Selenium binding proteins in rat tissues

The 100,000 × g extracts of rat intestine and colon were incubated $\text{in}\text{vitro}$ with Na₂[⁷⁵Se]O₃. Chromatography of this material on a Sephadex G-100 column produced three radioactive peaks corresponding to molecular weights of 17,000, 68,000 and > 90,000. The 17,000 peak corresponded to a protein which sedimented in the 2S region of a 5–20% ($\text{w}\text{v}$) linear sucrose density gradient. Selenium binding to this protein was specific, stable and sensitive to thiol inhibitors such as p-chloromercuriphenylsulfonic acid (1 mM) and iodoacetamide (2 mM). Chromatography of rat serum - [⁷⁵Se] complex on Sephadex G-100 yielded only two radioactive peaks that corresponded to molecular weights of 68,000 and > 90,000. The 2S selenium binding protein of intestine and colon may mediate the biological functions of selenium in those tissues.     

A precursor of globin messenger RNA

The size of pulse-labeled globin messenger RNA nucleotide sequences was investigated, to determine whether newly transcribed globin mRNA molecules are larger than steady-state globin mRNA. Molecular hybridization techniques were used to compare directly the sedimentation of steady-state (unlabeled) and pulse-labeled (radioactive) globin mRNA sequences in the same analytical sucrose gradient. In gradients containing 98% formamide, radioactive globin mRNA sequences from mouse fetal liver cells labeled for 15 to 20 minutes with [³H]uridine sediment in a broad band with a peak at approximately 14 S, while steady-state globin mRNA sediments at 10 S. The large radioactive RNA can be recovered from one gradient and recentrifuged in a second gradient, in which it again sediments in a broad band with a peak at 14 S. The large radioactive RNA is cleaved to 10 S during a 75-minute “chase” with either actinomycin D or unlabeled uridine plus cytidine. The estimated half-life of the precursor is 45 minutes or less under these conditions. A covalent RNA precursor larger than 18 S with a similar turnover rate is not observed.     

Effect of lead on the intestinal absorption of sodium selenite and selenomethionine (75Se) in chicks

The present study was designed to investigate the interactions of lead with the intestinal absorption of selenium in chicks. The absorption of⁷⁵Se-selenite and⁷⁵Se-methionine was determined in 3-wk-old white Leghorn cockerels using both thein situ ligated intestinal loop procedure and oral administration of the isotopes. The highest lead concentration (1 mM) significantly reduced the percentage of⁷⁵Se-selenite absorbed from thein situ ligated duodenal loop, but did not influence the retention of the orally administered isotope. Neither was the absorption of⁷⁵Se-methionine affected by the presence of Pb in the intraduodenal dosing solution. The above experiments suggest that the direct luminal interaction of lead with the absorption of selenium is not of practical importance. On the other hand, feeding 1000 ppm Pb in the diet prior to the measurement of selenium absorption significantly reduced the transfer of the radioactive selenite from the intestinal lumen to the body. The mechanism of this effect involved enhanced retention of the⁷⁵Se-selenite by the intestinal tissue. The inhibitory effect of lead exposure on selenium absorption also appeared to be alleviated by the increase in the dietary selenium content.     

Double Strandedness of Ribonucleic Acid of Bovine Ephemeral Fever Virus

Virus labeled with ³H-uridine or ³²P-orthophosphate was purified by CsCl equilibrium centrifugation of concentrated virus materials from infectious BHK21-WI2 cell culture fluids. A single clearly visible band formed in the gradient coinciding with a sharp peak of radioactivity having a buoyant density of 1.19 g/ml. Infectivity exhibited a broader distribution with a peak coinciding with the visible band, in which numerous virions of the virus were observed with the aid of an electron microscope using the phosphotungstic negative staining technique. Ribonucleic acid (RNA) extracted from the purified virus by the phenol method exhibited a rather broad distribution of radioactivity with a major peak at about 12 S when analyzed by the sucrose density gradient centrifugation technique. Viral RNA centrifuged in the gradient after ribonuclcase (RNase) treatment showed a single sharp peak at about 12 S. These findings seemed to indicate that the virion of this virus contained double-stranded RNA. Double strandedness of the viral RNA was further corroborated by an examination of the base composition, reduced resistance to RNase in low salt concentration and a sharp thermal transition with a relatively high melting temperature of 85 C. The virus could not be classified either in the rhabdovirus group, although the shape of the virion resembled that of the rhabdoviruses, or in the reovirus group since the virus was ether-sensitive. It seemed necessary to create a new genus for this virus.     

Characterization of the plasmid DNAs of Rhodopseudomonas capsulata

Presence of extrachromosomal DNa in Rhodopseudomonas capsulata strain BH9 was shown by the appearance of a satellite band in a dye-buoyant density gradient. Radioactively labelled DNA was prepared from this satellite band and examined on a 5–20% sucrose gradient. Three radioactive peaks with sedimentation coefficients of 100 S, 94 S, and 58–64 S, respectively, were consistently observed. Analysis of these sedimentation coefficients suggested that there are two species of plasmid DNA with molecular sizes of 94×10⁶ daltons (named pBH91) and 74×10⁶ daltons (named pBH92). The 58–64 S peak is attributed to open circular molecules. DNAs from each peak of the sucrose gradient were examined by electronmicroscopy, and the results agree closely with those of the sucrose gradient analysis. Reassociation kinetics of the plasmid DNA was also followed. Addition of total DNA of strain BH9 increased the renaturation rate of the plasmid DNA. It was calculated from the magnitude of the increase that approximately 10% of the BH9 total DNA may hybridize with the plasmid sequences. DNA prepared from the gene transfer agent (GTA) produced by R. capsulata increases the renaturation rate of the plasmid to the same extent as total DNA isolated from the GTA producing strain, Y262.     

Studies on the mechanism of DNA replication in Physarum polycephalum

The synthesis of single-stranded DNA subunits (4 × 10⁷ daltons) in Physarum polycephalum was studied by alkaline sucrose density gradient centrifugation. The results were compared with the synthesis of the double-stranded DNA molecules (2.3 × 10⁸ daltons) which they comprise, as determined from neutral sucrose density gradient centrifugation patterns. Although the initiation of synthesis of most double-stranded DNA molecules takes place relatively early in the S period, synthesis of the subunits within them is initiated throughout at least the first two hours of this period. Similarly, replicating (presumably forked) DNA molecules appear to split into daughter DNA molecules prior to the completion of synthesis of the subunits therein. The average rate of DNA chain elongation within subunits is 0.3 × 10⁶ daltons/minute. It is suggested that alkaline sucrose density gradient centrifugation may be a more sensitive method for determining the time required for the completion of replication than other methods based solely on the incorporation of radioactive DNA precursors into an acid-insoluble product.     

Separation of B and C Type Virions by Centrifugation in Gentle Density Gradients

Murine type B particles were separated from type C (Rauscher leukemia virus) by means of gentle (low-increment rate) density gradients. The best separation was obtained when the density ranged from 1.13 to 1.20 g/cm³ when sucrose was used and from 1.12 to 1.28 g/cm³ with CsCl. The buoyant densities of the B and C particle bands in sucrose were 1.18 and 1.16 g/cm³, respectively. The CsCl gradient gave a better separation with the B particles banding at a density of 1.20 g/cm³ and with the C particle density little different from its value in sucrose.     

Defective Friend Spleen Focus-Forming Virus: Interfering Properties and Isolation Free from Standard Leukemia-Inducing Helper Virus

Defective Friend spleen focus-forming virus (SFFV) is able to interfere with the ability of its naturally occurring leukemia-inducing helper virus (LLV-F) to induce XC plaque formation in several different strains of mouse embryo cells. This interference has been observed by using two different SFFV preparations, one contained in an NB-tropic stock of Friend virus (FV) complex, and the second present in a C57BL-adapted strain of FV complex containing an associated B-tropic LLV-F helper. The LLV-F in NB-tropic FV complex effectively induced XC plaques in C57BL/6 (Fv-1^bb; Fv-2^rr) mouse embryo fibroblasts (MEF) only in the absence of coinfecting SFFV, indicating that Fv-2-associated resistance to SFFV-induced focus formation in vivo does not necessarily extend to the restriction of SFFV function(s) in vitro (i.e., in Fv-2^rr C57BL MEF). SFFV interference appears to be an intracellular event since LLV-F can adsorb onto, penetrate, and rescue defective murine sarcoma virus (MSV) from transformed 3T3FL S⁺L⁻ cells with equal efficiency in the presence and absence of SFFV. However, significantly fewer LLV-infected S⁺L⁻ cells released LLV-F progeny if SFFV was present. These observations suggest that Friend SFFV may be classified as a defective, interfering (DI) particle. Further support for this conclusion has come from studies designed to investigate two physical properties of defective SFFV particles. SFFV layered onto a 0 to 20% sucrose sedimentation gradient was recovered as a symmetrical band of virus that sedimented more slowly than standard LLV-F particles. Pooled SFFV-containing gradient samples contained visualizable type C virus particles and occasionally small amounts of detectable LLV-F. In an attempt to determine the buoyant density of sedimentation gradient-purified SFFV, pooled SFFV samples were layered onto a 25 to 50% sucrose equilibrium density gradient and were centrifuged to equilibrium. Greater than 50% of the infectious SFFV originally layered onto this gradient was recovered and seen as a narrow symmetrical band with peak SFFV infectivity at a sucrose density of 1.14 g/ml. The observed difference between SFFV and LLV-F buoyant densities appears to be related to an inherent physical property of each virus. Mixtures of these two viruses express the buoyant density of that virus population which is in excess in fabricated FV complexes probably due to the formation of SFFV-LLV aggregates. Finally, gradient-purified SFFV failed to induce XC plaques in MEF and did not function to rescue MSV as expected since SFFV itself is replication defective.     

Density Gradient Centrifugation of Rubella Virus

Rubella virus was centrifuged in sucrose density gradients. One of two densities could be ascribed to the virus, depending upon the suspending medium used. The virus was found at a density of 1.16 g/cm³ after centrifugation for 18 hr in sucrose gradients prepared in distilled water. By contrast, when the sucrose gradients were prepared in tris(hydroxymethyl)aminomethane (Tris)buffer containing ethylenediaminetetraacetic acid (EDTA), the virus was found at a density of 1.18 g/cm³ after 18 hr of centrifugation. The virus banded at this higher density after only 2 hr of centrifugation when pretreated by overnight incubation in the Tris-EDTA buffer. A kinetic study showed that, in sucrose gradients containing this buffer, the virus gradually migrated as a single peak of infectivity from a density of 1.16 g/cm³ after 2 hr of centrifugation to the higher 1.18 g/cm³ density after 18 hr. The density change was shown to be reversible; after the removal of the Tris-EDTA buffer, rebanding of virus harvested at the heavy density resulted in its banding at the lower 1.16 g/cm³ density. The data indicate that density change could not be explained on the basis of the loss of some component from the virus or on the basis of the failure of the virus to reach equilibrium. However, it is possible that the two densities observed were a reflection of the existence of rubella virus in different hydration states in the presence and absence of Tris buffer containing EDTA.     

What is the origin of the lipids of potato tuber plasmalemma?

Plasmalemma isolated from potato tuber cells was prepared according to a flotation method in a sucrose density gradient. When incubated in vitro with various radioactive lipid precursors, the plasmalemma fragments were unable to synthesize any lipid. When labelled lipid precursors ([¹⁴C]acetate of [³²P]phosphate) were furnished to potato slices, radioactive fatty acids or phospholipids were integrated into plasmalemma lipids. When incubated in a suspension of [³²P]liposomes, plasmalemma fragments accepted labelled lipids from the suspension; this exchange process was markedly stimulated by the phospholipid exchange protein prepared from the potato cytoplasmic supernatant. These results suggest that potato tuber plasmalemma is dependent on other cellular fractions for lipid synthesis and exchange.     

Associations of maize protein bodies with cytoskeleton, membranes, and ribosomes in the endosperm of wild type and opaque-2 mutant

Maize endosperm was homogenized in a cytoskeleton-stabilizing buffer, filtered and layered on gradients of 20–80% sucrose and analyzed by monitoring their UV absorbance. A major peak of UV-light absorbing material was detected on the gradient, at about 60–65% sucrose (density of approximately 1.3 g·ml⁻¹). Biochemical, fluorescence microscopic, and immunoblot analyses of this peak showed that it consisted of protein bodies associated with actin, membranes, and RNA (ribosomes). Seeds of wild type and opaque-2 mutant were then homogenized, the homogenate was modified using detergents and/or cytoskeleton-disrupting agents, and centrifuged on sucrose gradients. In wild type maize endosperm, detergent treatment caused the major peak (protein bodies) to increase in density so that they sediment further down the gradient. However, in opaque-2 the protein bodies formed a broader, but smaller peak which, upon treatment with detergent, generated protein bodies which pelleted to the bottom of the gradient. Analysis of gradient fractions by gel electrophoresis and immuno-blotting showed that both the wild type and the mutant had cytoskeleton proteins in the upper regions (soluble, non-polymerized microfilaments and microtubules) as well as in the peak regions. Comparisons of both the UV-absorbance profiles and the immunoblot data suggest that the protein bodies from the two maize types associate differently with the membranes and the cytoskeleton.     

Isolation of 125I-concanavalin A-labeled plasma membrane from unfertilized mouse eggs

A procedure was developed for isolation of plasma membrane (PM) preparations from unfertilized mouse eggs. Zona-free mouse eggs prepared by the method of Boldt and Wolf (Gamete Res 13:213–222, 1986) were labeled with ¹²⁵I-concanavalin A (ConA) prior to sonication and fractionation on iso-osmotic self-generated Percoll density gradients. Experiments using the ConA-specific sugar α-methylmannoside (αMM) indicated that ¹²⁵I-ConA bound specifically to the egg PM. Greater than 95% of ¹²⁵I-ConA binding to zona-free eggs was blocked in the presence of 0.1 M αMM, and incubation of eggs in αMM after ¹²⁵I-ConA labeling caused release of 85–90% of bound label. Fractionation of ¹²⁵I-ConA-labeled eggs by Percoll density gradient centrifugation yielded a single radioactive peak at density = 1.025, corresponding to egg PM material. Prolonged incubation of ¹²⁵I-ConA-labeled eggs or egg sonicates prior to fractionation did not alter the location of the radioactive peak, indicating that ¹²⁵I-ConA did not label other organelles. As a control, human erythrocytes were labeled with ¹²⁵I-ConA and fractionated under identical experimental conditions and yielded a single radioactive peak at density (1.020) comparable to that observed for ¹²⁵I-ConA-labeled eggs. These results indicate that ¹²⁵I-ConA can be used as a specific marker to support PM isolation from small numbers of zona-free mouse eggs.     

Separation of the Mg-ATPases from the Ca-Phosphatase Activity of Microsomal Membranes Prepared from Barley Roots

Two methods for preparing membrane fractions from barley (Hordeum vulgare cv California Mariout 72) roots were compared in order to resolve reported differences between the characteristics of the plasma membrane ATPase of barley and that of other species. When microsomal membranes were prepared by a published procedure and applied to a continuous sucrose gradient, the membranes sedimented as a single broad band with a peak density of 1.16 grams per cubic centimeter (g/cm³). Activities of NADH cytochrome (Cyt) c reductase, Ca²⁺-ATPase, and Mg²⁺-ATPase were coincident and there was little ATP-dependent proton transport anywhere on the gradient. When the homogenization procedure was modified by increasing the pH of the buffer and the ratio of buffer to roots, the microsomal membranes separated as several components on a continuous sucrose gradient. A Ca²⁺-phosphatase was at the top of the gradient, NADH Cyt c reductase at 1.08 g/cm³, a peak of ATP-dependent proton transport at 1.09 to 1.12 g/cm³, a peak of nitrate-inhibited ATPase at 1.09 to 1.12 g/cm³, and of vanadate-inhibited ATPase at 1.16 g/cm³. The Ca²⁺-phosphatase had no preference for ATP over other nucleoside di- and tri-phosphates and was separated from the vanadate-inhibited ATPase on a sucrose gradient; approximately 70% of the Ca²⁺-phosphatase was removed from the microsomes by washing with 150 millimolar KCl. The vanadate-sensitive ATPase required Mg²⁺, was highly specific for ATP, and was not affected by the KCl wash. These results show that barley roots have a plasma membrane ATPase similar to that of other plant species.     

Fate of Tobacco Mosaic Virus after Entering the Host Cell

Diminutive viral RNAs recovered from tobacco leaves inoculated with ³²P-TMV were investigated. At 3.5 hr after inoculation, most of the viral RNA without coat protein revealed two peaks after sucrose density gradient analysis of SDS-extract from 12,000 × g leaf pellet. The first peak appeared between bacterial ribosomal RNA of 16 S and 5 S and the second peak was around 5 S. These two peaks were digestible with RNase and they appeared as early as 5 min after inoculation. These diminutive RNAs seemed to be derived from partially uncoated parental virus by abscission of their naked RNA tails. The active formation of these diminutive RNAs and their early appearance after inoculation seemed to indicate that most of the inoculated TMV received incomplete uncoating.     

Isolation of plasma membranes from corn roots by sucrose density gradient centrifugation: an anomalous effect of ficoll

An investigation was conducted into the isolation of plasma membrane vesicles from primary roots of corn (Zea mays L., WF9 × M14) by sucrose density gradient centrifugation. Identification of plasma membranes in cell fractions was by specific staining with the periodic-chromic-phosphotungstic acid procedure. Plasma membrane vesicles were rich in K⁺-stimulated ATPase activity at pH 6.5, and equilibrated in linear gradients of sucrose at a peak density of about 1.165 g/cc. It was necessary to remove mitochondria (equilibrium density of 1.18 g/cc) from the homogenate before density gradient centrifugation to minimize mitochondrial contamination of the plasma membrane fraction. Endoplasmic reticulum (NADH-cytochrome c reductase) and Golgi apparatus (latent IDPase) had equilibrium densities in sucrose of about 1.10 g/cc and 1.12 to 1.15 g/cc, respectively. A correlation (r = 0.975) was observed between K⁺-stimulated ATPase activity at pH 6.5 and the content of plasma membranes in various cell fractions. ATPase activity at pH 9 and cytochrome c oxidase activity were also correlated.     

The zonal centrifuge applied to the purification of a low-yield virus in human leukemia cells

The herpes-type virus found in certain cell cultures derived from Burkitt's lymphoma, other human leukemias, and normal human leukocytes, was concentrated and partially purified by large-volume density gradient centrifugation using zonal centrifuge systems. Using the Jiyoye (P-3) cell line as a model, rate-zonal runs on disrupted cell suspensions in sucrose gradients yielded concentrates with high virus particle counts when 10–15 ml of packed cells were processed per liter of gradient. Isolation and removal of cell nuclei or fluorocarbon treatment of cell sonicates permitted virus recovery from larger volumes of cells per experiment. Zonal centrifugation of concentrated cell-free spent media from highly infected cell cultures yielded more purified virus than obtained from cells. Viral concentrates were prepared with particle counts of 10¹⁰–10¹¹/ml and total protein concentrations of 0.2–0.5 mg/ml. Subsequent isopyenie-zonal centrifugation of the various high-count virus fractions from the zonal centrifuge showed a heterogeneity in buoyant virus density ranging from 1.18 to 1.27 in potassium tart rate. The spread in virus density was attributed to the different morphological forms of the virus observed by electron microscopy.     

Synthesis of glycinin in a wheat germ cell-free system.

Polyribosomes isolated from the cotyledons of developing soybean seeds were translated in a wheat germ cell-free system. When the radioactive translation products synthesized in the cell-free system were fractionated by centrifugation on sucrose density gradient, a radioactive peak which overlapped an authentic glycinin was detected. This radioactive co-sedimentable material was judged to be also a glycinin by its behavior toward polyacrylamide gel electrophoresis and immunoprecipitation.     

Intracellular localization of phosphatidylcholine and phosphatidylethanolamine synthesis in cotyledons of cotton seedlings

Subfractionation of clarified cotyledon homogenates of cotton (Gossypium hirsutum L.) seedlings on sucrose gradients revealed a single coincident peak of cholinephosphotransferase (EC 2.7.8.2) (CPT) and ethanolaminephosphotransferase (EC 2.7.8.1) (EPT) activities, which equilibrated with the main peak of Antimycin A-insensitive NADH:cytochrome c reductase (CCR) activity. The small percentage of CPT and EPT activities (less than 5% of the total) in glyoxysome-enriched pellets equilibrated with cytochrome c oxidase activity, not with catalase activity. Preincubation of microsomes (containing 83% of total CPT and EPT activities) in 0.2 millimolar MgCl₂ followed by subfractionation on sucrose gradients resulted in peak CPT and EPT activities equilibrating with peak CCR activity at 24% (w/w) sucrose. Preincubation of microsomes with ¹⁴C-CDPcholine (or ¹⁴C-CDPethanolamine) resulted in synthesis and incorporation of ¹⁴C-phosphatidylcholine (PC) (or ¹⁴C-phosphatidylethanolamine, PE) into membranes at the same density. Increasing the Mg²⁺ concentration to 2.0 millimolar facilitated binding of ribosomes and caused a concomitant shift in density (to 34% w/w sucrose) of peak CPT, EPT, and CCR activities. Under these conditions, newly synthesized and incorporated ¹⁴C-PC (or PE) was recovered in these membranes. Transmission electron microscopy of this fraction confirmed binding of ribosomes to membranes. Radiolabeling in vivo of cotyledons with [methyl-¹⁴C] choline chloride or [1,2 ethanolamine-¹⁴C] ethanolamine hydrochloride resulted in a linear incorporation of radiolabel into PC or PE in a time dependent manner. Subfractionation of homogenates of radiolabeled cotyledons on sucrose gradients showed that membranes sedimenting at 24% (w/w) sucrose (ER) contained the majority of radiolabeled PC and PE with a minor peak at 40% (w/w) sucrose (mitochondria), but no radioactive PC or PE was recovered in glyoxysomes. These results indicate that ER in cotyledons of germinated cotton seedlings is the primary subcellular site of PC and PE synthesis. This is similar to the situation in endosperm tissue but distinctly different from root and hypocotyl tissue where Golgi are a major subcellular site of PC and PE synthesis.     

Equilibrium centrifugation of measles virus

Measles virus has been centrifuged on different density gradients. It sediments at densities of 1,20 g/cm³ in K-tartrate, of 1,18–1,21 g/cm³ in sucrose, 1,19–1,23 g/cm³ in CsCl and 1,19 g/cm³ in metrizamide gradients. Metrizamide reduced measles virus infectivity. In sucrose gradients sometimes more than one infectious peak was observed. Control Vero cells produced particles of the same densities as measles virus peaks. These peaks did contain actin as the major protein. The relevance of this finding in relation to the presence of actin in measles virus is discussed.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: I. Identification and Characterization of an Anion-Sensitive H-ATPase

Microsomal membranes isolated from red beet (Beta vulgaris L.) storage tissue were found to contain high levels of ionophore-stimulated ATPase activity. The distribution of this ATPase activity on a continuous sucrose gradient showed a low density peak (1.09 grams per cubic centimeter) that was stimulated over 400% by gramicidin and coincided with a peak of NO₃⁻-sensitive ATPase activity. At higher densities (1.16-1.18 grams per cubic centimeter) a shoulder of gramicidin-stimulated ATPase that coincided with a peak of vanadate-sensitive ATPase was apparent. A discontinuous sucrose gradient of 16/26/34/40% sucrose (w/w) was effective in routinely separating the NO₃⁻-sensitive ATPase (16/26% interface) from the vanadate-sensitive ATPase (34/40% interface). Both membrane fractions were shown to catalyze ATP-dependent H⁺ transport, with the transport process showing the same differential sensitivity to NO₃⁻ and vanadate as the ATPase activity.

Characterization of the lower density ATPase (16/26% interface) indicated that it was highly stimulated by gramicidin, inhibited by KNO₃, stimulated by anions (Cl⁻ > Br⁻ > acetate > HCO₃⁻ > SO₄²⁻), and largely insensitive to monovalent cations. These characteristics are very similar to those reported for tonoplast ATPase activity and a tonoplast origin for the low density membrane vesicles was supported by comparison with isolated red beet vacuoles. The membranes isolated from the vacuole preparation were found to possess an ATPase with characteristics identical to those of the low density membrane vesicles, and were shown to have a peak density of 1.09 grams per cubic centimeter. Furthermore, following osmotic lysis the vacuolar membranes apparently resealed and ATP-dependent H⁺ transport could be demonstrated in these vacuole-derived membrane vesicles. This report, thus, strongly supports a tonoplast origin for the low density, anion-sensitive H⁺-ATPase and further indicates the presence of a higher density, vanadate-sensitive, H⁺-ATPase in the red beet microsomal membrane fraction, which is presumably of plasma membrane origin.