[1] Long-term preservation of liver for subcellular fractionation

Fleischer, Sidney; Kervina, Marija

doi:10.1016/0076-6879(74)31004-x

Cited by 39 publications

(11 citation statements)

References 3 publications

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“…A protease inhibitor mix (1 mM each of antipain, aprotinin, bestatin, chymostatin, pepstatin A, leupeptin, and phenylmethylsulfonyl fluoride) was added to the plasma membrane fractions that were stored at Ϫ70°C until used. The purity of plasma membrane fraction was assessed via marker enzyme activities of 5Ј-nucleotidase (8) and glucose 6-phosphatase (9 Ha-ras with a histidine tag affixed to its N terminus was a gift from Dr. Sandra L. Hoffmann (University of Texas Southwestern Medical Center, Dallas, TX). A BamHI/SalI fragment containing fulllength coding sequence of p21…”

Section: Nih3t3 Mouse Fibroblast Culture and Plasmamentioning

confidence: 99%

Evidence for a High Affinity, Saturable, Prenylation-dependent p21 Binding Site in Plasma Membranes

Siddiqui

Garland

Dalton

et al. 1998

Journal of Biological Chemistry

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Oncogenic p21ras proteins can only exert their stimulation of cellular proliferation when plasma membraneassociated. This membrane association has an absolute requirement for post-translational modification with isoprenoids. The mechanism by which isoprenoids participate in the specific association of p21 ras with plasma membranes is the subject of this report. We present in vitro evidence for a plasma membrane binding protein for p21ras that can recognize the isoprenoid substituent and, therefore, may facilitate the localization of p21 ras .Post-translational modification with isoprenoids results in considerable structural diversity at the carboxyl terminus of many proteins. Four such carboxyl-terminal structural motifs have been identified (for review see Ref. 1). These are farnesylated-methylated cysteine, geranylgeranylated-methylated cysteine, digeranylgeranylated vicinal cysteines of the -CC rab proteins, and digeranylgeranylated residue-interrupted cysteines of the -CXC rab proteins. The carboxyl-terminal cysteine of the -CXC rabs is methylated, whereas the carboxyl-terminal cysteine of the -CC rabs is not. Since these modifications always increase the hydrophobic character of the substituted proteins, it is generally assumed that they are involved in the association of proteins so modified with lipid bilayer membranes. This assumption is supported by the observation that most prenylated proteins are bound to cellular membranes, at least under some physiological conditions. A more recent hypothesis for the function of protein prenylation that is more in keeping with the observed structural diversity and membrane specificity is that these lipid modifications also serve to mediate protein-protein interactions (2). A well studied example of this function for protein prenylation is the heterodimeric association of rab proteins with GDP dissociation inhibitor molecules to form soluble complexes that appear to be dependent on the digeranylgeranylation of the rab proteins (3). Another recent example from our laboratory is the mechanism of endoproteolytic cleavage of the farnesylated prelamin A molecule, which is mediated by an enzyme that possesses a specific farnesyl binding site (4).It has been noted that it is possible that even membraneassociated prenylated proteins may utilize a polyisoprenoid-dependent protein-protein interaction for membrane binding (2). Recognition of the polyisoprenoid and other structural elements of the protein by membrane receptors could confer the necessary localization of particular prenylated proteins to particular subcellular compartments. Sequestration of the polyisoprenoid would be important in preventing nonspecific association of the protein with inappropriate membranes.Farnesylated proteins are found in a number of cellular compartments (1) including plasma membrane (p21 ras , ␥-transducin), peroxisomes (PxF) and nuclei (lamin B, prelamin A). For the naturally occurring forms of N, H, and K p21 ras , it is clear that plasma membrane localization shows an absolute requirement...

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Section: Nih3t3 Mouse Fibroblast Culture and Plasmamentioning

confidence: 99%

Evidence for a High Affinity, Saturable, Prenylation-dependent p21 Binding Site in Plasma Membranes

Siddiqui

Garland

Dalton

et al. 1998

Journal of Biological Chemistry

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“…Nuclei from adult rat liver were isolated following the procedure described by Fleischer and Kervina (1974). Briefly, rats were sacrificed by decapitation and liven were homogenized in nine volumes of 0.25 M sucrose-10 mM HEPES, pH 7.5.…”

Section: Isolation Of Nucleimentioning

confidence: 99%

Immunocytochemical and biochemical demonstration of formaldhyde dehydrogenase (class III alcohol dehydrogenase) in the nucleus.

Iborra

Renau‐Piqueras²,

Portolés³

et al. 1992

J Histochem Cytochem.

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Alcohol dehydrogenase (ADH), the major enzyme catalyzing the biological oxidation of ethanol in mammals, includes four classes with very different capacities for ethanol oxidation. Class III ADH is present in all the tissues and is well conserved throughout evolution. This enzyme has a low activity with ethanol, is specific for the glutathione-dependent oxidation of formaldehyde, and is therefore a formaldehyde dehydrogenase (FALDH). Until now there have been few and conflicting studies concerning its intracellular distribution, which is important for the understanding of its role in cell function. In the present work we used biochemical and immunocytochemical methods to assess the distribution of FALDH in rat hepatocytes and astroglial cells. With the glutathione-dependent formaldehyde dehydrogenase assay, we found the highest activity in the cytosol of hepatocytes and brain cells (12 and 2.6 mU/mg protein, respectively), but nuclei also exhibited significant activity (1.16 and 2.1 mU/mg protein, respectively). The immunocytochemical results showed the presence of FALDH binding sites in both the cytoplasm and the nucleus of the different cell types studied. Whereas no specific gold particle labeling was seen associated with any cytoplasmic component, in the nucleus the particles were found mainly over condensed chromatin and interchromatin regions. Finally, the gold particle density over both the nucleus and cytoplasm was greater in differentiated than in proliferating astrocytes in primary culture. In contrast, class I ADH, primarily responsible for ethanol metabolism, was found only in the cytoplasm of hepatocytes. We propose that one of the functions of FALDH is to protect cell structures, including DNA, from the toxic effects of endogenous formaldehyde, which is an intermediate in many metabolic process.

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“…Briefly, a rat liver (10-15 g) was homogenized in 5 vol of 0.25 M sucrose (buffered at pH 7 by 1 mM Tris-HCI) in a motor-driven PotterElvehjen homogenizer at 750 rpm, and employing pestles of 0.026-and 0.012-inch clearances, as specified (l0) . The postnuclear (2,000 g, 10 min) supernate and the postmitochondrial (11,000 g, 10 min) supernatant fractions were then prepared (10) . The cytosol (high-speed supernate) and microsome fraction were obtained by centrifuging the postmitochondrial supernate at 39,000 rpm for 50 min in the Beckman Ti50 rotor.…”

Section: Preparation Of Fractions Of Rat Livermentioning

confidence: 99%

“…To circumvent this difficulty, we have turned to rat liver, a tissue that has been successfully fractionated to yield all of the major organelles in both high yield and purity (10)(11)(12). We show here that the Golgi fraction and cruder subcellular fractions of rat liver will substitute for membranes of CHO cells as a source of transferase I and also act as acceptors of G protein donated by extracts of VSV-infected 15B cells .…”

mentioning

confidence: 99%

Transport of newly synthesized vesicular stomatitis viral glycoprotein to purified Golgi membranes.

Rothman¹,

Fries²

1981

The Journal of Cell Biology

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In a previous communication we reported that the newly synthesized membrane glycoprotein of vesicular stomatitis virus could be transported in crude extracts of CHO cells from endoplasmic reticulum-derived membranes to membranes of the Golgi complex. This conclusion was an indirect one, based on the terminal glycosylation of this glycoprotein, a reaction that was dependent upon a Golgi-specific enzyme, UDP-GlcNAc transferase I. We show here that the Golgi fraction of rat liver will substitute for members of CHO cells as a source of transferase I in this reaction. The use of highly purified fractions of liver Golgi membranes, coupled with the ability to recover these membranes from incubations, has now permitted a direct demonstration of net transport of G protein to these heterologous Golgi membranes. This transport reaction is specific, in that the smooth endoplasmic reticulum fraction will not substitute for the Golgi fraction, is quantitatively significant, involving at least 30% of the viral glycoprotein, and is sustained only in the presence of both ATP and a soluble, cytosol fraction of liver cells.

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[1] Long-term preservation of liver for subcellular fractionation

Cited by 39 publications

References 3 publications

Evidence for a High Affinity, Saturable, Prenylation-dependent p21 Binding Site in Plasma Membranes

Evidence for a High Affinity, Saturable, Prenylation-dependent p21 Binding Site in Plasma Membranes

Immunocytochemical and biochemical demonstration of formaldhyde dehydrogenase (class III alcohol dehydrogenase) in the nucleus.

Transport of newly synthesized vesicular stomatitis viral glycoprotein to purified Golgi membranes.

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