Toxoplasma gondii: large-scale purification by zonal density gradient centrifugation.

This study shows the feasibility of using density gradient centrifugation in the “A” zonal rotor for large-scale purification of Toxoplasma gondii tachyzoites from the host cells of mouse peritoneal exudate. Ficoll and Dextran 40 were used as gradients. Using Ficoll gradient, up to 70% of the toxoplasms were recovered by pooling purified fractions with the peak fractions giving recoveries around 38%. In the experiment using dextran gradients Toxoplasma recovery was of 41%, but the band of host cells was not so sharply formed.     

Fractionation of liver plasma membranes prepared by zonal centrifugation

1. Plasma membranes were isolated from crude nuclear sediments from mouse and rat liver by a rate-dependent centrifugation through a sucrose density gradient contained in the ;A' type zonal rotor. 2. The membranes were further purified by isopycnic centrifugation, and characterized enzymically, chemically and morphologically. 3. When the plasma-membrane fraction of sucrose density 1.17g/cm(3) was dispersed in a tight-fitting homogenizer, two subfractions of densities 1.12 and 1.18 were obtained by isopycnic centrifugation. 4. The light subfraction contained 5'-nucleotidase, nucleoside diphosphatase, leucine naphthylamidase and Mg(2+)-stimulated adenosine triphosphatase activities at higher specific activities than unfractionated membranes. The heavy subfraction was deficient in the above enzymes but contained higher Na(+)+K(+)-stimulated adenosine triphosphatase activity. 5. The light subfraction contained twice as much phospholipid and cholesterol, and three times as much N-acetylneuraminic acid relative to unit protein weight as the heavy subfraction. Polyacrylamide-gel electrophoresis indicated differences in protein composition. 6. Electron microscopy showed the light subfraction to be vesicular. The heavy subfraction contained membrane strips with junctional complexes in addition to vesicles.     

Preparative separation of DNA-ethidium bromide complexes by zonal density gradient centrifugation

Ethidium bromide-DNA complexes separated by rate-zonal sedimentation through a density gradient can be readily visualized and purified with little cross-contamination. The method is simple, rapid, and efficient.     

The characterization of rat kidney plasma membranes prepared by zonal centrifugation

1. Plasma membranes were isolated from a 10000g-min pellet prepared from a renal cortical homogenate in 20mm-NaHCO(3) by isopycnic centrifugation in a linear sucrose gradient in an ;A'-type zonal rotor. 2. The preparation was characterized by electron microscopy, and alkaline phosphatase, 5'-nucleotidase, l-leucine beta-naphthylamidase and l-leucine p-nitroanilidase activities were found to be selectively associated with the renal plasma membrane. 3. The preparation had a high degree of purity, as indicated by the presence of low activities of marker enzymes associated with subcellular organelles. A preliminary chemical analysis indicated that the chemical composition resembled that of plasma membranes of other tissues. 4. Plasma membranes were also prepared from tubular fragments and their enzyme contents were found to be similar to those of plasma membranes prepared from cortical homogenates. 5. l-Leucine beta-naphthylamidase, l-leucine p-nitroanilidase and 5'-nucleotidase were not enriched to the same extent as alkaline phosphatase in the preparation of plasma membranes from tubular fragments. A possible explanation for this finding is discussed.     

Isolation of intramitochondrial bodies in bovine adrenocortical cells by density gradient centrifugation.

Two method for isolating the intramitochondrial bodies from bovine adrenocortical cells are proposed. Electron microscopic examination shows that discontinuous sucrose density gradient centrifugation can separate the fraction rich in intramitochondrial bodies, but some indistinguishable fragments remain among them. Continuous sucrose density gradient centrifugation is probably superior to the former method in obtaining a highly purified fraction of the bodies. The amido black positive granules, presumed to be intramitochondrial bodies, are collected in the fractions of the sucrose density of around 1.27 (1.23--1.30), which lack cytochrome c oxidase activity.     

Isolation of intramitochondrial bodies in bovine adrenocortical cells by density gradient centrifugation

Summary Two methods for isolating the intramitochondrial bodies from bovine adrenocortical cells are proposed. Electron microscopic examination shows that discontinuous sucrose density gradient centrifugation can separate the fraction rich in intramitochondrial bodies, but some indistinguishable fragments remain among them. Continuous sucrose density gradient centrifugation is probably superior to the former method in obtaining a highly purified fraction of the bodies. The amido black positive granules, presumed to be intramitochondrial bodies, are collected in the fractions of the sucrose density of around 1.27 (1.23–1.30), which lack cytochrome c oxidase activity.This work was supported by a Scientific Research Grant, No. 144017, from the Ministry of Education of Japan to Professor M. Yasuda     

Purification of the protein crystal from Bacillus thuringiensis by zonal gradient centrifugation.

A method is described for the large-scale purification of the Bacillus thuringiensis protein crystal by zonal gradient centrifugation. NaBr gradients are employed in a Beckman J21-B centrifuge equipped with a JCF-Z rotor.     

Distribution of selected phospholipid modifying enzymes in rat brain microsomal subfractions prepared by density gradient zonal rotor centrifugation

A rat brain P₃ fraction enriched in ER derived microsomes was centrifuged through a 20–40% linear sucrose gradient in a Beckman Ti-14 Zonal rotor and 11 fractions were obtained. The distribution of marker enzyme activities and protein were determined in these 11 subfractions. NADPH-Cytochrome C reductase, choline phosphotransferase were employed for endoplasmic reticulum, Na⁺, K⁺-ATPase, 5-nucleotidase, and acetylcholinesterase were employed for plasma membrane, 2, 3-cyclic nucleotide phosphohydrolase was employed for myelin. The bulk of the protein was recovered in the 24–34% sucrose fractions, Na⁺, K⁺-ATPase, 5-nucleotidase, and acetylcholinesterase were in the 22–38% sucrose fractions while NADPH-cytochrome C reductase and CNPase were enriched in the 20–22% sucrose fractions. The ethanolamine and the serine base exchange activities had a bimodal distribution, with highest specific activities in sucrose fractions 32–34% and 20–24%. Choline base exchange activity was nearly undetectable in all the fractions. The specific activities of CDP-choline phosphotransferase, and phospholipid-N-methyltransferase were highest in the 20–22% sucrose fraction. Phospholipid-N-methyltransferase activity was significantly stimulated in the presence of exogenous phospholipid acceptors as phosphatidylethanolamine or phosphatidylmonomethylethanolamine or phosphatidyldimethylethanolamine, however, the greatest response was with phosphatidylmonomethylethanolamine. The rat brain P₃ fraction yielded a population of a membrane at the light end of the sucrose gradient which has a buoyant density similar to myelin but seemed to be enriched with NADPN cytochrome C reductase and phospholipid modifying enzymes. This is in contrast to liver microsomes submitted to a similar fractionation.     

Isolation of apical plasma membrane in rabbit gallbladder epithelium by Percoll density gradient centrifugation

The apical membranes of rabbit gallbladder epithelial cells were isolated by treating the homogenate with Ca2+ or Mg2+ and centrifuging the suspension in Percoll gradient. In this way brush-border membranes were obtained with enrichment factors ranging between 10 and 20 and yields of 15-30%. A second method is described with which membranes were isolated, without any preliminary treatment, first by differential centrifugation, then with Percoll gradient; the final membrane enrichment was over 15, however the yield was very low (3%). Many possible enzymatic markers of the apical plasma membrane were investigated: L-gamma-glutamyltransferase, alkaline phosphatase, leucine aminopeptidase, sucrase. The first appears to be that of choice. Apical membrane fraction could be also evidenced by autofluorescence or by labeling with Lotus tetragonolobus lectin. Preliminary experiments showed that apical plasma membranes isolated in this way form vesicles.