Isolation of a caveolae-enriched fraction from rat lung by affinity partitioning and sucrose gradient centrifugation

Caveolae were isolated from rat lungs by a combination of affinity partitioning and sucrose gradient centrifugation. After homogenization of the lungs directly in a polyethylene glycol-dextran two-phase system and conventional phase partitioning, the polyethylene glycol-rich top phase was affinity partitioned with fresh bottom phase containing dextran-linked wheat-germ agglutinin. The lectin selectively attracted plasma membranes to the bottom phase. The isolated plasma membrane fraction was treated with Triton X-100 or, alternatively, sonicated before centrifugation in a stepwise sucrose gradient. Caveolin-enriched material collected at the 5/24% sucrose boundary. This material also contained 5'-nucleotidase activity and actin. Electron microscopy showed the material to consist of a homogeneous population of 50- to 100-nm vesicles. This purification protocol should allow the facile purification of caveolae also from other tissues, facilitating structural and functional studies.     

Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient

A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al, (1955, Biochem. J. 60: 604-617), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction if set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10--12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane. The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.     

Buffer-induced rat liver mitochondrial aggregation during differential centrifugation

Use of buffers in homogenization media can result in loss of considerable particulate enzyme activity even with low-speed centrifugation. Addition of tris chloride buffer to 0.25 M sucrose homogenization media resulted in precipitation of 80 to 95% of the activity of two mitochondrial marker enzymes (3-hydroxy-3-methylglutaryl CoA lyase and citrate synthase) with the nuclear fraction during differential centrifugation. Lactate dehydrogenase, a cytoplasmic marker, was not precipitated under the same conditions, indicating that the precipitated enzymes were not associated with intact cells. Photomicrographs showed that tris chloride buffers resulted in mitochondrial aggregation. Isolated mitochondria resuspended in tris chloride or potassium phosphate buffer also aggregated, which resulted in a marked decrease in assayable mitochondrial enzyme activity.     

Isolation of a purified mitochondrial fraction from viable clonal insulin-producing cells (RINm5F) by percoll density gradient centrifugation

Summary A Percoll density gradient was employed for selecting large numbers of viable insulin-producing RINm5F cells. Homogenates of these cells were then subjected to gradient centrifugation and two clearly visible bands were obtained. The light fraction was essentially composed of mitochondria banded at a density of about 1.06 g/ml. The heavier fraction banded at 1.09 to 1.10 g/ml and contained lysosomes and a small number of secretory granules. The distribution of Percoll particles was restricted to the extracellular space and there was no adsorption to any membrane structures. The distribution pattern of marker enzymes for the mitochondria and lysosomes was similar to that of normal pancreatic β-cells. With the use of a Percoll density gradient it was thus possible to isolate a purified mitochondrial fraction from viable RINm5F cells. This work was supported by the Swedish Medical Research Council (03x-4, 12x-562, 12x-6240), the Swedish Diabetes Association, the Nordic Insulin Foundation, Syskonen Svenssons Foundation, and ?ke Wiberg’s Foundation. Per-Olof Berggren is a recipient of a postdoctoral fellowship from the Swedish Medical Research Council.     

Isolation of interstitial fluid from rat mammary tumors by a centrifugation method

Access to interstitial fluid is of fundamental importance to understand tumor transcapillary fluid balance, including the distribution of probes and therapeutic agents. Tumors were induced by gavage of 9,10-dimethyl-1,2-benzanthracene to rats, and fluid was isolated after anesthesia by exposing tissue to consecutive centrifugations from 27 to 6,800 g. The observed (51)Cr-EDTA (extracellular tracer) tissue fluid-to-plasma ratio obtained from whole tumor or from superficial tumor tissue by centrifugation at 27-424 g was not significantly different from 1.0 (0.92-0.99), suggesting an extracellular origin only. However, fluid collected from excised central tumor parts had a significantly lower ratio (0.66-0.77) for all imposed G forces, suggesting dilution by fluid deriving from a space unavailable for (51)Cr-EDTA. The colloid osmotic pressure in tumor fluid was generally higher than in fluid isolated from the subcutis, attributable to less selective capillaries and impaired lymphatic drainage in tumors. HPLC analysis of tumor fluid showed that low-molecular-weight macromolecules not present in arterial plasma were present in tumor fluid obtained by centrifugation and in venous blood draining the tumor, most likely representing proteins derived from tumor cells. We conclude that low-speed centrifugation may be a simple and reliable method to isolate interstitial fluid from tumors.     

Isolation of hexose-6-phosphate dehydrogenase from rat liver microsomal fraction by affinity chromatography

Hexose-6-phosphate dehydrogenase of rat liver microsomes was purified to an apparently homogeneous state with a recovery of about 36% using 8-aminooctyl Sepharose, DEAE-cellulose and 2′,5′-ADP Sepharose columns. This enzyme was insensitive to SH-reagent p-chloromercuribenzoate and oxidized galactose 6-phosphate, glucose 6-phosphate and glucose, with either NADP or NAD as an electron acceptor. The minimum molecular weight of this enzyme was estimated to be 104,000 in SDS-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol.     

Isolation of a Golgi apparatus-rich fraction from rat liver. IV. Thiamine pyrophosphatase

The thiamine pyrophosphatase (the enzyme [s] catalyzing the release of inorganic phosphate with thiamine pyrophosphate as the substrate) activities of Golgi apparatus-, plasma membrane-, endoplasmic reticulum-, and mitochondria-rich fractions from rat liver were compared at pH 8. Activity was concentrated in the Golgi apparatus fractions, which, on a protein basis, had a specific activity six to eight times that of the total homogenates or purified endoplasmic reticulum fractions. However, only 1–3% of the total activity was recovered in the Golgi apparatus fractions under conditions where 30–50% of the UDPgalactose:N-acetylglucosamine-galactosyl transferase activity was recovered. Considering both recovery of galactosyl transferase and fraction purity, we estimate that approximately 10% of the total thiamine pyrophosphatase activity of the liver was localized within the Golgi apparatus, with a specific activity of about ten times that of the total homogenate. Cytochemically, reaction product was found in the cisternae of the endoplasmic reticulum as well as in the Golgi apparatus. This is in contrast to results obtained in most other tissues, where reaction product was restricted to the Golgi apparatus. Thus, enzymes of rat liver catalyzing the hydrolysis of thiamine pyrophosphate, although concentrated in the Golgi apparatus, are widely distributed among other cell components in this tissue.     

Isolation of a fraction with Ca2+ ionophore properties from rat liver mitochondria

Isolation of a small protein with properties of a Ca2+ ionophore from calf heart mitochondria has recently been reported [A. Y. Jeng and A. E. Shamoo, 1980, J. Biol. Chem. 255, 6897, 6904]. We have isolated a fraction with similar physical and chemical properties from rat liver mitochondria. In particular, the hepatic preparation is able to bind Ca2+ with high affinity in such a fashion that the resultant complex is soluble in a hydrophobic phase. It will also transport Ca2+ through a stirred organic phase (Pressman cell). Interaction of the liver preparation with Ca2+ is sensitive to inhibitors of mitochondrial Ca2+ uptake. The hepatic preparation contains both protein and lipid components. The phospholipid components were identified and the behavior of a similar mixture of commercially available phospholipids was compared to that of the ionophore fraction from rat liver mitochondria. All of the Ca2+ binding properties of the rat liver preparation could be mimicked by the lipids. In a preliminary experiment, reduction of the phospholipid content of the preparation to less than one lipid phosphate per protein molecule (assuming a molecular weight of 3000 by analogy with the calf heart case) resulted in a protein that was unable to bind Ca2+. We, therefore, suggest that the ability of the preparation to interact with Ca2+ is due to the constituent phospholipids. Measurements of phospholipid-Ca2+ interactions in the model systems and under the conditions of low (microM) Ca2+ and phospholipid concentration utilized here demonstrated an affinity for Ca2+ (Ks approximately 1 microM) and a cation selectivity that have not previously been reported.     

Isolation of anucleated yeast protoplasts by means of density gradient centrifugation

An improved method for the preparation in high yield of anucleated Saccharomyces cerevisiae has been developed. This method is based on a two-stage centrifugation of the original protoplast mixture in linear density gradients (1–10%, w/v) of Ficoll 400. The yield of anucleated protoplasts was 5–9%, its value depended on the frequency of the nucleus-free protoplasts in the original mixture.The anucleated protoplasts were characterized by RNA, DNA and protein content, and by light and electron microscopy. The protoplasts lacking nuclei had about one third the diameter of the nucleated ones, and reduced of DNA, RNA and protein in comparison to normal protoplasts. Electron microscopy showed a typical yeast ultrastructure in anucleated protoplasts except that they lacked nuclei and exhibited a higher frequency of lipid granules and exocytotic electron-dense vesicles located close to the plasmalemma.     

Isolation of a mitochondrial factor from rat liver which potentiates the inactivation of glutamate dehydrogenase by lysosomes

Rat liver glutamate dehydrogenase (L-glutamate: NAD(P) oxidoreductase, deaminating) E.C. 1.4.1.3.) is inactivated by the mitochondrial matrix in combination with lysosomal preparations. Neither lysosomal or mitochondrial matrix extracts per se inactivate the enzyme appreciably under the conditions used. Fractionation of the matrix indicates that a low molecular weight factor is responsible for the potentiation of inactivation of glutamate dehydrogenase by lysosomes. Its absorption spectrum and chromatographic behaviour suggest that this factor is NADP.