Metrizamide Density Gradients Research Articles

Generation of oxygen free radicals from blood eosinophils from asthma patients after stimulation with PAF or phorbol ester

Eosinophils (EOS) may play an important role in the pathophysiology of bronchial asthma because they can release oxygen free radicals and several basic proteins which are cytotoxic to bronchial epithelium. We have studied the response of EOS isolated from the blood of atopic subjects with symptoms of asthma (AS, n = 7) or rhinitis (AR, n = 4) or without symptoms (AA, n = 5) and of subjects with the hypereosinophilic syndrome (HES, n = 5). EOS were separated using metrizamide density gradients and activated in vitro with platelet-activating factor (PAF, 100 nM) or phorbol 12-myristate-13-acetate (PMA, 100 nM). Oxygen free radical generation was assessed by a lucigenin-enhanced chemiluminescence (CL) assay. EOS purity was 83 +/- 1.7% (mean +/- SEM) with greater than 95% viability. Background CL responses of EOS from HES were significantly higher than those from AA (p less than 0.01) and AR (p less than 0.05). Normodense EOS from AS (PAF-induced CL = 90 +/- 27 mV) were more responsive to PAF than were those from AR (17 +/- 13 mV, p less than 0.01) and from AA (23 +/- 14 mV, p less than 0.01). Similar results were obtained with PMA. Hypodense EOS from HES subjects were as responsive as normodense EOS from AS to PMA and PFA. Thus, EOS from AS have an enhanced potential for activation, particularly by PAF; this may represent an important mechanism for the perpetuation of the inflammatory response in asthma, since EOS can also release PAF.

The isolation and partial characterization of bovine parathyroid secretory granules

We have developed a procedure which allows the isolation of secretion granules from fresh parathyroid glands. Following collagenase digestion of the tissue, the cells were broken with osmotic shock and a crude granule/mitochondrial pellet was obtained by differential centrifugation. Before loading this fraction onto a metrizamide density gradient it was subjected to brief sonication to disrupt the mitochondria. This procedure was necessary in order to achieve separation of the granules from the mitochondria during ultracentrifugation of the gradient. When the fractionated gradient was analysed for PTH by radioimmunoassay, three bands containing parathyroid hormone were found, at densities of 1.0, 1.05 and 1.18. Upon electron microscopic examination of the gradient fractions, granules were found only in those fractions containing hormone. A typical granule appearance was observed for two of the populations, but the third population (density 1.18), consisted of granules without membranes and which appeared less electron dense than those of populations 1 (density of 1.0) and 2 (density of 1.05). Moreover, the lack of a limiting membrane imparted a fuzzy appearance to the population 3 granules. When fresh tissue sections were examined as control samples, granules with and without membranes were also observed. Standard marker enzyme assays further confirmed that populations 2 and 3 were relatively free of other cellular contaminants, but population 1 contained endoplasmic reticulum and lysosomal material. Because the number of granules contained in this population is very small, we have not been successful in achieving further purification of population 1. Based on radioimmunoassay of extracts of each granule population, PTH was concentrated in population 3, while the other two contained lesser amounts. Interestingly, results obtained with a radioimmunoassay for SP-1 revealed a striking difference in the distribution of SP-1 in the three granule populations. This protein, which is also secreted by the parathyroid gland, was concentrated in populations 1 and 2. Only very low levels were found in population 3. Thus, the two major secretory products are localized in different granule populations. The isolated granules were stable to pH changes, cycles of freeze/thaw and sonication. The yields of PTH extracted from each of the granule populations by freezing and thawing in buffer or by Triton containing solutions were low. PTH was completely extracted from each population only by using 8 M urea in HCl. Lower concentrations of urea were less effective. These results indicate that the molecular architecture of the granules is highly resistant to disruption.

Chinese hamster ovary cells deficient in peroxisomes lack the nonspecific lipid transfer protein (sterol carrier protein 2).

Chinese hamster ovary cells deficient in intact peroxisomes were compared with wild type cells for the presence of the nonspecific lipid transfer protein (nsL-TP; sterol carrier protein 2). With the immunoblotting technique using the affinity purified antibody against rat liver nsL-TP, this protein was shown to be present in the homogenates from wild type cells, but could not be detected in mutant cells. In agreement with a previous study using livers from Zellweger patients it appears that there is a positive, as yet unknown, correlation between peroxisomes and the occurrence of nsL-TP in the cell. As a control using the affinity-purified antibody against the phosphatidylinositol transfer protein from bovine brain, levels of this protein were found to be normal in mutant cells. By use of metrizamide density gradients, nsL-TP was shown to cosediment with a membrane fraction different from peroxisomes. A protein of 58,000 daltons cross-reactive with the antibody against nsL-TP did cosediment with the peroxisomes in wild type cells and possibly with a "peroxisomal remnant" in the mutant cells. Incubation of wild type and mutant cells with L-[3-14C]serine showed that the biosynthesis of phosphatidylserine and the subsequent conversion into phosphatidylethanolamine was comparable in both cell types. This indicates that nsL-TP is not involved in the translocation of phosphatidylserine from the endoplasmic reticulum to the mitochondria as the site of decarboxylation.

Maturational changes in steroidogenic enzyme activities metabolizing testosterone and dihydrotestosterone in two populations of testicular interstitial cells

The present study examined changes in steroidogenic enzyme activities which metabolize testosterone or dihydrotestosterone between days 21-73 of maturation in Band 2 and Band 3 cells isolated by centrifugation of rat testicular interstitial cells on metrizamide density gradients. 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities increased progressively in Band 2 and Band 3 cells between days 21-35 of maturation, then both enzyme activities declined to reach low levels in adult Band 2 and Band 3 cells. The significantly higher activities of both enzymes in Band 3, which contains a greater concentration of Leydig cells at each age, suggest their localization in Leydig cells. 5 alpha-androstane-3 alpha- and 3 beta-hydroxysteroid dehydrogenase activities increased in both Band 2 and Band 3 cells between days 21-50 of maturation and remained elevated; however, dihydrotestosterone was metabolized primarily to 5 alpha-androstane-3 beta,17 beta-diol in Band 2 cells, while 5 alpha-androstane-3 beta,17 beta-diol was the major metabolite of dihydrotestosterone in Band 3 cells. These studies suggest that testosterone accumulation during sexual maturation can be influenced by changing patterns of 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities which metabolize testosterone, and of 5 alpha-androstane-3 alpha- and 3 beta-hydroxysteroid dehydrogenase activities which metabolize dihydrotestosterone in both Band 2 and Band 3 cells.

Phospholipid-Dependence of Plant UDP-Glucose Sterol β-d-Glucosyl Transferase

The phospholipid dependence of the UDP-glucose sterol glucosyl transferase (UDPG-SGTase) from maize coleoptiles was previously demonstrated using the partially purified and highly delipidated enzyme, in the presence of the detergent Triton X-100 (P Ullmann, P Bouvier-Navé, P Benveniste [1987] Plant Physiol 85: 51-55). We now report the reconstitution of the enzyme activity into unilamellar lipid vesicles. This was achieved by adding phospholipids, sterols and beta-octylglucoside to the solubilized enzyme and passing the mixture through Sephadex G-50. The treatment led to almost complete removal of the detergents. The incorporation of UDPG-SGTase in the lipid vesicles was demonstrated by (a) coelution of the enzyme activity with the labeled lipid vesicles (average diameter: 260A) on a Sephacryl S-1000 column and (b) flotation experiments on metrizamide density gradients. Release of dithiobis-(2-nitro-benzoic acid) (DTNB) from DTNB-preloaded vesicles was very slow, indicating good membrane integrity of the vesicles. Treatment of the intact vesicles with the nonpermeant reagent p-chloro-mercuribenzene sulfonate led to more than 95% inactivation of the total enzyme activity, i.e. the activity measured in the presence of Triton X-100 at permeabilizing concentration. This suggests an outward orientation for the active site of the enzyme. Finally, the enzyme was incorporated into vesicles of various phospholipid compositions and the kinetic parameters of the reactions were determined. Our results clearly show that the reconstituted UDPG-SGTase activity is stimulated to a large extent by negatively charged phospholipids.

Positively selected Leu-11a (CD16+) cells require the presence of accessory cells or factors for the lysis of herpes simplex virus-infected fibroblasts but not herpes simplex virus-infected Raji.

Previous studies from our laboratory indicated that human NK activity against HSV-infected fibroblasts (HSV-Fs) but not K562 targets was sensitive to treatment with anti-HLA-DR plus C. In the current study, we have selected Leu-11a+ (CD-16) cells by fluorescence activated cell sorting and found that although Leu-11a enriched populations lysed K562 targets in 14-h 51Cr-release assays, they were unable to kill HSV-Fs targets unless a Leu-11a-depleted population was added back to the effectors or unless known activators of NK cells (IFN-alpha or IL-2) were added to the assays. In contrast, Leu-11a-enriched populations were able to mediate ADCC against HSV-Fs in the presence of sera from HSV-seropositive individuals without the requirement for accessory cells. We have begun preliminary characterization of the accessory cells which allow lysis of HSV-Fs by NK cells: they are HLA-DR+ cells which enrich in the light density fractions of Metrizamide density gradients. They need be present in very small numbers for lysis to take place and are not MHC restricted in that heterologous add-backs between anti-HLA-DR plus C and anti-Leu-11b plus C-treated populations are capable of target cell lysis at levels similar to those achieved with the autologous add-backs. Further, the levels of lysis in heterologous add-back experiments reflected the lytic potential of the effector rather than the accessory cell donor. Finally, although the requirement for accessory cells for NK lysis has been demonstrated for fibroblasts infected with HSV-1, CMV, and VZV, lysis of HSV-infected Raji lymphoblastoid cells is relatively accessory-cell independent, indicating that the requirement for accessory cells for lysis by NK cells is not a property of all herpesvirus-infected targets.

Hormonal Regulation of Rat Testicular Arginine Vasopressin Receptors*

Previous studies have implicated arginine vasopressin (AVP) as a negative intratesticular modulator of androgen production. The present studies were undertaken to further define the testicular AVP system by investigating potential influences of pituitary hormones on testicular receptors for AVP. Binding of [3H]AVP to testicular membranes was time, temperature, and pH dependent, with optimum binding at 90 min, 22 C, and pH 8.0. Addition of divalent cations to the incubation mixture significantly increased binding by 4-, 10-, 15-, and 35-fold with Ca2+, Zn2+, Mg2+, and Mn2+, respectively. The influence of pituitary hormones on testicular AVP receptors was initially assessed by comparing binding parameters obtained by Scatchard analysis of saturation binding data between membranes prepared from intact or hypophysectomized rats. Testicular membranes from intact animals bound 66 +/- 2 fmol AVP/mg protein, whereas membranes from animals hypophysectomized 10 days previously showed a significantly lower binding capacity of 40 +/- 3 fmol AVP/mg protein; however, the affinity of the receptors for AVP did not differ between these two groups. A similar decrease was observed when binding was compared between purified Leydig cells isolated from intact and hypophysectomized animals by Metrizamide density gradients. Time-course analysis of this effect showed a continuous decline in total testicular AVP-binding capacity for the first 2 weeks after hypophysectomy and a slight increase at 3 weeks. Treatment of hypophysectomized animals with pituitary hormones beginning on the third day after hypophysectomy and continuing for either 4 or 7 days showed that LH and GH were able to dose-dependently restore testicular AVP-binding capacity to levels found in intact animals, whereas FSH and PRL were ineffective. These data indicate that testicular AVP receptors are under the control of LH and GH from the pituitary and suggest that pituitary hormones may impact on the efficacy of the testicular AVP system by regulating AVP receptor levels.

Properties and subcellular localization of myocardial fatty acyl-coenzyme A oxidase.

The properties and subcellular localization of fatty acyl-CoA oxidase (FAO) were studied in rat heart homogenates. After differential centrifugation, FAO was sedimentable and enriched in a "light-mitochondrial" fraction. FAO had a pH optimum of 8-9. Among straight-chain, saturated fatty acyl-CoAs, the enzyme showed a marked preference for medium chain substrates (C12 greater than C10 = C8 greater than C16 = C14 greater than C6) over a concentration range up to 100 microM. No activity was observed with C4-CoA. The apparent Michaelis constant (Km) for C12-CoA was 5-10 microM. After removal of nuclei by low-speed centrifugation, combined subcellular particle preparations were obtained by high-speed centrifugation and layered on linear density gradients of metrizamide. After density equilibration, FAO showed a symmetric distribution centered at p = 1.16-1.18, like that of the enzyme catalase, a marker for microperoxisomes. In contrast, enzyme markers for mitochondria, lysosomes, sarcolemma, and sarcoplasmic reticulum were recovered in low-density regions of the gradient. These results provide a direct demonstration of fatty acyl-CoA oxidase in cardiac tissue and its association with microperoxisomes.

Isolation and characterization of gonadotropin-rich secretory granules from rat: depletion by GnRH and by a high affinity agonist

Gonadotropin secretory granules were purified from rat pituitary tissue by differential centrifugation followed by a continuous metrizamide density gradient separation procedure. Fractions were characterized by radioimmunoassay, electron microscopy and polyacrylamide gel electrophoresis. The LH granule peak distribution across the gradient was biphasic with peaks at densities 1.287 and 1.275 (g/ml). Samples taken from the leading and trailing regions of the LH peak remigrated to similar positions in a second gradient indicating that the pattern was not an artifact caused by reversible aggregation during the isolation procedure. Reproducible separation of the LH granule peak distribution enabled us to assess the effect of secretagogues. GnRH (2 μg/rat) and a metabolically stable GnRH agonist (buserelin; 1 μg/rat) both caused a 6- to 8-fold increase in serum LH and a corresponding reduction (23–25%) in the amount of LH present in the granules. In both cases the reduction was more pronounced on the granules at the higher (1.287 g/ml) density and the bimodal LH distribution was shifted to lower densities (1.280 and 1.265 g/ml). Granules containing growth hormone and prolactin migrated in the denser (1.322-1.318 g/ml) region of the gradient whereas granules containing thyroid stimulating hormone and follicle stimulating hormone were present close to the LH peak. Electron microscopic inspection of ultrathin (60 nm) sections verified that the fractions enriched in immunoassayable gonadotropins contained predominantly granules. In the less dense (1.20 g/ml) regions, mitochondria and membranous debris were present. Fifteen days after ovariectomy, total granule LH content increased 6- to 7-fold and estrogen supplementation at 5 μg/rat, but not 1 μg/rat, reversed this effect to that of sham-operated animals.

Insulin-like growth factor-I augments gonadotropin-stimulated androgen biosynthesis by cultured rat testicular cells

In addition to the well-known growth stimulating effects of insulin-like growth factors (IGFs), recent studies suggest that these peptides may also modulate the differentiated functions of endocrine cells. Thus, in the present studies, we have investigated the actions of IGFs on androgen biosynthesis by cultured testicular cells. Treatment of cells obtained from adult hypophysectomized rats with LH (1 ng/ml) stimulated testosterone production 60-fold over basal levels. In contrast, treatment with either synthetic human IGF-I or IGF-II failed to stimulate androgen production. However, concomitant treatment of the LH-containing cultures with increasing doses of IGF-I (10–500 ng/ml) augmented testosterone production up to 70% over that seen with LH alone (ED 50 = 67 ng/ml). Similar effects were obtained with IGF-II but this peptide was about 10-fold less potent than IGF-I. In addition, these peptides also stimulated the accumulation of pregnenolone and progesterone in the culture medium. Additional studies demonstrated specific binding of [ 125I]iodo-IGF-I to testicular cells. This binding was competed by IGF-related peptides with the potency order IGF-II = IGF-I > insulin whereas unrelated peptides did not compete. The cellular localization of these receptors was examined in testicular cells separated on a metrizamide density gradient. IGF-I receptors were evenly distributed between two cell peaks containing subpopulations of Leydig cells whereas much less binding was found in other testicular cell types. Coupled with recent findings indicating testicular production of IGF-I, the present results suggest that this peptide may act as a positive intratesticular modulator of Leydig cell differentiation.

Purification of Ca 2+-activated K + channel protein on calmodulin affinity columns after detergent solubilization of luminal membranes from outer renal medulla

A method is developed for purification of the protein of the Ca 2+-activated K + channel from outer renal medulla of pig kidney. The response of this K + channel to physiological concentrations of Ca 2+ is important for regulation of transtubular NaCl transport. In reconstituted vesicles direct addition of calmodulin doubles Ca 2+ activation with sufficient affinity ( K 1 2 0.1 nM) for chromatographic purification of the protein. For purification luminal plasma membrane vesicles are isolated on metrizamide density gradients and solubilized in CHAPS. The fraction of soluble protein retained on calmodulin-Sepharose 4B columns in the presence of Ca 2+ and eluted by EGTA is 0.7%. The purified protein has high Ca 2+-activated K + channel activity after reconstitution into phospholipid vesicles. It distributes on two bands of 51 and 36 kDa after gel electrophoresis in SDS. The 36 kDa band is rapidly cleaved by trypsin and may be involved in Ca 2+ stimulation of the channel. Phosphorylation from cAMP-dependent protein kinase strongly stimulates Ca 2+-activated K + channel activity and labels the 51 kDa band suggesting that this protein is involved in regulation of K + channel opening.

Vasoactive intestinal peptide stimulates androgen biosynthesis by cultured neonatal testicular cells

Vasoactive intestinal peptide (VIP) was originally isolated from porcine duodenum and considered to be a gut hormone. Recent evidence indicates that it may also be involved in reproductive functions. In this study, a possible action of VIP on steroidogenesis by cultured testicular cells was investigated. Neonatal testicular cells were treated in vitro with hormones for 3 days and medium steroid or cAMP content was measured by radioimmunoassay. Treatment of cultured cells with VIP (10 −9 to 10 −6 M) increased the production of testosterone, progesterone, and pregnenolone in a dose-dependent fashion. Testosterone production in response to 10 −6 M VIP was about 5–10% of that maximally induced by LH. Addition of methyl-isobutyl-xanthine, a phosphodiesterase inhibitor, to the VIP-containing cultures significantly enhanced production of testosterone by 13-fold, of progesterone by 9-fold, and of pregnenolone by 2.5-fold as compared to treatment with VIP alone. Additional experiments also showed a dose-dependent stimulation of cAMP production by VIP. The VIP-related hormones PHM-27, secretin, and glucagon also stimulated progesterone and testosterone production with a potency order (PHM-27 > secretin > glucagon) consistent with that observed for other VIP receptor-mediated actions. A direct stimulatory effect of VIP on Leydig cells was indicated in studies on steroidogenesis by testicular cells separated on a metrizamide density gradient. In these studies, VIP stimulated androgen production in an LH-responsive subpopulation of testis cells but failed to affect steroid production in non-LH-responsive cells. Thus, these experiments demonstrate that VIP, independent of LH, stimulates steroidogenesis in cultured neonatal testicular cells and suggest a possible stimulatory role for VIP in testicular steroidogenesis by the developing testis.

An improved method for isolating type II cells in high yield and purity.

A method has been developed for isolating alveolar type II cells by digesting lung tissue with elastase and "panning" the resultant cell suspension on plates coated with IgG. This method provides both high yield and purity of type II cells. In 50 experiments with rats, we obtained 35 +/- 11 X 10(6) cells/rat, 89 +/- 4% of which were type II cells (mean +/- SD). Type II cells isolated by "panning" adhered more rapidly and completely in tissue culture than did cells isolated by centrifugation over discontinuous density gradients of metrizamide. The "panning" method is superior to other methods for isolating type II cells in that it provides a population of type II cells of both high yield and high purity. The method is fast, reproducible, and easily adaptable to isolating type II cells from species other than rats.

A platelet alpha granule membrane protein that is associated with the plasma membrane after activation. Characterization and subcellular localization of platelet activation-dependent granule-external membrane protein.

We have identified and purified a platelet integral membrane protein (140,000 mol wt), using the KC4 monoclonal antibody specific for activated platelets, that is internal in resting platelets but exposed on activated platelets (Hsu-Lin S.-C., C.L. Berman, B.C. Furie, D. August, and B. Furie, 1984, J. Biol. Chem. 259: 9121-9126.). The expression of the protein on the platelet surface is secretion-dependent. This protein has been named platelet activation-dependent granule-external membrane (PADGEM) protein. PADGEM protein is distinct from the surface glycoproteins of resting platelets, but identical to the S12 antigen, GMP-140. Using immunofluorescent staining, resting platelets failed to stain for PADGEM protein with the KC4 antibody, but after permeabilization showed a punctate staining of the cell interior. Thrombin-stimulated intact platelets stained with a peripheral rim pattern thus demonstrating the translocation of PADGEM protein from an internal location to the cell surface. PADGEM protein expression on the platelet surface at varying thrombin concentrations correlated with alpha granule release, as measured by the secretion of platelet factor 4. Further evidence for an alpha granule localization of PADGEM protein was provided by nitrogen cavitation of resting platelets followed by metrizamide density gradient centrifugation; PADGEM protein codistributed with platelet factor 4. Using immunoelectron microscopy, the protein was localized to the alpha granule in frozen ultrathin sections of resting platelets labeled using rabbit anti-PADGEM protein antibodies, whereas in thrombin-activated platelets, the plasma membrane was labeled. These studies indicate that PADGEM protein is a component of the alpha granule membrane of resting platelets and is incorporated into the plasma membrane upon activation and secretion.

Metabolic and ultrastructural characterization of guinea pig alveolar type II cells isolated by centrifugal elutriation

Direct biochemical studies of the whole lung have been quite misleading because of the heterogeneity of the lung cell types. One of the advantages of studying the isolated cells is to be able to correlate specific metabolic functions with intracellular molecular events and to differentiate factors that affect the type II cell function directly. In the present study we have isolated type II cells from guinea pig lung with elastase and purified them by centrifugal elutriation. These cells fluoresce with phosphine 3R as the dye is specifically taken up by the lamellar bodies. In the electron micrographs, the type II cells display punctate villi, which underwent fragmentation in those cases where metrizamide density gradient was used. Mitochondria are scattered throughout the cytoplasm, and smooth endoplasmic reticulum is sparse. Type II cells possess large irregularly shaped nuclei with peripheral areas of dense hemochromatin and at least one prominent nucleolus. Ovoid lamellar bodies are the most prominent cellular inclusions. These bodies are present throughout the cytoplasm and contain a substructure of whorling and concentric laminations. Biochemical studies indicate that type II cells prepared by centrifugal elutriation are metabolically well preserved as seen from incorporation of [ 14C]leucine into cellular proteins, methyl- 14C]choline into cellular disaturated phosphatidylcholine and CDP[ methy- 14C]choline into mitochondrial and microsomal phosphatidylcholine. Superiority of centrifugal elutriation over the commonly employed combination of discontinuous metrizamide gradient and cell elutriation is evident from the present study.

Subcellular distribution and properties of acyl/alkyl dihydroxyacetone phosphate reductase in rodent livers

On subcellular fractionation, the enzyme acyl/alkyl dihydroxyacetone phosphate (DHAP) reductase (EC 1.1.1.101) in guinea pig and rat liver was found to be present in both the light mitochondrial (L) and microsomal fractions. By using metrizamide density gradient centrifugation, it was shown that the alkyl DHAP reductase activity in the “L” fraction is localized mainly in peroxisomes. From the distribution of the marker enzymes it was calculated that about two-thirds of the liver reductase activity is in the peroxisomes and the rest in the microsomes. The properties of this enzyme in peroxisomes and microsomes are similar with respect to heat inactivation, pH optima, sensitivity to trypsin, and inhibition by NADP + and acyl CoA. The enzyme activity in the peroxisomes and microsomes from mouse liver is increased to the same extent by chronically feeding the animals clofibrate, a hypolipidemic drug. The kinetic properties of this enzyme in these two different organelles are also similar. From these results it is concluded that the same enzyme is present in two different subcellular compartments of liver.

Association of DNA with the nuclear lamina in Ehrlich ascites tumor cells.

We have studied in vitro binding of DNA to nuclear lamina structures isolated from Ehrlich ascites tumor cells. At low ionic strength in the presence of Mg++, they bind considerable amounts of mouse and bacterial DNA, forming complexes stable in 2 M NaCl. Single-stranded DNA and pulse-labeled DNA show higher binding efficiencies than native uniformly labeled DNA. When mixing occurs in 2 M NaCl, complex formation is inhibited. When nuclei are digested with DNAse I under conditions that favor chromatin condensation, DNA associated with matrices subsequently prepared from such nuclei is markedly enriched in satellite DNA. If digestion is carried out with DNAse II while nuclei are decondensed in EDTA, no enrichment in satellite DNA is observed. Preparations of purified, high-molecular weight, double-stranded DNA contain variable amounts of fast-sedimenting aggregates, which are insoluble in 2 M NaCl but are dispersed by DNA fragmentation or denaturation. These results point at some artifacts inherent in studies of DNA bound to residual nuclear structures in vivo and suggest conditions expected to avoid these artifacts. Further, using controlled digestion with DNAse II, we have studied the in vivo association of DNA with nuclear lamina isolated from Ehrlich ascites tumor cells. In the course of DNA fragmentation from above 50 kbp to about 20 kbp average size, the following events were observed. The DNA of high molecular weight (much longer than 50 kbp) behaved as if tightly bound to the nuclear lamina, as judged by sedimentation in sucrose and metrizamide density gradients, electron microscopy, and retention on glass fiber filters. As the size of DNA decreased, it was progressively detached from the nuclear lamina, and at about 20 kbp average length practically all DNA was released. The last 1-4% of DNA, although cosedimenting with the nuclear lamina in sucrose gradients, behaved as free DNA, banding at 1.14 g/cm3 in metrizamide density gradients and showing less than 4% retention on filters. At no stage of digestion did the DNA cosedimenting with nuclear lamina show changes in satellite DNA content relative to that of total DNA or enrichment in newly replicated DNA. It was shown, however, that digestion of nuclear lamina-DNA complex with EcoRI or Hae III led to the formation of DNA-protein aggregates, which banded at 1.35 g/cm3 in high salt containing metrizamide density gradients and which were strongly enriched in satellite DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification and reconstitution of a Na +/K +/Cl − cotransporter and K + channel from luminal membranes of renal red outer medulla

Electrophysiological studies on renal thick ascending limb segments indicate the involvement of a luminal Na +/K +/Cl − cotransport system and a K + channel in transepithelial salt transport. Sodium reabsorption across this segment is blocked by the diuretics furosemide and bumetanide. The object of our study has been to identify in intact membranes and reconstitute into phospholipid vesicles the Na +/K +/Cl − cotransporter and K + channel, as an essential first step towards purification of the proteins involved and characterization of their roles in the regulation of transepithelial salt transport. Measurements of 86Rb + uptake into membrane vesicles against large opposing KCl gradients greatly magnify the ratio of specific compared to non-specific isotope flux pathways. Using this sensitive procedure, it has proved possible to demonstrate in crude microsomal vesicle preparations from rabbit renal outer medulla two 86Rb + fluxes. (A) A furosemide-inhibited 86Rb + flux in the absence of Na + (K +-K + exchange). This flux is stimulated by an inward Na + gradient (Na +/K + cotransport) and is inhibited also by bumetanide. (B) A Ba 2+-inhibited 86Rb + flux, through the K + channel. Luminal membranes containing the Na +/K +/Cl − cotransporter and K + channels, and basolateral membranes containing the Na +/K + pumps were separated from the bulk of contaminant protein by metrizamide density gradient centrifugation. The Na +/K +/Cl − cotransporter and K + channel were reconstituted in a functional state by solubilizing both luminal membranes and soybean phospholipid with octyl glucoside, and then removing detergent on a Sephadex column.

Identification of subpopulations of rat granulosa cells: sedimentation properties and hormonal responsiveness.

Ovarian granulosa cells from small follicles have generally been considered to comprise a homogeneous cell population; however, stratified arrangements of hormone receptors have been found in antral and mural granulosa cells of Graafian follicles. Using cultured granulosa cells derived from immature, hypophysectomized, estrogen-treated rats, we have previously shown that vasoactive intestinal peptide (VIP) as well as FSH stimulates steroid production by these cells. Dose-response analysis indicated that the actions of these hormones were additive, suggesting the presence of subpopulations of granulosa cells. Using a continuous (0-30%) Metrizamide density gradient, we have identified three populations of granulosa cells with different sedimentation properties. After centrifugation for 15 min at 1500 X g, cells sedimented at Metrizamide concentrations of 13%, 18%, and 20% (peaks A, B, and C, respectively). The subpopulation with lowest density (peak A) comprised 5% of the total cells, whereas the remaining cells were distributed approximately equally in the other two peaks. The profiles of estrogen and progesterone production by these cells in response to FSH and VIP indicated that FSH preferentially stimulated steroid production in cells with the highest density (peak C), whereas VIP mainly induced steroidogenic responses in cells of intermediate density (peak B). In contrast, cells with the lowest density (peak A) were unresponsive to either hormone. Treatment with forskolin, a universal adenylate cyclase activator, induced steroid production in both subpopulations B and C. Further studies demonstrated that LH/human CG receptors were induced by FSH and forskolin in cells from peak C, whereas VIP treatment did not induce LH/human CG receptors in cells from peak B. In unfractionated cultured cells, GnRH potently antagonized FSH- but not VIP-induced steroidogenesis. Upon density-gradient fractionation, the profile of GnRH receptor content correlated well with GnRH effects since FSH-responsive cells (peak C) contained the majority of GnRH receptors. The present results demonstrate that granulosa cells from immature follicles are heterogeneous and consist of two major subpopulations of cells with differential responsiveness to FSH and VIP. These findings provide the basis for further morphological and biochemical analysis of subpopulations of granulosa cells during follicular development.

Preparative isolation of peroxisomes from liver and kidney using Metrizamide density gradient centrifugation in a vertical rotor

A method for the preparative isolation of peroxisomes from the livers of rat, guinea pig, and mouse, and also from rat kidney is described. The light mitochondrial fraction, i.e., particles sedimenting between 33,000 and 250,000 g-min, or the postnuclear supernatant of liver or kidney, is subjected to a 20–50% Metrizamide density gradient ultracentrifugation in a vertical rotor. After centrifugation, the peroxisomes (marker enzyme catalase and dihydroxyacetone phosphate acyltransferase) sedimented as a band near the bottom of the tube (ϱ = 1.22 g/ml). From the distribution of different marker enzymes and also from the morphometric examinations, it was demonstrated that the isolated peroxisomes are not contaminated with lysosomes, mitochondria, or microsomes.