scholarly journals A comparative study of ribonuclease hydrolysis of rat brain-cortex and liver membrane-bound ribosomes

1973 ◽  
Vol 135 (2) ◽  
pp. 299-305 ◽  
Author(s):  
H. Simpkins ◽  
E. Panko ◽  
S. Tay
Keyword(s):  
Endoplasmic Reticulum ◽  
Ribosomal Rna ◽  
Brain Cortex ◽  
Polyacrylamide Gel Electrophoresis ◽  
Endoplasmic Reticulum Membrane ◽  
Low Concentrations ◽  
Liver Membrane ◽  
Membrane Bound ◽  
Hydrolysis Of ◽  
Brain Ribosomes

Because it has been proposed that the ribosome–membrane interaction is different in endoplasmic reticulum derived from a non-secretory and secretory cell we undertook a study to determine whether attachment of the ribosome to the membrane involved ribosomal RNA and if the rRNA in ribosomes derived from the two classes of cell possessed an altered susceptibility to RNAase (ribonuclease) hydrolysis. We found that brain ribosomes appeared to possess more regions accessible to nuclease attack, independent of whether a sequence-dependent RNAase (T1) or a sterically hindered RNAase bound to Enzite polymer was employed. These results were independent of whether the ribosomes were membrane-bound or detached from the endoplasmic reticulum membranes, but at high RNAase concentration these differences became negligible. No conclusions, however, could be drawn as to whether ribosomal RNA is involved in the attachment of the ribosome to the endoplasmic reticulum membrane, because of the presence of endogeneous membrane-associated RNAases. Analysis of the rRNA fragments by polyacrylamide-gel electrophoresis suggests that the sites available for attack by low concentrations of nuclease in bound-ribosomes derived from brain cortex are different from those of liver.

scholarly journals Hydrolysis of histones by proteinases

1988 ◽  
Vol 250 (3) ◽  
pp. 859-864 ◽  
Author(s):  
R J Harvima ◽  
K Yabe ◽  
J E Fräki ◽  
K Fukuyama ◽  
W L Epstein
Keyword(s):  
Mast Cell ◽  
Cell Differentiation ◽  
Gel Electrophoresis ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Histone H2a ◽  
Cathepsin H ◽  
Low Concentrations ◽  
Hydrolysis Of ◽  
Epidermal Cell Differentiation

Hydrolysis of histones by proteinases from rat liver, skin and other sources was studied by using a rat thymus histone preparation as the substrate and polyacrylamide-gel electrophoresis and densitometric analysis as the methods to detect histone subtypes and their hydrolysis. The rat mast-cell proteinase I effectively hydrolysed histones except type H4. Thrombin hydrolysed effectively histones H1 and H2A, whereas plasmin hydrolysed all types of histones. Cathepsin D hydrolysed especially histone H2A. Cathepsins B and L hydrolysed all histones more slowly, and cathepsin H hydrolysed them extremely slowly. Epidermal aminoendopeptidase did not hydrolyse histones. Trypsin and chymotrypsin were used as reference enzymes, which hydrolysed all types of histones in very low concentrations. This study suggests that a variety of proteinases could play a role in histone hydrolysis. Hydrolysis of a specific subtype of histones, such as histone H2A at pH 6 by cathepsin D, may be directly involved in regulation of epidermal-cell differentiation.

scholarly journals Role of membrane-bound and free polyribosomes in the synthesis of cytochrome c in rat liver

1974 ◽  
Vol 140 (2) ◽  
pp. 157-167 ◽  
Author(s):  
Néstor F. González-Cadavid ◽  
Carmen Sáez De Córdova
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Rat Liver ◽  
Cell Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Sodium Dodecyl ◽  
Membrane Bound

The functional distinction of membrane-bound and free polyribosomes for the synthesis of exportable and non-exportable proteins respectively is not so strict as was initially thought, and it was therefore decided to investigate their relative contribution to the elaboration of an internal protein integrated into a cell structure. Cytochrome c was chosen as an example of a soluble mitochondrial protein, and the incorporation of [14C]leucine and δ-amino[14C]laevulinate into the molecule was studied by using different ribosomal preparations from regenerating rat liver. A new procedure was devised for the purification of cytochrome c, based on ion-exchange chromatography combined with sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. In spite of cytochrome c being a non-exportable protein, the membrane-bound polyribosomes were at least as active as the free ribosomes in the synthesis in vitro of the apoprotein and the haem moiety. The detergent-treated ribosomes could also effect the synthesis of cytochrome c, although at a lower rate. Since in liver more than two-thirds of the ribosomes are bound to the endoplasmic-reticulum membranes, it is considered that in vivo they are responsible for the synthesis of most of the cytochrome c content of the cell. This suggests that in secretory tissues the endoplasmic reticulum plays a predominant role in mitochondrial biogenesis, although free ribosomes may participate in the partial turnover of some parts of the organelle. The hypothesis on the functional specialization of the different kinds of ribosomes was therefore modified to account for their parallel intervention in the synthesis of proteins associated with membranous structures.

scholarly journals Preliminary characterization of two thymus-dependent xenoantigens from mouse lymphocytes

1977 ◽  
Vol 163 (2) ◽  
pp. 211-217 ◽  
Author(s):  
I S Trowbridge ◽  
M Nilsen-Hamilton ◽  
R T Hamilton ◽  
M J Bevan
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Membrane Vesicles ◽  
Sodium Deoxycholate ◽  
Pea Lectin ◽  
Preliminary Characterization ◽  
Low Concentrations ◽  
Membrane Bound

Preliminary characterization of two mouse thymus-dependent (T) lymphocyte xenoantigens, T25 and T200, which are selectively labelled by lactoperoxidase-catalysed iodination of T-cells, is described. Both molecules are membrane-bound glycoproteins. Fractionation of membrane vesicles prepared from BW5147 lymphoma cells by sedimentation through sucrose density gradients show that antigens T25 and T200 are in fractions enriched with plasma membrane. Moreover antigen T200 is partially degraded when viable cells are treated briefly with low concentrations of trypsin. Both molecules are efficiently solubilized in buffers containing sodium deoxycholate or Nonidet P-40, as measured by failure to sediment at 100000g for 60min. However, gel filtration on Sepharose 6B showed the presence of aggregated material in Nonidet P-40 extracts which was not found in deoxycholate-solubilized membranes. After solubilization in detergent, antigens T25 and T200 bind to, and may be specifically eluted from, columns of pea lectin--Sepharose or concanavalin A--Sepharose. Both molecules are heterogeneous when examined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. As judged by its binding to columns of pea lectin, at least part of the heterogeneity of mouse thymocyte antigen T25 resides in its carbohydrate moiety.

scholarly journals THE USE OF THIOL-SUBSTITUTED CARBOXYLIC ACIDS AS HISTOCHEMICAL SUBSTRATES

1965 ◽  
Vol 13 (8) ◽  
pp. 611-628 ◽  
Author(s):  
MARY BELL ◽  
RUSSELL J. BARRNETT
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Lipid Metabolism ◽  
Light Microscopy ◽  
Nuclear Envelope ◽  
Carboxylic Acids ◽  
Structural Level ◽  
Low Concentrations ◽  
Hydrolysis Of ◽  
Thiolacetic Acid

Thiobutyric, thiocaproic and thiocaprylic acids were synthesized, and enzyme histochemical methods were developed using these thiol-substituted carboxylic acids, as well as thiolacetic acid, as substrates with Pb++ as the capture reagent. The localization of final product of these histochemical reactions, PbS, was studied and compared in a variety of tissues with light microscopy. The enzymatic activities demonstrated were sensitive to low concentrations of E600. The localization of these reactions in several intensely reactive tissues, liver, testis and intestine, were also studied with electron microscopy. At a fine structural level, the final product was deposited primarily in relation to the membranous elements of the smooth and rough varieties of the endoplasmic reticulum, including the nuclear envelope, and of mitochondria. The results of these experiments are discussed, including the possible identity of the enzymes concerned with the hydrolysis of the thiol-substituted substrates. It was suggested that at least three activities were demonstrated in these experiments, one of which was the B type esterase of microsomes, and all of which functioned in lipid metabolism.

The Role of Membrane Proteins and Phospholipids in the Interaction of Ribosomes with Endoplasmic Reticulum Membranes

1975 ◽  
Vol 53 (9) ◽  
pp. 1039-1045 ◽  
Author(s):  
Serge Jothy ◽  
Jean-Louis Bilodeau ◽  
Henry Simpkins
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Binding Sites ◽  
Molecular Weights ◽  
Low Concentrations ◽  
Phospholipid Headgroups ◽  
Hydrolysis Of

Hydrolysis of the membrane proteins and phospholipid headgroups of rat liver rough endoplasmic reticulum membranes showed that the ribosomal binding sites involve membrane proteins susceptible to low concentrations of trypsin, chymotrypsin, and papain. Three membrane proteins having molecular weights of 120 000, 93 000 and 36 000 are found to be altered by trypsin and chymotrypsin treatment. Also the polar headgroup of phosphatidylinositol appears to play a role in the binding process.

scholarly journals Endoplasmic reticulum membrane-bound MoSec62 is involved in the suppression of rice immunity and is essential for the pathogenicity ofMagnaporthe oryzae

2016 ◽  
Vol 17 (8) ◽  
pp. 1211-1222 ◽  
Author(s):  
Zhuangzhi Zhou ◽  
Zhiqian Pang ◽  
Guihua Li ◽  
Chunhua Lin ◽  
Jing Wang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Membrane ◽  
Membrane Bound

scholarly journals The Conserved Npl4 Protein Complex Mediates Proteasome-dependent Membrane-bound Transcription Factor Activation

2001 ◽  
Vol 12 (10) ◽  
pp. 3226-3241 ◽  
Author(s):  
Amy L. Hitchcock ◽  
Heike Krebber ◽  
Seth Frietze ◽  
Andrew Lin ◽  
Martin Latterich ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Transcription Factors ◽  
Regulation Of Gene Expression ◽  
Fatty Acid Desaturase ◽  
Endoplasmic Reticulum Membrane ◽  
Desaturase Gene ◽  
Proteolytic Activation ◽  
Transcription Factor Activation ◽  
Membrane Bound ◽  
Fatty Acid Desaturase Gene

Proteolytic activation of membrane-bound transcription factors has emerged as an important mechanism for the regulation of gene expression. Two membrane-bound transcription factors regulated in this manner are the Saccharomyces cerevisiae proteins Mga2p and Spt23p, which direct transcription of the Δ9-fatty acid desaturase gene OLE1. We now show that a membrane-associated complex containing the highly conserved Npl4p, Ufd1p, and Cdc48p proteins mediates the proteasome-regulated cleavage of Mga2p and Spt23p. Mutations in NPL4,UFD1, and CDC48 cause a block in Mga2p and Spt23p processing, with concomitant loss of OLE1expression. Taken together, our data indicate that the Npl4 complex may serve to target the proteasome to the ubiquitinated endoplasmic reticulum membrane-bound proteins Mga2p and Spt23p. Given the recent finding that NPL4 is allelic to the ERAD geneHRD4, we further propose that this NPL4function extends to all endoplasmic reticulum-membrane–associated targets of the proteasome.

scholarly journals A sub-population of rat liver membrane-bound ribosomes that are detached in vitro by carcinogens and centrifugation

1978 ◽  
Vol 176 (1) ◽  
pp. 9-14 ◽  
Author(s):  
D N Palmer ◽  
B R Rabin ◽  
D J Williams
Keyword(s):  
Lipid Peroxidation ◽  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Chemical Carcinogen ◽  
Centrifugal Forces ◽  
In Vitro Incubation ◽  
Liver Membrane ◽  
Membrane Bound

The chemical-carcinogen-induced detachment of ribosomes from rat liver endoplasmic reticulum was studied in vitro. Incubation of postmitochondrial supernatant with 0.2 mM-diethylnitrosamine or N-2-acetylaminofluorene removed approx. 16% of membrane-bound ribosomes, measured as differences in RNA/protein values of membrane separated from unbound ribosomes by flotation. These ribosomes are also detached by exposure to high centrifugal forces (160000g) and are among those removed by NADPH-catalysed lipid peroxidation. Extensive lipid peroxidation prohibits any measurement. The ribosomes (polyribosomes) removed are not those detached from the membrane by exposure to high KC1 concentrations (loosely bound) or high KC1 concentrations in the presence of puromycin (tightly bound). It is concluded then that centrifugally labile and carcinogen-sensitive represent a previously unreported sub-population of membrane-bound ribosomes.

scholarly journals Adenosine triphosphatase activity in the neural lobe of the bovine pituitary gland

1974 ◽  
Vol 143 (1) ◽  
pp. 181-190 ◽  
Author(s):  
Hans Vilhardt ◽  
Derek B. Hope
Keyword(s):  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Subcellular Fractions ◽  
Neural Lobe ◽  
Bivalent Cations ◽  
Pituitary Glands ◽  
Membrane Bound ◽  
Triton X ◽  
Hydrolysis Of

1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential and density-gradient ultracentrifugation and the distribution of adenosine triphosphatase (ATPase) activity was studied. It was shown that all the activity was membrane-bound. 2. On the basis of ionic requirements the ATPase activity was grouped into three categories: (a) Mg2+-dependent, (b) Ca2+-dependent and (c) Mg2++Na++K+-dependent (ouabain-sensitive) ATPases. The activity in the absence of bivalent cations was negligible. The ratio between the activities of the three ATPases varied between the different subcellular fractions. 3. Preincubation of the subcellular fractions with deoxycholate increased the activity of the Mg2++Na++K+-dependent enzyme, whereas the Mg2+- and Ca2+-activated ATPases were either unaffected or slightly inhibited. Triton X-100 solubilized the Mg2+- and Ca2+-ATPases; however, the activity of the Mg2++Na++K+-ATPase was abolished by the concentration of Triton X-100 used. 4. All the subfractions displayed unspecific nucleotide triphosphatase activity towards GTP, ITP and UTP. These substrates inhibited the hydrolysis of ATP by all three ATPases. ADP also inhibited the ATPases. 5. Polyacrylamide-gel electrophoresis of extracts containing the Mg2+- and Ca2+-dependent ATPase activity solubilized by Triton X-100 revealed the presence of two enzymes; one activated by either Mg2+or Ca2+and the other activated only by Ca2+. 6. In sucrose density gradients the distribution of vasopressin was different from that of all three types of ATPases. It is therefore suggested that the neurosecretory granules do not possess ATPase activity.

scholarly journals Ultrastructural localization of coenzyme A phosphatase (CoA-Pase) activity to the GERL system in secretory ameloblasts of the rat incisor.

1981 ◽  
Vol 29 (11) ◽  
pp. 1243-1254 ◽  
Author(s):  
C E Smith
Keyword(s):  
Endoplasmic Reticulum ◽  
Coenzyme A ◽  
Secretory Granules ◽  
Ultrastructural Localization ◽  
Rat Incisor ◽  
Acetyl Coa ◽  
Selective Staining ◽  
Dense Bodies ◽  
Low Concentrations ◽  
Hydrolysis Of

Enzymatic hydrolysis of the monoester phosphate group from coenzyme A (CoA) was studied in rat incisor ameloblasts by incubating specimens from glutaraldehyde-fixed teeth in a cytochemical medium prepared with acetyl-CoA as substrate and lead ions as capture agent for phosphate. Ameloblasts incubated for 1 hr at 37 degrees C and at pH 5.0 in this medium showed reaction product localized almost exclusively along the trans (mature) aspect of the Golgi apparatus within a network of small granules and interconnecting tubular channels that comprise the GERL system in this cell. Reaction product was otherwise seen in trace amounts only within some Golgi saccules, a few lysosomal dense bodies and, in rare instances, within an occasional focal area of the endoplasmic reticulum. No selective staining of the GERL system was seen in control ameloblasts incubated at either pH 7.2 or pH 9.0 with acetyl-CoA as substrate, or incubated at pH 5.0 with dephospho-CoA as substrate. Control experiments at pH 5.0 also revealed that reaction product selectively stained the GERL system in ameloblasts when other molecules resembling CoA were used as substrate (e.g., crotonyl-CoA, 3'-NADP+), but not when adenosine 3'-monophosphate (3'-AMP) was used as substrate. That is, ameloblasts incubated at pH 5.0 with 3'-AMP showed heavy deposits of reaction product at many sites throughout the cell, including most lysosomal dense bodies, the Golgi saccules, the GERL system, most secretory granules, the nucleus, and extensively throughout the endoplasmic reticulum. These findings suggest that the GERL system of ameloblasts contains a CoA-specific phosphatase activity that may function to convert CoA to dephospho-CoA at acid pH. Biochemical studies included with this investigation further indicate that CoA-Pase activity saturates at exceptionally low concentrations of substrate (KM = 30 microM CoA) compared to other acid-dependent phosphatases.

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