyme. The nature of the association of acid phosphatase with potato shoot lysosomes is discussed.

Cerebral lysosomes. IV. The regional and intracellular distribution of arylsulfatase and evidence for two populations of lysosomes in rat brain

1. The activities of arylsulfatase and N-acetyl-β- d-glucosaminidase were determined in 6 regions of rat brain. 2. The degree of structural latency of the two hydrolases was estimated. 3. The intracellular distribution of arylsulfatase was studied in the cortex, thalamus and striatum and a lysosomal localization could be established. 4. Centrifugation of the cortical crude lysosomal fraction on continuous sucrose density gradients revealed the existence of a minority population of lysosomes which, unlike the bulk, sedimented through the gradient and could be recovered in pellet form. Proportionately more sulfatase than glucosaminidase was found in this lysosomal population. 5. The main lysosomal population was well separated from mitochondria but was contaminated by the membranes bearing acetylcholine esterase and soluble lactic dehydrogenase. The pellet fraction was virtually free of contamination. 6. Evidence for the presence of more than one population of lysosomes in the thalamus and the hypothalamus of rat brain was also obtained.