scholarly journals Immunolocalization of myosin I heavy chain kinase in Acanthamoeba castellanii and binding of purified kinase to isolated plasma membranes.

1991 ◽  
Vol 115 (1) ◽  
pp. 109-119 ◽  
Author(s):  
D Kulesza-Lipka ◽  
I C Baines ◽  
H Brzeska ◽  
E D Korn
Keyword(s):  
Plasma Membrane ◽  
Heavy Chain ◽  
Plasma Membranes ◽  
Acanthamoeba Castellanii ◽  
Permeabilized Cells ◽  
Electrophoretic Mobilities ◽  
Myosin I ◽  
Substrate Level ◽  
Membrane Bound ◽  
Acidic Phospholipids

The actin-activated Mg(2+)-ATPase activities of Acanthamoeba myosins I are known to be maximally expressed only when a single threonine (myosin IA) or serine (myosins IB and IC) is phosphorylated by myosin I heavy chain kinase. The purified kinase is highly activated by autophosphorylation and the rate of autophosphorylation is greatly enhanced by the presence of acidic phospholipids. In this paper, we show by immunofluorescence and immunoelectron microscopy of permeabilized cells that myosin I heavy chain kinase is highly concentrated, but not exclusively, at the plasma membrane. Judged by their electrophoretic mobilities, kinase associated with purified plasma membranes may differ from the cytoplasmic kinase, possibly in the extent of its phosphorylation. Purified kinase binds to highly purified plasma membranes with an apparent KD of approximately 17 nM and a capacity of approximately 0.8 nmol/mg of plasma membrane protein, values that are similar to the affinity and capacity of plasma membranes for myosins I. Binding of kinase to membranes is inhibited by elevated ionic strength and by extensive autophosphorylation but not by substrate-level concentrations of ATP. Membrane-bound kinase autophosphorylates to a lesser extent than free kinase and does not dissociate from the membranes after autophosphorylation. The co-localization of myosin I heavy chain kinase and myosin I at the plasma membrane is of interest in relation to the possible functions of myosin I especially as phospholipids increase kinase activity.

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 Quantification and localization of phosphorylated myosin I isoforms in Acanthamoeba castellanii.

1995 ◽  
Vol 130 (3) ◽  
pp. 591-603 ◽  
Author(s):  
I C Baines ◽  
A Corigliano-Murphy ◽  
E D Korn
Keyword(s):  
Plasma Membrane ◽  
Heavy Chain ◽  
Plasma Membranes ◽  
Phosphorylation Site ◽  
Acanthamoeba Castellanii ◽  
Single Site ◽  
Specific Antibodies ◽  
Large Vacuole ◽  
Myosin I ◽  
Phagocytic Cups

The actin-activated Mg(2+)-ATPase activities of the three myosin I isoforms in Acanthamoeba castellanii are significantly expressed only after phosphorylation of a single site in the myosin I heavy chain. Synthetic phosphorylated and unphosphorylated peptides corresponding to the phosphorylation site sequences, which differ for the three myosin I isoforms, were used to raise isoform-specific antibodies that recognized only the phosphorylated myosin I or the total myosin I isoform (phosphorylated and unphosphorylated), respectively. With these antisera, the amounts of total and phosphorylated isoform were quantified, the phosphomyosin I isoforms localized, and the compartmental distribution of the phosphomyosin isoforms determined. Myosin IA, which was almost entirely in the actin-rich cortex, was 70-100% phosphorylated and particularly enriched under phagocytic cups. Myosins IB and IC were predominantly associated with plasma membranes and large vacuole membranes, where they were only 10-20% phosphorylated, whereas cytoplasmic myosins IB and IC, like cytoplasmic myosin IA, were mostly phosphorylated (60-100%). Moreover, phosphomyosin IB was concentrated in actively motile regions of the plasma membrane. More than 20-fold more phosphomyosin IC and 10-fold more F-actin were associated with the membranes of contracting contractile vacuoles (CV) than of filling CVs. As the total amount of CV-associated myosin IC remained constant, it must be phosphorylated at the start of CV contraction. These data extend previous proposals for the specific functions of myosin I isozymes in Acanthamoeba (Baines, I.C., H. Brzeska, and E.D. Korn. 1992. J. Cell Biol. 119: 1193-1203): phosphomyosin IA in phagocytosis, phosphomyosin IB in phagocytosis and pinocytosis, and phosphomyosin IC in contraction of the CV.

scholarly journals Large-scale purification of presynaptic plasma membranes from Torpedo marmorata electric organ.

1985 ◽  
Vol 101 (5) ◽  
pp. 1757-1762 ◽  
Author(s):  
N Morel ◽  
J Marsal ◽  
R Manaranche ◽  
S Lazereg ◽  
J C Mazie ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Large Scale ◽  
Plasma Membranes ◽  
Electric Organ ◽  
Acetylcholinesterase Activity ◽  
Cholinergic Nerve Terminals ◽  
Membrane Bound ◽  
Torpedo Electric Organ ◽  
Presynaptic Plasma Membrane ◽  
Glycera Convoluta

The presynaptic plasma membrane (PSPM) of cholinergic nerve terminals was purified from Torpedo electric organ using a large-scale procedure. Up to 500 g of frozen electric organ were fractioned in a single run, leading to the isolation of greater than 100 mg of PSPM proteins. The purity of the fraction is similar to that of the synaptosomal plasma membrane obtained after subfractionation of Torpedo synaptosomes as judged by its membrane-bound acetylcholinesterase activity, the number of Glycera convoluta neurotoxin binding sites, and the binding of two monoclonal antibodies directed against PSPM. The specificity of these antibodies for the PSPM is demonstrated by immunofluorescence microscopy.

scholarly journals 5′-Nucleotidases of chicken gizzard and human pancreatic adenocarcinoma cells are anchored to the plasma membrane via a phosphatidylinositol–glycan

1989 ◽  
Vol 262 (1) ◽  
pp. 33-40 ◽  
Author(s):  
U Stochaj ◽  
K Flocke ◽  
W Mathes ◽  
H G Mannherz
Keyword(s):  
Plasma Membrane ◽  
Pancreatic Adenocarcinoma ◽  
Plasma Membranes ◽  
Chicken Gizzard ◽  
Human Pancreatic Adenocarcinoma ◽  
Adenocarcinoma Cells ◽  
Increased Sensitivity ◽  
Membrane Bound ◽  
Membrane Anchoring ◽  
Pancreatic Adenocarcinoma Cell Line

We have analysed the membrane anchorage of plasma-membrane 5′-nucleotidase, an ectoenzyme which can mediate binding to components of the extracellular matrix. We demonstrated that the purified enzyme obtained from chicken gizzard and a human pancreatic adenocarcinoma cell line were both completely transformed into a hydrophilic form by treatment with phospholipases C and D, cleaving glycosylphosphatidylinositol (GPI). These data indicate the presence of a glycolipid linker employed for membrane anchoring of the 5′-nucleotidase obtained from both sources. Incubation of plasma membranes under identical conditions revealed that about half of the AMPase activity was resistant to GPI-hydrolysing phospholipases. Investigation of the enzymic properties of purified chicken gizzard 5′-nucleotidase revealed only minor changes after removal of the phosphatidylinositol linker. However, cleavage of the membrane anchor resulted in an increased sensitivity towards inhibition by concanavalin A. After tissue fractionation, chicken gizzard 5′-nucleotidase could be obtained as either a membrane-bound or a soluble protein; the latter is suspected to be released from the plasma membrane by endogenous phospholipases. Higher-molecular-mass proteins immuno-cross-reactive with the purified chicken gizzard 5′-nucleotidase were detected as both soluble and membrane-bound forms.

Processing of receptor-bound somatostatin: internalization and degradation by pancreatic acini

1987 ◽  
Vol 252 (4) ◽  
pp. G535-G542 ◽  
Author(s):  
N. Viguerie ◽  
J. P. Esteve ◽  
C. Susini ◽  
N. Vaysse ◽  
A. Ribet
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Specific Radioactivity ◽  
Pancreatic Acinar Cells ◽  
Nuclear Fraction ◽  
Pancreatic Acini ◽  
Specific Binding Sites ◽  
Membrane Bound ◽  
Membrane Level

We have previously demonstrated the presence of specific binding sites for somatostatin on plasma membranes from pancreatic acinar cells. In the present study we attempted to characterize the fate of receptor-bound 125I-[Tyr11]somatostatin. Internalization of somatostatin was rapid (reaching a plateau at 20% of the cell-associated specific radioactivity) and temperature dependent. To follow the processing of bound somatostatin, acini were incubated with 125I-[Tyr11]somatostatin at 5 degrees C during 16 h then, after washing, incubated at 37 degrees C for 90 min in fresh medium. Surface-bound somatostatin decreased rapidly, whereas radioactivity increased in the cell interior and the incubation medium. Intracellular and membrane-bound radioactivity was mainly intact 125I-[Tyr11]somatostatin. Degradation occurred at the plasma membrane level and led to iodotyrosine production. After 15 min of incubation, 15% of the initially surface-bound 125I-[Tyr11]somatostatin was compartmentalized within the cell, mainly in the microsomal fraction. After 30 min, a significant increase in radioactivity appeared in the nuclear fraction. These results indicate that the major part of somatostatin cellular degradation takes place at the plasma membrane level. Within the cell, somatostatin is routed to the nucleus via particular fractions sedimenting with microsomal vesicles.

scholarly journals Levels of Human Erythrocyte Membrane-Bound and Cytosolic Glycohydrolases Are Associated with Oxidative Stress in Erectile Dysfunction Patients

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
L. Massaccesi ◽  
G. V. Melzi d’Eril ◽  
G. M. Colpi ◽  
G. Tettamanti ◽  
G. Goi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Plasma Membrane ◽  
Erectile Dysfunction ◽  
Plasma Membranes ◽  
Lag Time ◽  
Specific Marker ◽  
Human Erythrocyte Membrane ◽  
Key Enzyme ◽  
Membrane Bound

Oxidative stress (OS) and production of NO, by endothelium nitric oxide synthetase (eNOS), are involved in the pathophysiology of erectile dysfunction (ED). Moreover, OS induces modifications of the physicochemical properties of erythrocyte (RBC) plasma membranes and of the enzyme content of the same membranes. Due to their role in signalling early membrane alterations in OS-related pathologies, several plasma membrane and cytosolic glycohydrolases of human RBC have been proposed as new markers of cellular OS. In RBC, NOS can be activated and deactivated by phosphorylation/glycosylation. In this regulatory mechanism O-β-N-AcetylGlucosaminidase is a key enzyme. Cellular levels of O-GlcNAcylated proteins are related to OS; consequently dysfunctional eNOS O-GlcNAcylation seems to have a crucial role in ED. To elucidate the possible association between RBC glycohydrolases and OS, plasma hydroperoxides and antioxidant total defenses (Lag-time), cytosolic O-β-N-AcetylGlucosaminidase, cytosolic and membrane Hexosaminidase, membraneβ-D-Glucuronidase, andα-D-Glucosidase have been studied in 39 ED patients and 30 controls. In ED subjects hydroperoxides and plasma membrane glycohydrolases activities are significantly increased whereas Lag-time values and cytosolic glycohydrolases activities are significantly decreased. These data confirm the strong OS status in ED patients, the role of the studied glycohydrolases as early OS biomarker and suggest their possible use as specific marker of ED patients, particularly in those undergoing nutritional/pharmacological antioxidant therapy.

scholarly journals Oxidation-reduction properties of the cytochrome b found in the plasma-membrane fraction of human neutrophils. A possible oxidase in the respiratory burst

1981 ◽  
Vol 194 (2) ◽  
pp. 599-606 ◽  
Author(s):  
A R Cross ◽  
O T Jones ◽  
A M Harper ◽  
A W Segal
Keyword(s):  
Plasma Membrane ◽  
Cytochrome B ◽  
Plasma Membranes ◽  
Mixing Time ◽  
Human Neutrophils ◽  
Half Time ◽  
Sulphur Compounds ◽  
Midpoint Potential ◽  
Oxidation Reduction ◽  
Membrane Bound

The oxidation-reduction midpoint potential of the cytochrome b found in the plasma membrane of human neutrophils has been determined at pH 7.0 (Em,7.0) from measurements of absorption spectra at fixed potentials. In both unstimulated and phorbol myristate acetate-stimulated cells Em,7.0 was -245 mV. Changes in pH affected the Em of the cytochrome b, with a slope of approx. 25 mV/pH unit change. The Em,7.0 of the haem group(s) of the membrane-bound myeloperoxidase of human neutrophils was found to be +34 mV. The plasma membranes contained no detectable ubiquinone, and no iron-sulphur compounds were detected by e.p.r. spectroscopy at 5-20 K. No flavins were detected by e.p.r. spectroscopy. The cytochrome b-245 was not reduced by added NADH or NADPH. Dithionite-reduced cytochrome b-245 formed a complex with CO, supplied as a saturated solution, which was dissociated with 26 microseconds illumination from a xenon flash lamp, and the recombination with CO had a half-time of approx. 6 ms. Partly (80%) reduced cytochrome b-245 was oxidized by added air-saturated buffer with a half-time faster than 1 s at 20 degrees C, a resolution limited by mixing time. These results are compatible with cytochrome b-245 acting as an oxidase.

scholarly journals Basolateral plasma membranes of intestinal epithelial cells. Identification by lactoperoxidase-catalysed iodination and isolation after density perturbation with digitonin

1975 ◽  
Vol 152 (1) ◽  
pp. 71-84 ◽  
Author(s):  
B A Lewis ◽  
A Elkin ◽  
R H Michell ◽  
R Coleman
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Density Perturbation ◽  
Subcellular Fractionation ◽  
Mitochondrial Fraction ◽  
Intestinal Epithelial ◽  
Isolated Cells ◽  
Cell Sheets ◽  
Basolateral Plasma Membrane ◽  
Membrane Bound

1. Lactoperoxidase-catalysed iodination was used to label intestinal epithelial cell sheets with 125I. The iodination was carried out under conditions that allowed little penetration of lactoperoxidase into the cells and membrane-bound 125I therefore provided an effective marker for following plasma-membrane fragments through subcellular-fractionation procedures. 2. After homogenization and isopycnic zonal centrifugation through sucrose gradients two peaks of membrane-bound 125I were detected. One coincided with brush border enzymes such as alkaline phosphatase, disaccharidases and L-leucine β-naphthylamidase, whereas the other was coincident with the major peak of (Na++K+)-stimulated ATPase (adenosine triphosphatase), which has been thought to be concentrated in the basolateral plasma membranes of these cells. Neither peak of 125I reflected the distribution of any marker for an intracellular organelle. 3. A larger proportion of the (Na++K+)-stimulated ATPase, and thus of the basolateral plasma-membrane material, was found in a crude ‘mitochondrial’ fraction. It was not readiily separated from mitochondria by conventional techniques of subcellular fractionation. 4. Treatment of the ‘mitochondrial’ fraction with digitonin increased the density of basolateral plasma membrane but had little effect on mitochondrial density. A purified preparation of digitonin-loaded basolateral plasma membranes was isolated at a density of 1.20-1.22 by isopycnic centrifugation. 5. The enzymic composition of this preparation of basolateral plasma membranes is compared with previous preparations isolated from intestinal mucosal ‘scrape’ materials and from isolated cells.

scholarly journals Rapid Plasma Membrane Anchoring of Newly Synthesized p59  fyn: Selective Requirement for NH2-Terminal Myristoylation and Palmitoylation at Cysteine-3

1997 ◽  
Vol 136 (5) ◽  
pp. 1023-1035 ◽  
Author(s):  
Wouter van 't Hof ◽  
Marilyn D. Resh
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Northern Blotting ◽  
Lag Phase ◽  
Metabolic Labeling ◽  
Fatty Acylation ◽  
Ionic Detergent ◽  
Membrane Bound ◽  
Membrane Anchoring ◽  
Nih 3T3

The trafficking of Src family proteins after biosynthesis is poorly defined. Here we studied the role of dual fatty acylation with myristate and palmitate in biosynthetic transport of p59fyn. Metabolic labeling of transfected COS or NIH 3T3 cells with [35S]methionine followed by analysis of cytosolic and total membrane fractions showed that Fyn became membrane bound within 5 min after biosynthesis. Newly synthesized Src, however, accumulated in the membranes between 20– 60 min. Northern blotting detected Fyn mRNA specifically in soluble polyribosomes and soluble Fyn protein was only detected shortly (1–2 min) after radiolabeling. Use of chimeric Fyn and Src constructs showed that rapid membrane targeting was mediated by the myristoylated NH2-terminal sequence of Fyn and that a cysteine at position 3, but not 6, was essential. Examination of Gαo-, Gαs-, or GAP43-Fyn fusion constructs indicated that rapid membrane anchoring is exclusively conferred by the combination of N-myristoylation plus palmitoylation of cysteine-3. Density gradient analysis colocalized newly synthesized Fyn with plasma membranes. Interestingly, a 10–20-min lag phase was observed between plasma membrane binding and the acquisition of non-ionic detergent insolubility. We propose a model in which synthesis and myristoylation of Fyn occurs on soluble ribosomes, followed by rapid palmitoylation and plasma membrane anchoring, and a slower partitioning into detergent-insoluble membrane subdomains. These results serve to define a novel trafficking pathway for Src family proteins that are regulated by dual fatty acylation.

scholarly journals Calcium-calmodulin and regulation of brush border myosin-I MgATPase and mechanochemistry

1993 ◽  
Vol 122 (3) ◽  
pp. 613-621 ◽  
Author(s):  
JS Wolenski ◽  
SM Hayden ◽  
P Forscher ◽  
MS Mooseker
Keyword(s):  
Heavy Chain ◽  
Brush Border ◽  
Light Chains ◽  
Binding Domains ◽  
Myosin I ◽  
Acidic Phospholipids ◽  
Mgatpase Activity

We examined the Ca(2+)-dependent regulation of brush border (BB) myosin-I by probing the possible roles of the calmodulin (CM) light chains. BB myosin-I MgATPase activity, sensitivity to chymotryptic digestion, and mechanochemical properties were assessed using 1-10 microM Ca2+ and in the presence of exogenously added CM since it has been proposed that this myosin is regulated by calcium-induced CM dissociation from the 119-kD heavy chain. Each of these BB myosin-I properties were dramatically altered by the same threshold of 2-3 microM Ca2+. Enzymatically active NH2-terminal proteolytic fragments of BB myosin-I which lack the CM binding domains (the 78-kD peptide) differ from CM-containing peptides in that the former is completely insensitive to Ca2+. Furthermore, the 78-kD peptide exhibits high levels of MgATPase activity which are comparable to that observed for BB myosin-I in the presence of Ca2+. This suggests that Ca2+ regulates BB myosin-I MgATPase by binding directly to the CM light chains, and that CM acts to repress endogenous MgATPase activity. Ca(2+)-induced CM dissociation from BB myosin-I can be prevented by the addition of exogenous CM. Under these conditions Ca2+ causes a reversible slowing of motility. In contrast, in the absence of exogenous CM, motility is stopped by Ca2+. We demonstrate this reversible slowing is not due to the presence of inactive BB myosin-I molecules exerting a "braking" effect on motile filaments. However, we did observe Ca(2+)-independent slowing of motility by acidic phospholipids, suggesting that factors other than Ca2+ and CM content can affect the mechanochemical properties of BB myosin-I.

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