scholarly journals Water- and solute-accessible spaces of purified peroxisomes. Evidence that peroxisomes are permeable to NAD+

1983 ◽  
Vol 210 (3) ◽  
pp. 685-693 ◽  
Author(s):  
P Van Veldhoven ◽  
L J Debeer ◽  
G P Mannaerts
Keyword(s):  
Structural Integrity ◽  
Organic Layer ◽  
Peroxisome Proliferator ◽  
Marker Enzyme ◽  
Beta Oxidation ◽  
Percoll Gradients ◽  
Liver Homogenates ◽  
High Degree ◽  
Water Space ◽  
Isopycnic Centrifugation

Peroxisomes were purified from liver homogenates from rats, treated with the peroxisome proliferator clofibrate, by a combination of differential centrifugation and isopycnic centrifugation in iso-osmotic self-generating Percoll gradients. Structural integrity of the peroxisomes appeared to be preserved as evidenced by a high degree of catalase latency, the absence of catalase release during purification and the exclusion of inulin (mol.wt. +/- 5000). Spaces for water and solutes were measured after incubation of the peroxisomes in iso-osmotic sucrose with radioactive water or solutes and separation of the organelles from their media by centrifugation through an organic layer. Extraperoxisomal water was corrected for by the use of radioactive dextran or inulin. The sucrose, glucose, urea, methanol and acetate-accessible spaces were identical, suggesting that these spaces represent the volume in which molecules that can cross the membrane distribute. This volume equalled 50-65% of the water space. Urate and NAD+, a cofactor of peroxisomal beta-oxidation of fatty acids, also distributed in this volume, but were also partly bound. Urate and NAD+ binding was not abolished by sonication, which released the bulk of matrix catalase activity, but NAD+ binding was seriously diminished. The peroxisomal water and sucrose spaces were estimated to be 107 microliters and 55 microliters per g of liver tissue from a clofibrate-treated rat. From quantitative morphometric data [Anthony, Schmucker, Mooney & Jones (1978) J. Lipid Res. 19, 154-165] and our marker enzyme analyses, as well as from our experimentally determined water spaces of mitochondrial and microsomal fractions, it could be calculated that the volume contamination by lysosomes, mitochondria and microsomes did not exceed 1, 8 and 6% respectively. Our data indicate that apparently intact peroxisomes are permeable to a number of small molecules, including NAD+. Whether the NAD+-binding sites in sonicated peroxisomes mirror the likely existence of a membrane carrier requires further investigation.

scholarly journals Isolation of intracellular symbiotes by immune lysis of flagellate protozoa and characterization of their DNA.

1975 ◽  
Vol 65 (2) ◽  
pp. 309-323 ◽  
Author(s):  
R S Tuan ◽  
K P Chang
Keyword(s):  
Plasma Membranes ◽  
Structural Integrity ◽  
Light And Electron Microscopy ◽  
Heat Denaturation ◽  
Marker Enzyme ◽  
Genome Complexity ◽  
Numerical Abundance ◽  
Cellular Components ◽  
Isopycnic Centrifugation

A new method dependent on immune lysis is described for the isolation of intracellular symbiotes from two species of flagellate protozoa Blastocrithidia culicis and Crithidia oncopelti. The symbiote-containing flagellates are exposed to complement and antisera prepared in rabbits against symbiote-free organisms. The immune lysis seems to weaken the plasma membranes of the flagellates so that subsequent application of gentle shearing force liberates the intracellular entities in an undamaged condition. The symbiotes are then separated from other cellular components by DNAse digestion and differential centrifugation. The average recovery of symbiotes isolated by this method is 20%. Light and electron microscopy establishes the structural integrity and numerical abundance of isolated symbiotes in the final fractions. Integrity of symbiotes is further indicated by the high activity of a marker enzyme, uroporphyrinogen I synthetase. The DNA's of symbiote-containing and symbiote-free flagellates, and of isolated symbiotes were purified and compared after isopycnic centrifugation. The comparison establishes the presence of DNA's in symbiotes of both species. The guanine-cytosine (G-C) content of symbiote DNA differs from that of host DNA's in C. oncopelti, but resembles that of kinetoplast DNA in B. culicis. The latter observation was further shown by heat denaturation study. Renaturation kinetics indicate that the genome complexity of symbiote DNA in B. culicis is similar to that of bacteria.

Abstract 126: Peroxisomal Proliferator Activated Receptor Alpha Overexpression in Hypertrophied Cardiomyocytes Restores Mitochondrial Integrity and Function

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Sagartirtha Sarkar ◽  
Santanu Rana
Keyword(s):  
Energy Demand ◽  
Cardiac Tissue ◽  
Structural Integrity ◽  
Heart Function ◽  
Ros Generation ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Atp Production ◽  
Receptor Alpha ◽  
And Function

Cardiac tissue engineering is an interdisciplinary field that engineers modulation of viable molecular milieu to restore, maintain or improve heart function. Myocardial workload (energy demand) and energy substrate availability (supply) are in continual flux to maintain specialized cellular processes, yet the heart has a limited capacity for substrate storage and utilization during pathophysiological conditions. Damage to heart muscle, acute or chronic, leads to dysregulation of cardiac metabolic processes associated with gradual but progressive decline in mitochondrial respiratory pathways resulting in diminished ATP production. The Peroxisome Proliferator Activated Receptor Alpha ( PPARα ) is known to regulate fatty acid to glucose metabolic balance as well as mitochondrial structural integrity. In this study, a non-canonical pathway of PPARα was analyzed by cardiomyocyte targeted PPARα overexpression during cardiac hypertrophy that showed significant downregulation in p53 acetylation as well as GSK3β activation levels. Targeted PPARα overexpression during hypertrophy resulted in restoration of mitochondrial structure and function along with significantly improved mitochondrial ROS generation and membrane potential. This is the first report of myocyte targeted PPARα overexpression in hypertrophied myocardium that results in an engineered heart with significantly improved function with increased muscle mitochondrial endurance and reduced mitochondrial apoptotic load, thus conferring a greater resistance to pathological stimuli within cardiac microenvironment.

Assignment of estradiol-17β dehydrogenase and of estrone reductase to cytoplasmic structures of porcine endometrium cells

1989 ◽  
Vol 121 (2) ◽  
pp. 161-167 ◽  
Author(s):  
J. Adamski ◽  
W. D. Sierralta ◽  
P. W. Jungblut
Keyword(s):  
Cytochrome C ◽  
Kinetic Data ◽  
Reductase Activity ◽  
Particulate Fraction ◽  
Marker Enzyme ◽  
Structural Element ◽  
Cytochrome C Reductase ◽  
Percoll Gradients ◽  
Cytoplasmic Structures ◽  
Estradiol 17Β

Abstract. Homogenates of porcine endometrium contain substantial activity for the dehydrogenation of estradiol-17β but little for estrone reduction. Both activities are associated with cytoplasmic structures. The dehydrogenase is characterized by a pH 7.7 optimum, Km 2.2 × 10−7 mol/l for estradiol and Km 4.4 × 10−5 mol/l for the cosubstrate NAD+. The corresponding figures for the reductase are pH 6.6, Km 1.1 × 10−6 mol/1 for estrone and Km 2.1 × 10−5 mol/l for the cosubstrate NADPH. The (mitochondrial/lysosomal) 17 000 × g particulate fraction contains a 52-fold higher dehydrogenase than reductase activity. The (microsomal) 200000 × g particulate fraction is only 16-fold richer in dehydrogenase. Isopycnic centrifugations of the two fractions in Percoll gradients reveal that estrone reductase and the coequilibrating marker enzyme cytochrome c reductase occur in constant proportions, whereas the dehydrogenase/cytochrome c reductase ratios are different. Both, the kinetic data and the structural assignments speak in favour of individual enzymes catalyzing the dehydrogenation of estradiol and the reduction of estrone. All gradient fractions exhibiting dehydrogenase activity feature small, electrondense vesicles of 0.15–0.20 μm in diameter as a common structural element which might harbour the dehydrogenase.

In-vivo uptake of human growth hormone in male rat liver

1989 ◽  
Vol 121 (1) ◽  
pp. 19-25 ◽  
Author(s):  
F. Bullier-Picard ◽  
M. C. Postel-Vinay ◽  
C. Kayser
Keyword(s):  
Plasma Membranes ◽  
Human Growth ◽  
Rat Hepatocytes ◽  
Male Rats ◽  
Male Rat ◽  
Liver Homogenates ◽  
Isopycnic Centrifugation ◽  
Specific Labelling ◽  
In Vivo Uptake

ABSTRACT 125I-Labelled human GH (hGH) was injected i.v. to male rats and its subcellular distribution in the hepatocyte was examined using fractionation techniques. Uptake into liver homogenates was maximal by 15 min after injection and represented 24% of the injected radioactivity; it was markedly inhibited by coinjection of native hGH. 125I-Labelled hGH taken up by the liver underwent a time-dependent translocation process. The peak of specific labelling of plasma membranes occurred at 3 min whereas later on the radioactivity was concentrated in low-density structures present in Golgi-endosome fractions. To characterize the ligand-associated structures better, endosome-enriched fractions were prepared from a microsomal fraction by isopycnic centrifugation in a sucrose gradient and a Nycodenz gradient. The radioactivity was in one peak with a median density of 1·096 g/cm3 in the Nycodenz gradient fractions. The peak of radioactivity was distinct from that of galactosyltransferase activity which appeared at a median density of 1·114 g/cm3. The labelled material eluted from the various subcellular fractions appeared as intact hGH. Upon in-vivo interaction with male rat hepatocytes, 125I-labelled hGH was internalized with a sequential association with plasma membranes and endocytic structures distinct from Golgi elements. Journal of Endocrinology (1989) 121, 19–25

Lack of evidence for a hepatic peroxisome proliferator receptor and an explanation for the binding of hypolipidaemic drugs to liver homogenates

1988 ◽  
Vol 37 (5) ◽  
pp. 793-798 ◽  
Author(s):  
Mark N. Milton ◽  
Cliff R. ElcombeP ◽  
George E.N. Kass ◽  
G.Gordon Gibson
Keyword(s):  
Peroxisome Proliferator ◽  
Liver Homogenates

scholarly journals Mechanisms Mediating the Regulation of Peroxisomal Fatty Acid Beta-Oxidation by PPARα

2021 ◽  
Vol 22 (16) ◽  
pp. 8969
Author(s):  
Mounia Tahri-Joutey ◽  
Pierre Andreoletti ◽  
Sailesh Surapureddi ◽  
Boubker Nasser ◽  
Mustapha Cherkaoui-Malki ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Mammalian Cells ◽  
Atp Synthesis ◽  
Peroxisome Proliferator ◽  
Beta Oxidation ◽  
Peroxisome Proliferator Activated Receptor ◽  
Oxidation Pathway ◽  
Oxidative Phosphorylation Pathway ◽  
Peroxisome Proliferator Response Element ◽  
Peroxisomal Fatty Acid

In mammalian cells, two cellular organelles, mitochondria and peroxisomes, share the ability to degrade fatty acid chains. Although each organelle harbors its own fatty acid β-oxidation pathway, a distinct mitochondrial system feeds the oxidative phosphorylation pathway for ATP synthesis. At the same time, the peroxisomal β-oxidation pathway participates in cellular thermogenesis. A scientific milestone in 1965 helped discover the hepatomegaly effect in rat liver by clofibrate, subsequently identified as a peroxisome proliferator in rodents and an activator of the peroxisomal fatty acid β-oxidation pathway. These peroxisome proliferators were later identified as activating ligands of Peroxisome Proliferator-Activated Receptor α (PPARα), cloned in 1990. The ligand-activated heterodimer PPARα/RXRα recognizes a DNA sequence, called PPRE (Peroxisome Proliferator Response Element), corresponding to two half-consensus hexanucleotide motifs, AGGTCA, separated by one nucleotide. Accordingly, the assembled complex containing PPRE/PPARα/RXRα/ligands/Coregulators controls the expression of the genes involved in liver peroxisomal fatty acid β-oxidation. This review mobilizes a considerable number of findings that discuss miscellaneous axes, covering the detailed expression pattern of PPARα in species and tissues, the lessons from several PPARα KO mouse models and the modulation of PPARα function by dietary micronutrients.

Probing the effect of MODY mutations near the co-activator-binding pocket of HNF4α

2011 ◽  
Vol 31 (5) ◽  
pp. 411-419 ◽  
Author(s):  
Geun Bae Rha ◽  
Guangteng Wu ◽  
Young-In Chi
Keyword(s):  
Protein Function ◽  
Structural Integrity ◽  
Cell Function ◽  
Point Mutations ◽  
Binding Pocket ◽  
Peroxisome Proliferator ◽  
Loss Of Function ◽  
Peroxisome Proliferator Activated Receptor ◽  
Ligand Selectivity ◽  
Β Cell Function

HNF4α (hepatocyte nuclear factor 4α) is a culprit gene product for a monogenic and dominantly inherited form of diabetes, referred to as MODY (maturity onset diabetes of the young). As a member of the NR (nuclear receptor) superfamily, HNF4α recruits transcriptional co-activators such as SRC-1α (steroid receptor co-activator-1α) and PGC-1α (peroxisome-proliferator-activated receptor γ co-activator-1α) through the LXXLL-binding motifs for its transactivation, and our recent crystal structures of the complex provided the molecular details and the mechanistic insights into these co-activator recruitments. Several mutations have been identified from the MODY patients and, among these, point mutations can be very instructive site-specific measures of protein function and structure. Thus, in the present study, we probed the functional effects of the two MODY point mutations (D206Y and M364R) found directly near the LXXLL motif-binding site by conducting a series of experiments on their structural integrity and specific functional roles such as overall transcription, ligand selectivity, target gene recognition and co-activator recruitment. While the D206Y mutation has a subtle effect, the M364R mutation significantly impaired the overall transactivation by HNF4α. These functional disruptions are mainly due to their reduced ability to recruit co-activators and lowered protein stability (only with M364R mutation), while their DNA-binding activities and ligand selectivities are preserved. These results confirmed our structural predictions and proved that MODY mutations are loss-of-function mutations leading to impaired β-cell function. These findings should help target selective residues for correcting mutational defects or modulating the overall activity of HNF4α as a means of therapeutic intervention.

scholarly journals Specific changes in the protein composition of rat liver in response to the peroxisome proliferators ciprofibrate, Wy-14,643 and di-(2-ethylhexyl)phthalate

1985 ◽  
Vol 227 (3) ◽  
pp. 767-775 ◽  
Author(s):  
T Watanabe ◽  
N D Lalwani ◽  
J K Reddy
Keyword(s):  
Gel Electrophoresis ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferation ◽  
Peroxisome Proliferators ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Basic Proteins ◽  
Acidic Proteins ◽  
Liver Homogenates ◽  
Ethylhexyl Phthalate

The hypolipidaemic agents ciprofibrate and Wy-14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid) and the phthalate-ester plasticizer di-(2-ethylhexyl)-phthalate (DEHP), like other peroxisome proliferators, produce a significant hepatomegaly and induce the peroxisomal fatty acid beta-oxidation enzyme system together with profound proliferation of peroxisomes in hepatic parenchymal cells. Changes in the profile of liver proteins in rats following induction of peroxisome proliferation by ciprofibrate, Wy-14,643 and DEHP have been analysed by high-resolution two-dimensional gel electrophoresis. The proteins of whole liver homogenates from normal and peroxisome-proliferator-treated rats were separated by two-dimensional gel electrophoresis using isoelectric focusing for acidic proteins and nonequilibrium pH gradient electrophoresis for basic proteins. In the whole liver homogenates, the quantities of six proteins in acidic gels and six proteins in the basic gels increased following induction of peroxisome proliferation. Peroxisome proliferator administration caused a repression of three acidic proteins in the liver homogenates. By the immunoblot method using polyspecific antiserum against soluble peroxisomal proteins and monospecific antiserum against peroxisome proliferation associated Mr 80000 polypeptide (polypeptide PPA-80), the majority of basic proteins induced by these peroxisome proliferators appeared to be peroxisomal proteins. Polypeptide PPA-80 becomes the most abundant protein in the total liver homogenates of peroxisome-proliferator-treated rats. These results indicate that ciprofibrate, DEHP and Wy-14,643 induce marked changes in the profile of specific hepatic proteins and that some of these changes should serve as a baseline to identify a set of gene products that may assist in defining the specific ‘peroxisome proliferator domain’.

scholarly journals Functional plasticity and evolutionary adaptation of allosteric regulation

2020 ◽  
Vol 117 (41) ◽  
pp. 25445-25454
Author(s):  
Megan Leander ◽  
Yuchen Yuan ◽  
Anthony Meger ◽  
Qiang Cui ◽  
Srivatsan Raman
Keyword(s):  
Protein Function ◽  
Structural Integrity ◽  
Regulatory Mechanism ◽  
Current Knowledge ◽  
Functional Plasticity ◽  
Common View ◽  
Molecular Determinants ◽  
Thermodynamic Conditions ◽  
The Common ◽  
High Degree

Allostery is a fundamental regulatory mechanism of protein function. Despite notable advances, understanding the molecular determinants of allostery remains an elusive goal. Our current knowledge of allostery is principally shaped by a structure-centric view, which makes it difficult to understand the decentralized character of allostery. We present a function-centric approach using deep mutational scanning to elucidate the molecular basis and underlying functional landscape of allostery. We show that allosteric signaling exhibits a high degree of functional plasticity and redundancy through myriad mutational pathways. Residues critical for allosteric signaling are surprisingly poorly conserved while those required for structural integrity are highly conserved, suggesting evolutionary pressure to preserve fold over function. Our results suggest multiple solutions to the thermodynamic conditions of cooperativity, in contrast to the common view of a finely tuned allosteric residue network maintained under selection.

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