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.

In vivo assessment of microvascular nitric oxide production and its relation with blood flow

2001 ◽  
Vol 280 (3) ◽  
pp. H1222-H1231 ◽  
Author(s):  
X. F. Figueroa ◽  
A. D. Martínez ◽  
D. R. González ◽  
P. I. Jara ◽  
S. Ayala ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Blood Flow ◽  
Plasma Flow ◽  
Subcellular Fractionation ◽  
No Synthase ◽  
Cheek Pouch ◽  
No Production ◽  
Hamster Cheek Pouch ◽  
Membrane Bound

To assess the hypothesis that microvascular nitric oxide (NO) is critical to maintain blood flow and solute exchange, we quantified NO production in the hamster cheek pouch in vivo, correlating it with vascular dynamics. Hamsters (100–120 g) were anesthetized and prepared for measurement of microvessel diameters by intravital microscopy, of plasma flow by isotopic sodium clearance, and of NO production by chemiluminescence. Analysis of endothelial NO synthase (eNOS) location by immunocytochemistry and subcellular fractionation revealed that eNOS was present in arterioles and venules and was 67 ± 7% membrane bound. Basal NO release was 60.1 ± 5.1 pM/min ( n = 35), and plasma flow was 2.95 ± 0.27 μl/min ( n = 29). Local NO synthase inhibition with 30 μM N ω-nitro-l-arginine reduced NO production to 8.6 ± 2.6 pmol/min (−83 ± 5%, n = 9) and plasma flow to 1.95 ± 0.15 μl/min (−28 ± 12%, n = 17) within 30–45 min, in parallel with constriction of arterioles (9–14%) and venules (19–25%). The effects of N ω-nitro-l-arginine (10–30 μM) were proportional to basal microvascular conductance ( r = 0.7, P < 0.05) and fully prevented by 1 mM l-arginine. We conclude that in this tissue, NO production contributes to 35–50% of resting microvascular conductance and plasma-tissue exchange.

scholarly journals Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation.

1987 ◽  
Vol 105 (3) ◽  
pp. 1241-1251 ◽  
Author(s):  
J R Bartles ◽  
H M Feracci ◽  
B Stieger ◽  
A L Hubbard
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Subcellular Fractionation ◽  
Aminopeptidase N ◽  
Rat Hepatocyte ◽  
Dipeptidylpeptidase Iv ◽  
Plasma Membrane Fraction ◽  
Apical Domain ◽  
Basolateral Plasma Membrane

We have used pulse-chase metabolic radiolabeling with L-[35S]methionine in conjunction with subcellular fractionation and specific protein immunoprecipitation techniques to compare the posttranslational transport pathways taken by endogenous domain-specific integral proteins of the rat hepatocyte plasma membrane in vivo. Our results suggest that both apical (HA 4, dipeptidylpeptidase IV, and aminopeptidase N) and basolateral (CE 9 and the asialoglycoprotein receptor [ASGP-R]) proteins reach the hepatocyte plasma membrane with similar kinetics. The mature molecular mass form of each of these proteins reaches its maximum specific radioactivity in a purified hepatocyte plasma membrane fraction after only 45 min of chase. However, at this time, the mature radiolabeled apical proteins are not associated with vesicles derived from the apical domain of the hepatocyte plasma membrane, but instead are associated with vesicles which, by several criteria, appear to be basolateral plasma membrane. These vesicles: (a) fractionate like basolateral plasma membrane in sucrose density gradients and in free-flow electrophoresis; (b) can be separated from the bulk of the likely organellar contaminants, including membranes derived from the late Golgi cisternae, transtubular network, and endosomes; (c) contain the proven basolateral constituents CE 9 and the ASGP-R, as judged by vesicle immunoadsorption using fixed Staphylococcus aureus cells and anti-ASGP-R antibodies; and (d) are oriented with their ectoplasmic surfaces facing outward, based on the results of vesicle immunoadsorption experiments using antibodies specific for the ectoplasmic domain of the ASGP-R. Only at times of chase greater than 45 min do significant amounts of the mature radiolabeled apical proteins arrive at the apical domain, and they do so at different rates. Approximate half-times for arrival are in the range of 90-120 min for aminopeptidase N and dipeptidylpeptidase IV whereas only 15-20% of the mature radiolabeled HA 4 associated with the hepatocyte plasma membrane fraction has become apical even after 150 min of chase. Our results suggest a mechanism for hepatocyte plasma membrane biogenesis in vivo in which all integral plasma membrane proteins are shipped first to the basolateral domain, followed by the specific retrieval and transport of apical proteins to the apical domain at distinct rates.

scholarly journals Differential phosphorylation in vivo of cytoplasmic dynein associated with anterogradely moving organelles.

1994 ◽  
Vol 127 (6) ◽  
pp. 1671-1681 ◽  
Author(s):  
J F Dillman ◽  
K K Pfister
Keyword(s):  
Rat Brain ◽  
Cytoplasmic Dynein ◽  
Fast Axonal Transport ◽  
Brain Ventricles ◽  
Phosphorylation State ◽  
Tissue Extracts ◽  
Intracellular Movement ◽  
Membrane Bound ◽  
Differential Phosphorylation

Two microtubule-stimulated ATPases, cytoplasmic dynein, and kinesin, are believed to be responsible for the intracellular movement of membrane-bound organelles in opposite directions along microtubules. An unresolved component of this model is the mechanism by which cells regulate these two motors to direct various membrane-bound organelles to their proper locations. To determine if phosphorylation may play a role in the regulation of cytoplasmic dynein, the in vivo phosphorylation state of cytoplasmic dynein from two cellular pools was examined. The entire cellular pool of brain cytoplasmic dynein was metabolically labeled by the infusion of [32P]orthophosphate into the cerebrospinal fluid of rat brain ventricles. To characterize the phosphorylation of dynein associated with anterograde membrane-bound organelles, the optic nerve fast axonal transport system was used. Using a monoclonal antibody to the 74-kD polypeptide of brain cytoplasmic dynein, the native dynein complex was immunoprecipitated from the radiolabled tissue extracts. Autoradiographs of one and two dimensional gels showed labeling of nearly all of the polypeptide isoforms of cytoplasmic dynein from rat brain. These polypeptides are phosphorylated on serine residues. Comparison of the amount of 32P incorporated into the dynein polypeptides revealed differences in the phosphorylation of dynein polypeptides from the anterograde and the cellular pools. Most interestingly, the 530-kD heavy chain of dynein appears to be phosphorylated to a lesser extent in the anterograde pool than in the cellular pool. Since the anterograde pool contains inactive dynein, while the entire cellular pool contains both inactive and active dynein, these results are consistent with the hypothesis that phosphorylation regulates the functional activity of cytoplasmic dynein.

scholarly journals The Early Onset Dystonia Protein TorsinA Interacts with Kinesin Light Chain 1

2004 ◽  
Vol 279 (19) ◽  
pp. 19882-19892 ◽  
Author(s):  
Christoph Kamm ◽  
Heather Boston ◽  
Jeffrey Hewett ◽  
Jeremy Wilbur ◽  
David P. Corey ◽  
...  
Keyword(s):  
Rat Brain ◽  
Light Chain ◽  
Cortical Neurons ◽  
Early Onset ◽  
Subcellular Fractionation ◽  
Wild Type ◽  
Anterograde Transport ◽  
Glutamic Acid Residue ◽  
Carboxyl Terminal

Early onset dystonia is a movement disorder caused by loss of a glutamic acid residue (Glu302/303) in the carboxyl-terminal portion of the AAA+protein, torsinA. We identified the light chain subunit (KLC1) of kinesin-I as an interacting partner for torsinA, with binding occurring between the tetratricopeptide repeat domain of KLC1 and the carboxyl-terminal region of torsinA. Coimmunoprecipitation analysis demonstrated that wild-type torsinA and kinesin-I form a complexin vivo. In cultured cortical neurons, both proteins co-localized along processes with enrichment at growth cones. Wild-type torsinA expressed in CAD cells co-localized with endogenous KLC1 at the distal end of processes, whereas mutant torsinA remained confined to the cell body. Subcellular fractionation of adult rat brain revealed torsinA and KLC associated with cofractionating membranes, and both proteins were co-immunoprecipitated after cross-linking cytoplasmically oriented proteins on isolated rat brain membranes. These studies suggest that wild-type torsinA undergoes anterograde transport along microtubules mediated by kinesin and may act as a molecular chaperone regulating kinesin activity and/or cargo binding.

Saturable binding of [3H]phenytoin to rat brain membrane fraction

1981 ◽  
Vol 59 (4) ◽  
pp. 402-407 ◽  
Author(s):  
W. M. Burnham ◽  
L. Spero ◽  
M. M. Okazaki ◽  
B. K. Madras
Keyword(s):  
Rat Brain ◽  
Binding Sites ◽  
Membrane Fraction ◽  
Proteolytic Enzymes ◽  
Subcellular Fractionation ◽  
Brain Membrane ◽  
High Affinity ◽  
Number Of Binding Sites ◽  
Very High ◽  
Maximal Capacity

Preliminary studies indicate that [3H]phenytoin binds in a saturable and reversible fashion to at least two distinct sites in the membrane fraction of whole rat brain. One of these displays a high affinity (Kd = 6 nM) and a low maximal capacity (Bmax = 10 pmol/g protein). The other has a low affinity (Kd = 4.8 μM) and is estimated to have a very high maximal capacity. Phenytoin binding is reduced if the membrane fraction is preincubated with proteolytic enzymes and subcellular fractionation studies indicate that the P2 fraction has the largest number of binding sites. Competition experiments fail to reveal significant binding interactions with putative neurotransmitters or with other drugs except the hydantoins and anticonvulsant barbiturates. Although it is premature to speculate on the clinical significance of these findings, it is encouraging to note that the low affinity site has a Kd very similar to the therapeutic levels of phenytoin found in cerebrospinal fluid and that there seems to be some relationship between binding potency and anticonvulsant potency within the hydantoin series.

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 Retention of mRNA on the endoplasmic reticulum membranes after in vivo disassembly of polysomes by an inhibitor of initiation.

1976 ◽  
Vol 71 (1) ◽  
pp. 307-313 ◽  
Author(s):  
M Adesnik ◽  
M Lande ◽  
T Martin ◽  
D D Sabatini
Keyword(s):  
Messenger Rna ◽  
Membrane Fraction ◽  
Human Fibroblasts ◽  
Ribosomal Subunits ◽  
Run Off ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Polypeptide Chains ◽  
Extensive Release

Membrane-bound ribosomes and messenger RNA remained associated with the microsomal membranes of human fibroblasts after cultures were treated with Verrucarin A, an inhibitor of initiation which led to extensive run-off of ribosomes from polysomal structures. When a membrane fraction from Verrucarin-treated cells containing such inactive ribosomes and mRNA was suspended in a medium of high salt concentration, extensive release of ribosomal subunits occurred without the need for puromycin. The mRNA nevertheless remained associated with the membranes. These results add support to the conclusion that, in human fibroblasts, mRNA is bound directly to ER membranes, independently of the ribosomes and nascent polypeptide chains.

scholarly journals Chimeric constructs show that the unique N-terminal domain of the cyclic AMP phosphodiesterase RD1 (RNPDE4A1A; rPDE-IVA1) can confer membrane association upon the normally cytosolic protein chloramphenicol acetyltransferase

1995 ◽  
Vol 308 (2) ◽  
pp. 673-681 ◽  
Author(s):  
G Scotland ◽  
M D Houslay
Keyword(s):  
Cyclic Amp ◽  
Membrane Fraction ◽  
Structural Information ◽  
Subcellular Fractionation ◽  
Translation System ◽  
Membrane Association ◽  
Cytosol Fraction ◽  
Terminal Domain ◽  
Ionic Detergent ◽  
Membrane Bound

A novel plasmid was generated which allowed the expression of the cytosolic bacterial enzyme chloramphenicol acetyl transferase (CAT) in COS-7 cells. Upon transfection, the majority of the novel CAT activity was found in the cytosol fraction of COS cells. Chimeric molecules were made between N-terminal portions of the type IVA cyclic AMP-specific rat ‘dunce-like’ phosphodiesterase (RD1) (RNPDE4A1A; rPDE-IVA1) fused to CAT at its N-terminus. Expression in COS-7 cells of chimeras formed from 1-100RD1-CAT and 1-25RD1-CAT now showed CAT activity associated with the membrane fraction. In contrast, a chimera formed from 26-100RD1-CAT showed an identical expression pattern to native CAT, with the major fraction of CAT activity occurring in the cytosol fraction. Membrane-bound CAT activity provided by 1-100RD1-CAT and 1-25RD1-CAT was not released by either high-salt or washing treatments but was solubilized in a dose-dependent fashion by the non-ionic detergent Triton X-100. Subcellular fractionation of COS-7 cells showed that, as with RD1, the membrane-bound activity of the RD1-CAT chimera followed that of the plasma membrane marker 5′-nucleotidase. Plasmids containing chimeric cDNAs were exposed to a coupled transcription-translation system that, in addition to the full-length chimeras, was found to generate a range of N-terminal truncated species due to initiation at different methionine residues. Incubation of the mature protein products formed in this system with a COS cell membrane fraction showed that only those chimeric CAT constructs containing the first 25 amino acids of RD1 became membrane-associated. The unique 25 amino acid N-terminal domain of RD1 contains structural information that can confer membrane association upon an essentially soluble protein.

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