A dot-blot assay for quantitation of nanogram amounts of protein in the presence of carrier ampholytes and other possibly interfering substances

A method for protein determination in one- and two-dimensional electrophoresis sample buffer is presented. Accurate quantitation of protein in two-dimensional electrophoresis sample buffer (9.5 M urea, 2% Nonidet P-40, 2% carrier ampholytes, and 5% 2-mercaptoethanol) required removal of carrier ampholytes prior to the assay. This was made possible by taking advantage of the mutual solubility/insolubility of carrier ampholytes/proteins in saturated ammonium sulfate solution. In addition, improvement of protein determination in denaturing electrophoresis sample buffer containing the anionic detergent sodium dodecyl sulfate and the reducing agent 2-mercaptoethanol was achieved. The assay covers a range of sensitivity from 40 ng to 20 micrograms of protein. The procedure is applicable to large numbers of samples.     

Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis

A plasma membrane-enriched fraction prepared from barley roots was analyzed by two-dimensional gel electrophoresis. Four methods of sample solubilization were assessed on silver stained gels. When membranes were solubilized with 2% sodium dodecyl sulfate followed by addition of Nonidet P-40, gels had high background staining and few proteins because of incomplete solubilization. Gels of membranes solubilized in urea and Nonidet P-40 had a greater number of proteins but proteins with molecular weights greater than 85,000 were absent and proteins with low molecular weights were diffuse. High molecular weight proteins were present in gels of membranes solubilized in 4% sodium dodecyl sulfate followed by acetone precipitation but background staining and streaking remained a problem. Gels of the best quality were obtained when membrane proteins were extracted with phenol and precipitated with ammonium acetate in methanol; background staining and streaking were diminished and proteins were clearly resolved. This method makes possible the resolution required for meaningful qualitative and quantitative comparisons of protein patterns on two-dimensional gels of plant membrane proteins.     

Determination of protein covalently bound to agarose supports using bicinchoninic acid

A method using bicinchoninic acid (BCA) for the direct determination of protein covalently bound to agarose is described. The method involves the preparation of a standard curve using solubilized protein, the determination of the slurry concentration of the gel sample, the colorimetric assay of the gel slurry using BCA in a serum separator device, and the calculation of the amount of protein bound to the gel using the standard curve of the solubilized protein. The use of the BCA method for direct estimation of immobilized protein agrees well with the "balance" method which depends upon the measurement of protein depletion and yields highly reproducible results with a variety of coupling chemistries commonly used with agarose beads. The use of commercially available serum filters provides for a fast, less than 1 h, and convenient analytical format.     

A comparison of surface proteins in embryonal carcinoma cells and their differentiated derivatives

Surface proteins from five cell lines (three embryonal carcinoma cell lines (F9, PCC4 and PCC3), teratocarcinoma-derived endodermal cells (PYS) and fibroblasts (line 3/A/1-D-3 differentiated from PCC3)) were compared by two dimensional polyacrylamide gel electrophoresis after selective iodination with ¹²⁵I in the presence of lactoperoxidase. The labeled proteins were solubilized either in Nonidet P40/urea/ampholyte/mercaptoethanol solution or in Nonidet P40 only. In total, about thirty major ¹²⁵I-labeled surface proteins were identified by their isoelectric point and molecular weight. 14 proteins are present in all five cell types, although their quantity or accessibility for labeling differs between differentiated and undifferentiated cells. Three proteins (200, 160 and 150 kilodaltons) are present in undifferentiated cells only. Two of them (160 and 150 kilodaltons) were solubilized by Nonidet P40/urea/ampholyte/mercaptoethanol, but not by Nonidet P40. One protein (50 kilodaltons) was found in nullipotent F9 cells only. About 14–15 proteins (including fibronectin) were released by Nonidet P40/urea/ampholyte/mercaptoethanol but not by Nonidet P40. They are presumably bound to submembrane or cytoskeleton structures by non-covalent bonds.     

Dynamics of oligomer formation by denatured carbonic anhydrase II

Aggregation and subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. Many proteins unrelated to amyloidoses also fibrillate at the appropriate conditions. These proteins serve as a model for studying the processes of protein misfolding, oligomerization and fibril formation. The accumulated data support the model where protein fibrillogenesis proceeds via the formation of a relatively unfolded amyloidogenic conformation. The urea-induced unfolding of bovine carbonic anhydrase II, BCA II, is characterized by a combination of high-resolution NMR, circular dichroism spectroscopy and small angle X-ray scattering. It is shown that the formation of associates of protein molecules in complex with solvent (water and urea), APS, takes place in the presence of 4-6 M urea. The subsequent increase in urea concentration to 8 M is accompanied by a disruption of APS and leads to a complete unfolding of a protein molecule. Analysis of BCA II self-association in the presence of 4.2 M urea revealed that APS are relatively large mostly beta-structural blocks with the averaged molecular mass of 190-220 kDa. This work also demonstrates some novel NMR-based methodological approaches that provide useful information on protein self-association.     

Novel filaments 5 nm in diameter constitute the cytosolic ring of the plastid division apparatus

The plastid division apparatus (called the plastid-dividing ring) has been detected in several plant and algal species at the constricted region of plastids by transmission electron microscopy. The apparatus is composed of two or three rings: an outer ring in the cytosol, an inner ring in the stroma, and a middle ring in the intermembrane space. The components of these rings are not clear. FtsZ, which forms the bacterial cytokinetic ring, has been proposed as a component of both the inner and outer rings. Here, we present the ultrastructure of the outer ring at high resolution. To visualize the outer ring by negative staining, we isolated dividing chloroplasts from a synchronized culture of a red alga, Cyanidioschyzon merolae, and lysed them with nonionic detergent Nonidet P-40. Nonidet P-40 extracted primarily stroma, thylakoids, and the inner and middle rings, leaving the envelope and outer ring largely intact. Negative staining revealed that the outer ring consists of a bundle of 5-nm filaments in which globular proteins are spaced 4.8 nm apart. Immunoblotting using an FtsZ-specific antibody failed to show immunoreactivity in the fraction containing the filament. Moreover, the filament structure and properties are unlike those of known cytoskeletal filaments. The bundle of filaments forms a very rigid structure and does not disassemble in 2 M urea. We also identified a dividing phase-specific 56-kD protein of chloroplasts as a candidate component of the ring. Our results suggest that the main architecture of the outer ring did not descend from cyanobacteria during the course of endosymbiosis but was added by the host cell early in plant evolution.     

Evaluation of an effective sample prefractionation method for the proteome analysis of breast cancer tissue using narrow range two-dimensional gel electrophoresis

One method of improving the protein profiling of complex mammalian proteomes is the use of prefractionation followed by application of narrow pH range two dimensional (2-D) gels. The success of this strategy relies on sample solubilization; poor solubilization has been associated with missing protein fractions and diffuse, streaked, and/or trailing protein spots. In this study, I sought to optimize the solubilization of prefractionated human cancer cell samples using isoelectric focusing (IEF) rehydration buffers containing a variety of commercially available reducing agents, detergents, chaotropes, and carrier ampholytes. The solubilized proteins were resolved on 2-D gels and compared. Among five tested IEF rehydration buffers, those containing 3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS) and dithiothreitol (DTT) provided superior resolution, while that containing Nonidet P-40 (NP-40) did not significantly affect protein resolution, and the tributyl phosphine (TBP)-containing buffer yielded consistently poor results. In addition, I found that buffers containing typically high urea and ampholyte levels generated sharper 2-D gels. Using these optimized conditions, I was able to apply 2-D gel analysis successfully to fractionated proteins from human breast cancer tissue MCF-7, across a pH range of 4-6.7.     

Buffer optimization for high resolution of human lung cancer tissue proteins by two-dimensional gel electrophoresis

A problem in proteomic analysis of lung cancer tissue is the presence of complex components of different histological backgrounds (squamous cell carcinoma, small cell lung carcinoma, and adenocarcinoma). The efficient solubilization of protein components before two-dimensional electrophoresis (2-DE) is a very critical. Poor solubilization has been associated with a failure to detect proteins and diffuse, streaked and/or trailing protein spots. Here, we have optimized the solubilization of human lung cancer tissue to increase protein resolution. Isoelectric focusing (IEF) rehydration buffer containing a thiourea–urea mixture provided superior resolution, whereas a buffer without thiourea yielded consistently poor results. In addition, IEF rehydration buffers containing CHAPS and DTT gave superior resolution, whereas buffers containing Nonidet P-40 (NP-40) and/or Triton X-100 did not. A tributylphosphine-containing buffer gave consistently poor results. Using optimized conditions, we used 2-D gel analysis of human lung cancer tissue to identify 11 differentially-expressed protein spots by MALDI-mass spectrometry. This study provides a methodological tool to study the complex mammalian proteomes.     

Posttranslational processing of the Epstein-Barr virus-encoded p63/LMP protein.

In this paper we describe the posttranslational processing of the p63/LMP (latent membrane protein) encoded by Epstein-Barr virus in transformed B cells. Specifically, we show that after synthesis, free LMP disappeared with a half-life of about 0.5 h. This was caused by the association of LMP with an insoluble complex. All detectable LMP in the plasma membrane was insoluble. This interaction was resistant to nondenaturing detergents but readily dissociated with 8 M urea or by boiling in 0.5% sodium dodecyl sulfate, suggesting that LMP may be associated with cytoskeletal elements. Most of the Nonidet P-40-insoluble LMP was phosphorylated (ppLMP) primarily on serine but also on threonine residues. No phosphotyrosine was detected. Furthermore, greater than 90% of the ppLMP resided in the Nonidet P-40-insoluble fraction, suggesting a strong correlation between complexing and phosphorylation. Additionally, ppLMP was found to be associated with a 53,000-molecular-weight phosphoprotein (pp53) of unknown origin. Finally, LMP turned over extremely rapidly, with a half-life of about 2 h. Taken together, these properties suggest that although LMP falls broadly within the category of phosphorylated, cytoskeleton-associated oncoproteins, it is nevertheless clearly different from any previously described member of this family.     

The effect of phospholipids on the in vitro polymerization of rat brain tubulin

The association of brain tubulin, as measured by the temperature-dependent development of turbidity at 350 nm, is greatly stimulated by the detergent Nonidet P-40 in crude extracts of rat brain tissue. Stimulation of turbidity development is also obtained with partially purified rat brain tubulin treated with Nonidet or other detergents, or preincubated with phospholipase C or D; treatment with bovine pancreatic phospholipase A₂ produces an inhibition. Exogenous phospholipids, diglycerides, other related derivatives, and lipophilic extracts of tubulin and brain supernatants can also alter the turbidity development. In addition, microtubules arising from tubulin obtained in the presence of Tween-20 or Nonidet P-40 exhibit a 50 and 100% increased specific viscosity, respectively, over that of tubulin prepared in the absence of detergent or in the presence of Kyro or Triton N-101. The effectiveness of these detergents in removing phospholipids from tubulin preparations follows a similar pattern: Nonidet P-40 removes 80%, Tween-20 removes 50%, and Kyro or Triton N-101 removes none. The total mass of microtubule formed, as determined by sedimentation, is the same regardless of the effect of the detergents on the viscosity. The microtubules obtained in the presence of Nonidet P-40 have a normal appearance when examined by electron microscopy, and their composition on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is indistinguishable from that of standard tubulin, especially with regard to the minor protein bands always present in the tubulin preparations. The results obtained suggest that the phospholipids associated to brain tubulin preparations might have a role in determining the association of tubulin and/or the final dimensions of the assembled microtubules.     

Use of a scanning densitometer or an ELISA plate reader for measurement of nanogram amounts of protein in crude extracts from biological tissues

Protein contents of crude extracts from plant and animal tissues can be rapidly assayed using a Coomassie blue dye-binding procedure combined with scanning densitometry. Total protein is extracted from 100 mg of fresh-frozen or dried-ground tissue using 1 ml of extraction buffer. One-microliter aliquots of standard solutions or crude extracts are spotted in rows on a suitably sized sheet of Whatman 3MM chromatography paper. The dried samples are stained with Coomassie brilliant blue R-250 (0.2%, w/v, in acidified 50% MeOH) for 20 min and rinsed twice with acidified 20% MeOH. After drying, protein concentrations are read as reflectance using a scanning densitometer and peak heights or peak areas recorded using a digital integrator. In an alternative procedure, each spot is cut from the sample sheet and the dye-protein complex eluted in 1% sodium dodecyl sulfate (SDS) using an ultrasonic cleaner. Absorbance is subsequently read in a microwell sample holder at 590 nm with an enzyme-linked immunosorbent assay plate reader. Both procedures offer distinct advantages over previously reported methods. They are significantly faster when large numbers of samples are processed, they avoid interference by chlorophyll, dithiothreitol, SDS, 2-mercaptoethanol, Nonidet P-40, and phenylmethylsulfonyl fluoride (and other protease inhibitors) and they yield marked improvements in sensitivity, providing measurements of protein concentration below 100 and 200 ng.microliter-1, respectively.     

Solubilization of human prostatic 5 alpha-reductase

A sensitive assay for 5 alpha-reductase was introduced which is capable of detecting at least 0.2 U of activity per sample. The assay was used in developing a method for the solubilization of human prostatic 5 alpha-reductase. Homogenisation conditions were devised under which 95% of the total prostatic 5 alpha-reductase was released into the microsomal fraction. A combination of 0.1 M sodium citrate, 0.1 M KCl, 20% (v/v) glycerol, 0.5 mM NADPH and 1 microM testosterone was found to stabilise 5 alpha-reductase in the presence of detergents. The effect of the presence of low concentrations of detergents in the assay on the activity of 5 alpha-reductase was studied. Triton X-100, Lubrol PX and Nonidet P-40, caused a concentration-dependent inhibition of activity. The ability of several detergents (Triton X-100 MEGA-9, Tween 20, Tween 80, digitonin, Lubrol PX and Nonidet P-40) to solubilise 5 alpha-reductase was studied. All detergents caused a concentration-dependent solubilization of 5 alpha-reductase. Significant amounts of active solubilized enzyme were recovered only with Lubrol PX at concentrations less than 1.1 mg/ml. Seventy percent of the 5 alpha-reductase was solubilized in an active form by extracting the membranes 3 times with 0.8 mg/ml Lubrol PX.     

Comparison of chicken erythroid cell nuclear isolation methods using morphological,immunochemical and biochemical criteria

Chicken erythroid nuclei were prepared using four published methods. Our findings indicate that nuclei prepared by nitrogen cavitation are less likely to be contaminated with plasma membrane fragments than those made by procedures involving cell disruption by hypotonic lysis. However, globin gene sequences were much less sensitive to DNase I digestion in nuclei prepared by nitrogen cavitation. This suggests that the conformation of chromatin was altered by the cavitation procedure. Analysis of the proteins solubilized during limited DNase I digestion of nuclei prepared by both hypotonic lysis and cavitation revealed no appreciable differences in HMG proteins but a notable difference in the RNP-associated proteins and core histones.Abbreviations HMG high mobility group nonhistone chromosomal protein - RNP ribonucleoprotein - SSC 14 mM sodium citrate buffered saline pH 7.0 - PMSF phenylmethanesulfonyl fluoride - EDTA ethylenediaminetetraacetic acid - DTT dithiothreitol - PBS 10 mM sodium phosphate buffered saline pH 7.2 - NP-40 Nonidet P-40 (octylphenoxypolyethoxyethanol) - SS-DNA single-stranded DNA - RSB reticulocyte standard buffer, 0.01 M NaCl, 0.003 M MgCl₂, 0.01 M Tris-HCI, pH 7.4.     

Difference in extractability of estradiol- and tamoxifen-receptor complex in the nuclei from MCF-7 cells with nonidet P-40

The extraction of [³H]estradiol- and [³H]tamoxifen-receptor complex in the nuclei from MCF-7 cells with the nonionic detergent Nonidet P-40 has been studied. We found that there is a striking difference in the extractability of estradiol- and tamoxifen-receptor complex from nuclei with 0.5% Nonidet P-40. The nuclear bound estradiol-receptor complex is scarcely extractable with Nonidet P-40. In contrast, almost all of the nuclear bound tamoxifen-receptor complex is extractable. The nuclear [³H]tamoxifen-receptor complex extracted in the presence of Nonidet P-40 sediments in two peaks at 7 S and 5 S. The latter sedimentation rate is the same with that of the nuclear [³H]tamoxifen-receptor complex extracted with 0.4 M KC1. The nuclear [³H]estradiol-receptor complex extracted with 0.4 M KC1 sediments at 4 S.The results suggest that interaction of tamoxifen-receptor complex with chromatin is different from that of estradiol-receptor complex.     

Effects of nickel(II) on nuclear protein binding to DNA in intact mammalian cells

An intracellular effect of nickel(II) which may be involved in its carcinogenic action is the alteration of normal DNA-protein binding. This effect of ionic nickel was studied in Chinese hamster ovary cells using several chromatin isolation methods in combination with SDS-polyacrylamide gel electrophoresis. DNA from cells incubated with (35S)-methionine or (35S)-cysteine to radiolabel protein was prepared by three methods: (solation of nuclei or nucleoids followed by chloroform-isoamyl alcohol (24:1 v/v) extraction and in some cases an additional extraction in the absence or presence of 2M NaCl, 40 mM EDTA or SDS; by isopycnic centrifugation through Cs2SO4 gradients containing 0.8% sarkosyl, 2.2 MCs2SO4, 1 mM NaCl and 10 mM EDTA; or by chromatin disaggregation and denaturation using 9 M urea, 2% 2-mercaptoethanol, 4% Nonidet P-40 +/- 2 M NaCl. DNA from nickel-treated cells consistently had more (35S)-methionine radioactivity associated with it than did DNA from untreated cells. This radioactivity was resistant to ribonuclease but sensitive to protease. Differential extraction using denaturing agents and high ionic strength followed by SDS-polyacrylamide gel electrophoresis revealed that most of the tightly bound proteins were nonhistone chromosomal proteins, and possibly histone 1. The enhancement of DNA-protein binding from nickel-treated cells was disrupted by SDS, suggesting that nickel ions do not function as classical bifunctional crosslinking agents. Since regulation of DNA replication and gene expression is dependent upon DNA-protein interactions, the effect of nickel in altering the extent of DNA-protein binding may interfere with this regulation and may contribute to the carcinogenic activity of nickel compounds.     

Biosynthesis of glycoproteins in Candida albicans: activity of mannosyl and glucosyl transferases

The enzymes dolichol phosphate glucose synthase and dolichol phosphate mannose synthase (DPMS), which catalyze essential steps in glycoprotein biosynthesis, were solubilized and partially characterized in Candida albicans. Sequential incubation of a mixed membrane fraction with increasing concentrations of Nonidet P-40 released a soluble fraction that transferred glucose from UDP-Glc to dolichol phosphate glucose and minor amounts of glucoproteins in the absence of exogenous dolichol phosphate. Studies with the soluble fraction revealed that some properties were different from those previously determined for the membrane-bound enzyme. Accordingly, the soluble enzyme exhibited a twofold higher affinity for UDP-Glc and a sixfold higher affinity over the competitive inhibitor UMP, and the transfer reaction was fourfold more sensitive to inhibition by amphomycin. On the other hand, a previously described protocol for the solubilization of mannosyl transferases that rendered a fraction exhibiting both DPMS and protein mannosyl transferase (PMT) activities operating in a functionally coupled reaction was modified by increasing the concentration of Nonidet P-40. This resulted in a solubilized preparation enriched with DPMS and nearly free of PMT activity which remained membrane bound. DPMS solubilized in this manner exhibited an absolute dependence on exogenous Dol-P. Uncoupling of these enzyme activities was a fundamental prerequisite for future individual analysis of these transferases.     

Structural studies of retroviruses: characterization of oligomeric complexes of murine and feline leukemia virus envelope and core components formed upon cross-linking.

To examine the protein proximity and subunit organization of type C retroviruses, preparations of AKR murine leukemia virus were treated with bifunctional cross-linking reagents and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The cross-linked components obtained were characterized by immunoprecipitation with monospecific antisera against purified viral proteins, followed by SDS-PAGE analysis both before and after cleavage of the cross-links. With these procedures, complexes of both viral envelope and core components were identified. The major envelope subunit obtained was a large (apparent molecular weight of 450,000 to 500,000), glycosylated complex, composed of four to six gp70-p15(E) subunits. This complex was detected over a 100-fold range of cross-linker concentration and thus seems to represent a particularly stable viral substructure. The cross-linked complexes of the core proteins consisted of oligomers of p30 dimers, suggesting that the p30 dimer is a basic structural unit of the viral core. When virion preparations, which had previously been disrupted with the nonionic detergent Nonidet P-40, were cross-linked, the envelope complex was still observed, indicating that this structure is stable in the presence of Nonidet P-40. A similar envelope structure was observed for feline leukemia virus, suggesting that such a complex may be a conserved feature of oncornavirus structure.     

Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid.

The two envelope glycoproteins and the viral nucleocapsid of the coronavirus A59 were isolated by solubilization of the viral membrane with Nonidet P-40 at 4 degrees C followed by sucrose density gradient sedimentation. Isolated E2 consisted of rosettes of peplomers, whereas E1, the membrane glycoprotein, was irregular and amorphous. Under certain conditions significant interactions occurred between components of Nonidet P-40-disrupted virions. Incubation of the Nonidet P-40-disrupted virus at 37 degrees C resulted in formation of a complex between one of the viral glycoproteins, E1, and the viral nucleocapsid. This was caused by a temperature-dependent conformational change in E1, resulting in aggregation of E1 and interaction with the viral RNA in the nucleocapsid. E1 also bound rRNA. The E1-nucleocapsid complexes can be distinguished on sucrose and Renografin density gradients from native viral nucleocapsids. The separation of the membrane glycoprotein E1 from the peplomeric glycoprotein E2 permitted preparation of antisera against these isolated proteins. A model is proposed for the arrangement of the three major structural proteins in the coronavirus A59 virion in relation to the viral envelope and RNA.     

Protective monoclonal antibodies from mice vaccinated or chronically infected with Schistosoma mansoni that recognize the same antigens

An IgM monoclonal antibody, designated mAb 1.G1, has been generated from spleen cells of mice immunized with irradiated Schistosoma mansoni cercariae. As determined by indirect immunofluorescence, mAb 1.G1 binds to the surface membrane of schistosomula and to the ciliated plates of miracidia. mAb 1.G1 also binds to the protonephridial systems of live adult worms and denuded, acetone-fixed schistosomula. Western blot analysis shows that the target epitope of this mAb is found on Nonidet P-40-solubilized schistosomular antigens ranging in molecular size from 85 to 130 kDa and ciliated plate antigens of miracidia at 92, 95, and 102 kDa. The recognized epitope in an 8 M urea adult worm extract is found on a 97-kDa molecule. In addition, mAb 1.G1 mediates a high level of complement-dependent cytotoxic activity against schistosomula when used in an in vitro assay. In passive immunization experiments, approximately 40% protection was provided mice when mAb 1.G1 was administered either at the time of challenge or when given 8 days postchallenge. However, when administered 15 days postchallenge, mAb 1.G1 failed to mediate passive protection. The ability of mAb 1.G1 to mediate protection in vivo correlates with its recognition of epitopes on the surfaces of live schistosomula up to 8 days but not at 15 days. Western blot analysis showed that the antigens were contained within Nonidet P-40 extracts of schistosomula during the same time period. Furthermore, a second monoclonal antibody (mAb 4.4B) derived from mice chronically infected with S. mansoni exhibits the identical properties as described for mAb 1.G1.     

Characterization of protein tyrosine kinase activity in murine Leydig tumor cells

The activity of protein tyrosine kinase (EC 2.7.1.37) was characterized from Leydig tumor cells (M5480A) using the synthetic peptide NH2-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly-COOH as a substrate. Relatively high tyrosine-specific protein kinase activity (about 135 pmol/mg protein per min) was detected in a particulate fraction (30 000 X g pellet) and was found to be linear as a function of time and protein concentration. The enzymic activity in the particulate fraction was stimulated 1.4-fold by 0.02% Nonidet P-40 as judged by 32PO4 incorporated into the peptide. Phosphorylation of endogenous proteins in M5480A particulate fractions with [gamma-32P]ATP resulted in several alkali-resistant radiolabeled bands in polyacrylamide gels in the presence of sodium dodecyl sulfate. Included in this group was a major radiolabeled doublet with an apparent molecular-weight in the range of 50 000-54 000. Phosphoamino acid analysis of hydrolysates of these eluted proteins indicated the presence of phosphotyrosine. Several alkali-resistant radio-labeled bands, including a major doublet with an apparent molecular-weight of 32 000, were also detected after culturing M5480A cells in the presence of 32PO4. These studies demonstrate the presence of high levels of protein tyrosine kinase activity in Leydig tumor cells and of endogenous protein substrates for this enzyme activity.