Presence de glycosyltransferases a accepteurs chromatiniens dans les membranes nucleaires d'hepatocytes de singeThe presence of glycosyltransferases on chromatin acceptors in monkey liver nuclear membranes

Nuclei were prepared from monkey hepatocytes by centrifugation of the homogenate on a cushion of 2.3 M sucrose, during 45 min at 100 000 × g. The yield was 2.2 · 10⁷ nuclei per g of liver, and 70% of the homogenate DNA was recovered in these nuclei. An electron microscopic study as well as a biochemical analysis of marker enzymes showed that the nuclei are not contaminated by other subcellular fractions, especially endoplasmic reticulum. A mannosyltransferase and an N-acetylglucosaminyltransferase, working on endogenous glycoproteic acceptors, are present in the nuclei for 1.4 and 6.5% of the homogenate activities, respectively.The nuclei are hydrolysed by DNAase I. The suspension, adjusted in 1.9 M sucrose, was centrifuged for 2 h at 100 000 × g, under a buffer layer. Purified nuclear membranes were collected at the interface. These membranes did not contain any more endoplasmic reticulum enzyme activities, but the mannosyl and N-acetylglucosaminyltransferase activities were still present. They essentially work on an exogenous chromatin acceptor, prepared by lysis of the nuclei. The eventual role of these glycosyltransferases in the glycosylation of non-histone proteins is discussed.     

Gonadotropin and prostaglandins binding sites in rough endoplasmic reticulum and Golgi fractions of bovine corpora lutea.

Highly purified rough endoplasmic reticulum and three subfractions of golgi were prepared from 105,000g pellet of the homogenate by centrifugation in floatation and sedimentation discontinuous sucrose gradients. Highly purified plasma membranes were also prepared from 9,000g pellet of the same homogenates for assessment under the same experimental conditions. Although 5′-nucleotidase, a marker for plasma membranes, was markedly enriched in plasma membranes, very little or none of this enzyme activity was found in other fractions. Very little or no NADH cytochrome c reductase activity, a marker for rough endoplasmic reticulum, was found in fractions other than rough endoplasmic reticulum. Galactosyl transferase, a marker for golgi, was found and enriched in all the fractions; however, enrichment in golgi fractions was higher than in other fractions. Very little or no lysosomal marker activity, i.e., acid phosphatase, was found in rough endoplasmic reticulum or golgi fractions as compared to lysosomes. These marker enzyme data suggested that rough endoplasmic reticulum and golgi fractions were relatively pure with little or no cross contamination with other organelles. The [¹²⁵I]human choriogonadotropin ([¹²⁵I]hCG), [³H]prostaglandin (PG)E₁, and [³H]PGF_2a specifically bound to rough endoplasmic reticulum and golgi fractions in addition to plasma membranes. The enrichments of binding in the former two fractions, in some cases, were as high as plasma membranes itself. The specific binding of some of the ligands was found to be partially latent in rough endoplasmic reticulum and golgi fractions but not in plasma membranes. Marker enzyme data, ratio between bindings and marker enzyme activities (an index of organelle contamination), and partial latency of binding suggest that rough endoplasmic reticulum and golgi fractions intrinsically contain gonadotropin and PGs binding sites.     

Woronin bodies fromPenicillium chrysogenum: Isolation and characterization by analytical subcellular fractionation

Differential centrifugation of whole homogenates ofPenicillium chrysogenum, disrupted by a modified Ballotini bead method, resulted in the enrichment of Woronin bodies between 800g (5 minutes) and 6000g (10 minutes). Isolated Woronin bodies are membrane-bounded, electron-opaque, approximately spherical organelles, 0.11 to 0.29 μm in diameter. Woronin bodies have a buoyant density (ϱ) of 1.21 g cm⁻³ and S_20,w values of 6300 to 37,600 in sucrose gradients. Analytical subcellular fractionation of whole homogenates in a zonal rotor showed that Woronin bodies did not cosediment with marker enzymes for lysosomes (acid phosphatase), peroxisomes (catalase), mitochondria (cytochrome c oxidase), or endoplasmic reticulum (NADPH cytochrome c reductase).     

Boophilus microplus: characterization of larval phosphomonoesterases and isolation of subcellular fractions with high phosphatase activity.

Phosphomonoesterase activity was determined for a 115,000g pellet and soluble fractions resulting from a subcellular fractioning of a homogenate of larval Boophilus microplus. Both fractions showed maximum phosphatase activity at pH 5.5 and 10. Acid phosphatase (EC 3.1.3.2) activity was found to be greatest in the soluble fraction. When the reaction rate was plotted against homogenate concentration, the soluble acid phosphatase deviated from the linear relationship. For both fractions different thermostability patterns were obtained, inactlvation beginning for the alkaline phosphatase (EC 3.1.3.1) at 45–55 C. When the effect of substrate concentration on activity was studied, deviations from the typical hyperbolic behavior were observed. Homogenization of larvae with 5 mm EDTA buffer failed to yield a low-speed pellet with high alkaline phosphatase activity, as it is expected if absorptive structures sediment. Moreover, total alkaline phosphatase activity recovered by this method is significantly lower than activity recovered when homogenization is carried out without EDTA. Alternately, homogenization with 10 mM Tris buffer and 0.25 M sucrose gave 27,000g and 115,000g fractions with high phosphatase activity when fractioned by centrifugation. Alkaline treatment of the 115,000g fraction with 10 mM Tris buffer, pH 7.8, failed to separate endoplasmic reticulum contaminants without loss of phosphatase activity. When the 115,000g fraction was centrifuged in a sucrose density gradient, two activity peaks, coincident for both acid and alkaline phosphatases, were obtained. Antigenic analysis showed the existence of similar antigenic determinants in both peaks “immunologically” presented in different ways.     

Androgen receptors in rat testis

Testosterone (T) and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one; DHT) are bound to specific cytoplasmic receptors (CR) in 105, 000 × g supernatant fractions of seminiferous tubules from hypophysectomized rats following the intravenous injection of [1, 2-³h]testosterone. CR is clearly different from the testicular androgen binding protein (ABP) by electrophoretic mobility, temperature stability and rate of dissociation of steroid-CR complex, but very similar to the cytoplasmic receptors of epididymis and ventral prostate. Under these labeling conditions, the nuclei of seminiferous tubules also contain radioactive T and DHT bound to protein. These androgen-protein complexes, which can be extracted with 0.4 M ? 1 M KC1, have a sedimentation coefficient of 3–4 S. Binding of radioactive T and DHT to both cytoplasmic and nuclear receptors $\text{in vivo}$ is specific for androgen target tissues and abolished by simultaneous injection of unlabeled T, DHT and cyproterone acetate (1, 2-α-methylene-6-chloro-pregn-4, 6-diene-17α-o1–3, 20-diene-17-acetate), but not by cortisol. It is suggested that receptors for testosterone and DHT in the seminiferous tubules are involved in the mediation of the androgenic stimulus to the germ cells.     

Androgen receptors in rat testis

Testosterone (T) and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one; DHT) are bound to specific cytoplasmic receptors (CR) in 105,000 × g supernatant fractions of seminiferous tubules from hypophysectomized rats following the intravenous injection of [1,2-³H]testosterone. CR is clearly different from the testicular androgen binding protein (ABP) by electrophoretic mobility, temperature stability and rate of dissociation of steroid-CR complex, but very similar to the cytoplasmic receptors of epididymis and ventral prostate. Under these labeling conditions, the nuclei of seminiferous tubules also contain radioactive T and DHT bound to protein. These androgen-protein complexes, which can be extracted with 0.4 M — 1 M KC1, have a sedimentation coefficient of 3–4 S. Binding of radioactive T and DHT to both cytoplasmic and nuclear receptors $\text{in vivo}$ is specific for androgen target tissues and abolished by simultaneous injection of unlabeled T, DHT and cyproterone acetate (1,2-α-methylene-6-chloro-pregn-4, 6-diene-17α-ol-3,20-diene-17-acetate), but not by cortisol. It is suggested that receptors for testosterone and DHT in the seminiferous tubules are involved in the mediation of the androgenic stimulus to the germ cells.     

Studies on isolated subcellular components of cat pancreas

Summary Pancreas of the cat was fractionated into its subcellular components by centrifugation through an exponential ficoll-sucrose density gradient in a zonal rotor. This enables a preparation of four fractions enriched in plasma membranes, endoplasmic reticulum, mitochondria and zymogen granules, respectively. The first fraction, enriched by 9- to 15-fold in the plasma membrane marker enzymes, hormone-stimulated adenylate cyclase, (Na⁺K⁺)-ATPase, and 5-nucleotidase, is contaminated by membranes derived from endoplasmic reticulum but is virtually free from mitochondrial and zymogen-granule contamination. The second fraction from the zonal gradient shows only moderate enrichment of the above marker enzymes but contains a considerable quantity of plasma membrane marker enzymes and represents mostly rough endoplasmic reticulum. The third fraction contains the bulk of mitochondria and the fourth mainly zymogen granules as assessed by electron microscopy and marker enzymes for both mitochondria and zymogen granules, namely succinic dehydrogenase, trypsin and amylase. Further purification of the plasma membrane fractions by differential and sucrose step-gradient centrifugation yields plasma membrane enriched 40-fold in basal and hormone-stimulated adenylate cyclase and (Na⁺K⁺)-ATPase.     

The subcellular distribution of carotenoids in Neurospora crassa

The intracellular localization of carotenoids in the fungus Neurospora crassa was examined after completion of photoinduced biosynthesis of these pigments. Differential centrifugation of cell homogenates yielded subcellular fractions which were characterized by activities of several marker enzymes for cell constituents and in part purified by subsequent sucrose density gradient centrifugation. Most (ca 58%) of the carotenoids were found to be localized in lipid globules, but substantial amounts are also associated with two membrane fractions that were rich in membranes of the endoplasmic reticulum as indicated by high activities of NADPH- and NADH—cytochrome c reductase. These results, along with the coincidence in the distribution of both carotenoids and activities of specific marker enzymes in the sucrose density gradients, led to the conclusion that apart from lipid globules, carotenoids are also localized in membranes of the endoplasmic reticulum.     

Intracellular localization of N-formyl chemotactic receptor and Mg2+ dependent ATPase in human granulocytes

Human granulocytes were disrupted by nitrogen cavitation and the lysates fractionated by sucrose density gradient centrifugation at 83 000 × g for 20 min (rate zonal) or 3.5 h (isopycnic). The distribution of marker enzymes allowed the identification of the following subcellular components: plasma membrane, Golgi, endoplasmic reticulum, azurophil granules, specific granules, mitochondria and cytosol. Examination of the gradient fractions by electron microscopy confirmed the biochemical marker analysis. The protocol permitted isolation of vesicles highly enriched in either plasma membrane or Golgi (galactosyl transferase) activities. Absolute plasma membrane yields of 40–60% were achieved with a 20–70-fold increase in specific activity of surface marker over the cells. Plasma membrane sedimented to an average density of 1.14 g·cm^?3. Galactosyl transferase activity was bimodal in distribution. The denser peak cosedimanted with specific granules (g9 = 1.19). The lighter peak sedimented to unique position at an average density of 1.11, was enriched 18-fold over the low speed supernatant, and contained structures resembling Golgi. N-Formyl-Met-Leu-Phe binding and Mg²⁺ -ATPase activities cosedimented with the plasma membrane as well as specific granule and/or high density galactosyl transferase fractions. These findings suggest that Mg²⁺ -ATPase and N-formyl chemotactic peptide receptor activities may be localized in an internal pool of membranes as well as in the plasma membrane and that Golgi may have been a contaminant of previous granulocyte plasma membrane or specific granule preparations.     

Characterization of a Membrane Fraction Containing a b-type Cytochrome

The various components obtained from etiolated corn (Zea mays L.) coleoptiles were fractionated by differential or sucrose gradient centrifugation. The endoplasmic reticulum, proplastids, Golgi, and mitochondria were localized by enzymic or other markers in the various fractions. A fifth fraction was also characterized. It contains glucan synthetase II activity, binding sites for N-naphthylphthalamic acid, NADH dehydrogenase activity which is both antimycin A- and cyanide-insensitive, and a b-type cytochrome. It is possible that this fraction is plasma membrane and that it may contain the blue-ultraviolet photoreceptor for phototropism in corn.     

Failure of lactoperoxidase to iodinate specifically the plasma membrane of cucurbita tissue segments

An attempt has been made to use lactoperoxidase-catalyzed iodination of excised Cucurbita hypocotyl hooks to monitor the distribution of plasma membrane fragments relative to that of phytochrome in particulate fractions from this tissue. Upon fractionation, the iodinated tissue yields a 20,000g pellet which contains 58% of the trichloroacetic acid-precipitable ¹²⁵I at a specific radioactivity 12 times that of the proteins in the supernatant. On sucrose gradients, the labeled fraction has a mean isopycnic density of 1.15 g · cm⁻³. The distribution profile is distinct from that of the total particulate protein and does not coincide with either mitochondrial or endoplasmic reticulum markers. These observations satisfy operational criteria commonly accepted in other systems as indices of selective labeling of the cell surface. The sucrose gradient profiles of the phytochrome and ¹²⁵I in the 20,000g pellets are noncoincident. In the absence of more direct evidence, this is readily interpreted to indicate a lack of association of the pigment with the plasma membrane.     

Effect of calcium on subcellular distribution of peroxidases

About 40% of the peroxidase activity extracted from hypocotyl hooks of etiolated Cucurbita pepo was pelletable at 20000 g. The activity in the pellet was partially solubilized by the addition of 5 mM EGTA. This effect of EGTA was reversed by Ca²⁺, but not by Mg²⁺. Reassociation of EGTA-solubilized peroxidases to 4 cellular fractions obtained by centrifugation on discontinuous sucrose gradients was assayed. It appeared that the enzymes could be linked to ribosomes or RNP particles through Ca²⁺. Two fractions, identified as smooth and rough endoplasmic reticulum, also bound peroxidases and, in this case, the Ca²⁺-mediated binding involved a loss of the enzyme activity. The fraction containing mitochondria and plasmalemma exhibited a slight binding capacity. Isoelectric focusing in thin layer polyacrylamide gel showed that only 5 out of the 10 isoperoxidases present in hypocotyl hooks had their pelletability level changed by Ca²⁺.     

Location of rat liver iodothyronine deiodinating enzymes in the endoplasmic reticulum

The iodothyronine-deiodinating enzymes (iodothyronine-5- and 5′-deiodinase) of rat liver were found to be located in the parenchymal cells. Differential centrifugation of rat liver homogenate revealed that the deiodinases resided mainly in the microsomal fraction. The subcellular distribution pattern of these enzymes correlated best with glucose-6-phosphatase, a marker enzyme of the endoplasmic reticulum. Plasma membranes, prepared by discontinuous sucrose gradient centrifugation, were found to contain very little deiodinating activity. Analysis of fractions obtained during the course of plasma membrane isolation showed that the deiodinases correlated positively with glucose-6-phosphates (r >/ 0.98) and negatively with the plasma membrane marker 5′-nucleotidase (r ranging between ?0.88 and ?0.97). It is concluded that the iodothyronine-deiodinating enzymes of rat liver are associated with the endoplasmic reticulum.     

Subcellular fractionation of actively growing protoplasts of Saccharomyces cerevisiae

Cell homogenates obtained from partially regenerated Saccharomyces cerevisiae protoplasts were fractionated by a procedure using a combination of continuous and discontinuous sucrose gradients, under experimental conditions that minimize possible artifacts due to centrifugation and resuspension. At least five different membranous organelle fractions (plasma membrane, mitochondria, rough endoplasmic reticulum, smooth endoplasmic reticulum-like structures and small-sized particulated structures) were isolated. Subcellular fractions were characterized by assaying established marker enzymes. Radioactive labelled ([U-³H]uracil) ribosomes were also used as a further characterization criterion of the rough endoplasmic reticulum. Comparative SDS-polyacrylamide gel electrophoresis of the protein constituents of the isolated membrane-bound organelles suggest that the polypeptide pattern could also be used as an additional marker for some of these structures. Finally, subcellular distribution of chitin synthase was determined using this fractionation procedure, and two partially zymogenic enzyme pools (one inside the cell associated to particles which sediments at high speed, and the second one associated to the plasma membrane) were found.     

Effects of centrifugal force and centrifugation time on the sedimentation of plant organelles

The effect of centrifugal force and length of centrifugation time on the sedimentation of plant organelles was determined for corn (Zea mays L.) root homogenates. A centrifugal force of 6000g for at least 20 minutes was necessary to pellet 90% of the mitochondrial marker (cytochrome c oxidase). This initial centrifugation step is optimal for separating mitochondria from microsomes, since cross-contamination of endoplasmic reticulum and plasma membrane vesicles with mitochondria is minimized. Centrifugal forces of 8000g or 10,000g for 20 minutes and 13,000g for 15 minutes pellet 90% of the mitochondrial marker; however, these centrifugation conditions also sediment more plasma membrane and endoplasmic reticulum.     

Physiological role for androgen binding protein-steroid complex in testis?

The concentration of $\text{unbound}$ androgens in the lumen of rat testis seminiferous tubules is not dependent on the presence of the extra-cellular androgen binding protein (ABP); other parameters such as permeability properties, fluid dynamics and metabolic activities in different testicular compartments appear to have a much greater influence. It has been shown that in other target tissues the metabolic effects of steroids on cells depend on the concentration of the steroids which are not bound to extra-cellular proteins. It is therefore unlikely that the physiological function of ABP is related to the accumulation of androgens around germinal cells.     

Characterization of cell membrane and sarcoplasmic reticulum from bovine uterine smooth muscle

Subcellular fractions, enriched in sarcoplasmic reticulum or in cell membrane, were separated from one another. Starting material was a microsomal pellet (15–40 × 1000g) obtained by differential centrifugation from the uteri of close-to-term pregnant cows. A microsomal fraction enriched in ATP-dependent calcium accumulation was shown to contain sarcoplasmic reticulum and cell membrane. Only 8% or less of the protein in this fraction could be recovered, using affinity chromatography on Sepharose 6MB wheat germ agglutinin. The small yields did not allow extensive characterization. A method was developed to separate sarcoplasmic reticulum from cell membrane using discontinuous sucrose density gradient centrifugation. Protein was collected at the 24–28, the 28–33, and the 33–45% sucrose interfaces. Characterization was by enzyme assays and by specific receptor assay. ATP-dependent calcium accumulation was fourfold greater in the 24–28% sucrose layer than in the 33–45% layer. In contrast, 5′-nucleotidase was more than threefold as high in the 33–45% sucrose layer as in the 24–28% layer. Ouabain-inhibited p-nitrophenylphosphatase doubled and ouabain-inhibited Na,K-ATPase tripled in the 28–33% layer, compared with the 24–28% layer, specific ouabain binding was also doubled in the 28–33% sucrose layer. ¹²⁵I-Labeled wheat germ agglutinin binding was greatest in the 33–45% sucrose layer. It is concluded that the 24–28% layer consists primarily of sarcoplasmic reticulum, whereas the 28–33 and the 33–45% layers are concentrated in the cell membrane. Specific prostaglandin (PGE₂) binding was found to be a property of the cell membrane.     

Subcellular location of adenylate cyclase in rat cardiac muscle

Crude homogenates of rat cardiac muscle were fractionated in order to examine the subcellular location of adenylate cyclase in this tissue. The fractionation procedure employed differential centrifugation of homonized material, followed by collagenase treatment, centrifugation on a discontinuous sucrose density gradient and extraction with 1 M KCl. The particulate fraction obtained by this procedure contained a high specific activity and yield of adenylate cyclase, moderate levels of mitochondria and low levels of sarcoplasmic reticulum and contractile protein as judged by marker enzyme activities. Adenylate cyclase was purified 20-fold with a 33% yield from the crude homogenate, while mitochondrial, sarcoplasmic reticulum and contractile protein yields were 5, 0.4 and 0.7% respectively. The membrane fractions prepared in this manner were examined by sodium dodecyl sulfate · gel electrophoresis.Adenylate cyclase copurified with ouabain-sensitive (Na⁺ + K⁺)-ATPase, a plasma membrane marker enzyme, and not with Ca²⁺-accumulating activity, which is associated with the sarcoplasmic reticulum. The distribution of marker enzyme activities indicates that heart adenylate cyclase is not located in the sarcoplasmic reticulum but is localized predominantly, if not exclusively, in the plasma membrane.     

Heterogeneous distribution of glycosyltransferases in the endoplasmic reticulum of castor bean endosperm

Endoplasmic reticulum membranes stripped of attached ribosomes were isolated from homogenates of germinating castor bean (Ricinus communis L.) endosperm by sucrose density gradient centrifugation. The isolated endoplasmic reticulum fraction was further separated into two major membrane subfractions by centrifugation on a flotation gradient. Both subfractions appeared to be derived from the endoplasmic reticulum inasmuch as they share several enzymic markers including cholinephosphotransferase, NADH-cytochrome c reductase, and glycoprotein fucosyl-transferase and phase separation of membrane polypeptides using Triton X-114 revealed a striking similarity in both their hydrophilic and hydrophobic protein components. The endoplasmic reticulum membrane subfractions contain glycoproteins which were readily labeled by incubating intact endosperm tissue with radioactive sugars prior to fractionation.

Castor bean endosperm endoplasmic reticulum apparently exhibits a degree of enzymic heterogeneity, however, since the enzymes responsible for the synthesis of dolicholpyrophosphate N-acetylglucosamine and dolicholmonophosphate mannose together with their incorporation into the oligosaccharide-lipid precursor of protein N-glycosylation were largely recovered in a single endoplasmic reticulum subfraction.

     

Intracellular localization of heat shock proteins in maize

The intracellular distribution of the maize root heat shock proteins (hsp) was studied as a step toward understanding their physiological function. Linear sucrose density centrifugation was employed to separate organelles so the relative quantities of hsp in different subcellular compartments could be analyzed in a single preparation. Gradient fractions were assayed for the presence of hsp by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and for marker enzyme activities. Analyses of 15 to 60% gradients showed five hsp to be organelle associated. Hsp 25 and 72 were in fractions containing closely equilibrating Golgi and endoplasmic reticulum marker activities, while hsp 18, 29, and 72 were in fractions containing overlapping plasma membrane, mitochondria, and glyoxysomal marker activities. Hsp larger than 72 kilodaltons were not present in gradient fractions. A second fractionation scheme achieved better separation of the two sets of closely equilibrating organelles. When a 13,000g centrifugation step to remove mitochondria was employed prior to gradient centrifugation, hsp 29 was absent from the gradient fractions. If the buoyant density of the endoplasmic reticulum was shifted by either maintaining the ribosomes on the membrane or removing them, a corresponding shift in the equilibrium positions of hsp 25 and 72 occurred. Hsp 18 and 70 remained in plasma membrane-containing fractions irrespective of these treatments.