The Purification and Biochemical Characterization of Bovine Liver Nuclear Membranes

Berezney, Ronald; Macaulay, L.K.; Crane, F.L.

doi:10.1016/s0021-9258(20)81140-5

Cited by 108 publications

(19 citation statements)

References 52 publications

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“…Nuclear membranes were isolated as described by Berezney et al (9,19) with slight modifications. The isolated nuclei were suspended in 0.25 M sucrose TM buffer at a protein concentration of 2 mg/ml, and digested for 12-14 h at 2~ with 25/~g of DNase (Worthington Biochemical Corp., Freehold, N.…”

Section: Residual Nuclear Membranesmentioning

confidence: 99%

“…Assays for various electron transport components were performed as previously reported (19). NADHcytochrome c reductase activity was assayed by following the reduction of cytochrome c at 550 nm.…”

Section: Electron Transport Componentsmentioning

confidence: 99%

“…Succinate dehydrogenase activity was assayed by measuring the reduction of phenazine methosulfate spectrophotometrically at 600 nm coupled to 2,6-dichlorophenolindophenol. All spectrophotometric determinations were performed with a Gilford Model 2000 recording spectrophotometer at 30~ Cytochrome bs was determined by difference spectra in a Cary-15 double beam spectrophotometer (Cary Instruments, Monrovia, Calif.) after reduction by dithionite or NADH (19). Cytochrome c oxidase activity was measured polarographically by a modification of the assay of Chuang et al (27) as previously described (19).…”

Section: Electron Transport Componentsmentioning

confidence: 99%

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Nuclear matrix: isolation and characterization of a framework structure from rat liver nuclei

Berezney

Coffey²

1977

The Journal of Cell Biology

576

261

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A nuclear framework structure termed the nuclear matrix has been isolated and characterized. This matrix forms the major residual structure of isolated nuclei and consists largely of protein with smaller amounts of RNA, DNA, carbohydrate, and phospholipid. The nuclear matrix can be further resolved by combined treatment with DNase and RNase. The remaining nuclear protein structure, after extraction of 90% of the nuclear protein, 99.9% of the DNA, and 98% of the RNA and phospholipid, is termed the nuclear protein matrix. Electron microscopy of this final nuclear protein matrix reveals an interior framework structure composed of residual nucleolar structures associated with a granular and fibrous internal matrix structure. The internal matrix framework is derived from the interchromatinic structures of the nucleus, and is connected to a surrounding residual nuclear envelope layer containing residual nuclear pore complex structures.Sodium dodecyl sulfate-acrylamide gel electrophoresis of the nuclear matrix proteins demonstrates three major polypeptide fractions, P-l, P-2, and P-3, with average molecular weights of -69,000, 66,000 and 62,000, as well as several minor polypeptides which migrate at -50,000 and at higher molecular weights (>100,000). Polypeptides with molecular weights identical to those of P-l, P-2, and P-3 are also components of isolated nuclear envelopes and nucleoli, whereas isolated chromatin contains no detectable matrix polypeptides. This suggests that the major matrix polypeptides are localized in specific structural regions of the nucleus, i.e., nuclear envelope, nucleoli, and interchromatinic structures. The presence of cytochrome oxidase activity in the isolated nuclear matrix indicates that at least some integral proteins of the nuclear membrane are associated with the matrix.Preliminary communications from our laboratory (12-14) have reported the isolation of a residual framework structure derived from rat liver nuclei, and termed the nuclear protein matrix, or more generally the nuclear matrix. Aside from serving as a skeletal framework, the nuclear matrix may

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Section: Residual Nuclear Membranesmentioning

confidence: 99%

Section: Electron Transport Componentsmentioning

confidence: 99%

Section: Electron Transport Componentsmentioning

confidence: 99%

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Nuclear matrix: isolation and characterization of a framework structure from rat liver nuclei

Berezney

Coffey²

1977

The Journal of Cell Biology

576

261

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“…Intact nuclei and nuclear membranes were isolated by the procedure of Berezney et al . (5), or sonicated to include nuclear organelles . Intact mitochondria were isolated and purified according to the procedure of Bustamente et al (10) .…”

Section: Incubation Of Microtubule Protein With Liver Membranesmentioning

confidence: 99%

Effect of various hepatic membrane fractions on microtubule assembly-with special emphasis on the role of membrane phospholipids.

Reaven¹,

Azhar²

1981

The Journal of Cell Biology

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This report describes an interaction between rat brain microtubule protein and various hepatic fractions in vitro . Purified preparations of Golgi membranes, plasma membranes, rough and smooth endoplasmic reticulum, nuclear membranes, and mitochondria were obtained from the livers of 200-g rats . Several concentrations of fresh or sonicated frozen membranes were incubated with twice-cycled rat brain microtubule protein in a microtubule assembly buffer for 60 min at 30°C. Changes in microtubule assembly were assessed either by quantitative electron microscopy on negatively stained samples or by spectrophotometric methods. The results show that all the tested membranes "bound" microtubule protein, preventing assembly : Golgi and plasma membranes, as well as mitochondria, were especially potent in this regard . To identify the membrane-associated components responsible for microtubule protein binding, the membranes were extracted with methanol-chloroform, and liposomes were prepared from the resulting lipids . Microtubule protein incubated with these liposomes showed a differential ability to assemble that was similar to the effect obtained with intact membranes . Membrane-extracted phospholipids were identified as the lipid component responsible for these changes, with the negatively charged phospholipids (cardiolipin and phosphatidylserine) being uniquely active . These findings indicate that hepatic membranes differentially interact with brain microtubule protein ; this interaction may be dependent on membrane phospholipids .In a previous in vivo study (32), we noted a unique association between microtubules and Golgi complexes in rat hepatocytes following colchicine treatment. This led to the suggestion that microtubules may normally aid in maintaining the structural integrity of the cisternal membranes in Golgi complexes and, in so doing, play a crucial, if permissive, role in hepatic lipoprotein secretion. In the present study, we wish to see whether isolated Golgi complexes show any special interactions with microtubule protein in vitro. Accordingly, Golgi complexes and various other hepatic membranes were isolated and incubated with twice-cycled brain microtubule protein . The results show that all liver membranes "bind" microtubule protein, but that some membranes, such as those derived from the Golgi complex and cell surface, are particularly effective in this regard . Subsequent incubation with the lipids of the various membrane preparations show an effect similar to that obtained with the intact membranes themselves and suggests that mem-300 brane phospholipids may play an important role in microtubule protein-membrane interactions . MATERIALS AND METHODS Incubation of Microtubule Protein with Liver MembranesVarious concentrations of fresh, intact, or frozen-sonicated hepatic membranes were preincubated with twice-cycled rat brain microtubule protein (120 pg per incubation vial or 460 kg/ml) for 30 min at 30°C, pH 6.4, in the following buffer : 100 mM 2-[N-morphohno]ethane sulfonic acid [MES]; ...

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“…low cholesterol and sphingo myelin; little, if any, cardiolipin; little, if any, glycolipid; high phosphatidylcholine; e.g. Gurr, Finean & Hawthorne 1963;Lemarchal & Bornens 1969;Keenan, Berezney, Funk & Crane 1970;Kleinig 1970;K handaw ala & Kasper 1971;Kleinig, Zen Gomes & Stadler 1971;Zentgraf, Deumling, Jarasch & Franke 1971;Keenan, Berezney & Crane 1972;Sato, Fujii, M atsuura & U eda 1972;Franke 1974;Kasper 1974), and by its protein and enzyme activity pattern (table 1, figures 5 and 6; Kashnig & Kasper 1969;Zbarsky et al 1969;Berezney et al 1970;Berezney, M acaulay & Crane, 1972;Kasper 1971Kasper , 1974Zentgraf et al 1971;Kay, Fraser & Johnston 1972;M onneron et al 1972;Zbarsky 1972aKartenbeck, Jarasch & Franke 1973;Jarasch & Franke 1974).…”

Section: Escription Of Plate 24mentioning

confidence: 99%

Nuclear envelopes: Structure and biochemistry of the nuclear envelope

1974

Phil. Trans. R. Soc. Lond. B

118

100

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The ultrastructure of the nuclear evelope is described in various cell types with special emphasis on its pore complexes (p.c.). The architecture of the p.c. is defined against the properties of other membranous pore formations. Evidence is presented that the non-membranous p.c. components contain ribonucleoproteins but do not represent the attachment sites of nuclear chromatin. The possible dynamic nature of the p.c. material is discussed in relation to nucleocytoplasmic translocation processes. DNA of the nuclear genome is firmly attached to interporous sections of the inner nuclear membrane. The stability of this attachment is demonstrated, and chemical and conformational characteristics as well as periods and kinetics of replication are given for both isolated membrane DNA and the corresponding chromatin in situ . The membrane-associated chromatin is dominated by a heterochromatinous character; it does not represent a transitory membrane interaction of replicating DNA. It is hypothesized that membraneattachment of specific regions of the chromosomes are a means to their ordered arrangement during interphase and prophase. Structure, lipid, protein and enzyme pattern of the nuclear membranes, as well as the incorporation kinetics, underline the relationship to the endoplasmic reticulum.

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The Purification and Biochemical Characterization of Bovine Liver Nuclear Membranes

Cited by 108 publications

References 52 publications

Nuclear matrix: isolation and characterization of a framework structure from rat liver nuclei

Nuclear matrix: isolation and characterization of a framework structure from rat liver nuclei

Effect of various hepatic membrane fractions on microtubule assembly-with special emphasis on the role of membrane phospholipids.

Nuclear envelopes: Structure and biochemistry of the nuclear envelope

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