Effects of Phenobarbital on the Synthesis and Degradation of the Protein Components of Rat Liver Microsomal Membranes

Kuriyama, Yoshiaki; Omura, Tsuneo; Siekevitz, Philip; Palade, George E.

doi:10.1016/s0021-9258(18)94361-9

Cited by 210 publications

(20 citation statements)

References 31 publications

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“…Thus, the PBinduced increase in NADPH: cytochrome P-450 reductase activity observed by ourselves and others (Orrenius & Ernster, 1964;Orrenius et al, 1965) can be accounted for entirely by an increase in the amount of the protein and not to any change in its activity state. This result is supported by the findings that both the rate of synthesis of the protein and the amount of its mRNA are increased by PB treatment (Kuriyama et al, 1969;Gonzalez & Kasper, 1980Shephard et al, 1982). NF treatment neither increased the activity nor the amount of NADPH: cytochrome P-450 reductase.…”

Section: Discussionmentioning

confidence: 72%

Quantification of NADPH: cytochrome P-450 reductase in liver microsomes by a specific radioimmunoassay technique

Shephard¹,

Phillips²,

Bayney³

et al. 1983

Biochemical Journal

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We have developed a specific radioimmunoassay to quantify NADPH: cytochrome P-450 reductase. The assay is based on the use of 125I-labelled NADPH: cytochrome P-450 reductase as the radiolabelled antigen and can detect quantities of this protein in amounts as low as 30 pg. The results of the radioimmunoassay demonstrates that the 2.7-fold increase in enzyme activity in rat liver microsomal membranes after phenobarbital treatment is due to increased amounts of the protein. beta-Naphthoflavone treatment, however, did not alter the activity or the quantity of this enzyme in microsomes. The quantification of NADPH: cytochrome P-450 reductase in the microsomes isolated from control and phenobarbital- and beta-naphthoflavone-treated animals permits the calculation of the ratio of this protein to that of total cytochromes P-450. A molar ratio of 15:1 (cytochromes P-450/NADPH: cytochrome P-450 reductase) was calculated for control and phenobarbital-treated animals. This ratio increased to 21:1 after beta-naphthoflavone treatment. Thus the molar ratio of these proteins in liver microsomes can vary with exposure of the animals to particular xenobiotics.

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Section: Discussionmentioning

confidence: 72%

Quantification of NADPH: cytochrome P-450 reductase in liver microsomes by a specific radioimmunoassay technique

Shephard¹,

Phillips²,

Bayney³

et al. 1983

Biochemical Journal

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“…* mg/g liver . ular constituents, i .e ., lipids and enzymes, of the same membrane turn over at different rates (31) . In addition, many membrane components turn over by exchange reactions not associated with degradation (51) .…”

Section: Distribution Of Protein and Marker Enzymes In Isolated Subcellular Fractionsmentioning

confidence: 99%

Isolation of autophagic vacuoles from rat liver: morphological and biochemical characterization.

Marzella¹,

Ahlberg²,

Glaumann³

1982

The Journal of Cell Biology

141

110

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The induction of autophagy caused by vinblastine (VBL) has been found to be concomitant with a stimulation of proteolysis in a mitochondrial-lysosomal (ML) fraction from the rat liver (Marzella and Glaumann, 1980, Lab. Invest., 42 : 8-17 . Marzella and Glaumann, 1980, Lab. Invest., 42 :18-27) . In this fraction the enhanced proteolysis is associated with a threefold increase in the relative fractional volume of autophagic vacuoles (AVs) . In an attempt to isolate the AVs, we subfractionated the ML suspension at different intervals after the induction of autophagy by VBL by centrifugation on a discontinuous Metrizamide gradient ranging from 50% to 15%. The material banding at the 24 to 20% and the 20 to 15% interphases was collected .Morphological analysis reveals that 3 h after induction of autophagy these fractions consist predominantly (-90%) of intact autophagic vacuoles . These autophagic vacuoles contain cytosol, mitochondria, portions of endoplasmic reticulum, and occasional very low density lipoprotein particles either free or in Golgi apparatus derivatives, in particular secretory granules . The sequestered materials show ultrastructural signs of ongoing degradation . In addition to containing typical autophagic vacuoles, the isolated fractions consist of lysosomes lacking morphologically recognizable cellular components . Contamination from nonlysosomal material is only a few percent as judged from morphometric analysis .Typical lysosomal "marker" enzymes are enriched 15-fold, whereas the proteolytic activity is enriched 10-to 20-fold in the isolated AV fraction as compared to the homogenate . Initially, the yield of nonlysosomal mitochondrial and microsomal enzyme activities increases in parallel with the induction of autophagy but, later on, decreases with advanced degradation of the sequestered cell organelles . Therefore, in the case of AVs the presence of nonlysosomal marker enzymes cannot be used for calculation of fraction purity, since newly sequestered organelles are enzymatically active . Isolated autophagic vacuoles show proteolytic activity when incubated in vitro. The comparatively high phospholipid/protein ratio (0 .5) of the AV fraction suggests that phospholipids are degraded more slowly than proteins . It is concluded that AVs can be isolated into a pure fraction and are the subcellular site of enhanced protein degradation in the rat liver after induction of autophagy.We have used the stimulation of autophagy as a means of studying mechanisms of sequestration and degradation of intracellular proteins in the lysosomal compartment (36, 37). To this end, vinblastine (VBL) was administered, which depolymerizes microtubules (30,35) and induces autophagy in the rat liver (6, 37). The induction of autophagy enhances the proteolytic rates of mitochondrial-lysosomal (ML) fractions threefold (36). The stimulation of proteolysis in the ML fractions correlates with a three-to fourfold increase in the fractional volume of autophagic vacuoles (AVs) and residual bodies. In 144 contrast, an ...

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“…Similar findings have been shown by Svensson et al (15) who demonstrated that the membrane fragments resulting from sonicated rough microsomes all move in a gradient of higher density than that used in this study. The pellet was resuspended in 0.15 M KCI-10 mM EDTA and centrifuged at 105,000 g for 90 min to ensure maximum detachment of "nonmembranous" material from the fractions (16). The ensuing pellets were designated "membrane fractions" as an operational term.…”

Section: Washing Proceduresmentioning

confidence: 99%

“…Chemical analysis of the supernate disclosed significant amounts of lipids (mainly TG and some PLP, but very little cholesterol), indicating that Tris washing is also effective in detaching adsorbed, nonmembranous lipids as well as in removing loosely attached proteins (13,14). After Tris washing and an additional KC1-EDTA wash (16), sonication was performed to rupture the vesicles and cisternae and release their contents. The chemical composition of the membranes is illustrated in Table II.…”

Section: Glaumann Et Al Synthesis and Lntracellular Transport Of Lipoprotein Particlesmentioning

confidence: 99%

Studies on the synthesis and intracellular transport of lipoprotein particles in rat liver.

Glaumann¹,

Bergstrand²,

Ericsson³

1975

The Journal of Cell Biology

151

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Lipoprotein particles (d < 1.03 g/ml) were isolated from rough and smooth microsomes and from the Golgi apparatus of rat liver, and were characterized chemically and morphologically. The rough endoplasmic reticulum (ER) particles were rich in protein (50%) and contained phospholipids (PLP) and triglycerides (TG) in smaller amounts, whereas the lipoprotein particles emanating from the smooth ER, and especially the Golgi apparatus, were rich in TG and PLP, resembling very low density lipoproteins (VLDL) of serum. The difference in chemical composition among the particles was associated with change in size both in situ and in isolated lipoprotein fractions. The rough ER particles were 200-800 A in diameter (mean ,-,420 A); the smooth ER particles 200-900 ~ (mean ~520 A); the Golgi particles 350-950 A (mean ~ 580A); and serum VLDL 300-800 A (mean ~450 A). Generally, lipoprotein particles were rare in the rough ER, frequent but diffusely dispersed in smooth ER, and occurring mainly in clusters in "secretory vesicles" of the Golgi complex. They were seldom observed in the cisternal compartments of the Golgi complex.At short intervals (~ 15 rain), intravenously injected radioactive glycerol was preferentially channelled into TG, whereas at later time points the majority of the isotope was recovered in the PLP. Three TG pools were distinguished: (a) a cytoplasmic pool with a slow turnover rate; (b) a membrane-associated TG pool; and (c) a pool corresponding to the TG moiety of lipoprotein particles, which showed the highest initial rate of labeling and fastest turnover.When, after pulse labeling, the appearance of incorporation of radioactive glycerol into TG or PLP of isolated lipoproteins was followed from one subcellular fraction to the other, a sequence of labeling was noted. During the first interval, TG from both rough and smooth microsomal lipoproteins displayed a high rate of labeling with peak value at 6 min, followed by a quick fall-off, while the Golgi lipoproteins reached maximal level at 10-20 min after administration. There was an interval of 10-15 min before the appearance of labeled VLDL in serum.It is concluded that the assembly of the apoproteins and lipid moieties into lipoprotein particles--presumed to be precursors of liver VLDL--begins in the rough ER and continues in the smooth ER. Also, there is a parallel change in chemical composition and size of the lipoprotein particles as they make their way through the ER and the Golgi apparatus. Some remodeling of the particles may take place in the Golgi apparatus before discharge into the circulation.

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Effects of Phenobarbital on the Synthesis and Degradation of the Protein Components of Rat Liver Microsomal Membranes

Cited by 210 publications

References 31 publications

Quantification of NADPH: cytochrome P-450 reductase in liver microsomes by a specific radioimmunoassay technique

Quantification of NADPH: cytochrome P-450 reductase in liver microsomes by a specific radioimmunoassay technique

Isolation of autophagic vacuoles from rat liver: morphological and biochemical characterization.

Studies on the synthesis and intracellular transport of lipoprotein particles in rat liver.

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