Enriched Plasma Membrane Research Articles

Preparation of glial plasma membrane from a cell fraction enriched in astrocytes

A technique for obtaining glial plasma membrane has been developed, starting with a bulk-prepared glial cell-enriched fraction from rabbit cerebral cortex. The astrocytic-enriched fraction was hand-homogenized in isotonic sucrose media, and the crude membrane fraction sedimented at 3,000g. The isolation of a membrane-enriched fraction was accomplished with sucrose density gradient centrifugation. The plasma membrane fraction was collected at the interphase between 31.5% and 25.5% sucrose. Enzymatic and electron-microscopical analyses indicated a 4-7-fold enrichment in plasma membrane, and a 15-20% contamination with microsomal and mitochondrial material. Some multilaminar membrane structures were also seen in the fraction.

Adipocyte beta-adrenergic receptors. Identification and subcellular localization by (-)-[3H]dihydroalprenolol binding.

(--)-[3H]Dihydroalprenolol, a potent beta-adrenergic antagonist, was used to identify binding sites which have the characteristics of beta-adrenergic receptors in membranes from rat adipocytes. The subcellular distribution of the (--)-[3H]Dihydroalprenolol binding sites was examined. The binding sites were predominantly in the plasma membrane fraction, consistent with the proposal that the physiologically significant beta-adrenergic receptors are localized in the adipocyte plasma membrane. Binding of (--)-[3H]dihydroalprenolol to unfractionated adipose membranes was saturable with 0.24 pmol bound/mg of protein at saturation. Half-maximal saturation occurred at 15 nM providing an estimate of the equilibrium dissociation constant, KD, for the interaction of (--)-[3H]dihydroalprenolol with its adipocyte receptor. Kinetic analysis of (--)-[3H]dihydroalprenolol binding provided a value of 2.4 X 10(7) M-1 min-1 for the forward bimolecular rate constant, k1. Dissociation of (--)-[3H]dihydroalprenolol was a first order reaction with a rate constant, k2, of 2.94 X 10(-1) min-1. The ratio k2/k1 = 12 nM provides an independent measurement of the KD for the interaction of (--)-[3H]dihydroalprenolol with its receptor which is in good agreement with the values obtained by steady state analysis (12 to 15 nM). Beta-Adrenergic agonists and antagonists competed for the binding sites in unfractionated adipocyte membranes with a typical beta1-adrenergic specificity. The order of potency of agonists was (--)-isoproterenol greater than (--)-norepinephrine congruent to (--)-epinephrine. The beta-adrenergic antagonist, (--)-propranolol, potently competed for the binding sites with a KD of 17 nM. Compounds such as dihydroxyphenylaline, dihydroxymandelic acid, normetanephrine, pyrocatechol, and phentolamine which are structurally related to beta-adrenergic agents, but are devoid of beta-adrenergic physiologicl effects in adipocytes, did not compete for the binding sites. Binding was highly stereospecific, the (+) isomers of adrenergic agonists and antagonists requiring 23- to 330-fold higher concentrations to half-maximally inhibit binding than the corresponding (--) stereoisomers. (--)-[3H]Dihydroalprenolol binding was examined highly enriched plasma membrane, mitochondrial, and microsomal (endoplasmic reticulum) fractions of adipocytes. In the presence of 12 nM (--)-[3H]dihydroalprenolol, the specific activity of binding in the plasma membrane fraction was 5-fold higher than that of the mitochondrial fraction and 8-fold higher than that of the microsomal (endoplasmic reticulum) fraction. The specificity and affinity characteristics of the plasma membrane binding sites were found to be virtually identical with those of the unfractionated adipocyte membranes. The observation that (--)-[3H]dihydroalprenolol binding sites are predominantly localized in the plasma membrane fraction suggests the potential usefulness of this ligand as a marker for adipocyte plasma membranes.

Chemical composition of a cellular fraction enriched in plasma membranes from sugarcane

The protein-lipid ratio in the enriched plasma membrane fraction of sugarcane leaves is almost 1:1. Virtually no phospholipid is present in the lipid fraction of the membranes. The major lipid is digalactosy diglyceride with smaller amounts of sterol glycosides, sterol esters, and acyl sterol glycosides. The potential importance of the glycolipids and two detected glycoproteins in the recognition phenomenon in plant diseases is discussed.

Plasma membrane associated enzymes of mammary tumours as the biochemical indicators of metastasizing capacity. Analyses of enriched plasma membrane preparations

Plasma membranes from 6 spontaneously metastasizing and 4 non-metastasizing rat mammary carcinomata were isolated by discontinuous sucrose density gradient centrifugation of microsomal pellets. The starting microsomal fraction contained 40-50% plasma membranes as determined by the levels of 5'-nucleotidase activity, with a negligible amount of nuclear (1%), mitochondrial (5%) and lysomal (7%) contamination. Five distinct fractions (F1-F5) were banded at densities 1 X 09, 1 X 13, 1 X 15, 1 X 17 and 1 X 21 at 25 degrees C, in addition to a pellet (F6) obtained by centrifuging at 76,000 g for 17 h. The fractions F1 through F5, all contained various concentrations of membranous structures, while the pellet (F6) contained only amorphous materials as evidenced by electron microscopy. The F3 fraction at the gradient 1 X 15 had the highest specific as well as total activity of the plasma membrane marker enzyme, with aggregates of the least contaminated plasma membranes in vesicular forms. This fraction also had the lowest specific activity for glucose-6-phosphatase (smooth ER marker) and for beta-D-glucuronidase (lysomal marker), and therefore was considered to be the "cleanest" plasma membrane fraction. When the activity of 4 additional plasma membrane marker enzymes, i.e., alkaline phosphatase, phosphodiesterase I, nucleotide pyrophosphatase and alkaline ribonuclease was determined in the same F3 fraction, their levels were significantly lower in every metastasizing tumour than in the non-metastasizing ones, with the enzyme activity decreasing in direct proportion to the metastasizing capacity. On the other hand, the marker enzymes were high in all non-metastasizing tumours, with the activity seemingly increasing with the immunogenicity of tumour cells. There was no significant difference between the 2 groups of mammary tumours in the levels of sialic acid, hexosamine, phospholipid or cholesterol in the plasma membranes. Thus, the level of plasma membrane marker enzymes is considered an accurate indicator for metastasizing capacity in the rat mammary tumour system.

Isolation of membranes from sugarcane cell suspensions: Evidence for a plasma membrane enriched fraction

Membranes in a post-mitochondrial supernanant from sugarcane cells grown in suspension culture were separated by sucrose density centrifugation and examined for presence of plasma membrane. Membrane vesicles from the fraction differed in size, binding properties toward naphthylphthalamic acid (NPA) or helminthosporoside and in some marler enzyme activities. Phosphotungstic acid (PTA) staining failed to distinguish between plasma membrane and other types of membranes in these preparations, but binding and enzymatic activities indicated enrichment of plasma membrane in the 38% sucrose fraction.

Transfer of toxin susceptibility to plant protoplasts via the Helminthosporoside binding protein of sugarcane

The eyespot disease of sugarcane is caused by Helminthosporium sacchari . Helminthosporoside, a host-specific toxin produced by H. sacchari , is essential for the pathogenicity of this fungus. The presence of the helminthosporoside-binding protein in sugarcane likewise appears to be essential for susceptibility to the toxin. The results of this report show that leaf cell protoplasts of tobacco and toxin resistant sugarcane effectively adsorbed the toxin-binding protein derived from membranes of susceptible sugarcane. These protoplasts then became susceptible to the helminthosporoside. They also functioned to takeup raffinose, a trisaccharide structurally related to the toxin. Tobacco protoplasts were treated with [ 14C] - binding protein, ruptured, and fractionated on a sucrose density gradient column. A peak of radioactivity was associated with the enriched plasma membrane fraction. The results support the hypothesis that the binding protein is the primary recognition site governing susceptibility of sugarcane to helminthosporoside.

The isolation of plasma membrane from frog cardiac muscle cells

A vesicular preparation consisting largely of the plasma membrane of frog cardiac cells was isolated and its enzymatic activities and lipid content were investigated. The enriched plasma membrane preparation was obtained by (1) mildly homogenizing washed ventricles, (2) separating away the cellular debris by low speed differential centrifugation, (3) separating the plasma membrane fraction from other membranous components by centrifugation to equilibrium in various sucrose gradients. The frog cardiac plasma membranes were found to be concentrated between specific gravities of 1.07 and 1.11. In the membrane fraction the specific activities of membrane marker enzymes: 5′-nucleotides (EC 3.1.3.5), alklaline phosphatase (EC 3.1.3.1) and Na +: K +)-activated ATPase were, respectively, 19, 15, and 14 times greater than in the homogenate. Activities of mitochondrial marker enzymes were either very low or absent. No unusual lipid types were found. Cardiolipin was less than 0.1% (by wt) in the membrane fraction. The molar ratio of phospholipid to cholesterol was approximatley 3 : 2.

Wax ester biosynthesis by isolated membrane fractions from calanoid copepods

1. 1. A plasma membrane rich fraction was isolated from two species of calanoid copepods by density gradient ultracentrifugation. 2. 2. Membrane fractions were identified by electron microscopy and the following marker enzyme analyses: NaK activated ATPase, 5′-nucleotidase, glucose-6-phosphatase and succinic dehydrogenase. 3. 3. Membrane fractions were incubated with 1- 14C palmitic acid and subsequently analyzed for synthesis of wax esters. Greater wax ester biosynthesis was observed to coincide with plasma membrane enrichment.

Localization of an Adrenocorticotropic Hormone Receptor on Bovine Adrenal Cortical Membranes

Abstract The preparation of a subcellular fraction from bovine adrenal cortex is described. Characterization by electron microscopy, electron microscopic cytochemistry, and enzymatic assay indicates that this fraction is enriched plasma membranes. The preparation binds the steroidogenically active [Gln5] [14C-Phe7] corticotropin1–20 amide. The specificity of this binding is demonstrated by competitive binding studies with pig corticotropin as well as adrenocorticotropic hormone (ACTH) fragments and analogs. The membrane preparation contains an adenylate cyclase sensitive to both sodium fluoride and corticotropin1–24. Corticotropin11–20 amide binds to the membranes but fails to activate adenylate cyclase. This membrane preparation provides a means to correlate peptide structure and in vivo corticotropic activity with selective binding and adenylate cyclase activation. The observation that corticotropin11–20 amide binds to the membranes but does not activate the adenylate cyclase supports the previous hypothesis that major binding sites of the ACTH molecule reside in this portion of the sequence. Two peptides exhibiting significantly higher in vivo adrenocorticotropic activity than ACTH from natural sources were only weakly active in stimulating the membrane adenylate cyclase. The experimental results strongly suggest that the membrane preparation contains an ACTH receptor.

The lipid content of musculature and of tissue preparations with enriched plasma membranes in Paramyotonia congenita

In einer Biopsieprobe menschlicher Muskulatur bei Paramyotonia congenita wurden Neutrallipide und Phosphatide im Gesamtmuskel und in Präparationen mit angereicherten Plasmamembranen bestimmt und mit den entsprechenden Werten in normaler Muskulatur verglichen. Dabei findet sich eine Erhöhung der freien Fettsäuren in den Plasmamembranen bei Paramyotonie.