scholarly journals Turkey erythrocytes possess a membrane-associated inositol 1,4,5-trisphosphate 3-kinase that is activated by Ca2+ in the presence of calmodulin

1987 ◽  
Vol 248 (2) ◽  
pp. 489-493 ◽  
Author(s):  
A J Morris ◽  
C P Downes ◽  
T K Harden ◽  
R H Michell
Keyword(s):  
Membrane Protein ◽  
Membrane Fraction ◽  
Inositol 1,4,5 Trisphosphate ◽  
Membrane Bound

Turkey erythrocytes contain soluble and particulate kinase activities which catalyse the ATP-dependent phosphorylation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. The particle-bound activity accounts for approximately one-quarter of the total cellular Ins(1,4,5)P3 kinase, when assayed at a [Ca2+] of 10 nM. The particle-bound Ins(1,4,5)P3 kinase is not washed from the membrane by 0.6 M-KCl, yet may be solubilized by a variety of detergents. This suggests that it is an intrinsic membrane protein. The product of the membrane-bound Ins(1,4,5)P3 kinase is inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], identifying the enzyme as an Ins(1,4,5)P3 3-kinase. In the presence of calmodulin, the membrane-associated Ins(1,4,5)P3 3-kinase is activated as [Ca2+] is increased over the range 0.2-1.0 microM. Under these conditions, the rates of dephosphorylation of Ins(1,3,4,5)P4 and Ins(1,4,5)P3 by phosphatases in the membrane fraction are unchanged.

scholarly journals d-myo-inositol 1,4,5-trisphosphate phosphatase in skeletal muscle

1988 ◽  
Vol 254 (2) ◽  
pp. 525-529 ◽  
Author(s):  
D Milani ◽  
P Volpe ◽  
T Pozzan
Keyword(s):  
Skeletal Muscle ◽  
Membrane Fraction ◽  
Main Pulmonary Artery ◽  
Total Activity ◽  
Rabbit Brain ◽  
Contraction Coupling ◽  
Inositol 1,4,5 Trisphosphate ◽  
Membrane Bound ◽  
Pulmonary Artery Smooth Muscle ◽  
Fast Twitch

The presence and subcellular distribution of D-myo-inositol 1,4,5-trisphosphate phosphatase (InsP3ase) in rabbit fast-twitch skeletal muscle were investigated. A specific InsP3ase was found in both sarcotubular-membrane and soluble fractions. Membrane-bound InsP3ase accounted for 60-65% of total activity. The InsP3ase was detected both on the surface membranes and on the InsP3-sensitive intracellular Ca2+ store, i.e. the sarcoplasmic reticulum. The Km for inositol 1,4,5-trisphosphate (InsP3) ranged between 15 and 18 microM, and the highest Vmax. (19.6 nmol of InsP3 hydrolysed/min per mg of protein) was measured in a membrane fraction enriched in transverse tubules. Several known inhibitors of InsP3ase, e.g. 2,3-bisphosphoglycerate, CdCl2 and EDTA, were active on skeletal-muscle InsP3ase. Total InsP3ase activity of both rabbit and frog skeletal muscle was comparable with that of rabbit brain, liver and main pulmonary artery (smooth muscle). The present results are consistent with the hypothesis that InsP3 plays a role in excitation-contraction coupling in skeletal muscle [Volpe, Salviati, Di Virgilio & Pozzan (1985) Nature (London) 316, 347-349].

scholarly journals The state of phosphorylation in vivo of membrane-bound phosphoproteins in rat brain

1973 ◽  
Vol 133 (2) ◽  
pp. 387-389 ◽  
Author(s):  
M. Weller ◽  
R. Rodnight
Keyword(s):  
Membrane Protein ◽  
Rat Brain ◽  
Protein Phosphatase ◽  
Membrane Fraction ◽  
Subcellular Fractionation ◽  
Control Value ◽  
P Content ◽  
Membrane Bound ◽  
Labile P

The alkali-labile P content of membrane protein prepared from rapidly frozen rat brain was measured, CuSO4 being used to inhibit protein phosphatase activity during subcellular fractionation. The P content of the membrane fraction was significantly increased (+12%) over the control value by incubation of homogenates with ATP before fractionation. This suggests that the membrane protein in rat brain is normally only partially phosphorylated.

scholarly journals Subcellular localization of the enzymes that dephosphorylate myo-inositol polyphosphates in human platelets

1988 ◽  
Vol 255 (3) ◽  
pp. 795-800 ◽  
Author(s):  
L Molina Y Vedia ◽  
R D Nolan ◽  
E G Lapetina
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Membrane Fraction ◽  
Specific Activity ◽  
Human Platelets ◽  
Inositol Polyphosphates ◽  
Plasma Membrane Fraction ◽  
Inositol 1,4,5 Trisphosphate ◽  
Membrane Bound ◽  
Hydrolysis Of

The phosphatase-induced hydrolysis of [3H]inositol 1,4-bisphosphate [Ins(1,4)P2)] and [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was studied in platelet subcellular fractions. The activity that hydrolyses Ins(1,4)P2 is cytosolic, whereas the activity that hydrolyses Ins(1,4,5)P3 is present in both particulate and cytosolic fractions. The cytosolic Ins(1,4)P2 phosphatase hydrolyses the 1-phosphate of Ins(1,4)P2, whereas the cytosolic and membrane-bound Ins(1,4,5)P3 phosphatases hydrolyse the 5-phosphate of Ins(1,4,5)P3. In the presence of ATP, it is possible to observe a cytosolic Ins(1,4,5)P3 3-kinase that phosphorylates Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate. Apparent Km values for the particulate and the cytosolic Ins(1,4,5)P3 phosphatases are 100 microM and 40 microM respectively. A large proportion of the membrane-associated Ins(1,4,5)P3 phosphatase can be extracted with 1 M-NaCl, and the Mr of this enzyme, as determined by hydrodynamic studies, is 49,000, whereas that of the cytosolic enzyme is 59,000. The Km values for the cytosolic Ins(1,4)P2 phosphatase is 40 microM; this enzyme has an Mr of 49,000. The highest specific activity of the Ins(1,4,5)P3 phosphatase is present in a highly purified plasma-membrane fraction.

scholarly journals Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jasmine M. Hershewe ◽  
Katherine F. Warfel ◽  
Shaelyn M. Iyer ◽  
Justin A. Peruzzi ◽  
Claretta J. Sullivan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Synthetic Biology ◽  
Membrane Protein ◽  
Membrane Vesicles ◽  
Processing Methods ◽  
Glycoprotein Synthesis ◽  
On Demand ◽  
Free Gene ◽  
Membrane Bound

AbstractCell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

scholarly journals Contamination of free-ribosome fractions by detached previously membrane-bound ribosomes (Short Communication)

1974 ◽  
Vol 138 (2) ◽  
pp. 305-307 ◽  
Author(s):  
K. O'Toole
Keyword(s):  
Rat Liver ◽  
Membrane Fraction ◽  
Short Communication ◽  
Free Ribosome ◽  
Membrane Bound ◽  
Free Polyribosome

A rough-membrane fraction isolated from rat liver by a procedure designed to prevent membrane denaturation was subjected to the gradient treatment normally used to isolate free ribosomes. Under these conditions, at most 20% of the ribosomes were detached from membrane with less than 5% sedimenting into the free-polyribosome pellet.

scholarly journals The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria

2018 ◽  
Vol 29 (20) ◽  
pp. 2386-2396 ◽  
Author(s):  
Braulio Vargas Möller-Hergt ◽  
Andreas Carlström ◽  
Katharina Stephan ◽  
Axel Imhof ◽  
Martin Ott
Keyword(s):  
Membrane Protein ◽  
Mitochondrial Gene ◽  
Ribosomal Subunit ◽  
Mitochondrial Translation ◽  
Protein Insertion ◽  
Mitochondrial Ribosomes ◽  
Protein Biogenesis ◽  
Membrane Bound ◽  
Highly Hydrophobic ◽  
Soluble C

Mitochondrial gene expression in Saccharomyces cerevisiae is responsible for the production of highly hydrophobic subunits of the oxidative phosphorylation system. Membrane insertion occurs cotranslationally on membrane-bound mitochondrial ribosomes. Here, by employing a systematic mass spectrometry–based approach, we discovered the previously uncharacterized membrane protein Mrx15 that interacts via a soluble C-terminal domain with the large ribosomal subunit. Mrx15 contacts mitochondrial translation products during their synthesis and plays, together with the ribosome receptor Mba1, an overlapping role in cotranslational protein insertion. Taken together, our data reveal how these ribosome receptors organize membrane protein biogenesis in mitochondria.

Membrane protein import in yeast mitochondria

2000 ◽  
Vol 28 (4) ◽  
pp. 495-499 ◽  
Author(s):  
K. Tokatlidis ◽  
S. Vial ◽  
P. Luciano ◽  
M. Vergnolle ◽  
S. Clémence
Keyword(s):  
Membrane Protein ◽  
Molecular Mechanism ◽  
Protein Import ◽  
Mitochondrial Matrix ◽  
Yeast Mitochondria ◽  
Inner Membrane ◽  
Protein Insertion ◽  
The Past ◽  
Membrane Protein Insertion ◽  
Membrane Bound

The protein import pathway that targets proteins to the mitochondrial matrix has been extensively characterized in the past 15 years. Variations of this import pathway account for the sorting of proteins to other compartments as well, but the insertion of integral inner membrane proteins lacking a presequence is mediated by distinct translocation machinery. This consists of a complex of Tim9 and Tim10, two homologous, Zn2+-binding proteins that chaperone the passage of the hydrophobic precursor across the aqueous inter-membrane space. The precursor is then targeted to another, inner-membrane-bound, complex of at least five subunits that facilitates insertion. Biochemical and genetic experiments have identified the key components of this process; we are now starting to understand the molecular mechanism. This review highlights recent advances in this new membrane protein insertion pathway.

scholarly journals Plasma membrane association of Acanthamoeba myosin I.

1989 ◽  
Vol 109 (4) ◽  
pp. 1519-1528 ◽  
Author(s):  
H Miyata ◽  
B Bowers ◽  
E D Korn
Keyword(s):  
Plasma Membrane ◽  
Binding Site ◽  
Heavy Chain ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Actin Binding ◽  
Plasma Membrane Fraction ◽  
Myosin I ◽  
Membrane Bound ◽  
Actin Binding Site

Myosin I accounted for approximately 2% of the protein of highly purified plasma membranes, which represents about a tenfold enrichment over its concentration in the total cell homogenate. This localization is consistent with immunofluorescence analysis of cells that shows myosin I at or near the plasma membrane as well as diffusely distributed in the cytoplasm with no apparent association with cytoplasmic organelles or vesicles identifiable at the level of light microscopy. Myosin II was not detected in the purified plasma membrane fraction. Although actin was present in about a tenfold molar excess relative to myosin I, several lines of evidence suggest that the principal linkage of myosin I with the plasma membrane is not through F-actin: (a) KI extracted much more actin than myosin I from the plasma membrane fraction; (b) higher ionic strength was required to solubilize the membrane-bound myosin I than to dissociate a complex of purified myosin I and F-actin; and (c) added purified myosin I bound to KI-extracted plasma membranes in a saturable manner with maximum binding four- to fivefold greater than the actin content and with much greater affinity than for pure F-actin (apparent KD of 30-50 nM vs. 10-40 microM in 0.1 M KCl plus 2 mM MgATP). Thus, neither the MgATP-sensitive actin-binding site in the NH2-terminal end of the myosin I heavy chain nor the MgATP-insensitive actin-binding site in the COOH-terminal end of the heavy chain appeared to be the principal mechanism of binding of myosin I to plasma membranes through F-actin. Furthermore, the MgATP-sensitive actin-binding site of membrane-bound myosin I was still available to bind added F-actin. However, the MgATP-insensitive actin-binding site appeared to be unable to bind added F-actin, suggesting that the membrane-binding site is near enough to this site to block sterically its interaction with actin.

scholarly journals Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV

1983 ◽  
Vol 210 (2) ◽  
pp. 389-393 ◽  
Author(s):  
E M Danielsen ◽  
H Sjöström ◽  
O Norén
Keyword(s):  
Organ Culture ◽  
Membrane Fraction ◽  
Bound State ◽  
Dipeptidyl Peptidase ◽  
Dipeptidyl Peptidase Iv ◽  
Soluble Form ◽  
Membrane Bound ◽  
Aminopeptidase A ◽  
High Mannose ◽  
Small Intestinal

The biogenesis of three intestinal microvillar enzymes, maltase-glucoamylase (EC 3.2.1.20), aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. The earliest detectable forms of the enzymes were polypeptides of Mr 225000, 140000 and 115000 respectively. These were found to represent the enzymes in a ‘high-mannose’ state of glycosylation, as judged by their susceptibility to treatment with endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96). After about 40-60 min of chase, maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV were further modified to yield the mature polypeptides of Mr 245000, 170000 and 137000 respectively, which were expressed at the microvillar membrane after 60-90 min of chase. The fact that the enzymes before reaching the microvillar membrane were found in a Ca2+-precipitated membrane fraction (intracellular and basolateral membranes), but not in soluble form, indicates that during biogenesis maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV are transported and assembled in a membrane-bound state.

scholarly journals Making two organelles from one: Woronin body biogenesis by peroxisomal protein sorting

2008 ◽  
Vol 180 (2) ◽  
pp. 325-339 ◽  
Author(s):  
Fangfang Liu ◽  
Seng Kah Ng ◽  
Yanfen Lu ◽  
Wilson Low ◽  
Julian Lai ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Neurospora Crassa ◽  
Filamentous Fungi ◽  
Dense Core ◽  
Cell Cortex ◽  
Protein Matrix ◽  
Woronin Body ◽  
Membrane Bound ◽  
And Control ◽  
Dual Functions

Woronin bodies (WBs) are dense-core organelles that are found exclusively in filamentous fungi and that seal the septal pore in response to wounding. These organelles consist of a membrane-bound protein matrix comprised of the HEX protein and, although they form from peroxisomes, their biogenesis is poorly understood. In Neurospora crassa, we identify Woronin sorting complex (WSC), a PMP22/MPV17-related membrane protein with dual functions in WB biogenesis. WSC localizes to large peroxisome membranes where it self-assembles into detergent-resistant oligomers that envelop HEX assemblies, producing asymmetrical nascent WBs. In a reaction requiring WSC, these structures are delivered to the cell cortex, which permits partitioning of the nascent WB and WB inheritance. Our findings suggest that WSC and HEX collaborate and control distinct aspects of WB biogenesis and that cortical association depends on WSC, which in turn depends on HEX. This dependency helps order events across the organellar membrane, permitting the peroxisome to produce a second organelle with a distinct composition and intracellular distribution.

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