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.

The Occurrence of Phenylalanine Ammonia-Lyase and Cinnamic Acid p-Hydroxylase on the Endoplasmic Reticulum of Cell Suspension Cultures of Glycine max

1978 ◽  
Vol 33 (1-2) ◽  
pp. 65-69 ◽  
Author(s):  
C. Postius ◽  
H. Kindi
Keyword(s):  
Endoplasmic Reticulum ◽  
Glycine Max ◽  
Golgi Apparatus ◽  
Cinnamic Acid ◽  
Time Course ◽  
Phenylalanine Ammonia Lyase ◽  
Plasma Membranes ◽  
Microsomal Fraction ◽  
Activity Increase ◽  
Membrane Bound

Abstract 1. The time course of activity of soluble and microsomal phenylalanine ammonia-lyase (PAL) was studied in dark grown cell cultures of soybean (Glycine max). A distinct activity increase of PAL in the soluble and microsomal fraction occurred prior to the stationary phase of the cell culture. Cinnamic acid p-hydroxylase and NADH : cytochrome c reductase, too, exhibited maximal activity in the log phase, 5 days after the transfer of soybean cells to fresh culture medium.2. Upon subfractionation of the once washed microsomal fraction by sedimentation velocity centrifugation on a sucrose gradient, membranes of the endoplasmic reticulum could be separated from fractions containing mainly membranes from the Golgi apparatus or plasma membranes, respectively. PAL and cinnamic acid p-hydroxylase were found in fractions of endoplasmic reticulum whereas no activity of either enzymes could be detected in fractions containing Golgi apparatus or plasma membranes.3. Repeated washing of microsomal fractions led to a residual membrane-bound PAL representing about 1% of the total PAL activity of the cells. This residual membrane-bound activity could be solubilized almost completely by Triton X-100 or digitonin at concentrations of 0.5 - 5%.

scholarly journals Incorporation of [14C]glucosamine and [14C]leucine into mouse kidney β-glucuronidase induced by gonadotrophin

1970 ◽  
Vol 117 (1) ◽  
pp. 161-167 ◽  
Author(s):  
Keitaro Kato ◽  
Hiroyuki Ide ◽  
Tsuranobu Shirahama ◽  
William H. Fishman
Keyword(s):  
Endoplasmic Reticulum ◽  
Microsomal Fraction ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Mouse Kidney ◽  
Differential Centrifugation ◽  
Freezing And Thawing ◽  
Sucrose Density Gradient Centrifugation ◽  
Glucuronidase Activity

Male BALB/C mice were injected intraperitoneally with 2.5 i.u. of gonadotrophin. After the injection, increase of β-glucuronidase activity was first observed in the microsomal fraction. By 36h 45–50% of the total homogenate activity was found in the microsomal fraction compared with 20–25% in the control microsomal fraction. From 36 to 80h not only microsomal β-glucuronidase but also lysosomal β-glucuronidase increased progressively. After 69h stimulation with 2.5 i.u. of gonadotrophin, d-[1-14C]glucosamine or l-[U-14C]leucine was injected intraperitoneally. After a further 3h the kidneys were homogenized and five particulate fractions were prepared by differential centrifugation. The β-glucuronidase in the microsomal and lysosomal fractions was released respectively by ultrasonication and by freezing and thawing treatment. The enzyme was purified by organic-solvent precipitation and by sucrose-density-gradient centrifugation. The results demonstrated the incorporation of these two labels into the mouse renal β-glucuronidase. The microsomal β-glucuronidase was much more radioactive than the lysosomal enzyme and approx. 80% of the newly synthesized enzyme appeared in microsomes and approx. 20% of that was found in lysosomes at this period. These results suggest that the mouse renal β-glucuronidase is a glycoprotein and that the newly synthesized enzyme is transported from endoplasmic reticulum to lysosomes.

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 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 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.

Is Smooth Endoplasmic Reticulum Involved in Hepatic Glycogen Synthesis in the Fetal Mouse?

1985 ◽  
Vol 43 ◽  
pp. 496-497
Author(s):  
Joanette S. Breslin ◽  
Robert R. Cardell
Keyword(s):  
Endoplasmic Reticulum ◽  
Time Course ◽  
Smooth Endoplasmic Reticulum ◽  
Periodic Acid ◽  
Glycogen Synthesis ◽  
Microscopic Analysis ◽  
Hepatic Glycogen ◽  
Periodic Acid Schiff ◽  
Glycogen Deposition ◽  
Glycogen Particles

Considerable evidence suggests that hepatic smooth endoplasmic reticulum (SER) functions in both glycogen deposition and depletion and is closely associated with glycogen particles during both processes in the adult rodent liver. In this study we have investigated the time course of hepatic glycogen deposition and examined the association of SER with glycogen particles during fetal glycogen synthesis, i.e., from day 15 to day 19 of gestation (plug day = day 1).Livers were removed from fetal ICR mice and processed for either light (LM) and electron microscopy (EM) or biochemical determination of glycogen. Biochemical analysis of glycogen concentrations in each liver revealed an average of 0.1% glycogen in day 15 and day 16 fetal livers, 0.6% in those from day 17, 2.0% on day 18 and nearly 5.0% by day 19. Light microscopic analysis of periodic acid-Schiff (PAS) stained semi-thin (1.0μm) sections confirmed the presence of increasing amounts of glycogen beginning on day 16 and reaching a maximum on day 19 of gestation.

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.

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 The submicrosomal localization of acyl-coenzyme A–cholesterol acyltransferase and its substrate, and of cholesteryl esters in rat liver

1978 ◽  
Vol 174 (3) ◽  
pp. 863-872 ◽  
Author(s):  
Santhirasegaram Balasubramaniam ◽  
Soundararajan Venkatesan ◽  
Konstantinos A. Mitropoulos ◽  
Timothy J. Peters
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Microsomal Fraction ◽  
Smooth Endoplasmic Reticulum ◽  
Cholesterol Acyltransferase ◽  
Specific Radioactivity ◽  
Cholesteryl Esters ◽  
Distribution Profile

To determine the submicrosomal distribution of acyl-CoA–cholesterol acyltransferase and of cholesteryl esters, the microsomal fraction and the digitonin-treated microsomal preparation of rat liver were subjected to analytical centrifugation on sucrose density gradients. With untreated microsomal fractions the distribution profile and the median density of acyl-CoA–cholesterol acyltransferase were very similar to those of RNA. This is in contrast with hydroxymethylglutaryl-CoA reductase and cholesterol 7α-hydroxylase, which are confined to endoplasmic reticulum membranes with low ribosomal coating. In digitonin-treated microsomal preparations activity of acyl-CoA–cholesterol acyltransferase was not detectable. The labelling of untreated microsomal fractions with trace amounts of [14C]cholesterol followed by subfractionation of the labelled microsomal fraction showed that the specific radioactivity of cholesteryl esters obtained in vitro by the various subfractions was similar with all subfractions but different from the specific radioactivity of the 7α-hydroxycholesterol obtained in vitro by the same subfraction. These results demonstrate the existence of two pools of cholesterol confined to membranes from the endoplasmic reticulum, one acting as substrate for cholesterol 7α-hydroxylase and the other acting as substrate for acyl-CoA–cholesterol acyltransferase. The major part of cholesteryl esters present in both untreated and digitonin-treated microsomal fractions was distributed at densities similar to those of membranes from the smooth endoplasmic reticulum and at densities lower than those of smooth membranes from Golgi apparatus. The ratio of the concentrations of non-esterified to esterified cholesterol in the subfractions from both untreated and digitonin-treated microsomal fractions was highest at the maximum distribution of plasma membranes.

scholarly journals EVIDENCE FOR THE PARTICIPATION OF THE GOLGI APPARATUS IN THE INTRACELLULAR TRANSPORT OF NASCENT ALBUMIN IN THE LIVER CELL

1970 ◽  
Vol 47 (3) ◽  
pp. 555-567 ◽  
Author(s):  
Hans Glaumann ◽  
Jan L. E. Ericsson
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Liver Cell ◽  
Intracellular Transport ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Parenchymal Liver Cell ◽  
Parenchymal Liver ◽  
Membrane Bound

A comparative biochemical and radioautographic in vivo study was performed to identify the site of synthesis and route of migration of albumin in the parenchymal liver cell after labeling with leucine-14C or leucine-3H via the portal vein. Free cytoplasmic ribosomes, membrane-bound ribosomes, rough- and smooth-surfaced microsomes, and Golgi membranes were isolated. The purity of the Golgi fraction was examined morphologically and biochemically. After administration of leucine-14C, labeled albumin was extracted, and the sequence of transport was followed from one fraction to the other. Approximately 2 min after the intravenous injection, bound ribosomes displayed a maximal rate of leucine-14C incorporation into albumin. 4 min later, a peak was reached for rough microsomes. Corresponding maximal activities for smooth microsomes were recorded at 15 min, and for the Golgi apparatus at ∼20 min. The relative amount of albumin, calculated on a membrane protein basis, was higher in the Golgi fraction than in the microsomes. By radioautography the silver grains were preferentially localized over the rough-surfaced endoplasmic reticulum at the 5 min interval. Apparent activity in the Golgi zone was noted 9 min after the injection; at 15 and 20 min, the majority of the grains were found in this location. Many of the grains associated with the Golgi apparatus were located over Golgi vacuoles containing 300–800 A electron-opaque bodies. It is concluded that albumin is synthesized on bound ribosomes, subsequently is transferred to the cavities of rough-surfaced endoplasmic reticulum, and then undergoes migration to the smooth-surfaced endoplasmic reticulum and the Golgi apparatus. In the latter organelle, albumin can be expected to be segregated together with very low density lipoprotein in vacuoles known to move toward the sinusoidal portion of the cell and release their content to the blood.

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