Zonal Gradient Centrifugation Research Articles

Purification of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) and analyses of the structural proteins

The Kaposis's sarcoma-associated herpesvirus (KSHV) infected BCBL-1 cell line, adapted to and grown in medium containing 10% horse serum, was induced to lytic replication with 12- O-tetradecanoylphorbol-13-acetate (TPA) for virus production. Supernatants from induced cells were filtered through a 0.45- μm filter and virions were concentrated by polyethylene glycol extraction and high speed centrifugation. The virus was purified by a glycerol gradient zonal centrifugation step followed by isopycnic separation using positive density–negative viscosity gradients. Two visible bands were detected after the final centrifugation step: an upper band that contained a homogenous population of purified virions and a lower band that contained aggregates of purified virus and other cellular debris. Fractionation of purified virion preparations by SDS-PAGE revealed 32 bands with estimated molecular weights between 19 and 280 K in silver stained gels. The glycoprotein bands in purified virus were identified with biotinylated lectins and horseradish peroxidase-labeled streptavidin. Two lectins were used to identify the KSHV glycoproteins: concanavalin A and Ricinus communis agglutinin I. Eight distinct glycoproteins were detected with these lectins. In addition, antisera from KS patients were used to detect immunoreactive proteins in purified virions. An apparent immunodominant band of M r 94 000 (94 K) was recognized by patients' antisera. Other proteins detected with some of the KS antisera tested corresponded to molecular weights of 57 K, 70 K, 180 K, 200 K and 240 K. The 94 K band was identified as gp94 by Endo F digestion.

Chapter 67 Isolation of Inner- and Outer-Arm Dyneins

Publisher Summary Dyneins are the main constructive body part of the inner and outer arms of eukaryotic flagella and cilia. The composition of axonemal dyneins varies from species to species and within a single axoneme. Within an axoneme, outer-arm dyneins are thought to be homogenous in form, whereas the inner-arm dyneins consist of at least three subforms. Dyneins were first separated from other axonemal components by selective extraction in low-ionic-strength solution containing ethylenediaminetetraacetic acid; however, this procedure causes dynein molecules to dissociate into subunits. It is now most common to use high-ionic-strength (elevated NaCl or KCl) solution to extract dynein from axonemes. Biochemical quantities of dynein can be obtained from five different sources. This chapter describes the procedures for isolation of axonemes and outer-arm dynein from the flagella of the sea urchin Strongylocentrotus purpuratus and for the isolation of inner-arm dynein from the flagella of the green alga Chlamydomonas reinhurdtii . Similar procedures are used to isolate dynein from a number of sources. The chapter discusses isolation of sea urchin sperm axonemes and dynein—collection of sperms, isolation and demembranation of flagella (by osmotic shock and with detergent), and isolation of outer-arm dynein from sea urchin sperm flagella. Because exposure of sea urchin dynein to detergent nonphysiologically activates the dynein ATPase activity, it is advised to demembranate by osmotic shock with high sucrose for investigations into the regulation of axonemes and dynein. There are details on Isolation of Chlamydomonas inner-arm dynein and fractionation of dyneins by sucrose density gradient zonal centrifugation. The existence of numerous outer-arm and inner-arm mutants and the ability to extract both inner and outer arms with high-salt solutions make Chlamydomonas an excellent system for purification of inner-arm dyneins. To isolate inner-arm dyneins from Chlamydomonas , a cell line genetically lacking the entire outer-arm structure is used.

The association of mannitol oxidase with a distinct organelle in the digestive gland of the terrestrial slugArion ater

Post nuclear supernatants prepared from homogenates of the digestive gland of the slugArion ater were analysed by rate-dependent and density-dependent density gradient zonal centrifugation. Mannitol oxidase carrying structures exhibited distinct centrifugal behaviour sedimenting more slowly than mitochondria, lysosomes and peroxisomes but faster than microsomes, and banding sharply at a density of 1.15 g/ml in sucrose gradients. By combining rate and isopycnic centrifugations mannitol oxidase carrying structures were largely separated from contaminating organelles. Electron microscopic examination of such mannitol oxidase-enriched fractions revealed the predominant presence of distinct structures of tubular form. SDS PAGE analysis indicated that the major polypeptide present had a mass of ∼ 68 kDa corresponding to the major subunit previously reported for partially purified mannitol oxidase ofHelix aspersa.

Biochemical characterization of the central nervous system myelin proteins of the rainbow trout, Salmo gairdneri

Central nervous system myelin isolated from the rainbow trout ( Salmo gairdneri) displays a very low median density on zonal gradient centrifugation, banding at approximately 0.32 M sucrose. Its proteins consist of a 36 K (36,000 mol.wt.) component, two Concanavalin A-reactive intermediate proteins IP1 (23,000 mol.wt.) and IP2 (26,200 mol.wt.), and two basic proteins BP1 and BP2, of which the latter co-migrates with rat SBP while BP1 is of slightly smaller size. The trout myelin proteins electrofocus at pH positions similar to those of their mammalian counterparts. Immunoblotting shows that antibodies against rat PNS myelin P 0 glycoprotein are bound by IP1 and IP2, but not by 36K. None of the trout myelin proteins react with anti-rat CNS myelin proteolipid protein (PLP) antiserum. The basic proteins BP1 and BP2 bind strongly to antibodies directed against human myelin basic protein. In vivo injection of tritiated fucose or palmitate leads to radiolabeling of IP1 and IP2. Under autolytic in situ conditions the appearance of a glycosylated 20,000 mol.wt. component (IP0) is noted, with parallel reduction of both IP1 and IP2, indicating sequence homologies between IP1 and IP2. The 36K protein is not affected by autolysis.

Purification by sucrose density gradient zonal centrifugation and affinity column chromatography of antigenic substances from the livers of mice infected with Tyzzer's disease.

Antigenic substances from livers of mice infected with Tyzzer's disease were purified by means of sucrose density gradient zonal centrifugation and affinity column chromatography using antiserum and checking antigenicity with the complement fixation test. Fractions obtained from zonal centrifugation fell into three main groups with different molecular weights, two of which (Fr. I and Fr. II) positively reacted with antiserum in the complement fixation tests. Both fractions were further purified by affinity column chromatography. The molecular weights of the main antigenic substances derived from Fr. I and Fr. II were determined to be about 52 000 and 66 000, respectively, by means of SDS-PAGE.

Distribution of PNS Myelin proteins and membrane enzymes in fractions isolated by continuous gradient zonal centrifugation

Myelin was purified from adult rabbit sciatic nerve by two procedures: discontinuous gradient centrifugation and continuous gradient zonal centrifugation. Two fractions were obtained from the discontinuous gradient. The fraction floating on 0.32 M sucrose and the fraction recovered from the 0.32/0.85 M sucrose interface showed typical myelin membranes by electron microscopy and typical myelin proteins by gel electrophoresis. The specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) decreased from the top to the bottom of the discontinuous gradient. The myelin separated by zonal centrifugation on a continuous sucrose gradient showed three distinct peaks (on monitoring optical density) at 0.10, 0.30 and 0.57 M sucrose. The latter peak yielded 92% of the material applied. The two minor peaks of low density exhibited high CNP and acetylcholinesterase (AChE) activities but the specific activity of both enzymes increased markedly at the heavy end of the gradient. The zonal fractions showed typical myelin proteins in all fractions by polyacrylamide gel electrophoresis but with important quantitative differences. These results indicate that PNS myelin shows significant heterogeneity.

Carbonic anhydrase activity in myelin fractions from rat optic nerves

The carbonic anhydrase activity of myelin fractions isolated from the optic nerves of adult and immature (20-day-old) rats was examined. The specific activity in both total homogenate and myelin fractions was about 2-fold higher in adult than in immature animals and at both ages, the activity in the homogenate was higher than in myelin. After subfractionation by zonal gradient centrifugation, it was shown that carbonic anhydrase activity was greatest in the heaviest myelin particles at both ages. These data are consistent with the hypothesis that a small proportion of the total enzyme activity is localised in myelin.

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.

LOCALIZATION OF 2′,3′‐CYCLIC NUCLEOTIDE‐3′‐PHOSPHOHYDROLASE OF RABBIT BRAIN BY SEDIMENTATION IN A CONTINUOUS SUCROSE GRADIENT1

Abstract— The enzyme 2′,3′‐cyclic nucleotide‐3′‐phosphohydrolase (CNP) has been assayed in fractions from a continuous sucrose density gradient zonal centrifugation of rabbit brain homogenates. Basic protein (BP) was also assayed by a radioimmunomethod. Fractions were examined by SDS‐polyacrylamide gel electrophoresis and by electron microscopy. These studies show that the major membrane fractions in the gradient differ greatly in the content of CNP and BP, and of high molecular weight proteins (HMW). The lightest membrane fractions contained numerous multilamellae, the highest content of BP and the lowest content of CNP and HMW, while the heaviest membrane fractions contained single membrane fragments and vesicles of unknown origin, the lowest content of BP and the highest content of CNP and HMW. The fraction containing the largest amount of membrane measured by turbidity, protein content, and water‐washed dry weight contained only half the CNP specific activity of a denser fraction in the gradient. CNP specific activity in the lightest fractions was insignificant compared to that of denser fractions. Thus, we conclude that this enzyme may be absent from the typical multilamellar myelin structures but present in the single‐membrane structures associated with myelin, such as the glial membrane and the paranodal segments of myelin adjacent to the axon. BP appears to occupy the opposite positions, highest in the multilamellae and lowest in the single‐membrane structures of myelin. These studies do not exclude the possibility that CNP may not be bound to myelin membranes, but rather to a membrane of different origin. Evidence that this enzyme is a myelin‐marker enzyme is circumstantial. Our evidence indicates the enzyme could be present either in a unique portion of myelin membranes or in another membrane structure.

Newcastle disease virus-specific RNA: poly(A)-containing and poly(A)-deficient transcripts as revealed by chromatography on poly(U)-sepharose.

Total [3H]uridine-labeled, virus-specific RNA from Newcastle disease virus-infected cells was fractionated by poly(U)-sepharose chromatography and analyzed by rate zonal gradient centrifugation. The sedimentation pattern of both eluted and nonadsorbed RNA resembled that of the total RNA. However, nonadsorbed RNA was enriched in 50S material, ant its 18S peak was broader and slightly shifted towards the top of the gradient. Poly(U-sepharose chromatography of isolated 18S RNA and 24S RNA resulted in the separation of poly(A)-containing RNA and poly(A)-deficient RNA. In the former the percentage of adenosine content represented by poly(A) sequences was estimated as 10 to 12% (for 18S RNA) or approximately 6.0% (for 24S RNA). The size of poly(A) fragments as measured by their sedimentation rate was the same for 18S and 24S RNA. Polyacrylamide gel electrophoresis of poly(A)-containing RNA revealed a characteristic pattern closely resembling the pattern of nonchromatographed 18S and 24S RNA. The pattern of poly(A)-deficient RNA was heterogenous, and for 18S RNA it shifted towards the anode. The possible origin of poly(A)-deficient transcripts is discussed.

An automated procedure for the extraction and fluorimetric determination of 5-hydroxytryptamine in platelet subcellular fractions

A fully automated procedure for the determination of 5-hydroxytryptamine is described. The specificity and sensitivity of the assay depend upon a differential solvent extraction procedure to remove interfering 5-hydroxy-indoles and the measurement of the fluorescence of the amine after reaction with ninhydrin. The method is highly suitable for the analysis of tissue subcellular fractions since there is no interference by sucrose, and after a preliminary deproteinisation of the sample with ZnSO 4 NaOH , performed manually, the rest of the procedure is fully automated. The sensitivity of the assay depends upon the quality of the fluorimeter used but is better than 5 ng/ml of sample with a double monochromator spectrofluorimeter. There is no interference by 5-hydroxytryptophan, 5-hydroxyindole-3-acetic acid, or 5-hydroxytryptophol. The suitability of the procedure for the routine analysis of 5-hydroxytryptamine in platelet subcellular fractions prepared by zonal gradient centrifugation has been explored and the soluble phase and particulate bound amine stores can be identified.

Synchronous mating in yeast.

Homogeneous a and alpha unbudded yeast cells in logarithmic phase, grown in supplemented minimal medium and isolated by zonal gradient centrifugation, are used for mating. When these cells are resuspended in aerated defined medium, highly synchronous mating rapidly occurs. Within 20 min of incubation at 30 degrees early sexual pairing is evident; extensive agglutination is observed by 60 min, and cell fusion and bud initiation in zygotes occurs after 60-140 min. Sorbitol gradient fractionation of mating mixtures taken at various times during incubation allows the isolation of zygotes or unmated cells. Zygote preparations 90-95% purified are obtained in quantities suitable for genetic and biochemical analysis. The mating procedure is predictable and reproducible.

Composition, particle size and role in dietary fat transport of two different lipoproteins of the intestinal lymph.

AbstractRat intestinal lymph very low density lipoproteins have been separated by cellulose acetate electrophoresis. After administration of a meal including natural fats and labeled fatty acids, two labeled bands were detected on the electrophoregraphs. Further separations of these two kinds of particles were performed by density gradient zonal centrifugation. The isolated lipoproteins were analyzed for chemical composition and investigated by electron microscopy and electrophoresis. The moving particles had a higher protein and cholesterol content and a smaller diameter than the particles which remained at the origin. It has also been shown that the major part of the labeled lymph lipids were transported by the smaller particles.

Purification of large quantities of coxiella burnetii rickettsia by density gradient zonal centrifugation.

The purification of large quantities of inactivated, phase II Coxiella burnetii by isopycnic zonal centrifugation for use as diagnostic antigen and as a vaccine is described. The fractionation of egg yolk sac-derived C. burnetii vaccine resulted in the separation of two distinct populations of organisms, each devoid of microscopically and serologically recognizable components of egg yolk sac. One population of organisms, characterized by an equilibrium density of 1.240, was rod shaped (1.0 by 0.5 mumole) with a thick, densely strained wall and prominent central body. The second population, with an equilibrium density of 1.280, had a coccobacillary shape (approximately 1 mumole in diameter), granular, sometimes fibrillar cytoplasm, thin cellular walls, and lacked a prominent nucleoid.

DNA from anaerobic adult Ascaris lumbricoides and Hymenolepis diminuta mitochondria isolated by zonal centrifugation

1. Reorienting gradient zonal centrifugation was used to isolate large quantities of mitochondria from the parasitic helminths, Ascaris lumbricoides and Hymenolepis diminuta. This technique may be particularly useful in obtaining mitochondria or other organelles present in small quantities from any tissue. 2. Electron micrographs of mitochondrial DNA released from osmotically shocked mitochondria and from purified mitochondrial DNA revealed a circular molecule with an average contour length of 4.76 ± 0.03 μ m in H. diminuta and 4.79 ± 0.03 μ m in A. lumbricoides. 3. Mitochondrial DNA was subsequently isolated from both helminths and characterized using equilibrium centrifugation in CsCl. The buoyant density of mitochondrial DNA was 1.690 g/cm 3 in A. lumbricoides and 1.691 g/cm 3 in H. diminuta. Estimates made from buoyant density measurements and thermal transition values indicated a base composition of 31 % G + C for A. lumbricoides and 32 % for H. diminuta mitochondrial DNA.

Purification of Large Quantities of Coxiella burnetii Rickettsia by Density Gradient Zonal Centrifugation

The purification of large quantities of inactivated, phase II Coxiella burnetii by isopycnic zonal centrifugation for use as diagnostic antigen and as a vaccine is described. The fractionation of egg yolk sac-derived C. burnetii vaccine resulted in the separation of two distinct populations of organisms, each devoid of microscopically and serologically recognizable components of egg yolk sac. One population of organisms, characterized by an equilibrium density of 1.240, was rod shaped (1.0 by 0.5 mumole) with a thick, densely strained wall and prominent central body. The second population, with an equilibrium density of 1.280, had a coccobacillary shape (approximately 1 mumole in diameter), granular, sometimes fibrillar cytoplasm, thin cellular walls, and lacked a prominent nucleoid.

A functional equivalence of top component a RNA and coat protein in the initiation of infection by alfalfa mosaic virus

The nucleoproteins of alfalfa mosaic virus (AMV) were purified by zonal gradient centrifugation, and their homogeneity and RNA contents were established by gel electrophoresis. Each component was shown to contain one type of RNA of a specific length. Combinations of purified preparations were infectious only when the bottom component (B), middle component (M), and the top component b (Tb) nucleoproteins were present. However, no infectivity was found in a mixture of the three corresponding RNAs. Infection could be started by adding to this mixture a fourth RNA, the top component a RNA (Ta-RNA). The fact that a mixture of B, M, and Tb nucleoprotein is infectious while the corresponding nucleic acid mixture is not, is explained by the finding that the latter becomes infectious upon addition of low amounts of AMV coat protein. Evidence is presented that the activity of AMV coat protein is not due to protection of the RNAs against degrading enzymes. The function of Ta-RNA and its translation product—the coat protein—in the onset of infection is discussed.

Zonal centrifugation of mammalian DNA

A modified alkaline sucrose gradient technique for the fractionation of single-stranded mammalian DNA has been developed. The method involves the use of a reorienting gradient zonal centrifugation system of large sample zone capacity. The data obtained support the concept that the DNA in mammalian chromosomes exists as a linked array of subunits (10).

The Metabolism of Mitochondrial Proteins: III. The Dynamic State of Rat Liver Mitochondria

The turnover rates of six protein fractions of rat liver mitochondria were estimated from the incorporation of 14C-carbonate given continuously. The rates were similar and little heterogeneity was evident. When expressed as half-lives, the values ranged from 4 to 6 days. However, the turnover rates of two inducible mitochondrial enzymes, alanine and ornithine aminotransferase, were much faster. The half-lives ranged from 0.73 to 1.03 days. Zonal gradient centrifugation of mitochondria from treated animals indicated that the changes in enzyme activity were occurring in all of the mitochondria at the same rapid rate. The evidence from these experiments suggests that all mitochondria are in a dynamic state and that alteration of the enzyme complement is independent of mitochondrial genesis.