The isolation of lysosomes from normal rat liver by affinity chromatography

Lysosomes from normal rat liver were isolated by affinity chromatography using Sepharose-bound Ricinus communis agglutinins I + II. Characterization of the lysosomal fraction by marker enzymes showed--compared with the homogenate--an enrichment in: acid phosphatase and arylsulfatase about 30- to 60-fold, the tartrate-sensitive acid phosphatase about 95-fold, whereas beta-D-glucosidase, beta-D-galactosidase and sphingomyelinase showed a much higher enrichment of 170- to 260-fold. Marker enzymes for other cell organelles were not detectable. The phospholipid pattern and optical control with electron microscopy gave further indications that the isolated fractions were very rich in lysosomes. A comparison of the phospholipid compositions of plasma membranes isolated from normal rat liver and membranes from the isolated fractions of lysosomes, showed that they were quite different; in particular bis(monoacylglycero)phosphate, which we found to be a typical lysosomal phospholipid, was absent in plasma membranes.     

A detergent-independent procedure for the isolation of gap junctions from rat liver

In this paper, the isolation of rat liver gap junctions from alkali-extracted rat liver plasma membranes is described. The purification is significantly more rapid than the commonly used detergent-based approaches and is subject to less variability. The gap junctions isolated by this method are comprised of a 27,000-Da polypeptide previously identified as the major gap junction polypeptide. The isolated gap junctions have the characteristic double-membrane organization and subunit structure observed in vivo. The protein yield is from 8 to 10 micrograms/g of liver (wet weight), about a 10-fold increase in recovery over that of earlier isolation procedures. With the availability of increased amounts of material, antibodies were raised to the liver gap junction polypeptide. Immunofluorescence localization of these antibodies on rat liver sections revealed a distribution consistent with that expected from electron microscopic analysis of liver thin sections. Double diffusion of antibody against solubilized gap junctions in detergent-containing gels resulted in the formation of precipitin arcs, suggesting response to multiple determinants. Antibody binding to the 27,000-Da gap junction polypeptide was demonstrated by immunoblot analysis of sodium dodecyl sulfate-polyacrylamide gels containing rat liver plasma membranes and isolated gap junctions. These results confirm the identification of the 27,000-Da polypeptide as the major protein component of gap junctions.     

A simple and rapid procedure for high-yield isolation of essentially undegraded free and membrane-bound polysomes from rat liver

A procedure is described for the preparation of free and membrane-bound polysomes from rat liver. The procedure involves: differential centrifugation of liver homogenate to separate free and membrane-bound polysomes; treatment of the membrane-bound polysome fraction with a detergent to release bound polysomes from membranes; and magnesium precipitation of both classes of polysomes. Free and bound polysomes prepared in this manner were essentially undegraded and highly active in cell-free protein synthesis. The recovery of polysomes was nearly quantitative and the distribution between the free and membrane-bound state was 41 and 59%, respectively. Polypeptides synthesized in vitro by the free and membrane-bound polysomes were quite different. The majority (81-84%) of mRNA activities of two secretory proteins (albumin and transferrin) were recovered in the membrane-bound polysomes, whereas the majority (81-85%) of mRNA activities of two cytosolic [aldolase B, EC 4.1.2.13, and argininosuccinate synthetase, EC 6.3.4.5], one mitochondrial [ornithine carbamoyltransferase, EC 2.1.3.3] and one peroxisomal [catalase, EC 1.11.1.6] proteins were recovered in the free polysomes. A polysome class synthesizing ornithine carbamoyltransferase was purified 42-fold from the free polysomes by immunoprecipitation. The procedure is rapid (4-5 h) and reproducible, and provides a nearly quantitative means of separating the two classes of polysomes.     

Isolation of highly purified lysosomes from rat liver: identification of electron carrier components on lysosomal membranes.

Using Percoll density gradient centrifugation after treatment of the postnuclear supernatant (PNS) with 1 mM Ca2+ to swell and lighten mitochondria, we isolated highly purified lysosomes (dextranosomes) in high yield (25%) from the livers of rats to which dextran had been administered. The lysosomal fraction obtained by this method was enriched more than 100-fold in N-acetyl-beta-glucosaminidase and arylsulfatase and 40-fold in acid phosphatase and beta-glucosidase. Electron microscopic examination and measurement of marker enzyme activity for various subcellular organella indicated that the lysosomal fraction was essentially free from contamination by other organella. Flavins, ubiquinones, and hemochromes were found on lysosomal membranes and investigated. The FAD and ubiquinone-9 contents of the purified lysosomal membranes were 0.118 and 6.93 nmol/mg of protein, respectively. Hemochromes in lysosomes showed spectra similar to that of a b-type cytochrome, with the alpha-peak at 562 nm and the gamma-peak at 436 nm.     

A simple procedure for isolation of microsomes from human placenta

A method for rapid isolation of human placenta microsomes, which does not require facilities for ultracentrifugation was described. Such microsomes were compared with microsomes prepared by conventional ultracentrifugation technique. Both microsomal preparations were tested for protein, RNA and phospholipid content as well as for sulphohydrolase activities and glucose-6-phosphatase activity. The degree of contamination with other subcellular particles were tested as well. The data presented showed that microsomal proteins precipitated at pH 5.3 may be conveniently used for preparative separation of microsomal enzymes.     

A modified procedure for the isolation of a pore complex-lamina fraction from rat liver nuclei

A modified procedure for the isolation of a nuclear pore complex-lamina fraction from rat liver nuclei is described. Evidence is provided that the isolated lamina, a 150-A thick, proteinaceous structure, apposes the inner nuclear envelope membrane, connecting nuclear pore complexes and surrounding the entire nucleus.     

A rapid procedure for the isolation of lysosomes from kidney cortex by Percoll density gradient centrifugation

Highly pure lysosomes were isolated from buffalo(Bubalus bubalis) kidney cortex by a procedure involving differential and isopycnic Percoll density gradient centrifugations. Arylsulphatase, N-acetyl-Β-glucosamindase and cathepsin D in the lysosomal preparation were 26–45-fold enriched over the homogenate. The purified lysosomes contained less than 0·06% of mitochondrial, microsomal and peroxisomal marker enzymes. In the electron micrographs the particles appeared as large dense granules of size 0·3-1·9 μm with no apparent structural features belonging to mitochondria or microsomes. The isolation procedure was also found to be suitable to obtain highly pure lysosome particles from renal cortex of other sources such as rat, lamb and beef. No ultracentrifugation steps were involved in the procedure