Subcellular fractionation of tissue culture cells

Subcellular fractionation has two major steps, (1) the homogenization of the cells and (2) the subsequent separation of the organelles. The homogenization step is discussed with reference to the problems encountered using tissue culture cells. Promising techniques for the isolation of specific compartments are illustrated using the isolation of the endosomal compartment as the example.     

Nucleosome-associated protein kinases in murine erythroleukemia cells.

Chromatin subunits from murine erythroleukemia cells were prepared by a method which releases actively transcribing genes. Two casein kinase activities (CK₁ and CK₂) were isolated from these nucleosomes by gel nitration in 0.5 m NaCl. CK₁ (M_r ~ 200,000) and CK₂ (M_r ~ 35,000) were further purified by phosphocellulose chromatography and characterized with regard to several parameters which may regulate their activity in vivo. CK₁ has an NaCl optimum of 0.14 m, utilizes GTP as phosphate donor ~25% as efficiently as ATP, and phosphorylates a discrete group of high molecular weight nonhistone proteins in the unfractionated chromatin starting material. CK₂ has an NaCl optimum of 0.24 m, cannot utilize GTP, and modifies a different group of nonhistones. Both kinases are inhibited by concentrations of hemin (<50 μm) which efficiently induce globin gene expression in erythroleukemia cells. A histone kinase resolved during the gel filtration step is unaffected by hemin. An investigation of the mode of hemin inhibition reveals that CK₁ and CK₂ interact in different fashions with the inhibitor.     

Differential hemoglobin synthesis in murine erythroleukemic cells: hemin effects

The addition of butyric acid to murine erythroleukemic cells (clone T3Cl2) induced the cells to differentiate, producing adult hemoglobin (A, alpha 2,beta 2) and an embryonic hemoglobin (E2, alpha 2Y2). The subsequent addition of hemin to the differentiating cells increased the synthesis of adult hemoglobin four-fold and the synthesis of embryonic hemoglobin two-fold; the relative synthesis of the alpha and beta globins increased more than the y globin. The embryonic hemoglobin was expressed prior to the adult hemoglobin in differentiating cells.     

Differentiation of murine erythroleukemic cells during exposure to microwave radiation

Cultures of murine erythroleukemic cells undergoing erythroid differentiation in response to induction by hexamethylene bisacetamide (HMBA) were exposed to 1180-MHz microwave (MW) radiation for 48 h while maintained at 37.4 degrees C by variable-temperature air flow. Exposures at 1180 MHz were at 5.5, 11, and 22 mW/cm2 with a normalized specific absorption rate of 3.32 W/kg per mW/cm2. HMBA-induced control cells were incubated in a 37.4 degrees C water bath. Mean cell doubling time was 16.5 h in both the irradiated cultures and the control cultures. About 65% of the cells of irradiated cultures and control cultures were benzidine-positive differentiated cells. Both the irradiated cultures and the control cultures contained approximately 58 micrograms of hemoglobin/mg total cytoplasmic protein. The absence of any change in these parameters suggests that MW radiation at 1180 MHz and similar frequencies exerts no effect on proliferation and differentiation of mammalian cells in the absence of hyperthermia.     

Effects of murine tumor necrosis factor on Friend erythroleukemic cells

Tumor necrosis inducing factor (TNF), a 140,000 molecular weight glycoprotein present in the serum of Corynebacterium parvum endotoxin-treated mice, was cytotoxic toward Friend virus-transformed erythroleukemic cells (FELC). These cells grow in culture as undifferentiated pro-erythroblasts but can be induced to differentiate in a limited fashion along the erythroid pathway to orthochromatic normoblasts by various agents such as dimethylsulfoxide (DMSO). Partially and highly purified preparations of TNF were cytotoxic toward logarithmically growing FELC whereas a comparable serum protein fraction from C. parvum treated mice or endotoxin from E. coli had no effect upon FELC viability. DMSO-induced cells were more sensitive to the action of TNF requiring only about half the concentration needed to produce 50% kill in noninduced cells. Inhibition of hemoglobin formation was TNF dose-related and could be decreased by 94%. TNF was also cytotoxic toward DMSO-induced cells in stationary phase and mitomycin C treated noninduced FELC. Neuraminidase modification of the surface of FELC increased the cytotoxicity of TNF by 50%. These results demonstrate that TNF destroys FELC whether they are nondividing, dividing or partially differentiated and suggest that TNF may accomplish this by affecting cell metabolism after internalization.     

Subcellular fractionation enhances proteome coverage of pancreatic duct cells

ObjectivesSubcellular fractionation of whole cell lysates offers a means of simplifying protein mixtures, potentially permitting greater depth of proteomic analysis. Here we compare proteins identified from pancreatic duct cells (PaDC) following organelle enrichment to those identified from PaDC whole cell lysates to determine if the additional procedures of subcellular fractionation increase proteome coverage.MethodsWe used differential centrifugation to enrich for nuclear, mitochondrial, membrane, and cytosolic proteins. We then compared – via mass spectrometry-based analysis – the number of proteins identified from these four fractions with four biological replicates of PaDC whole cell lysates.ResultsWe identified similar numbers of proteins among all samples investigated. In total, 1658 non-redundant proteins were identified in the replicate samples, while 2196 were identified in the subcellular fractionation samples, corresponding to a 30% increase. Additionally, we noted that each organelle fraction was in fact enriched with proteins specific to the targeted organelle.ConclusionsSubcellular fractionation of PaDC resulted in greater proteome coverage compared to PaDC whole cell lysate analysis. Although more labor intensive and time consuming, subcellular fractionation provides greater proteome coverage, and enriches for compartmentalized sub-populations of proteins. Application of this subcellular fractionation strategy allows for a greater depth of proteomic analysis and thus a better understanding of the cellular mechanisms of pancreatic disease.     

Subcellular distribution of protein kinase C in rat adrenal glomerulosa cells

With the aid of a synthetic nonapeptide which is a selective substrate for protein kinase C the activity of this enzyme was determined in the crude cytosolic and particulate fractions of rat adrenal glomerulosa cells. When the cells were sonicated in the presence of Ca2+ chelators 65 per cent of their total protein kinase C activity was found in the cytosolic extract. The treatment of cells with angiotensin II under conditions where the maximal stimulation of inositol-lipid hydrolysis was observed did not cause a statistically significant change in the apparent subcellular distribution of protein kinase C. However, when the cytosolic extract was prepared in the presence of Ca2+ the protein kinase C activity was recovered nearly exclusively from the particulate fraction.     

Subcellular fractionation of pig platelets

Subcellular components were obtained from pig platelets, disrupted by means of a French press and separated into 4 primary fractions. The granule fraction (10 000 g) was subjected to a sucrose gradient fractionation. Primary fractions and the granule subfractions were studied electron microscopically and biochemically by following the distribution of markers of membranes, lysosomes of α-granules, mitochondria and dense granules. With this technique of platelet homogenization, 80% of the serotonin and 93% of the β-N-acetylglucosaminidase were found to be particulate. In the gradient, mitochondria were sharply banded in a fraction (density 1.16–1.17) having a specific activity 10–100 times higher than the other fractions of the gradient. Serotonin-containing granules were found in a pellet of density greater than 1.27 and contained 60% of the serotonin and adenine nucleotides of the granule fraction. The lysosome markers that were monitored, acid phosphatase and β-N-acetylglucosaminidase, exhibited different distribution patterns. Acid phosphatase showed the highest specific activity in the microsomal fraction with only 2.8% in the granule fraction, and this latter amount also appeared to be associated with membranes upon further fractionation. β-N-Acetylglucosaminidase was present in both the granule fraction and in the microsomal fraction with nearly the same specific activity. However, that present in the granule fraction was clearly associated with granules that distributed over a wide range of densities on a sucrose gradient. The calcium distribution was followed to attempt to determine its subcellular location; 19% was found in the same subfraction as the serotonin-containing granules, but at least 50% of the particulate calcium was associated with granules distinctly separate from the storage granules.     

Subcellular fractionation of postmortem brain

Abstract— Procedures used to separate subcellular organelles from fresh brain were applied to brains which had been removed from guinea pigs (1) immediately after death; (2) after the dead animal was maintained at room temperature for 3 h, followed by 16–17 h at 4°C; or (3) after the dead animal was maintained for 19–20 h at room temperature. Subcellular fractionation of the brains in 0.32 M sucrose was followed by discontinuous density gradient centrifugation of the crude mitochondrial fraction. After overnight storage of brains at room temperature, there was a moderate shift in succinate dehydrogenase activity from sub-fraction C (mitochondria) to subfraction B (synaptosomes). There was little change in the distribution of galactolipid among particulate subfractions. There was little change in distributions of monoamine oxidase or acetylcholinesterase activities. Under the less extreme postmortem conditions, there were no shifts in the subcellular distributions of brain enzymes. Ultrastructural changes were much more profound and consisted of losses of identifiable mitochondria and synaptosomes and a progressive increase in very dense bodies. Our results suggest that in spite of the marked morphological changes, meaningful separation of subcellular organelles can be achieved with postmortem tissue.     

Subcellular fractionation and distribution of cholinergic binding sites in fetal human brain

Conventional subcellular fractionation techniques have been applied to human fetal brain (13–15 weeks gestation) and the fractions have been characterized by assaying for marker enzymes, cholinergic binding sites and electron microscopy. Fractionation of the homogenate resulted in a nuclear pellet (P₁), a crude mitochrondrial pellet (P₂) and a supernatant (S₂). Further resolution of the P₂ fraction by density gradient centrifugation resulted in two bands at the gradient interfaces and a pellet. The P₂ and subsequently the P₂B fraction contained intact plasma membrane profiles as judged by the predominance of adenylate cyclase activity and the presence of occluded lactate dehydrogenase which constituted over 70% of the total activity in these fractions. Morphological examination of the gradient fractions revealed that the P₂B fraction contains membrane bound structures which resembie synaptosomes prepared from neonatal rat brain. These structures have a granular matrix in which mitochondria and frequently, neurofilaments were observed. Very few synaptic vesicles were present and there was no evidence for post synaptic attachments. The cholinergic markers choline acetyltransferase, acetylcholinesterase and receptor sites defined by quinuclidinyl benzilate and -bungarotoxin binding were enriched in fractions P₂ and P₂B which contained the bulk of nerve ending particles. This enriched preparation of fetal synaptosomes may be valuable for functional studies on pre-synaptic terminals in developing brain.Special Issue dedicated to Prof. Eduardo De Robertis.     

Subcellular fractionation of WI-38 fibroblasts: Compatison between young and old cells

WI-38 fibroblasts cultivated in vitro were homogenized and their subcellular organelles analysed by the techniques of differential centrifugation and isopycnic equilibration in density gradient. In these experiments, the assayed enzymes were known to be specifically associated with subcellular components in other cells types. In most cases, their behaviour and properties corresponded with observations made in earlier studies and we could consider them as being representative of the specific subcellular organelles.Some significant differences were observed between young and old fibroblasts. The specific activity of alkaline phosphodiesterase was lower in the old cells whereas for the other enzymes it was identical or higher, especially for the 5′-nucleotidase; also the particulate fractions obtained by differential centrifugation contained more material. After equilibration in density gradient, the average density of the 5′-nucleotidase, alkaline phosphodiesterase and $\text{N-}\text{acetyl}\text{-β-}\text{D}\text{-glucosaminidase}$ was less in the old than in the young cells, whereas that of the galactosyltransferase of Golgi apparatus was greater. For mitochondria, endolasmic reticulum and peroxisomes, the differences observed were small.     

Subcellular distribution of newly synthesized virus-specific polypeptides in Moloney murine leukemia virus infected cells.

Immune precipitation analysis of pulse-labeled proteins present in subcellular fractions of mouse embryo cells infected with Moloney murine leukemia virus showed the presence of anti-gp70 serum-precipitable viral envelope gene products mainly in the microsomal fractions of these cells. In contrast, anti-p30 serum-specific gag (group specific antigen) gene products were found to be distributed in similar amounts in both the microsomal and postmicrosomal supernatant fractions of pulse-labeled cells.     

Antitumor activity of normal human globulin: effects on murine and human erythroleukemic cells

The effect of the antitumor fraction isolated from the alpha 2-globulin region of normal human serum (NHG-I) upon murine (FELC) and human (K562) erythroleukemic cells in vitro was determined. NHG-I inhibited the growth of both actively growing FELC and K562 cells in a dose-dependent manner. However, it has no effect upon mitomycin C treated FELC which were unable to divide nor upon dimethylsulfoxide-induced FELC in stationary phase. These results indicate that NHG-I has a cytostatic effect upon cell growth and suggests that its action may be dependent upon DNA synthesis. This is in marked contrast to TNF, the alpha 2-globulin factor obtained from murine serum which is also not species-specific but whose action upon these cell lines is cytotoxic.     

IgE-binding protein. Subcellular location and gene expression in many murine tissues and cells

We show that IgE-binding protein (epsilon BP) is found primarily in the cytoplasm of rat basophilic leukemia (RBL) cells and COS-1 cells transfected with epsilon BP cDNA. Antibodies to a synthetic peptide internal to epsilon BP were generated that specifically recognized epsilon BP by protein immunoblotting. These antibodies also bind the surface of RBL cells. Surprisingly, blot hybridization analysis of RNA from nine various normal rat tissues showed that the epsilon BP gene is transcribed in all the tissues tested as well as in a mouse macrophage-like cell line.