Acetylated tubulin associates with the fifth cytoplasmic domain of Na+/K+-ATPase: possible anchorage site of microtubules to the plasma membrane

2009 ◽  
Vol 422 (1) ◽  
pp. 129-137 ◽  
Author(s):  
Guillermo G. Zampar ◽  
María E. Chesta ◽  
Agustín Carbajal ◽  
Natalí L. Chanaday ◽  
Nicolás M. Díaz ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Catalytic Activity ◽  
Molecular Mass ◽  
Cytoplasmic Domain ◽  
Membrane Preparation ◽  
Acetylated Tubulin ◽  
Exclusion Chromatography ◽  
Molecular Exclusion Chromatography

We showed previously that NKA (Na+/K+-ATPase) interacts with acetylated tubulin resulting in inhibition of its catalytic activity. In the present work we determined that membrane-acetylated tubulin, in the presence of detergent, behaves as an entity of discrete molecular mass (320–400 kDa) during molecular exclusion chromatography. We also found that microtubules assembled in vitro are able to bind to NKA when incubated with a detergent-solubilized membrane preparation, and that isolated native microtubules have associated NKA. Furthermore, we determined that CD5 (cytoplasmic domain 5 of NKA) is capable of interacting with acetylated tubulin. Taken together, our results are consistent with the idea that NKA may act as a microtubule–plasma membrane anchorage site through an interaction between acetylated tubulin and CD5.

Steroid sequestration within the rat adrenal zona glomerulosa

1988 ◽  
Vol 117 (2) ◽  
pp. 191-NP ◽  
Author(s):  
S. M. Laird ◽  
G. P. Vinson ◽  
B. J. Whitehouse
Keyword(s):  
Plasma Membrane ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Zona Glomerulosa ◽  
Membrane Preparation ◽  
The Media ◽  
Considerable Concentration ◽  
Highly Correlated ◽  
Plasma Membrane Preparation

ABSTRACT Accumulated data from in-vitro experiments have suggested that 18-hydroxysteroids may be stored within the intact rat adrenal zona glomerulosa. The phenomenon was further investigated by comparing the amount of steroid remaining in the zona glomerulosa tissue with that secreted into the media during incubation in vitro. The results showed that 18-hydroxydeoxycorticosterone (18-OH-DOC) and 18-hydroxycorticosterone (18-OH-B) were retained within the tissue against a considerable concentration gradient, with smaller amounts of aldosterone and corticosterone. Lysis of the intact zona glomerulosa, by preincubation in distilled water, yielded an enriched plasma membrane preparation. After subsequent incubation in Krebs–Ringer bicarbonate this preparation contained significantly more 18-OH-DOC than did the intact tissue, suggesting that tissuesequestered 18-OH-DOC is normally metabolized to other products. These may include 18-OH-B and aldosterone. Fractionation of homogenized intact zona glomerulosa and the enriched plasma membrane preparation by density gradient centrifugation showed that tissue 18-OH-DOC banded in fractions of density 1·063– 1·21 g/ml and that its distribution was highly correlated with protein. Corticosterone, 18-OH-B and aldosterone banded like added free [3H]18-OH-DOC in fractions of density < 1·006 g/ml. The results suggest that 18-OH-DOC is the major sequestered steroid within the rat adrenal zona glomerulosa and that this sequestration is attributable to the association of 18-OH-DOC with a high-density component of the plasma membrane. J. Endocr. (1988) 117, 191–196

scholarly journals Trefoil Factor Family (TFF) Modules Are Characteristic Constituents of Separate Mucin Complexes in the Xenopus laevis Integumentary Mucus: In Vitro Binding Studies with FIM-A.1

2020 ◽  
Vol 21 (7) ◽  
pp. 2400 ◽  
Author(s):  
René Stürmer ◽  
Jana Reising ◽  
Werner Hoffmann
Keyword(s):  
Molecular Mass ◽  
Complex I ◽  
Size Exclusion ◽  
Trefoil Factor ◽  
Binding Studies ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Exclusion Chromatography ◽  
Trefoil Factor Family

The skin of the frog Xenopus laeevis is protected from microbial infections by a mucus barrier that contains frog integumentary mucins (FIM)-A.1, FIM-B.1, and FIM-C.1. These gel-forming mucins are synthesized in mucous glands consisting of ordinary mucous cells and one or more cone cells at the gland base. FIM-A.1 and FIM-C.1 are unique because their cysteine-rich domains belong to the trefoil factor family (TFF). Furthermore, FIM-A.1 is unusually short (about 400 amino acid residues). In contrast, FIM-B.1 contains cysteine-rich von Willebrand D (vWD) domains. Here, we separate skin extracts by the use of size exclusion chromatography and analyze the distribution of FIM-A.1 and FIM-C.1. Two mucin complexes were detected, i.e., a high-molecular-mass Complex I, which contains FIM-C.1 and little FIM-A.1, whereas Complex II is of lower molecular mass and contains the bulk of FIM-A.1. We purified FIM-A.1 by a combination of size-exclusion chromatography (SEC) and anion-exchange chromatography and performed first in vitro binding studies with radioactively labeled FIM-A.1. Binding of 125I-labeled FIM-A.1 to the high-molecular-mass Complex I was observed. We hypothesize that the presence of FIM-A.1 in Complex I is likely due to lectin interactions, e.g., with FIM-C.1, creating a complex mucus network.

scholarly journals Dihydrolipoamide dehydrogenase from Trypanosoma brucei. Characterization and cellular location

1987 ◽  
Vol 243 (3) ◽  
pp. 661-665 ◽  
Author(s):  
M J Danson ◽  
K Conroy ◽  
A McQuattie ◽  
K J Stevenson
Keyword(s):  
Plasma Membrane ◽  
Catalytic Activity ◽  
Chemical Modification ◽  
Trypanosoma Brucei ◽  
Disulphide Bond ◽  
Bloodstream Form ◽  
Membrane Preparation ◽  
Dihydrolipoamide Dehydrogenase ◽  
Cellular Location ◽  
Multienzyme Complexes

Dihydrolipoamide dehydrogenase has been discovered in the bloodstream form of the eukaryotic African parasite, Trypanosoma brucei. The enzyme catalysed the stoichiometric oxidation of dihydrolipoamide by NAD+ and exhibited a hyperbolic dependence of catalytic activity on the concentrations of both dihydrolipoamide and NAD+. Chemical modification with the tervalent arsenical reagent p-aminophenyldichloroarsine indicates the involvement in catalysis of a reversibly reducible disulphide bond. Plasma-membrane sheets were purified from T. brucei, and it was shown that virtually all the dihydrolipoamide dehydrogenase remained closely associated with this membrane preparation. T. brucei apparently lacks the 2-oxoacid dehydrogenase multienzyme complexes of which dihydrolipoamide dehydrogenase is usually an integral component. In the context of this absence, the possible function of trypanosomal dihydrolipoamide dehydrogenase is discussed, with particular reference to its cellular location in the plasma membrane.

scholarly journals Role of P-Selectin Cytoplasmic Domain in Granular Targeting In Vivo and in Early Inflammatory Responses

1998 ◽  
Vol 143 (4) ◽  
pp. 1129-1141 ◽  
Author(s):  
Daqing W. Hartwell ◽  
Tanya N. Mayadas ◽  
Gaëtan Berger ◽  
Paul S. Frenette ◽  
Helen Rayburn ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Inflammatory Responses ◽  
Cytoplasmic Domain ◽  
Leukocyte Rolling ◽  
Storage Pool ◽  
Storage Granules

P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (ΔCT mice). The deletion did not affect the sorting of P-selectin into α-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The ΔCT–P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the ΔCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

In-vitro effect of phalloidin on a plasma membrane preparation from rat liver

1972 ◽  
Vol 59 (11) ◽  
pp. 521-522 ◽  
Author(s):  
V. M. Govindan ◽  
H. Faulstich ◽  
Th. Wieland ◽  
B. Agostini ◽  
W. Hasselbach
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Membrane Preparation ◽  
Vitro Effect ◽  
Plasma Membrane Preparation

The Kidney and Fibrinolysis

1970 ◽  
Vol 24 (01/02) ◽  
pp. 026-032 ◽  
Author(s):  
N. A Marsh
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Enzyme System ◽  
Normal Animal ◽  
Fibrinolytic Enzyme ◽  
The Other ◽  
Exclusion Chromatography ◽  
Normal Urine ◽  
Renal Origin ◽  
Molecular Exclusion Chromatography

SummaryMolecular exclusion chromatography was performed on samples of urine from normal and aminonucleoside nephrotic rats. Normal urine contained 2 peaks of urokinase activity, one having a molecular weight of 22,000 and the other around 200,000. Nephrotic urine contained three peaks of activity with MW’s 126,000, 60,000 and 30,000. Plasma activator determined from euglobulin precipitate had a MW. in excess of 200,000. The results indicate that in the normal animal, plasma plasminogen activator does not escape into the urine in substantial quantities but under the conditions of extreme proteinuria there may be some loss through the kidney. The alteration in urokinase output in nephrotic animals indicates a greatly disordered renal fibrinolytic enzyme system.The findings of this study largely support the hypothesis that plasma plasminogen activator of renal origin and urinary plasminogen activator (urokinase) are different molecular species.

CHARACTERIZATION OF A NEW LCAT MUTATION CAUSING FAMILIAL LCAT DEFICIENCY (FLD) AND THE ROLE OF APOE AS A MODIFIER GENE OF THE FLD PHENOTYPE

2009 ◽  
Vol 32 (6S) ◽  
pp. 3
Author(s):  
A Baass ◽  
H Wassef ◽  
M Tremblay ◽  
L Bernier ◽  
R Dufour ◽  
...  
Keyword(s):  
Modifier Gene ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Lipoprotein Profile ◽  
Exclusion Chromatography ◽  
Low Hdl ◽  
Lcat Deficiency ◽  
Apoe Genotypes

Introduction: LCAT (lecithin:cholesterol acyltransferase ) is an enzyme which plays an essential role in cholesterol esterification and reverse cholesterol transport. Familial LCAT deficiency (FLD) is a disease characterized by a defect in LCAT resulting in extremely low HDL-C, premature corneal opacities, anemia as well as proteinuria and renal failure. Method: We have identified two brothers presenting characteristics of familial LCAT deficiency. We sequenced the LCAT gene, measured the lipid profile as well as the LCAT activity in 15 members of this kindred. We also characterized the plasma lipoproteins by agarose gel electrophoresis and size exclusion chromatography and sequenced several candidate genes related to dysbetalipoproteinemia in this family. Results: We have identified the first French Canadian kindred with familial LCAT deficiency. Two brothers affected by FLD, were homozygous for a novel LCAT mutation. This c.102delG mutation occurs at the codon for His35 causing a frameshift that stops transcription at codon 61 abolishing LCAT enzymatic activity both in vivo and in vitro. It has a dramatic effect on the lipoprotein profile, with an important reduction of HDL-C in both heterozygotes (22%) and homozygotes (88%) and a significant decrease in LDL-C in heterozygotes (35%) as well as homozygotes (58%). Furthermore, the lipoprotein profile differed markedly between the two affected brothers who had different APOE genotypes. We propose that APOE could be an important modifier gene explaining heterogeneity in lipoprotein profiles observed among FLD patients. Our results suggest that a LCAT-/- genotype associated with an APOE ?2 allele could be a novel mechanism leading to dysbetalipoproteinemia.

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