THE ISOLATION OF A CELL MEMBRANE FRACTION FROM RAT LIVER

A procedure is described for isolating cell membranes from rat liver homogenates. 20 gm. of rat liver was homogenized in a Dounce homogenizer in ice cold water buffered to pH 7.5 with NaHCO₃, rupturing all of the cells and most nuclei. The diluted homogenate was filtered through cheesecloth to remove precipitated nucleoprotein and centrifuged at 1500 g, 10 minutes, to sediment a crude membrane fraction. The membrane containing sediment was recentrifuged 3 times in conical tubes (1220 g, 10 minutes), the top layer of the 2-layered sediment being retained. Flotation in a sucrose solution d = 1.22 freed the preparation from contaminating cell fragments and nuclear membranes not previously disintegrated. The floating material ~0.4 ml. was quite homogeneous and consisted of thin amorphous membranes. Electron micrographs revealed numerous double profiles similar in shape and dimensions to apposed liver cell membranes in intact tissue.     

ISOLATION OF A GOLGI APPARATUS-RICH FRACTION FROM RAT LIVER : I. Method and Morphology

Golgi apparatus were released without fixatives from rat hepatocytes by gentle homogenization, concentrated by differential centrifugation, and purified by sucrose gradient centrifugation. Examination of sections of purified fractions by electron microscopy showed fields of morphologically intact units of Golgi apparatus consisting of stacks of parallel flattened cisternae, secretory vesicles, and small vesicular profiles. Negative staining of unfixed pellets revealed a complex network of anastomotic tubules continuous with platelike structures and secretory vesicles. These structures corresponded, respectively, to the small vesicular profiles and parallel flattened cisternae with attached secretory vesicles of sectioned material. Small fragments of granular endoplasmic reticulum were often closely associated with the peripheral tubules, suggesting sites of continuity in intact hepatocytes.     

ISOLATION OF A GOLGI APPARATUS-RICH FRACTION FROM RAT LIVER : II. Enzymatic Characterization and Comparison with Other Cell Fractions

Enzymatic activities associated with Golgi apparatus-, endoplasmic reticulum-, plasma membrane-, mitochondria-, and microbody-rich cell fractions isolated from rat liver were determined and used as a basis for estimating fraction purity. Succinic dehydrogenase and cytochrome oxidase (mitochondria) activities were low in the Golgi apparatus-rich fraction. On the basis of glucose-6-phosphatase (endoplasmic reticulum) and 5'-nucleotidase (plasma membrane) activities, the Golgi apparatus-rich fraction obtained directly from sucrose gradients was estimated to contain no more than 10% endoplasmic reticulum- and 11% plasma membrane-derived material. Total protein contribution of endoplasmic reticulum, mitochondria, plasma membrane, microbodies (uric acid oxidase), and lysosomes (acid phosphatase) to the Golgi apparatus-rich fraction was estimated to be no more than 20–30% and decreased to less than 10% with further washing. The results show that purified Golgi apparatus fractions isolated routinely may exceed 80% Golgi apparatus-derived material. Nucleoside di- and triphosphatase activities were enriched 2–3-fold in the Golgi apparatus fraction relative to the total homogenate, and of a total of more than 25 enzyme-substrate combinations reported, only thiamine pyrophosphatase showed a significantly greater enrichment.     

ISOLATION OF SMOOTH VESICLES AND FREE RIBOSOMES FROM RAT LIVER MICROSOMES

Microsomes, isolated from rat liver homogenate in 0.88 M sucrose, have been fractionated by differential centrifugation. The 2nd microsomal fraction, sedimented between 60 minutes at 105,000 g and 3 hours at 145,000 g, consists mainly of smooth vesicles, free ribosomes, and ferritin. By utilizing the differences in density existing between the membranes and the granular elements it has been possible to separate the smooth membranes from the free ribosomes and ferritin. The procedure is to resuspend the 2nd microsomal fraction in a sucrose solution of 1.21 or 1.25 density and centrifuge it at 145,000 g for 20 or 40 hours. A centripetal migration of membranes and a centrifugal sedimentation of granular elements are obtained. Phospholipids, as well as the enzymatic activities DPNH-cytochrome c reductase, glucose-6-phosphatase and esterase are localized in the membranes. The free ribosomes have been purified by washing. A concentration of 200 µg RNA per mg nitrogen has been reached. RNA is also present in the membranes. These results are discussed in relation to current views on microsomal structure and chemistry.     

QUANTITATIVE ISOLATION AND PROPERTIES OF NEARLY HOMOGENEOUS POPULATIONS OF UNDEGRADED FREE AND BOUND POLYSOMES FROM RAT BRAIN1

Abstract— A procedure is described for the preparation of free and bound polysomes from whole homogenate of rat brain tissue. Brain is homogenized in a sucrose-polysome buffer medium high in KCl (250 mm). After a 12-min centrifugation at 135,000 g, the free polysomes in the supernatant are decanted and saved, while the membrane bound polysomes in the pellet are resuspended in homogenizing medium, homogenized in the presence of detergent (Triton X-100), centrifuged for 5min at 1470 g to remove nuclei, decanted, treated with deoxycholate and centrifuged for 10 min at 24,000 g to remove deoxycholate-insoluble material. Polysomes in the two supernatants are harvested by centrifugation through sucrose gradients prepared in high KCl polysome buffer, and with or without cell sap. Free and bound polysomes prepared in this manner are undegraded, equally active in cell-free protein synthesis, and largely free of the usual contaminants. Cross-contamination is minimal (>10%). The recovery of polysomes is at least 95%. The distribution of ribosomes and polysomes in rat brain is 58% free and 42% membrane-bound. The distribution of rat brain RNA is 65% ribosomal and 35% non-ribosomal. Conditions are described for the visualization and analysis of the entire complement of free and bound ribosomes. The size fractionation procedure is rapid and reproducible, requires much less ultracentrifugation than the density-gradient technique, and provides a nearly quantitative means of isolating undegraded free and bound polysomes of rat brain tissue.     

用DNA过量核酸分子杂交法对大鼠肝和大鼠肝癌细胞核RNA的比较研究

用DNA过量核酸分子杂交法观察到,大鼠肝癌细胞中重复频率在10~4以上的Poly(A)-核RNA的频率和种类,均比大鼠正常肝细胞有所增加。大鼠肝癌细胞中重复频率在1—4×10~2的Poly(A)-核RNA和Poly(A)~+核RNA的重复频率,比大鼠正常肝细胞减少,而RNA种类则增加。大鼠肝癌细胞中,接近单拷贝的Poly(A)-核RNA和Poly(A)~+核RNA,其频率比大鼠正常肝细胞略有增加,而种类则减少。用总细胞核RNA得类似结果。     

ISOLATION OF POSTSYNAPTIC DENSITIES FROM RAT BRAIN

Most synapses in the central nervous system exhibit a prominent electron-opaque specialization of the postsynaptic plasma membrane called the postsynaptic density (PSD). We have developed a procedure for the isolation of PSDs which is based on their buoyant density and their insolubility in N-lauroyl sarcosinate. Treatment of synaptic membranes with this detergent solubilizes most plasma membranes and detaches PSDs from the plasma membrane so that they can be purified on a density gradient. Isolated PSDs appear structurally intact and exhibit those properties which characterize them in tissue. The isolated PSDs are of the size, shape, and electron opacity of those seen in tissue; they stain with both ethanolic phosphotungstic acid and bismuth iodide-uranyl lead and the fraction contains cyclic 3',5'-phosphodiesterase activity. Quantitative electron microscope analysis of the PSD fraction gives an estimated purity of better than 85%. Inasmuch as the PSD is associated primarily with dendritic excitatory synapses, our PSD fraction represents the distinctive plasma membrane specialization of this specific synaptic type in isolation.     

SUBFRACTIONATION OF SMOOTH MICROSOMES FROM RAT LIVER

Total smooth microsomes from rat liver isolated on a Cs⁺-containing sucrose gradient were concentrated and subsequently fractionated by zone centrifugation on a stabilizing sucrose gradient. The prerequisite for fractionation is to prepare total smooth microsomes in a nonaggregated condition, as well as to utilize a procedure which counteracts enzyme inactivation. The median equilibrium density of the various smooth microsomal vesicles ranges from 1.10 to 1.18. The phospholipid/protein ratio is identical in all subfractions, but cholesterol, on a PLP basis, is enriched in the subfractions with the highest sedimentation velocity. The enzyme distribution pattern reveals a pronounced heterogeneity. A number of NADH- and NADPH-oxidizing enzymes are concentrated in the upper part of the gradient and exhibit a certain degree of separation from G6Pase. Mg⁺⁺-ATPase and AMPase are enriched in the lower part of the gradient. No specific enrichment of newly synthesized NADPH-cytochrome c reductase activity occurs in any of the subfractions after phenobarbital treatment. These data demonstrate that smooth microsomes, by adequate fractionation procedure, can be separated into subfractious of heterogeneous composition.     

ISOLATION AND ELECTROPHORETIC IDENTIFICATION OF HISTONES OF RAT BRAIN AND LIVER

Abstract—

• 1 Relatively pure brain nuclei were prepared on a discontinuous sucrose gradient which gave yields of between 40 and 60 per cent.
• 2 An extraction procedure has been developed for the isolation of total histones from brain and liver nuclei. This procedure is also applicable to the isolation of histones directly from whole brain and liver.
• 3 The electrophoretic pattern of histones prepared from brain and liver by the above procedure was similar to that of calf thymus histones.

     

THE ISOLATION OF CELL NUCLEI FROM RAT BRAIN

A method for preparing highly pure cell nuclei from adult rat brain, using both differential and isopycnic centrifugation in sucrose media, is described. The morphology of these preparations was examined by both phase contrast and electron microscopy. The isolated nuclei retained many aspects of their in situ morphology; in particular, the nuclear envelope was double layered and interrupted by pore-like discontinuities, and the nucleoli consisted of irregular masses of densely packed granules. Analyses of these nuclear preparations for cytochrome oxidase and cholinesterase activity, as well as RNA/DNA ratio, indicated minimal contamination with mitochondria and microsomes. Problems involving the homogenization technique, choice of ionic conditions in the homogenization medium, and choice of optimal density of the sucrose solution used for the final purification of nuclei are discussed. Results of application of the technique to isolation of adult rat liver nuclei are also reported.     

GLYCOPROTEINS OF THE NUCLEAR MATRIX FROM CHICKEN LIVER CELLS

The nuclear matrix from chicken liver cells contains a small amount of glycoproteins recognized by Concanavalin A. These proteins are present not only in the peripheral matrix, but also in the internal one. In this latter localization many glycoprotein species appear, by cross-linking experiments, to be placed in the proximity of DNA. The effect of a partial enzymatic deglycosylation of matrix preparations suggests that these proteins contribute to the stabilization of the native matrix structure.     

CYTOCHEMISTRY OF GOLGI FRACTIONS PREPARED FROM RAT LIVER

Cytochemical tests for several marker enzymes were applied to liver tissue and to the three Golgi fractions (GF₁, GF₂, GF₃) separated by the procedure of Ehrenreich et al. from liver homogenates of alcohol-treated rats. 5'-Nucleotidase (AMPase) reaction product was found in all three fractions but in different locations: It occurred along the inside of the membrane of VLDL-filled vacuoles in GF₁ and GF₂, and along the outside of the cisternal membranes in GF₃. In the latter it was restricted to the dilated cisternal rims and was absent from the cisternal centers. The AMPase activity found in the fractions by biochemical assay is therefore indigenous to Golgi components and is not due to contamination by plasma membrane. Acid phosphatase (AcPase) reaction product was detected within lysosomal contaminants in GF₁ and within many VLDL-filled vacuoles in GF₁ and GF₂, indicating that AcPase activity is due not only to contaminating lysosomes, but also to enzyme indigenous to Golgi secretory vacuoles. G-6-Pase reaction product was present in GF₃ and within contaminating endoplasmic reticulum fragments, but not in other fractions. Thiamine pyrophosphatase (TPPase) was localized to some of the VLDL-filled vacuoles and cisternae in GF₁ and GF₂, and was not found in the cisternae in GF₃. The results demonstrate the usefulness of cytochemical methods in monitoring the fractionation procedure: They have (a) allowed a reliable identification of contaminants, (b) made possible a distinction between indigenous and contaminating activities, and (c) shown, primarily by the results of the TPPase test, that the procedure achieves a meaningful subfractionation of Golgi elements, with GF₁ and GF₃, representing primarily trans-Golgi elements from the secretory Golgi face, and GF₃ consisting largely of cis-Golgi components from the opposite face.     

ELECTRON MICROSCOPY OF LYSOSOME-RICH FRACTIONS FROM RAT LIVER

A preliminary electron microscope study has revealed the presence in lysosome-rich fractions, isolated from rat liver, of hitherto undescribed cytoplasmic particles, called "dense bodies." Approximately 0.37 µ in length, the dense bodies often possess an internal cavity and external membrane. They contain many electron-dense granules 55 to 77 A, or less, in diameter. Such dense bodies are also visible in electron micrographs of parenchymatous cells in liver sections. The correlations between dense bodies and lysosomes are listed, but until pure preparations are available it is not possible to assert that dense bodies and lysosomes are identical.     

PERMEABILITY OF MICROSOMAL MEMBRANES ISOLATED FROM RAT LIVER

Water compartments, permeability, and the possible active translocation of various substances in rat liver microsomes were studied by using radioactive compounds and ultracentrifugation. The total water of the microsomal pellet, 3.4 µl/mg dry weight, is the sum of water in the extramicrosomal and intramicrosomal spaces, or 56 and 44%, respectively. Sucrose space accounts for 77% of the intramicrosomal water and the hydration water ~ 14%, leaving almost no sucrose-impermeable space when using the ultracentrifugation approach. With increasing sucrose concentration, microsomes do not show an osmotic response. The intramicrosomal water decreases greatly in the presence of Cs⁺ and Mg⁺⁺ in rough but not in smooth microsomes. Uncharged substances of molecular weight of up to at least 600 freely penetrate microsomal membranes, which already become impermeable to charged substances at a molecular weight of 90. These substances also induce an osmotic response. The vesicles can be made permeable to charged substances after water treatment and cooling, which, however, does not increase glucose-6-phosphatase and inosine diphosphatase (IDPase) activities, and these enzymes can still be activated by deoxycholate. IDPase, reduced nicotinamide adenine dinucleotide-cytochrome c reductase, and reduced nicotinamide adenine dinucleotide phosphate-dependent hydroxylation reactions, performed in vitro, also disproved the hypothesis of an accumulation of charged substances inside of vesicles of being a major pathway. The products of the enzymic reactions as well as the glucuronidated form of a hydroxylated product can be recovered on the cytoplasmic side of membranes, and little accumulation occurs in the intravesicular compartment.     

从大鼠肝染色质富集具有转录活性的DNA

采用DNase消化法从大鼠肝染色质分离得到富有转录活性的DNA(sDNA)。sDNA琼脂糖凝胶电泳,在0.5—6Kb范围内背景有一片荧光,小于1Kb范围内,出现明显区带。sDNA为探针与大鼠正常肝核RNA杂交百分数(29.5%),为以总核DNA为探针杂交百分数(8.2%)的3.6倍,并高于sDNA与大鼠肝癌核RNA杂交百分数(16.4%).