Preparation Of Membrane Fractions Research Articles

Preparation of Membrane Fractions (Envelope, Thylakoids, Grana, and Stroma Lamellae) from Arabidopsis Chloroplasts for Quantitative Proteomic Investigations and Other Studies.

Chloroplasts are semiautonomous organelles found in plants and protists. They are surrounded by a double membrane system, or envelope. These envelope membranes contain machineries to import nuclear-encoded proteins, and transporters for ions or metabolites, but are also essential for a range of plastid-specific metabolisms. The inner membrane surrounds a stroma, which is the site of the carbon chemistry of photosynthesis. Chloroplasts also contain an internal membrane system, or thylakoids, where the light phase of photosynthesis occurs. The thylakoid membranes themselves have a bipartite structure, consisting of grana stacks interconnected by stroma lamellae. These thylakoid membranes however form a continuous network that encloses a single lumenal space. Chloroplast-encoded or targeted proteins are thus addressed to various sub-compartments that turn out to be flexible systems and whose main functions can be modulated by alterations in the relative levels of their components. This article describes procedures developed to recover highly purified chloroplast membrane fractions (i.e., envelope, crude thylakoid membranes, as well as the two main thylakoid subdomains, grana and stroma lamellae), starting from Percoll-purified Arabidopsis chloroplasts. Immunological markers are also listed that can be used to assess the purity of these fractions and reveal specific contaminations by other plastid membrane compartments. The methods described here are compatible with chloroplast proteome dynamic studies relying on targeted quantitative proteomic investigations.

Lost in centrifugation: accounting for transporter protein losses in quantitative targeted absolute proteomics.

In drug development, considerable efforts are made to extrapolate from in vitro and preclinical findings to predict human drug disposition by using in vitro-in vivo extrapolation (IVIVE) approaches. Use of IVIVE strategies linked with physiologically based pharmacokinetic (PBPK) modeling is widespread, and regulatory agencies are accepting and occasionally requesting model analysis to support licensing submissions. Recently, there has been a drive to improve PBPK models by characterizing the absolute abundance of enzymes, transporters, and receptors within mammalian tissues and in vitro experimental systems using quantitative targeted absolute proteomics (QTAP). The absolute abundance of proteins relevant to processes governing drug disposition provided by QTAP will enable IVIVE-PBPK to incorporate terms for the abundance of enzymes and transporters in target populations. However, most studies that report absolute abundances of enzymes and transporter proteins do so in enriched membrane fractions so as to increase the abundance per sample, and thus the assay's sensitivity, rather than measuring the expected lower abundance in the more biologically meaningful whole cells or tissues. This communication discusses the balance between protein enrichment and potential loss during the preparation of membrane fractions from whole cells or tissues. Accounting for losses with recovery factors throughout the fractionation procedure provides a means to correct for procedural losses, thereby enabling the scaling of protein abundance from subcellular fractions to whole-cell or organ abundances. PBPK models based on corrected abundances will more closely resemble biological systems and facilitate development of more meaningful IVIVE scaling factors, producing more accurate quantitative predictions of drug disposition.

Association of Paracellin-1 with ZO-1 Augments the Reabsorption of Divalent Cations in Renal Epithelial Cells

Paracellin-1 (PCLN-1) belongs to the claudin family of tight junction proteins and possibly plays a critical role in the reabsorption of magnesium and calcium. So far, the physiological properties of PCLN-1 have not been clarified. In the present study, we investigated whether PCLN-1 is associated with ZO-1. We also investigated whether (45)Ca(2+) transport across the paracellular barrier is affected by this association. In vitro binding analysis using glutathione S-transferase fusion protein showed that the C-terminal TRV sequence, especially Thr and Val residues, of PCLN-1 interacts with ZO-1. Next, PCLN-1 was stably expressed in Madin-Darby canine kidney cells using a FLAG tagging vector. ZO-1 was co-immunoprecipitated with the wild-type PCLN-1 and the alanine substitution (TAV) mutant. However, mutants of the deletion (Delta TRV) and the alanine substitution (ARV and TRA) inhibited the association of PCLN-1 with ZO-1. Confocal immunofluorescence demonstrated that the wild-type PCLN-1 and the TAV mutant localized in the tight junction along with ZO-1, but the Delta TRV, ARV, and TRA mutants were widely distributed in the lateral membrane including the tight junction area. Interestingly, monolayers of cells expressing the wild-type PCLN-1 and the TAV mutant showed higher activities of (45)Ca(2+) transport from apical to basal compartments, compared with those expressing the Delta TRV, ARV, and TRA mutants and the mock cells. (45)Ca(2+) transport was inhibited by increased magnesium concentration suggesting that magnesium and calcium were competitively transported by PCLN-1. It was noted that a positive electrical potential gradient enhanced (45)Ca(2+) transport from apical to basal compartments without affecting the opposite direction of transport. Thus, PCLN-1 localizes to the tight junction followed by association with ZO-1, and the PCLN-1.ZO-1 complex may play an essential role in the reabsorption of divalent cations in renal epithelial cells.

Preparation of membrane fractions from human neutrophil granules: A simple method

The surface composition of the azurophilic granule of human neutrophils is largely unknown. To understand the role played by the membrane components of these organelles a simple methodology for their disruption and the isolation of suitable amounts of granule membranes is required. The method of disruption of azurophilic granules available up to now consists of seven steps of freezing-thawing and sonication, a method time-consuming when large amount of granule needs to be processed. In this paper we describe a simple, one-step method based on the granule damaging capacity of Percoll which is suitable for the disruption of large amounts of azurophilic granules. The components of granule lysates obtained with this method were resolved on sucrose gradients. Two major bands were obtained, which are shown to be different from each other judging from their content of selective markers.

Exercise training prevents maturation-induced decrease in insulin sensitivity.

We examined the effects of exercise training initiated before maturation or after maturation on insulin sensitivity and glucose transporter GLUT-4 content in membrane fractions of skeletal muscle. Female Wistar rats (4 wk of age) were divided into sedentary and exercise-trained groups. At 12 wk of age, a subset of the trained animals (Tr) was killed along with a subset of sedentary controls (Sed). One-half of the remaining sedentary animals remained sedentary (Sed-Sed) while the other half began exercise training (Sed-Tr). The remaining rats in the original trained group continued to train (Tr-Tr). Euglycemic clamp (insulin infusion rate at 6 mU.kg body wt-1. min-1) was performed at 4, 12, and 27 wk. After euglycemic clamp in all animals except the 4-wk-old, hindlimb (gastrocnemius and part of quadriceps) muscles were removed for preparation of membrane fractions. In sedentary rats, glucose infusion rate (GIR) during euglycemic clamp was decreased from 15.9 mg.kg-1.min-1 at 4 wk of age to 9.8 mg.kg-1.min-1 at 12 wk of age and 9.1 mg.kg-1.min-1 at 27 wk of age. In exercise-trained rats, the GIR was not significantly decreased by maturation (at 12 wk) and further aging (at 27 wk). Initiation of exercise after maturation restored the GIR at 27 wk of age to the same levels as these for the corresponding exercise-trained rats. GLUT-4 content in plasma and intracellular membrane fractions of hindlimb muscle obtained just after euglycemic clamp showed the same trend as the results of GIR. These results suggest that exercise training prevented the maturation-induced decrease in insulin sensitivity. Improvement of insulin sensitivity caused by exercise training was attributed, at least in part, to the increase in insulin-sensitive GLUT-4 on the plasma membrane in skeletal muscle.

The lymphocyte-specific protein LSP1 is associated with the cytoskeleton and co-caps with membrane IgM.

LSP1 is a lymphocyte-specific intracellular Ca2(+)-binding protein. We found previously that a fraction of the total cellular pool of LSP1 protein accumulates at or near the cytoplasmic face of the plasma membrane. LSP1 protein was also shown to be present in the cytoplasm. Here we report that approximately 10% of the total intracellular LSP1 protein is associated with the Nonidet P-40 insoluble cytoskeleton of the mIgM+, mIgD+ B lymphoma cell line BAL17. Variation in conditions of extraction did not alter this value. To rule out the possibility that LSP1 associates with the nucleus that is also present in the detergent insoluble pellet, we prepared a separate nuclear fraction essentially free of cytoskeletal material and found only trace amounts of LSP1 protein. After accounting for yield losses during subcellular fractionation by measuring the recovery of 125I-labeled membrane IgM, or of the cytoplasmic marker enzyme lactate dehydrogenase activity, the LSP1 in membrane fractions was calculated to represent approximately 30% of the total cellular LSP1 and cytoplasmic LSP1 accounted for approximately 55% of the total. Approximately 75% of the plasma membrane LSP1 protein was soluble in 1% Nonidet P-40 containing buffer, indicating that the majority of the LSP1 in the plasma membrane fraction was distinct from the cytoskeletal LSP1 protein. The preparation of membrane fractions in the presence of 1 M NaCl, or washing of membranes in 3 M KCl did not diminish the levels of membrane LSP1. These results show the existence of three discrete intracellular LSP1 pools. Double label immunofluorescence studies showed that the peripheral ring-like distribution of LSP1 in BAL17 cells became a distinct cap upon cross-linking the mIgM. These intracellular LSP1 caps were always found to be located directly underneath the mIgM caps.

On in vitro and in vivo binding of tobacco mosaic virus to cytoplasmic membranes

Summary TMV contents were determined in 20 fractions separated in 10 to 50% saccharose gradients of resuspended 1,000 to 20,000 · g pellets from TMV-infected leaves of Nicotiana tabacum L. “Samsun” A maximum content was observed in the fraction containing 27 % saccharose. This fraction is characterized by a high proportion of smooth cytoplasmic (SC) membranes. These SC membranes proved are to be able in in vitro binding of considerable quantities of TMV. Bovine serum albumin (BSA) prevents the adsorption of subsequently added TMV to SC membranes but cannot detach previously bound TMV. When BSA was added before the preparation of membrane fractions, the SC membranes from TMV-infected plants remained contaminated with TMV. This results provides evidence for TMV binding to SC membranes already in vivo. Using 3 M KCl and subsequent dialysis or 0.1 % Triton X-100 suggested that ionic forces as well as hydrophobic interactions may be involved in the binding of TMV to SC membranes.

Preparation of membrane fraction from herpes simplex virus-infected cells which induce cytotoxic T lymphocytes.

The immunogenic capacity of herpes simplex virus (HSV)-infected cells and their subcellular membrane fractions was investigated by assessing the anti-HSV cytotoxic T lymphocyte (CTL) response in cultures of spleen lymphocytes from HSV-primed BALB/c mice. Methylchloranthrane-induced fibrosarcoma (Meth A) cells infected with HSV (HSV-Meth A) were fixed either with glutaraldehyde or by heating at 56 C to preserve their immunogenic competence and then used as a stimulator. Microsomes and plasma membranes were prepared from HSV-Meth A and their immunogenic activities were determined. Through the recovery of stimulatory activity in the plasma membrane fraction was half of that in the microsome fraction, the activity in the former was much more stable than in the latter and the plasma membrane fraction proved to be well qualified as an immunogen for anti-HSV CTL induction. Upon purification, the specific activity of the membrane fraction, on the basis of protein concentration, increased 43-fold.

31] d-Gluconate dehydrogenase from bacteria, 2-keto-d-gluconate-yielding, membrane-bound

This chapter describes an assay method for the isolation of D-gluconate dehydrogenase from bacteria. D-gluconate dehydrogenase occurs on the outer surface of cytoplasmic membrane of oxidative bacteria, such as Pseudomonas, Klebsiella, Serratia , and acetic acid bacteria. The enzyme activity is linked to the electron transport chain in the cytoplasmic membrane constituting a D-gluconate oxidase system. The assay is performed spectrophotometrically at 25°C by measuring the decrease of absorbance at 600 nm of 2,6-dichlorophenolindophenol (DCIP) mediated with phenazine methosulfate (PMS). The activity is also measured with PMS, DCIP, ferricyanide, or coenzyme Q (CoQ) as an electron acceptor. The purification procedures of the enzyme from P. fluorescens and K. pneumoniae are described in the chapter. Potassium phosphate buffer, pH 6.0, containing 5 m M MgCl 2 is used throughout the purification of P. fluorescens . The steps involved in the purification procedure are (1) the preparation of membrane fraction, (2) the solubilization of enzyme, (3) ammonium sulfate fractionation, and (4) hydroxyapatite column chromatography. Purified D-gluconate dehydrogenase shows a visible absorption spectrum of the cytochrome c type having an asymmetrical α-peak.

91] Formate dehydrogenase from Escherichia coli

This chapter describes the procedure used for the purification of formate dehydrogenase in a form suitable for use in the reconstruction of the formate–nitrate reductase activity. Formate dehydrogenase is measured by spectrophotometric assay of the reduction of dichlorophenolindophenol in the presence of phenazine methosulfate in an anaerobic cuvette. The bacteria for large-scale purification are grown in 45-liter batches in 12-gallon carboys, which are capped with rubber stoppers fitted with a sintered-glass sparger tube and a gas exit port. The steps involved in the purification of formate dehydrogenase are (1) the preparation of membrane fraction, (2) solubilization and salt fractionation, (3) agarose gel chromatography, and (4) anion-exchange chromatography. All steps are carried out at 0-5°C. After the disruption of the bacterial cells, these steps are carried out in closed vessels under an atmosphere of N 2 . Strict anaerobic conditions are not required; the major precautions include the use of deaerated solutions and providing an atmosphere of N 2 above the solutions at all times. The enzyme has the oxidized and reduced spectra of a b -type cytochrome. The cytochrome of formate dehydrogenase is completely reduced in the presence of formate.

24] d-Glucose dehydrogenase from Pseudomonas fluorescens, membrane-bound

Publisher Summary This chapter describes an assay method for the synthesis of D-glucose dehydrogenase from Pseudomonas fluorescens . D-Glucose dehydrogenase occurs on the outer surface of the cytoplasmic membrane of oxidative bacteria, such as Pseudomonas and Gluconobacter species, and initiates a direct oxidation of D-glucose through an electron transport chain. The enzyme is solubilized from the membrane and further purified to a homogeneous state. A spectrophotometric assay at 25° measures the decrease of absorbance at 600 nm of 2,6-dichlorophenolindophenol (DCIP) mediated with phenazine methosulfate (PMS). The activity can also be measured with PMS, DCIP, Wurster's blue (WB), ferricyanide, or coenzyme Q (CoQ) as an electron acceptor. Many strains of Pseudomonas aeruginosa and P. fluorescens are suitable sources of D-glucose dehydrogenase. The steps involved in the purification procedure are (1) the preparation of membrane fraction, (2) the solubilization of enzyme, (3) polyethylene glycol fractionation, (4) ethanol fractionation, (5) hydroxyapatite column chromatography, and (6) phenyl-sepharose column chromatography. All procedures are carried out at 0–5°C. D-Glucose dehydrogenase has a dual pH optimum depending on the electron acceptor used.

Preparation of Membrane Fractions With Enhanced Tumor-Transplantation-Antigen Activity From Tumor Cells Infected With Influenza Virus

Preparation of Membrane Fractions With Enhanced Tumor-Transplantation-Antigen Activity From Tumor Cells Infected With Influenza Virus

Preparation of Membrane Fraction and Its DNA Polymerase Activity in Vegitative Cells of Bacillus subtilis

Preparation of Membrane Fraction and Its DNA Polymerase Activity in Vegitative Cells of Bacillus subtilis

Caesium chloride in the preparation of membrane fractions from human cerebral tissue.

THE standard procedure for the isolation of myelin and other membrane fractions involves differential and density gradient centrifugation in sucrose solutions. Two bands of myelin, light and heavy1 or small and large2, and many bands of microsomes3 have been obtained by these techniques when applied to cerebral tissues, and a similar multiplicity of bands has been noted with the use of ‘Ficol’ gradients4. One explanation may be that the membranes exist as vesicles, which are impermeable to sucrose, and thus are separated by buoyant density rather than matrix density5. The two bands of myelin obtained show an almost identical lipid composition6.