Zonal Centrifugation In Sucrose Density Gradients Research Articles

Vacuolar storage proteins and the putative vacuolar sorting receptor BP-80 exit the golgi apparatus of developing pea cotyledons in different transport vesicles

In the parenchyma cells of developing legume cotyledons, storage proteins are deposited in a special type of vacuole, known as the protein storage vacuole (PSV). Storage proteins are synthesized at the endoplasmic reticulum and pass through the Golgi apparatus. In contrast to lysosomal acid hydrolases, storage proteins exit the Golgi apparatus in 130-nm-diameter electron-dense vesicles rather than in clathrin-coated vesicles. By combining isopycnic and rate zonal sucrose density gradient centrifugation with phase partitioning, we obtained a highly enriched dense vesicle fraction. This fraction contained prolegumin, which is the precursor of one of the major storage proteins. In dense vesicles, prolegumin occurred in a more aggregated form than it did in the endoplasmic reticulum. The putative vacuolar sorting receptor BP-80 was highly enriched in purified clathrin-coated vesicles, which, in turn, did not contain prolegumin. The amount of BP-80 was markedly reduced in the dense vesicle fraction. This result was confirmed by quantitative immunogold labeling of cryosections of pea cotyledons: whereas antibodies raised against BP-80 significantly labeled the Golgi stacks, labeling of the dense vesicles could not be detected. In contrast, 90% of the dense vesicles were labeled with antibodies raised against alpha-TIP (for tonoplast intrinsic protein), which is the aquaporin specific for the membrane of the PSV. These results lead to the conclusions that storage proteins and alpha-TIP are delivered via the same vesicular pathway into the PSVs and that the dense vesicles that carry these proteins in turn do not contain BP-80.

Vaccination of rabbits with a bovine herpesvirus type 1 subunit vaccine: adjuvant effect of ISCOMs

Bovine herpesvirus type 1 has two major immunogenic surface glycoproteins: a 90 kDa haemagglutinin and the 130 (74 + 54) kDa glycoprotein. These proteins were purified by rate zonal sucrose density gradient centrifugation after extraction with nonionic detergent Triton X-100. For the preparation of ISCOMs, the glycoproteins were further adsorbed during a second rate zonal centrifugation, to micelles of Quil A glycoside, already added to the gradient. Haemagglutinating peaks were collected and used as subunit vaccine in rabbits: groups of three animals were injected with 50, 10 and 5 μg protein. Seroconversion was followed by ELISA, haemagglutination inhibition, neutralizing and plaque reduction assays. ISCOMs and unadsorbed subunits were efficient in inducing neutralizing as well as haemagglutination inhibiting antibodies: ISCOMs gave a higher level of response. The efficiency demonstrated by ISCOMs suggests their potential as a subunit vaccine.

Purification and initial characterization of a protein from skeletal muscle that caps the barbed ends of actin filaments.

We describe herein the purification of a protein from skeletal muscle that binds to ("caps") the morphologically defined barbed end of actin filaments. This actin-capping protein appeared to be a heterodimer with chemically and immunologically distinct subunits of Mr = 36,000 (alpha) and 32,000 (beta), Rs = 37 A, s20,w = 4.0 S, and a calculated native molecular weight of approximately 61,000. The protein was obtained in milligram quantities at greater than 95% purity from acetone powder of chicken skeletal muscle by extraction in 0.6 M KI, precipitation with ammonium sulfate, sequential chromatographic steps on DEAE-cellulose, hydroxylapatite, and Sephacryl S-200, followed by preparative rate zonal sucrose density gradient centrifugation. In immunoblots of myofibrillar proteins, affinity-purified antibodies selectively recognized protein bands of the same molecular weight as the subunits of the capping protein to which they were made, indicating that the isolated capping protein is a native myofibrillar protein, and not a proteolytic digestion product of a larger muscle protein. A specific interaction of the capping protein with the barbed end of actin filaments was indicated by its ability to inhibit actin filament assembly nucleated by spectrin-band 4.1-actin complex in 0.4 mM Mg2+, accelerate actin filament formation and increase the critical concentration of actin in 2-5 mM Mg2+, 75-100 mM KCl, and inhibit the addition of actin monomers to the barbed end of heavy meromyosin-decorated actin filaments as determined by electron microscopy. All of these effects occurred at nanomolar concentrations of capping protein and micromolar concentrations of actin, suggesting a high affinity interaction.

Protein and glycoprotein electrophoretic patterns of enriched fractions of primary and secondary granules from guinea pig polymorphonuclear leukocytes.

The postnuclear supernatant fraction of sucrose homogenates of guinea pig polymorphonuclear leukocytes (PMNL) was subjected to differential centrifugation to obtain a total particulate fraction, a particle-free supernatant fraction, highly enriched fractions of primary and secondary granules, and a membrane-rich fraction. The various fractions were solubilized in buffer containing sodium dodecyl sulfate (SDS) and analyzed for protein and glycoproteincomponents by SDS -polyacrylamide gel electrophoresis. The major glycoprotein components of the postnuclear supernatant fraction were found mainly associated with the enriched fraction of secondary granules and, to a lesser extent, with the membrane-rich fraction. No major glycoprotein components were visible in the polypeptide electrophoretic patterns of the primary granule fraction or of the particle-free supernate. Attempts at separation of guinea pig granules by zonal sucrose density gradient centrifugation were only partially successful. Data supporting a species difference in this regard between rabbit and guinea pig PMNL granules are presented.

Properties of ATPase of gastric mucosa V. Preparation of membranes and mitochondria by zonal centrifugation

Using zonal sucrose density gradient centrifugation methods to fractionate the subcellular components in gastric mucosal homogenates have been developed. Methods are described which give high yield or rapidity in fractionation. A procedure is described which enables large-scale preparations of smooth walled vesicular membranes containing HCO 3 −-ATPase activity free from mitochondrial contamination as assessed by electron microscopic morphology and undetectable succinic dehydrogenase or monoamine oxidase activity. A method to purify gastric mitochondria is also described.

Purification and characterization of rice tungro virus by analytical density-gradient centrifugation

The leafhopper-borne rice tungro virus (RTV) was purified by heating leaf extracts in 0.01 M EDTA, pH 7–8, at 40° for 1 hour followed by differential centrifugation and by rate zonal centrifugation in sucrose density-gradients. Some of its physicochemical characteristics were determined by analytical density-gradient centrifugation. The virus withstood temperatures below 63° for 10 minutes, pH up to 9, without apparent denaturation and did not denature in vitro for more than 24 hours at room temperature. The virus concentration in the plant was low. The particle was a polyhedron of 30–33 mμ diameter with a sedimentation coefficient of 175 ± 5 S. Infectivity was associated with the zone in the sucrose density-gradient columns containing these particles. The feasibility is emphasized of determining physicochemical characteristics by analytical density-gradient centrifugation for viruses difficult to transmit mechanically.

Studies on Ribonucleic Acid Synthesis in Tadpole Liver during Metamorphosis Induced by Thyroxine: II. ANALYSIS OF RIBONUCLEIC ACID FRACTIONS

Abstract A method of preparing ribonucleic acid from tadpole liver has been described. It was found that tadpole liver RNA is unstable during the isolation procedure. Various factors, including temperature, the concentration of Mg++ in the homogenizing medium, and the presence of ribonuclease inhibitors, were found to be of importance in protecting RNA during its preparation. Three peaks with sedimentation coefficients of 29 S, 18 S, and 6 S were obtained by zonal sucrose density gradient centrifugation. The 6 S RNA was found in the purified ribosomes from mitochondrial and postmitochondrial fractions. The base ratios of 6 S RNA were different from those of the 18 S and 29 S fractions and the bulk RNA, showing in particular a low concentration of UMP. Some properties of RNase from tadpole liver have been described and discussed in relation to the base composition of 6 S RNA. The most rapidly labeled RNA appeared in the transfer RNA fraction when orotic-acid-14C was administered to untreated tadpoles. The RNA heavier than transfer RNA has the highest specific radioactivity 2 days after thyroxine treatment. The base composition of this RNA was analyzed with the use of 32Pi and found to be high in uridine 5'-phosphate (25 to 35%) in spite of the low value of the UMP in bulk RNA in this area. Rapidly labeled RNA which appears in the 6 to 10 S area has the properties of messenger RNA needed for induction of the enzymes in liver associated with metamorphosis.