The proteins associated with the soluble form of p36, the main target of the src oncogene product in chicken fibroblasts, are glycolytic enzymes

One of the main targets of pp60v-src tyrosine kinase is a 34 to 39-kilodalton protein of chicken embryo fibroblasts called p36 or calpactin I. We have previously reported an association of the cytoplasmic fraction of p36 (10-20% of the total cellular p36) with three chicken polypeptides named p32, p48, and p54. We have now raised and affinity-purified antibodies against each of these proteins. This has allowed their identification: p32 is lactate dehydrogenase, p48 is enolase, and p54 is phosphoglucose isomerase. An association between p36 and two other known substrates of pp60v-src, the glycolytic enzymes enolase and lactate dehydrogenase, suggests a cellular organization of the various targets of the oncogene tyrosine kinases. Furthermore, a possible relationship between p36 and glycolysis is questioned.     

Association of pp60src and src protein kinase activity with the plasma membrane of nonpermissive and permissive avian sarcoma virus-infected cells.

The intracellular localization of pp60src and src protein kinase activity in avian sarcoma virus (ASV)-infected chicken embryo fibroblasts and transformed and morphologically reverted field vole cells was examined by subcellular fractionation procedures. Fractionation by differential centrifugation of Dounce-homogenized cellular extracts prepared from vole cells showed that 83 to 91% of pp60src sedimented with particulate subcellular components from both transformed and revertant vole cells. A slightly lesser amount (60 to 70%) of pp60src was found associated with the particulate fraction from ASV-infected chicken embryo fibroblasts. The distribution of src protein kinase activity in the cytosol and particulate cell fractions was identical to that of pp60src, indicating no detectable differences in the activity of cytosol- and particulate-associated pp60src. When subcellular components of the cell were fractionated by discontinuous sucrose gradient centrifugation, similar amounts of both pp60src and src protein kinase activity cosedimented with the plasma membrane fractions from both transformed and revertant vole cells, as well as from ASV-infected chicken embryo fibroblasts. src protein kinase activity associated with plasma membrane fractions prepared from vole cells and ASV-infected chicken embryo fibroblasts was resistant to extraction with high salt concentrations, but partial elution was achieved with nonionic detergent. Thus, in both transformed and morphologically reverted vole cells, pp60src is intimately associated with the plasma membrane. Since transforming virus can be rescued from revertant vole cells by fusion to chicken embryo fibroblasts, revertant vole cell pp60src is capable of inducing morphological transformation. Thus, although the data presented herein suggest that transformation requires the association of pp60src with the plasma membrane, the binding of pp60src to the plasma membrane per se is insufficient to induce morphological transformation and requires the additional interaction with a specific target membrane protein which appears to be defective in revertant vole cells.     

Identification of RNAase-sensitive 20S RNA in a cell culture infected with Sindbis virus]

RNAse-sensitive 20S RNA component with molecular weight of 0.7-10(6) is found when analysing virus-specific RNAs isolated from cultured chicken embryo fibroblasts infected with Sindbis virus by means of gradient centrifugation and polyacrylamide gel electrophoresis.     

Transformation by avian sarcoma viruses leads to phosphorylation of multiple cellular proteins on tyrosine residues

Phosphoamino acid compositions were determined for 10 size classes of cellular proteins, separated by electrophoresis through one-dimensional sodium dodecyl sulfate-polyacrylamide gels. Phosphotyrosine-containing proteins were observed in uninfected chicken embryo fibroblasts in every size class analyzed, ranging from approximately 20,000 to greater than 200,000 daltons. Transformation of chicken embryo fibroblasts by Rous sarcoma virus or PRC II avian sarcoma virus led to increases in phosphorylation of proteins at tyrosine residues in all of these size classes. A large fraction of the phosphotyrosine-containing protein molecules observed in Rous sarcoma virus-transformed cells was larger than 100,000 daltons with a second broad peak in the 35,000- to 60,000-dalton range. This study suggests that there are a number of substrates of viral or cellular tyrosine-specific protein kinases, which have not yet been identified by other methods.     

Adrenal Chromaffin Cell Calmodulin: Its Subcellular Distribution and Binding to Chromaffin Granule Membrane Proteins

Bovine adrenal medullae were homogenized in the presence or in the absence of EGTA and different subcellular fractions were prepared by differential and density gradient centrifugations. In the presence of the chelating agent, 69% of the total calmodulin, measured by radioimmunoassay, was present in the cytosol; the rest was bound to different membrane-containing fractions (nuclei, microsomal, and crude granule fraction). When the chelating agent was omitted, 43% of the calmodulin was present in the cytosol, the remaining calmodulin being membrane-bound. Further resolution of the crude granule fraction by sucrose density centrifugation demonstrated that the distribution of calmodulin in the density gradient was similar to the distribution of chromaffin granules rather than to that of mitochondria, Golgi elements, and lysosomes. In this case, there was also more calmodulin bound to chromaffin granules when EGTA was omitted from the density gradient. Experiments with 125I-calmodulin indicated the presence of high-affinity binding sites (KD = 1.3 X 10(-8) M; Bmax = 30 pmol/mg protein) for calmodulin in chromaffin granule membranes. Further, photoaffinity crosslinking experiments with 125I-calmodulin followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography indicated the presence of three calmodulin-binding polypeptide complexes (84,000; 41,000; and 38,000 daltons) in chromaffin granule membranes. These polypeptides were not labelled when either Ca2+ was omitted or an excess of nonradioactive calmodulin was present in the photolysis buffer, indicating the Ca2+ dependency and the specificity of the interaction. On the basis of the results described, it is suggested that the cellular levels of Ca2+ control the cellular distribution of calmodulin and its binding to specific chromaffin granule membrane proteins. Further, it is also suggested that the interactions between calmodulin and granule proteins might play a role in stimulus-secretion coupling.     

Identification of a membrane protein responsible for ribosome binding in rough microsomal membranes

A membrane protein fraction was obtained from rat liver rough microsomes by affinity chromatography on a concanavalin A-Sepharose column and then a chelating-Sepharose column. This protein fraction comprised about 2% of the total membrane proteins of rough microsomes and the ribosome-binding activity of ribosome-stripped rough microsomes was predominantly found in this protein fraction, as determined with a liposome assay system. To identify the essential components responsible for the ribosome binding, two approaches were employed. Trypsin treatment of liposomes reconstituted with this protein fraction resulted in the loss of the ribosome-binding activity in parallel with the loss of a dominant band, estimated Mr 34,000, in SDS-polyacrylamide gels. Next, the direct interaction between the binding sites on the membrane of reconstituted liposomes and 60S ribosomal subunits was investigated by photocrosslinking using sulfosuccinimidyl 2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (SAND). The photocrosslinked complex was formed between 60S ribosomal subunits pretreated with SAND and binding-site proteins on the membrane of the liposomes. Then, after the liposomes were solubilized, the complex was isolated by sucrose gradient centrifugation of the binding mixture. The crosslinked proteins were released from 60S ribosomal subunits by cleavage of of crosslinks with beta-ME and analyzed by SDS-polyacrylamide gel electrophoresis and 125I-autoradiography. The 34-kDa protein (p34) was the predominant component that crosslinked to the 60S ribosomal subunits and was found in proportion to the amount of 60S ribosomal subunits added to the system. The p34 was distinguishable by immunoblot analysis from urate oxidase, which is the 34-kDa protein of peroxisomal cores contaminating rough microsomes. These results suggest that the present p34 is a likely candidate molecule for the ribosome-binding activity of rough microsomes.     

ATP-dependent calcium uptake activity associated with a disk membrane fraction isolated from bovine retinal rod outer segments

Ca2+ sequestration and release from disks of rod outer segments may play a critical role in visual transduction. An ATP-dependent Ca2+ uptake activity has been identified in association with purified disks of bovine rod outer segments. A crude preparation of osmotically active disks was obtained from rod outer segments by hypoosmotic shock and subsequent flotation on a 5% Ficoll 400 solution. These "crude" disks were further purified by separation into two distinct components by centrifugation in a linear Ficoll gradient. Disks comprised the major component; at least 60% of the protein was rhodopsin. This fraction also contained a Ca2+ uptake activity stimulated approximately 4-fold by ATP. The initial rate was approximately 0.21 nmol of Ca2+ (mg of protein)-1 min-1. Most of the ATP-dependent accumulation of 45Ca2+ was released by the calcium ionophore A23187. The uptake activity was sensitive to vanadate (Ki approximately 20 microM) and insensitive to the mitochondrial Ca2+ uptake inhibitor ruthenium red (10 microM). The ATP-dependent Ca2+ uptake exhibited Michaelis-Menten activation kinetics with respect to [Ca2+] (Km approximately 6 microM). The osmotic properties of the sealed disk membranes were exploited to determine whether the association of Ca2+ transport activity with the disks was merely coincidental. The sedimentation properties of these disks, upon centrifugation on a second Ficoll linear density gradient, varied with the osmolarity of the gradient solution. In several separate gradient solutions of differing osmotic and ionic strengths, the Ca2+ uptake activity always comigrated with the disks. These results indicate that the ATP-dependent Ca2+ uptake activity was physically associated with sealed native disk membranes. The characteristics of the Ca2+ uptake activity suggest that it may play a major role in the regulation of cytosolic Ca2+ levels in rod cells in vivo.     

Calmodulin and Ca2+- and Calmodulin-Dependent Protein Kinase in Rat Anterior Pituitary Gland

Calmodulin and Ca2+- and calmodulin-dependent protein kinase were identified in the rat anterior pituitary gland. The concentration of calmodulin was 1.18 +/- 0.11 microgram/mg protein (n = 7) in the cytosol fraction. The calmodulin of the anterior pituitary gland co-migrated with brain calmodulin on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The Ka value of the partially purified enzyme for Ca2+ was 3.3 microM in the presence of 0.30 microM calmodulin. Trifluoperazine and chlorpromazine, calmodulin-interacting agents, inhibited enzyme activity, with Ki values of 1.3 and 2.6 X 10(-5) M, respectively. The enzyme was resolved into two peaks of activity, with sedimentation coefficients of 5.5 S and 16.5 S, by sucrose density gradient centrifugation. At least nine proteins were phosphorylated by the enzyme in a Ca2+- and calmodulin-dependent manner. In light of these results, the possibility that calmodulin and the calmodulin-activatable protein kinase system are involved in the mediation of the Ca2+ effect on hormone release from the anterior pituitary gland must be given consideration.     

Analysis of structural proteins of purified murine cytomegalovirus.

Murine cytomegalovirus propagated in mouse embryo fibroblasts was purified by the following procedures. (i) Extracellular virus was concentrated by centrifugation at 100,000 x g for 90 min. (ii) The concentrated virus was passed through a Bio-Rad Bio-Gel A-15m column to eliminate contaminating materials smaller than 15 x 10(6) daltons. Most of the virus was recovered in the void volume of the column. (iii) Two consecutive centrifugations through 20 to 50% potassium tartrate gradients were performed. After the second tartrate gradient centrifugation, symmetrical, coinciding peaks of plaque titer, protein, and radioactivity were found at a density between 1.20 g/cm3 and 1.21 g/cm3. To establish purification criteria, virus was purified from two different mixtures: [35S]methionine-labeled extracellular virus, mixed with an equal volume of unlabeled normal culture fluid, and unlabeled extracellular virus mixed with an equal volume of [35S]methionine-labeled normal culture fluid. At the end of the procedure, the extent of purification, as judged by the ratio of cellular to viral radioactivity was at least 70-fold. Virus proteins were analyzed by electrophoresis on a 5 to 20% gradient polyacrylamide gel slab. After gel electrophoresis,, Coomassie brilliant blue staining profiles and autoradiograms of the purified virus preparations were compared. At least 33 virus structural protein bands were present. The molecular weights of these proteins ranged from 11,500 to 255,000. The sum of the molecular weights of the virus structural proteins was 2,462,000. Autoradiograms obtained from electrophoresis of purified [14C]glucosamine-labeled virus showed that at lease 6 of the 33 viral structural proteins were glycoproteins.     

Association of a protease (plasminogen activator) with a specific membrane fraction isolated from transformed cells

The intracellular distribution of specific protease, plasminogen activator (PA), has been examined in Rous sarcoma virus-transformed chick embryo fibroblasts (RSV-CEF). Cellular homogenates were fractionated by differential centrifugation followed by sucrose gradient centrifugation. The activities and the percent distribution of a series of marker enzymes, specific for different subcellular organelles, were compared to those of PA. Normal CEF have been similarly fractionated and the relatively low amount of PA activity present in these cells has been analyzed in terms of its subcellular distribution. A membrane fraction was isolated from the RSV-CEF that contained the bulk of the PA activity and less than 8% of the total cellular protein. The specific activity of the PA in this fraction is 40-fold higher than that of a comparable fraction isolated from companion cultures of normal cells. This fraction contains little or no nuclear and cytoplasmic material and is contaminated only to a relatively small degree with mitochondria, lysosomes, endoplasmic reticulum. Significant amounts of a putative Golgi membrane marker are present in this fraction. The relatively high specific activities of Na+,K+-ATPase, 5'-nucleotidase, and [3H]fucose indicate that the fraction is enriched in surface membrane. Further purification of the fraction by equilibrium centrifugation on shallow sucrose gradients reduces further the contaminating activities and results in a PA distribution that closely parallels the distribution of the membrane enzyme, 5'-nucleotidase. PA was not released from its membrane association by hypotonic and hypertonic extraction and ultrasonication, while granule-bound enzymes were released by these treatments. The PA activity from hamster SV40 cells fractionated the same way as that of RSV-CEF. These results suggest that a protease that is dramatically enhanced upon malignant transformation is associated with "plasma membrane-like" elements of the cell and may serve as an intrinsic modifier of cell surface proteins after malignant transformation.     

Twenty-eight-kilodalton phosphorylatable calcium- and lipid-binding proteins purified from Physarum plasmodium.

Two 28-kDa calcium- and lipid-binding proteins were isolated from a detergent-insoluble fraction of the Physarum plasmodium. Both proteins have molecular masses of approximately 28 kDa by SDS-PAGE. The protein designated 28K-I has a slightly lower mobility than that designated 28K-II. The purified 28K-I has a dissociation constant of 1.0 microM for Ca2+ ions, while the 28K-II has two different dissociation constants: one of 0.32 microM and the other of 3.2 mM. The 28K-I binds to liposomes at Ca2+ concentrations higher than 1.0 microM and has a dissociation constant for lipid of 34 micrograms/ml at 10 microM Ca2+. The 28K-II binds to liposomes at concentrations of Ca2+ above the mM range and has a dissociation constant of 36 micrograms/ml for lipid at 2 mM Ca2+. There is no evidence of actin-binding activity by either of the 28-kDa (28K) proteins. The 28K proteins crossreacted with an antiserum against chicken brush border calpactin I. The two proteins have quite different phosphorylation levels between a fraction prepared from the cytosolic endoplasm and a fraction prepared from the whole cell. The 28K proteins may play some role in the membrane structure dynamics of the cortical gel layer.     

Calcium-dependent isolation of the 36-kilodalton substrate of pp60src-kinase. Fractionation of the phosphorylated and unphosphorylated species

In this paper, we present a new and simple purification of the 36-kDa protein, a major substrate of both viral and growth factor-receptor associated tyrosine protein kinases, and its complex from normal and Schmidt-Ruppin strain Rous sarcoma virus-transformed chicken embryo fibroblasts that employs a DEAE-Sephacel column and introduces the calcium-dependent adsorption of 36-kDa protein. The use of EGTA step gradients differentially elutes the 36-kDa molecules from the DEAE-Sephacel column. An average total yield of the 36-kDa protein in all fractions approached 80% and represented 0.78% of the [35S] methionine-labeled cellular protein. A purity of 95-99% was obtained with a yield of 60% in the central elution fractions from normal or Rous sarcoma virus-transformed chicken embryo fibroblasts. Furthermore, 2 mM EGTA elutes poorly phosphorylated molecules while heavily phosphorylated 36-kDa protein requires 4 or 6 M EGTA; a small residual fraction is released at 8-10 mM EGTA. If the EGTA step gradients were neutralized with Ca2+ ion, elution of the 36-kDa protein is inhibited. The complex of the 36-kDa protein and the 6-10-kDa protein may not be dependent on the phosphorylation as the associated 6-10-kDa peptide is seen in all fractions containing the 36-kDa protein. Tyrosine phosphorylation of the 36-kDa protein is increased 2-3-fold following a short term incubation of whole cells with micromolar vanadate. The elution pattern (but not intensity) of the 36-kDa protein obtained from lysates of vanadate-treated cells was identical to untreated cell lysates. The additional phosphorylation appears to result from a recruitment of unphosphorylated 36-kDa protein as the position (but not intensity) of the phosphorylated tryptic peptides is unchanged. We conclude that the function of the 36-kDa protein may be calcium ion-dependent and may be influenced by the phosphorylation state of the protein.     

Isolation and characterization of subcellular membranes from canine stomach smooth muscle

Subcellular membrane fractions were isolated from the circular muscle of the corpus of canine stomach by differential and isopycnic sucrose density gradient centrifugation. Differential centrifugation gave a mitochondrial fraction enriched (fourfold) in cytochrome c oxidase and a microsomal fraction enriched (fourfold) in 5'-nucleotidase and NADPH-cytochrome c reductase over postnuclear supernatant. On the basis of a study using continuous gradient, a discontinuous sucrose density gradient was prepared to yield F1 to F5 fractions. The F3 fraction at the interface of 18-32% (w/w) sucrose was maximally enriched (13-fold) in 5'-nucleotidase. The fraction contained very low levels of cytochrome c oxidase but did contain NADPH-cytochrome c reductase (eightfold enrichment). The F4 fraction, at the interface of 32-40% (w/w) sucrose, was maximally enriched in NADPH-cytochrome c reductase (12-fold) and cytochrome c oxidase (6-fold). The distribution of the azide-insensitive. ATP-dependent Ca2+ uptake correlated very well with that of 5'-nucleotidase but less well with NADPH-cytochrome c reductase and not at all with cytochrome c oxidase. Sodium azide and ruthenium red inhibited the ATP-dependent Ca2+ uptake by the mitochondrial fraction and postnuclear supernatant, but not by the F3 fraction. ATP-dependent Ca2+ uptake by the F3 fraction was inhibited by calcium ionophores A23187 and ionomycin, but not by the sodium ionophore, monensin. These results are consistent with the hypothesis that the plasma membrane plays a major role ih regulating intracellular Ca2+ concentration in canine corpus circular muscle.     

The purified Ca2+ antagonist receptor from skeletal muscle: subunit structure, photoaffinity labeling and endogenous protein kinase activity

Tritiated analogues of the Ca2+ channel blockers such as [3H] PN200-110, [3H] verapamil and [3H] diltiazem have been used to identify and isolate Ca2+ antagonist receptors. The Ca2+ antagonist binding sites were solubilized from skeletal muscle transverse tubules with the detergent CHAPS and purified by wheat germ lectin column chromatography and sucrose density gradient centrifugation. The isolated proteins retained their ability to bind the various classes of Ca2+ channel blockers. Polypeptides of 170, 150, 108, 56, and 32 kDa were found to be present in the purified receptor fraction when analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis under non-reducing conditions. The apparent molecular weight of the 170 kDa polypeptide changed to 145 kDa in the presence of reducing agents, as where the apparent molecular weight of the 150, 108, 56 and 32 kDa peptides remained unchanged. An endogenous protein-kinase present in the original membranes, co-purified with the receptor and stimulated the phosphorylation of the 150 and 56 kDa polypeptides in the isolated fraction.     

Cellular localization of c-fps gene product NCP98.

We compared the intracellular location of the product of the c-fps proto-oncogene, NCP98, with that of its viral homolog P140, the transforming protein of Fujinami sarcoma virus. Using the technique of biochemical subcellular fractionation, we determined that 60 to 90% of NCP98 and its associated kinase activity are in the soluble fraction of a chicken myeloblast cell line. This fractionation behavior differs from that of P140, which is found predominantly in the particulate fraction, both in Fujinami sarcoma virus-infected chicken embryo fibroblasts and in Fujinami sarcoma virus-infected myeloblasts. The fractionation behavior of NCP98 is, however, similar to that of the P140 encoded by a temperature-sensitive strain of Fujinami sarcoma virus in infected cells grown at the nonpermissive temperature. The absence of gag sequences from NCP98 is not responsible for the difference in fractionation behavior: the v-fps transforming protein of strain F36, P91, which lacks gag sequences, is also predominantly particulate. These results indicate that association with cellular structural components correlates with the transforming activity of proteins containing fps sequences.     

Preparation of a highly concentrated, completely monomeric, active sarcoplasmic reticulum Ca2+-ATPase

Sarcoplasmic reticulum vesicles from fast skeletal muscle were partially delipidated with sodium cholate at high ionic strength and sedimented in a discontinuous sucrose gradient. Phospholipid content was reduced from 0.777 mumol/mg protein to 0.242 mumol/mg protein. As judged from gel electrophoresis and high pressure liquid gel chromatography, accessory proteins were removed during centrifugation and the Ca2+-ATPase was obtained in an almost pure form. Addition of myristoylglycerophosphocholine (1 mg/mg protein) reactivates ATPase and dinitrophenylphosphatase activity to the same degree obtained with native vesicles. Using the analytical ultracentrifuge it could be demonstrated that the reactivated Ca2+-ATPase was present exclusively in a monomeric state. These results were obtained at high and low ionic strength and up to a protein concentration of 10 mg/ml. Therefore this preparation should be very useful to investigate differences between oligomeric and monomeric Ca2+-ATPase.     

Identification of phosphotyrosine-containing proteins in untransformed and Rous sarcoma virus-transformed chicken embryo fibroblasts

Phosphorylation on tyrosine residues mediated by pp60src appears to be a primary biochemical event leading to the establishment of the transformed phenotype in Rous sarcoma virus (RSV)-infected cells. To identify the cellular proteins that undergo tyrosine phosphorylation during transformation, a 32P-labeled RSV-transformed chicken embryo cell extract was analyzed by electrophoresis on a polyacrylamide gel. After slicing the gel into approximately 60 slices, phosphoamino acid analyses were carried out on the protein recovered from each gel slice. Phosphotyrosine was found in every gel slice, with two major peaks of this phosphoamino acid around M(r)'s of 59 and 36 kilodaltons. When the same analysis was performed with cells infected with a transformation-defective src deletion mutant of RSV (tdNY101), significant and reproducible peaks of phosphotyrosine were found in only 2 of 60 gel slices. These gel slices corresponded to M(r)'s of 42 and 40 kilodaltons. Identical results were obtained with normal uninfected chicken embryo fibroblasts. We conclude from these observations that pp60src or the combined action of pp60src and pp60src-activated cellular protein kinases cause the tyrosine-specific phosphorylation of a very large number of cellular polypeptides in RSV-transformed cells. In addition, untransformed cells appear to possess one or more active tyrosine-specific protein kinases which are responsible for the phosphorylation of a limited number of proteins. These proteins are different from the major phosphotyrosine-containing proteins of the transformed cells.     

An analytical approach to the preparation and characterization of subcellular membranes from canine mesenteric arteries

Subcellular membrane fractions were isolated from dog mesenteric arteries by differential and isopynic sucrose density gradient centrifugations. Isolated membrane fractions were characterized by marker enzyme activities, morphological features and sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns. Our results show that the microsomal fraction isolated by conventional differential centrifugation was highly heterogenous and contained substantial amount of plasma membranes which could be further enriched as a light density membrane fraction on a discontinuous sucrose density gradient. The microsomal fraction and its subfractions were vesicular in appearance under electron microscope and were capable of binding and actively transporting Ca2+. The binding of Ca2+ and ATP-supported Ca2+-transport in the presence or absence of oxalate paralleled the distribution of plasma membrane marker enzyme activities suggesting that plasma membranes in vascular smooth muscle may play a major role in handling Ca2+ and thus the control of contractile function.     

Microtubules in mouse embryo fibroblasts extracted with Triton X-100

Treatment of mouse embryo fibroblasts with 1% Triton X-100 at 37 degrees C in the presence of 4M glycerol and 1 mM EGTA results in the extraction of about 80% cellular proteins. Indirect immunofluorescent staining with monospecific antibodies against tubulin showed that extracted cultures contained a well developed system of cytoplasmic microtubules, indistinguishable from a system of control non-extracted cells. Microtubules in extracted cells were sensitive to Ca2+ ions, and to cold or prolonged incubation in a glycerol-free buffer. Sodium dodecylsulphate-polyacrilamide gel electrophoresis revealed proteins co-electrophoresed with tubulin and actin in Triton-treated cultures. Electron microscopy demonstrated the presence of both microtubules and microfilament bundles in the extracted cells, but complete dissolution of plasma and intracellular membranes.     

Isolation and characterization of Sertoli cell plasma membranes and associated plasminogen activator activity

Plasma membranes were isolated from the cultured Sertoli cells of 20-day-old rat testes by differential centrifugation and sucrose density fractionation. The distribution and purity of subcellular components was determined by marker enzyme analysis of gradient fractions. The plasma membrane fraction showed an enrichment in two plasma membrane marker enzymes, 5'-nucleotidase and ouabain-sensitive Na+/K+-ATPase-specific activities, of 9- and 23-fold, respectively. Forty-two percent and 52% of the total cellular 5'-nucleotidase and ouabain-sensitive Na+/K+-ATPase activities, respectively, were found in the membrane fraction. The protein yield of plasma membrane was approximately 6% of the total cellular protein. Two-dimensional polyacrylamide gel electrophoresis was used to compare [35S] methionine- and [3H] glucosamine-labeled membrane proteins. The incorporation of [35S] methionine and [3H] glucosamine was increased in several proteins when the cultured Sertoli cells were treated with follicle-stimulating hormone, insulin, retinol, and testosterone. Isolated Sertoli cell membranes contained a membrane-associated form of plasminogen activator. Analysis of this plasminogen activator demonstrated that the membrane-associated enzyme existed primarily as a single 38,000-40,000-Mr form.