scholarly journals Proteolytic activation and solubilization of endoplasmic-reticulum cyclic AMP phosphodiesterase activity

1983 ◽  
Vol 213 (1) ◽  
pp. 99-105 ◽  
Author(s):  
S R Wilson ◽  
M D Houslay
Keyword(s):  
Endoplasmic Reticulum ◽  
Proteinase Inhibitors ◽  
Smooth Endoplasmic Reticulum ◽  
Limited Proteolysis ◽  
Proteolytic Activation ◽  
Freeze Thaw ◽  
Membrane Bound ◽  
Time Courses ◽  
Triton X ◽  
Thiol Proteinase

Dithiothreitol led to the activation and solubilization of the cyclic nucleotide phosphodiesterase activities associated with the smooth and various rough subfractions of rat liver endoplasmic reticulum. The activity in each of the subfractions exhibited somewhat different time courses, and sensitivities to dithiothreitol concentration, in respect of their solubilization and activation. Both activation and solubilization by dithiothreitol could be blocked by either thiol proteinase inhibitors or excess bovine serum albumin. Freeze-thaw solubilization was not blocked by the thiol proteinase inhibitor antipain and did not lead to the activation of the enzyme. After dithiothreitol-induced solubilization, all of the enzymes exhibited non-linear Lineweaver-Burk plots indicative of apparent negative co-operativity. In contrast, after freeze-thaw solubilization the enzyme in the smooth-endoplasmic-reticulum-plus-Golgi fraction still obeys Michaelis kinetics, as does the membrane-bound enzyme. It is possible to mimic the action of dithiothreitol in solubilizing and activating the enzyme by limited proteolysis with trypsin. Triton X-100 is highly efficient at solubilizing these enzymes, yet has little effect on their activities. Charged detergents exhibit highly selective effects on the enzymes as regards their solubilization and activity expressed.

scholarly journals Quantification of apolipoprotein B-48 and B-100 in rat liver endoplasmic reticulum and Golgi fractions

1992 ◽  
Vol 285 (1) ◽  
pp. 153-159 ◽  
Author(s):  
I J Cartwright ◽  
J A Higgins
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Protein A ◽  
Subcellular Fractions ◽  
Apo B ◽  
Sds Page ◽  
Membrane Bound ◽  
Triton X ◽  
Quantitative Immunoblotting

We have developed a method for measurement of apolipoprotein (apo) B-48 and apo B-100 in blood and subcellular fractions of rat liver based on SDS/PAGE followed by quantitative immunoblotting using 125I-Protein A. Standard curves were prepared in each assay using apo B prepared from total rat lipoproteins by extraction with tetramethylurea. Subcellular fractions (rough and smooth endoplasmic reticulum and Golgi fractions) were prepared from rat liver and separated into membrane and cisternal-content fractions. For quantification, membrane fractions were solubilized in Triton X-100, and the apo B was immunoprecipitated before separation by SDS/PAGE and immunoblotting. Content fractions were concentrated by ultrafiltration and separated by SDS/PAGE without immunoprecipitation. Quantification of apo B in subcellular fractions and detection of apo B by immunoblotting yielded consistent results. In all fractions apo B-48 was the major form, accounting for approximately three-quarters of the total apo B. By using marker enzymes as internal standards, it was calculated that all of the apo B was recovered in the endoplasmic reticulum and Golgi fractions, with approximately 80% of each form of apo B in the endoplasmic reticulum. More than 90% of the apo B of the rough- and smooth-endoplasmic-reticulum fractions was membrane-bound, whereas approx. 33 and 15% of the apo B of the cis-enriched Golgi fractions and trans-enriched Golgi fractions respectively were membrane-bound.

Studies on the site of addition of sialic acid and glucosamine to rat α1-acid glycoprotein

1977 ◽  
Vol 55 (4) ◽  
pp. 408-414 ◽  
Author(s):  
J. C. Jamieson
Keyword(s):  
Endoplasmic Reticulum ◽  
Sialic Acid ◽  
Rat Liver ◽  
Golgi Complex ◽  
Smooth Endoplasmic Reticulum ◽  
Main Site ◽  
Acid Glycoprotein ◽  
Membrane Bound ◽  
Polypeptide Chains

Ultrasonic extracts of rough and smooth endoplasmic reticulum fractions and Golgi fractions from rat liver were examined by immunoelectrophoresis using antiserum to α1-acid glycoprotein. Rough endoplasmic reticulum fractions contained only sialic acid free α1-acid glycoprotein, whereas smooth endoplasmic reticulum and Golgi fractions also contained sialic acid containing α1-acid glycoprotein. Determination of the sialic acid contents of immune precipitates isolated from the extracts suggested that the Golgi complex was the main site of addition of sialic acid to α1-acid glycoprotein. Immunological studies on puromycin extracts of polyribosomes showed that polypeptide chains of α1-acid glycoprotein and albumin were assembled mainly on membrane-bound polyribosomes. Evidence is presented from incorporation studies with labelled leucine and glucosamine that initial glycosylation of α1-acid glycoprotein occurs mainly or entirely after release of nascent polypeptide from the ribosomal site.

scholarly journals Properties of membrane-bound bilirubin UDP-glucuronyltransferase in rough and smooth endoplasmic reticulum and in the nuclear envelope from rat liver

1989 ◽  
Vol 259 (3) ◽  
pp. 659-663 ◽  
Author(s):  
F Vanstapel ◽  
L Hammaker ◽  
K Pua ◽  
N Blanckaert
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Membrane System ◽  
Permeability Barrier ◽  
Membrane Bound ◽  
Marker Studies ◽  
High Degree ◽  
Regulatory Properties

We examined regulatory properties of bilirubin UDP-glucuronyltransferase in sealed RER (rough endoplasmic reticulum)- and SER (smooth endoplasmic reticulum)-enriched microsomes (microsomal fractions), as well as in nuclear envelope from rat liver. Purity of membrane fractions was verified by electron microscopy and marker studies. Intactness of RER and SER vesicles was ascertained by a high degree of latency of the lumenal marker mannose-6-phosphatase. No major differences in the stimulation of UDP-glucuronyltransferase by detergent or by the presumed physiological activator, UDPGlcNAc, were observed between total microsomes and RER- or SER-enriched microsomes. Isolated nuclear envelopes were present as a partially disrupted membrane system, with approx. 50% loss of mannose-6-phosphatase latency. The nuclear transferase had lost its latency to a similar extent, and the enzyme failed to respond to UDPGlcNAc. Our results underscore the necessity to include data on the integrity of the membrane permeability barrier when reporting regulatory properties of UDP-glucuronyltransferase in different membrane preparations.

scholarly journals Polarized compartmentalization of organelles in growth cones from developing optic tectum.

1985 ◽  
Vol 101 (4) ◽  
pp. 1473-1480 ◽  
Author(s):  
T P Cheng ◽  
T S Reese
Keyword(s):  
Endoplasmic Reticulum ◽  
Growth Cone ◽  
Smooth Endoplasmic Reticulum ◽  
Freeze Substitution ◽  
Growth Cones ◽  
Distal Segment ◽  
Computer Assisted ◽  
Chemical Fixation ◽  
Coated Pits ◽  
Membrane Bound

We have used computer-assisted reconstructions of continuous serial sections to study the cytoplasmic organization of growth cones in vivo. Optic tecta from 6.25-6.5-d-old chicken embryos were quick-frozen and then freeze-substituted in acetone-osmium tetroxide or, for comparison, prepared by conventional fixation. Images of eight freeze-substituted and two conventionally fixed growth cones were reconstructed from aligned serial micrographs. After freeze-substitution, numerous lumenless membrane-bound sacs arrayed in multilamellar stacks appear to replace the abundant smooth endoplasmic reticulum found after chemical fixation. Microtubule fascicles progressively diverge from their typical fascicular organization in the initial segment of the growth cone and are absent in the varicosity and the more distal segment. Mitochondria, in contrast, are concentrated in the proximal segment of the varicosity; multilamellar stacks and endosome-like vacuoles are in the distal segment; and coated pits and vesicles are concentrated near the terminal filopodium, which is the most distal and organelle-poor domain of the growth cone. These observations suggest that dilation and fusion of the lumenless, membrane-bound sacs that occurs during chemical fixation give rise to the network of smooth endoplasmic reticulum. The three-dimensional reconstructions show that the cytoplasmic components of growth cones, including the membrane-bound sacs and multilamellar stacks revealed by freeze substitution, are polarized along the axis of these growth cones, which suggests that they have a role in recycling of membrane during elongation of the growth cone.

Pink-eyed Dilution Protein Controls the Processing of Tyrosinase

2002 ◽  
Vol 13 (6) ◽  
pp. 1953-1964 ◽  
Author(s):  
Kun Chen ◽  
Prashiela Manga ◽  
Seth J. Orlow
Keyword(s):  
Endoplasmic Reticulum ◽  
Culture Medium ◽  
Posttranslational Processing ◽  
Bafilomycin A1 ◽  
Wild Type ◽  
P Protein ◽  
Membrane Bound ◽  
Triton X ◽  
Pink Eyed Dilution ◽  
Perinuclear Area

The processing of tyrosinase, which catalyzes the limiting reaction in melanin synthesis, was investigated in melan-p1 melanocytes, which are null at the p locus. Endoglycosidase H digestion showed that a significant fraction of tyrosinase was retained in the endoplasmic reticulum. This retention could be rescued either by transfection of melan-p1 cells with an epitope-tagged wild-typep transcript or by treatment with either bafilomycin A1 or ammonium chloride. We found that the endoplasmic reticulum contains a significant amount of p protein, thus supporting a role for p within this compartment. Using immunofluoresence, we showed that most mature full-length tyrosinase in melan-p1 cells was located in the perinuclear area near the Golgi, in contrast to its punctate melanosomal pattern in wild-type melanocytes. Expression of p in melan-p1 cells restored tyrosinase to melanosomes. Triton X-114 phase separation revealed that an increased amount of tyrosinase was proteolyzed in melan-p1 cells compared with wild-type melanocytes. The proteolyzed tyrosinase was no longer membrane bound, but remained enzymatically active and a large proportion was secreted into the culture medium of melan-p1 cells. We conclude that p regulates posttranslational processing of tyrosinase, and hypopigmentation in melan-p1 cells is the result of altered tyrosinase processing and trafficking.

scholarly journals Studies on the glycosylation of hydroxylysine residues during collagen biosynthesis and the subcellular localization of collagen galactosyltransferase and collagen glucosyltransferase in tendon and cartilage cells

1975 ◽  
Vol 152 (2) ◽  
pp. 291-302 ◽  
Author(s):  
Richard Harwood ◽  
Michael E. Grant ◽  
David S. Jackson
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Specific Activity ◽  
Smooth Endoplasmic Reticulum ◽  
Gradient Centrifugation ◽  
Subcellular Fractions ◽  
Anterior Lens Capsule ◽  
Cartilage Cells ◽  
Membrane Bound ◽  
And Cartilage

1. The glycosylation of hydroxylysine during the biosynthesis of procollagen by embryonic chick tendon and cartilage cells was examined. When free and membrane-bound ribosomes isolated from cells labelled for 4min with [14C]lysine were assayed for hydroxy[14C]lysine and hydroxy[14C]lysine glycosides, it was found that hydroxylation took place only on membrane-bound ribosomes and that some synthesis of galactosylhydroxy[14C]lysine and glucosylgalactosylhydroxy[14C]lysine had occurred on the nascent peptides. 2. Assays of subcellular fractions isolated from tendon and cartilage cells labelled for 2h with [14C]lysine demonstrated that the glycosylation of procollagen polypeptides began in the rough endoplasmic reticulum. 14C-labelled polypeptides present in the smooth endoplasmic reticulum and Golgi fractions were glycosylated to extents almost identical with the respective secreted procollagens. 3. Assays specific for collagen galactosyltransferase and collagen glucosyltransferase are described, using as substrate chemically treated bovine anterior-lens-capsule collagen. 4. When homogenates were assayed for the collagen glycosyltransferase activities, addition of Triton X-100 (0.01%, w/v) was found to stimulate enzyme activities by up to 45%, suggesting that the enzymes were probably membrane-bound. 5. Assays of subcellular fractions obtained by differential centrifugation for collagen galactosyltransferase activity indicated the specific activity to be highest in the microsomal fractions. Similar results were obtained for collagen glucosyltransferase activity. 6. When submicrosomal fractions obtained by discontinuous-sucrose-density-gradient-centrifugation procedures were assayed for these enzymic activities, the collagen galactosyltransferase was found to be distributed in the approximate ratio 7:3 between rough and smooth endoplasmic reticulum of both cell types. Similar determinations of collagen glucosyltransferase indicated a distribution in the approximate ratio 3:2 between rough and smooth microsomal fractions. 7. Assays of subcellular fractions for the plasma-membrane marker 5′-nucleotidase revealed a distribution markedly different from the distributions obtained for the collagen glycosyltransferase. 8. The studies described here demonstrate that glycosylation occurs early in the intracellular processing of procollagen polypeptides rather than at the plasma membrane, as was previously suggested.

scholarly journals FINE STRUCTURE OBSERVATIONS OF THE UPTAKE OF INTRAVENOUSLY INJECTED PEROXIDASE BY THE RAT CHOROID PLEXUS

1967 ◽  
Vol 15 (3) ◽  
pp. 160-165 ◽  
Author(s):  
NORWIN H. BECKER ◽  
ALEX B. NOVIKOFF ◽  
H. M. ZIMMERMAN
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Cerebrospinal Fluid ◽  
Choroid Plexus ◽  
Smooth Endoplasmic Reticulum ◽  
Multivesicular Bodies ◽  
Adult Rats ◽  
Dense Bodies ◽  
Pinocytotic Vesicles ◽  
Membrane Bound

The uptake by the choroid plexus of adult rats of intravenously injected horseradish peroxidase has been investigated by electron microscopy. Within 4 min, the injected protein passes the capillary and is rapidly distributed through extracellular space and choroidal cells. Peroxidase enters the choroidal cells within coated vesicles which act as pinocytotic vesicles. At 15 min, peroxidase activity is present in numerous membrane-bound vesicles, multivesicular bodies, dense bodies and what appear to be segments of smooth endoplasmic reticulum. None of the peroxidase-containing organelles is seen to empty to the ventricular surface. Egress of the extracellular peroxidase into the cerebrospinal fluid is apparently blocked by apical zonulae occludentes between the choroidal cells.

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.

Localization of various phosphatase activities within the golgi apparatus and lysosomes of rat leydig cells

1986 ◽  
Vol 44 ◽  
pp. 294-295
Author(s):  
M. F. Lalli ◽  
V. Lacroix ◽  
L. Hermo ◽  
Y. Clermont ◽  
C. E. Smith
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Leydig Cells ◽  
Smooth Endoplasmic Reticulum ◽  
Fluid Phase ◽  
Multivesicular Bodies ◽  
Golgi Stack ◽  
Dense Membrane ◽  
Membrane Bound ◽  
Adsorptive Endocytosis

The testosterone-secreting Leydig cells contain an abundance of smooth endoplasmic reticulum, peroxisomes, mitochondria as well as a large, spheroidal, juxtanuclear Golgi apparatus composed of interconnected stacks of saccules (Figs. 1,2). Each Golgi stack appears to be composed of between 5 to 7 saccules or sacculo-tubular elements (Figs.1,2). These cells also possess pale and dense multivesicular bodies and dense membrane-bound bodies identified assecondary lysosomes, all of which have been shown to be involved in fluid phase and adsorptive endocytosis as well as in receptor mediated endocytosis. The purpose of the present study was to characterize the reactivity of Golgi saccules, multivesicular bodies and lysosomes of Leydig cells for different phosphatases.

scholarly journals Intracellular transport of mouse kidney β-glucuronidase induced by gonadotrophin

1972 ◽  
Vol 127 (2) ◽  
pp. 425-435 ◽  
Author(s):  
Keitaro Kato ◽  
Itsuyo Hirohata ◽  
William H. Fishman ◽  
Hisao Tsukamoto
Keyword(s):  
Endoplasmic Reticulum ◽  
Intracellular Transport ◽  
Time Course ◽  
Microsomal Fraction ◽  
Smooth Endoplasmic Reticulum ◽  
Mouse Kidney ◽  
Lysosomal Fraction ◽  
Glucuronidase Activity ◽  
Maximum Value ◽  
Membrane Bound

1. The response of renal β-glucuronidase with time to the injection of gonadotrophin was investigated in each submicrosomal fraction of rough and smooth microsomal fractions of mouse kidney homogenate. 2. The increase in β-glucuronidase activity appeared initially in membranes of the rough microsomal fraction, 24h after injection. 3. Afterwards the newly synthesized enzyme appeared in the contents of the rough microsomal fraction and was subsequently found in the smooth microsomal fraction, reaching a maximum concentration in this fraction at 72h. 4. At this juncture, a decrease in the enzyme activity was observed in rough microsomal contents whereas the lysosomal fraction had reached its maximum value. 5. The time-course of the appearance of β-glucuronidase in the submicrosomal fractions after the gonadotrophin stimulation suggests that the newly synthesized enzyme at the site of membrane-bound ribosomes is transferred across the membrane into cisternae of the rough endoplasmic reticulum, and then is transported into lysosomes via the smooth endoplasmic reticulum. 6. The properties of microsomal and lysosomal β-glucuronidases were compared.

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