Flagellar motility requires the cAMP-dependent phosphorylation of a heat-stable NP-40-soluble 56 kd protein, axokinin

Using NP-40-treated dog sperm as a model, the stimulatory effect of cAMP upon reactivated flagellar motility has been shown to be dependent upon the cAMP-dependent phosphorylation of a heat-stable NP-40-soluble protein of 56 kd. Examination by two-dimensional polyacrylamide gel electrophoresis of NP-40 extract proteins phosphorylated with gamma-32P-ATP revealed a major cAMP-dependent phosphopeptide at 56 kd. This is the only cAMP-dependent phosphoprotein common to NP-40 extracts of all tissues that show cAMP-dependent stimulation of flagellar motility. These cells and tissues include sea urchin, dog, and human sperm, as well as dog trachea and retina. Moreover, this phosphoprotein is absent in nonstimulatory extracts from tissues such as skeletal muscle, brain, and liver. We conclude that the cAMP-dependent phosphorylation of the 56 kd peptide represents a major regulatory component of not only sperm but other types of axonemal motility as well.     

The Dictyostelium discoideum 30,000-dalton protein is an actin filament- bundling protein that is selectively present in filopodia

The interaction with actin and intracellular localization of the 30,000-D actin-binding protein from the cellular slime mold Dictyostelium discoideum have been investigated to analyze the potential contributions of this protein to cell structure and movement. The formation of anisotropic cross-linked filament networks (bundles) containing actin and the 30,000-D protein has been observed by electron microscopy, light scattering, viscometry, and polarization microscopy. Cosedimentation experiments indicate that a maximum of one molecule of the 30,000-D protein can bind to 10 actin monomers in filaments with an apparent association constant of 1 X 10(7) liters/mol. Inhibition of the interaction of the 30,000-D protein with actin by either magnesium or calcium was observed by viscometry, light scattering, polarization microscopy, and direct binding assays. However, the concentration of magnesium required to diminish the interaction is greater than 100 times greater than that of calcium. The association constant of the 30,000-D protein for actin is 4.2 X 10(6) liters/mol, or less than 1 X 10(5) liters/mol in the presence of increased concentrations of either Mg2+ or Ca2+, respectively. Enzyme-linked immunoassays indicate that the 30,000-D protein comprises 0.04% of the protein in D. discoideum. Extensive interaction of the 30,000-D protein with actin in cytoplasm is predicted from these measurements of the concentration of this protein and its affinity for actin. The distribution of the 30,000-D protein was analyzed by immunofluorescence microscopy using mono-specific affinity-purified polyclonal antibody. The 30,000-D protein exhibits a diffuse distribution in cytoplasm, is excluded from prominent organelles, and is quite prominent in fine extensions protruding from the cell surface. The number, length, and distribution of these extensions containing the 30,000-D protein are similar to those of filopodia observed by scanning electron microscopy. To analyze the effects of cell thickness and the distribution of organelles on the immunofluorescence localization, fluorescein-labeled BSA was incorporated into the cytoplasm of living cells before fixation and staining using a sonication loading technique. The results indicate that the 30,000-D protein is selectively incorporated into filopodia. These results provide a clear distinction between the multiple actin-cross-linking proteins present in D. discoideum, and suggest that the 30,000-D protein contributes to organization of bundles of actin filaments in filopodia.     

c-Abl proto-oncoprotein is expressed and tyrosine phosphorylated in human sperm cell

The presence and possible role of c-Abl proto-oncoprotein was investigated in human sperm cell. The c-Abl monoclonal antibody (mAb), against the protein tyrosine kinase domain of v-Abl protein, reacted specifically with the acrosomal region of methanol-fixed capacitated and non-capacitated human sperm cell in the indirect immunofluorescence technique. The c-Abl mAb predominantly recognized two protein bands of 145 kD and 95 kD in detergent-solubilized (Triton X-100 and NP-40) sperm and testes preparations in the Western blot procedure. The 95 kD protein band reacted stronger than the 145 kD band and was the only band detected in the lithium diiodosalicylate (LIS)–solubilized sperm preparation, and even in the Triton X-100/NP-40 extracts of sperm of some men. In the in vitro kinase assay using the Triton X-100–solubilized capacitated sperm preparation, the 95 kD protein was autophosphorylated at the tyrosine residues, which was inhibited in the presence of c-Abl mAb. The tyrosine phosphorylation of sperm proteins, especially of the 95 kD protein, has been shown to have a vital role in human sperm function, namely, the sperm capacitation/acrosomal exocytosis and binding to zona pellucida of oocyte. These findings suggest that the c-Abl or c-Abl-like proteins are present in mature sperm cells that are tyrosine autophosphorylated and may have a role in human sperm cell function. Mol. Reprod. Dev. 51:210–217, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Actin and myosin: control of filament assembly

Actin filaments, assembled from highly purified actin from either skeletal muscle or Dictyostelium amoebae, are very stable under physiological ionic conditions. A small and limited amount of exchange of actin filament subunits for unpolymerized actin or subunits in other filaments has been measured by three techniques: fluorescence energy transfer, incorporation of 35S-labelled actin monomers into unlabelled actin filaments, and exchange of [14C]ATP with filament-bound ADP. A 40 kDa protein purified from amoebae destabilizes these otherwise stable filaments in a Ca2+-dependent manner. Myosin purified from Dictyostelium amoebae is phosphorylated both in the tail region of the heavy chain and in one of the light chains. Phosphorylation appears to regulate myosin thick-filament formation.     

Actin filaments, localized to the region of the developing acrosome during early stages, are lost during later stages of guinea pig spermiogenesis

The presence and localization of actin was investigated in guinea pig spermatogenic cells and cauda epididymal sperm (CauE). Staining with rhodamine-phalloidin demonstrated the presence of actin filaments in the region of the developing acrosome in guinea pig spermatids. The actin filaments were visualized predominantly in the region of the inner acrosomal membrane in both round and elongating spermatids. As development progressed, the intensity of the staining diminished. No rhodamine-phalloidin staining was found in testicular sperm lacking a residual body or in CauE sperm. Analysis of actin levels by immunoblotting with an anti-actin monoclonal antibody showed that the disappearance of actin filaments is accompanied by a decrease in the level of actin per cell. By using immunoblotting techniques, actin was readily detected in preparations of purified spermatogenic cells, but not in preparations of purified CauE sperm. Actin was also not detected in cauda sperm by indirect immunofluorescence (IIF) with anti-actin antibodies or examination of whole cell extracts by two-dimensional gel electrophoresis.     

Actin polymerization in boar spermatozoa: Fertilization is reduced with use of cytochalasin D

The aggregational state of actin in boar spermatozoa after capacitation and the acrosome reaction has been examined by several methods. In vitro fertilization (IVF) experiments were conducted in the presence and absence of cytochalasin D (CD) to evaluate the role of actin polymerization in the events of fertilization. The fertilizing capacity was very high in controls, but, when CD (an inhibitor of the polymerization of actin) was added to the capacitation medium, there was a marked decrease in the fertilizing capacity of the boar spermatozoa. There was a further decrease when CD was present during both capacitation and fertilization processes. In addition to the IVF tests, biochemical and immunoelectron microscopic methods were used to analyze the state of aggregation of actin in boar spermatozoa after capacitation, and the acrosome reaction. By immunoelectron microscopy with a phalloidin probe, there were no gold particles, indicating the presence of F-actin on boar sperm heads capacitated and acrosome-reacted in media containing CD. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis there were differences in NP-40 solubility, reflecting actin polymerization, between CD-treated and untreated sperm. These results suggest that actin polymerizes during capacitation and the acrosome reaction and that this polymerization is essential to the fertilization process. © 1993 Wiley-Liss, Inc.     

Isolation from human erythrocytes of a new membrane protein which inhibits the formation of complement transmembrane channels

A protein which inhibited complement channel formation was isolated from extracts of papain-digested human erythrocyte membranes using DEAE-Sephacel, Bio-Gel A0.5m column chromatographies, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose paper and elution with 2% NP-40 solution. The purified protein showed a molecular weight of 18 kDa, and efficiently inhibited hemolysis of EC5-7 cells with C8 and C9, but did not show any decay-accelerating activity to C5 convertase. Immunochemical analysis of native membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the antibody against this protein gave a single band having the same mobility as this protein; papain did not eliminate a significant portion of this protein.     

Identification and characterization of major bovine serum tyrosine-O-sulfate-binding protein as a complement factor H

A major tyrosine-O-sulfate (TyrS)-binding protein present in bovine serum was purified to electrophoretic homogeneity using a combination of TyrS-Affi-Gel 10 affinity chromatographyy, DEAE-Bio-Gel A ion-exchange chromatography, and hydroxylapatite chromatography. The purified TyrS-binding protein migrated as doublet protein bands with apparent molecular weights of ca. 160, 000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. N-termini of the two forms of purified TyrS-binding protein contain most likely identical sequence for the first fifteen amino acids residues, which displays a high degree of homology to those of human and mouse complement factor H. Furthermore, the purified TyrS-binding protein exhibited immunologic cross-reactivity with anti-human complement factor H. These results indicate the identity of the purified TyrS-binding protein being bovine complement factor H. The two forms of the purified bovine factor H were investigated with respect to the sensitivity to limited trypsin digestion. The high-molecular weight form was cleaved into two fragments with apparent molecular masses of, respectively, 45 kD and 125 kD. The low-molecular weight form was cleaved in a different manner to generate three major fragments with molecular masses of 25 kD, 45 kD and 100 kD, respectively. Limited V8 protease mapping of the two forms yielded similar, yet unidentical, peptide band patterns. Purified bovine factor H appeared to bind agarose-bonded heparin through its anion-binding domain and the binding was inhibited by the presence of free heparin or dextran sulfate.Abbreviations HEPES N-2-hydroxylpiperazine-N-2-ethanesulfonic acid - NP-40 Nonidet P-40 - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TyrS tyrosine-O-sulfate     

F-actin in acrosome-reacted boar spermatozoa.

Biochemical and immunoelectron microscopic methods have been used to analyze the distribution of actin in boar spermatozoa and its state of aggregation before and after acrosome reaction. F-actin was detected on sperm head and tail by electron microscopy using an improved phalloidin probe: incubation with a fluorescein-phalloidin complex and an anti-fluorescein antibody, followed by labeling with protein A-gold complex. Gold particles, indicating the presence of F-actin, were localized on the sperm surface of the acrosome-reacted spermatozoa. Specific labeling was localized (1) between the outer acrosomal membrane and the plasma membrane in the equatorial region, (2) between the outer surface of the fibrous sheath and the plasma membrane in the postacrosomal region, (3) around the connecting piece and the neck region, and (4) on the external surface of the fibrous sheath in the principal piece of the tail. Furthermore, after NP-40 extraction, the SDS-PAGE revealed a difference in solubility between reacted and unreacted boar spermatozoa, reflecting actin polymerization. We conclude that most actin in the acrosome reacted boar spermatozoa is polymeric.     

THE POLYMERIZATION OF ACTIN: ITS ROLE IN THE GENERATION OF THE ACROSOMAL PROCESS OF CERTAIN ECHINODERM SPERM

When Asterias or Thyone sperm come in contact with egg jelly, a long process which in Thyone measures up to 90 µm in length is formed from the acrosomal region. This process can be generated in less than 30 s. Within this process is a bundle of microfilaments. Water extracts prepared from acetone powders of Asterias sperm contain a protein which binds rabbit skeletal muscle myosin forming a complex whose viscosity is reduced by ATP. Within this extract is a protein with the same molecular weight as muscle actin. It can be purified either by collecting the pellet produced after the addition of Mg⁺⁺ or by reextracting an acetone powder of actomyosin prepared by the addition of highly purified muscle myosin to the extract. The sperm actin can be polymerized and by electron microscopy the polymer is indistinguishable from muscle F-actin. The sperm actin was shown to be localized in the microfilaments in the acrosomal processes by: (a) heavy meromyosin binding in situ, (b) sodium dodecyl sulfate (SDS) gel electrophoresis of the isolated acrosomal processes and a comparison to gels of flagella which contain no band corresponding to the molecular weight of actin, and (c) SDS gel electrophoresis of the extract from isolated acrosomal caps. Since the precursor for the microfilaments in the unreacted sperm appears amorphous, we suspected that the force for the generation of the acrosomal process is brought about by the polymerization of the sperm actin. This supposition was confirmed, for when unreacted sperm were lysed with the detergent Triton X-100 and the state of the actin in the sperm extract was analyzed by centrifugation, we determined that at least 80% of the actin in the unreacted sperm was in the monomeric state.     

Thymosin beta 4 and Fx, an actin-sequestering peptide, are indistinguishable

At least 50% of the actin in resting human platelets is unpolymerized, and the bulk of this unpolymerized actin is complexed with a recently identified acidic, heat-stable 5-kDa peptide, named "Fx." Purified Fx binds stoichiometrically to muscle G-actin, forming a complex identifiable by nondenaturing polyacrylamide gel electrophoresis. Formation of the complex inhibits salt-induced polymerization of G-actin. Here we report that Fx is indistinguishable from thymosin beta 4, an acidic, heat-stable 5-kDa peptide first isolated from calf thymus and thought to be a thymic hormone. The complete amino acid sequence of Fx was determined and was found to be identical with that of thymosin beta 4. Authentic thymosin beta 4 is functionally equivalent to Fx, forming a 1:1 complex with actin monomers and inhibiting polymerization. The widespread distribution and high intracellular concentration of thymosin beta 4 (Fx) strongly suggest that it plays a significant role in regulating actin polymerization in many cell types.     

Immunocytochemical localization of actin and tubulin in rat testis and spermatozoa

Summary Using commercial monoclonal antibodies against actin and tubulin ( and ), the respective antigens were localized on semithin and ultrathin sections of the rat testis. Tubulin immunofluorescence was found in the socalled manchette surrounding the heads of the maturating spermatids as well as the sperm tail. The distribution pattern varied with sperm development. Modified Sertoli cells found at the transition between the seminiferous tubules and the rete testis displayed much filamentous tubulin-reactive material. The immunofluorescence findings could be confirmed at the ultrastructural level using the indirect immunogold method. Actin immunofluorescence was demonstrated in vascular smooth muscle cells, interstitial macrophages and — most intensely — in peritubular cells. Inside the seminiferous tubules the Sertoli cell junctions and the ectoplasmic specializations of the Sertoli cells that follow the outer contour of spermatid heads displayed distinct actin immunofluorescence. In addition to the locations mentioned, actin-like immunoreactivity was visualized at the ultrastructural level in the chromatoid body and the subacrosomal space of spermatids as well as on the outer dense fibers of the sperm tail.Immunoblotting experiments with actin antibodies showed that in extracts from testicular spermatozoa, intact or fragmented into heads and tails, from isolated Sertoli cells grown in vitro, and from testis tissue in addition to authentic actin a protein was present in sperm tail extracts that strongly bound the actin antibody. This protein may be an actin-related protein and may be responsible for the actin-like immunoreactivity of the outer dense fibers of the sperm tail.     

The effects of insulin,cycloheximide and phalloidin on the content of actin and p35 in extracts prepared from the nuclear fraction of Krebs II ascites cells

The nuclear fraction isolated from Krebs II ascites cells following cell disruption by nitrogen cavitation was separated into four fractions by salt/detergent extraction: NP-40 soluble fraction, 130 mM KCl extract, DOC/Triton × 100 soluble fraction and salt/detergent treated nuclei. The protein composition of the individual fractions was studied by SDS-PAGE and the relative amounts of actin and a 35 kDa protein (p35) were measured from gel scans. There was a time-dependent shift of actin from the 130 mM KCl extract to the NP-40 soluble fraction upon storage of the nuclear fraction on ice, indicating a progressive depolymerization of microfilaments. Compared with actin there was a slower release of p35 into the NP-40 soluble fraction. The results suggest that p35 is not integrated in the microfilament network. Phalloidin, which stabilizes the microfilaments, enriched the amount of both proteins in the 130 mM KCl extracts, together with a series of other proteins in the range 50–205 kDa. The presence of phalloidin also resulted in a large increase in the actin content in both the DOC/Triton × 100 extract and the fraction containing salt/detergent treated nuclei. Incubation of cells with insulin and/or cycloheximide enriched the amount of actin in the 130 mM KCl fraction. The results show that short term incubation of cells with phalloidin, insulin or cycloheximide increases the actin content of the nuclear fraction and also affects the presence of several other proteins.     

Axokinin phosphorylation by cAMP-dependent protein kinase is sufficient for activation of sperm flagellar motility

Using a selective inhibitor of cAMP-dependent protein kinase, N-[2(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), the requirement for cAMP-dependent phosphoproteins in the initiation of dog sperm flagellar motility was examined. H-8 inhibited motility of live as well as reactivated sperm in a dose-dependent manner. The half-maximal inhibition of reactivated motility (32 microM) paralleled the inhibition of pure catalytic subunit of cAMP-dependent protein kinase (50 microM) measured under the same conditions. H-8 inhibited protein phosphorylation both in whole models and in isolated Nonidet P-40 (NP-40) extracts of sperm. Axokinin, the heat-stable NP-40-soluble protein whose phosphorylation is required for flagellar reactivation, represented 97% of the de novo phosphate incorporation in the NP-40 extract after stimulation by cAMP. 500 microM H-8 inhibited axokinin phosphorylation by 87%. When sperm were reactivated in the presence of up to 5 mM H-8 with NP-40 extract that had been prephosphorylated with cAMP-dependent protein kinase, then neither cAMP nor cAMP-dependent protein kinase activity was required for full flagellar reactivation. If sperm were rendered completely immotile by pretreatment with H-8, then the resulting model remained immotile in the continued presence of H-8 unless prephosphorylated axokinin was added. These results suggest that phosphorylated axokinin is not only required for flagellar reactivation but is sufficient as well.     

Identification and purification of calcium ion dependent modulators of actin polymerization from bovine thyroid

We describe the purification of Ca2+-dependent actin modulator proteins from bovine thyroid using DNase I affinity chromatography and diethylaminoethylcellulose chromatography. The 40K actin modulator has been purified to 98% homogeneity. It is a single polypeptide chain with a molecular weight of approximately 40 000 and an isoelectric point of 8.1. Its amino acid composition is different from previously described actin-associated proteins and thyroid actin. On the basis of the centrifugation assay and the DNase I inhibition assay, the actin complexed with the 40K protein is G-actin in its conformation rather than F-actin oligomers. Substoichiometric concentrations of the 40K protein rapidly inhibit actin polymerization in the presence of physiological concentrations of Ca2+ and Mg2+. An 80K actin modulator also has been purified to 98% homogeneity. It is a single polypeptide chain with a molecular weight of approximately 80 000 and an isoelectric point of 6.35-7.0. Its amino acid composition is different from those of villin, gelsolin, and leukocyte actin polymerization inhibitor. On the basis of the DNase inhibition assay and the centrifugation assay, the nonprecipitable actin associated with the 80K protein was F-actin in its conformation. The 80K protein acts very efficiently as a Ca2+-dependent nucleator for actin assembly and reduces its viscosity. In addition to the 40K and 80K actin modulators, 91K and 95K actin-associated proteins were partially purified. The 91K-95K fraction has similar activity to the 80K protein regarding precipitation of F-actin. The 125I-G-actin polyacrylamide gel overlay technique [Snabes, M. C., Boyd, A.E., & Bryan, J. (1981) J. Cell Biol. 90, 809-812] revealed that both the 91K and 95K proteins bind 125I-actin after sodium dodecyl sulfate (NaDodSO4) electrophoresis while the 80K and 40K proteins do not. Thyroid 91K protein comigrated with a human platelet 91K actin binding protein on NaDodSO4 gels and may be similar to macrophage gelsolin. The 95K protein may be similar to villin, the intestinal cytoskeletal protein.     

The bulk of unpolymerized actin in Xenopus egg extracts is ATP-bound.

Non-muscle cells contain 15-500 microM actin, a large fraction of which is unpolymerized. Thus, the concentration of unpolymerized actin is well above the critical concentration for polymerization in vitro (0.2 microM). This fraction of actin could be prevented from polymerization by being ADP bound (therefore less favored to polymerize) or by being ATP bound and sequestered by a protein such as thymosin beta 4, or both. We isolated the unpolymerized actin from Xenopus egg extracts using immobilized DNase 1 and assayed the bound nucleotide. High-pressure liquid chromatography analysis showed that the bulk of soluble actin is ATP bound. Analysis of actin-bound nucleotide exchange rates suggested the existence of two pools of unpolymerized actin, one of which exchanges nucleotide relatively rapidly and another that apparently does not exchange. Native gel electrophoresis of Xenopus egg extracts demonstrated that most of the soluble actin exists in complexes with other proteins, one of which might be thymosin beta 4. These results are consistent with actin polymerization being controlled by the sequestration and release of ATP-bound actin, and argue against nucleotide exchange playing a major role in regulating actin polymerization.     

Purification and location of the major glycoprotein of vaccinia virus.

A glycoprotein component of vaccinia virus was extracted with a non-ionic detergent NP-40 (Nonidet P-40) and purified by gel chromatography. The single antigen extracted by the detergent had a molecular weight estimated between 100,000 and 200,000 daltons. This glycoprotein was found to contain less than 1% hexosamine which would correspond to 5--10 sugar residues per molecule. Antibodies produced against this glycoprotein were able to neutralize vaccinia virus. Using immunoelectron microscopy, this molecule was found to be located in the outer layer of the virion. These results further suggest that this protein called complex E (for external) is a surface component of vaccinia virus.     

A high molecular weight actin binding protein: Its localization in the cortex of the sea urchin egg

From the Triton-treated cortex fraction of sea urchin eggs, a high molecular weight actin binding protein (260K protein) was solubilized by a high salt solution and purified. A cosedimentation assay revealed that the 260K protein binds to actin filaments in a concentration-dependent manner. The low-shear viscosity of actin solutions largely increased in a concentration-dependent manner after addition of 260K protein. Electron microscopy showed that this protein induces the formation of large curled bundles of actin filaments. Different from fascin-induced actin bundles, no clear striations were observed within the actin bundles formed by the 260K protein. Antibodies against the 260K protein were raised in a rabbit and affinity purified. Immunoblotting analysis of Triton-solubilized cortex and various subcellular fractions showed that first only a single band reacted with the antibody and second that the 260K protein exclusively localized in the cortex fraction. Indirect immunofluorescence microscopy localized the protein in the cortex and the region of the cleavage furrow. After double staining, the fluorescence images for actin filaments and the 260K protein well correlate with each other.     

Immunopurification, characterization, and nature of membrane association of human melanoma-associated oncofetal antigen gp87 defined by monoclonal antibody 140.240

A melanoma-associated oncofetal antigen, gp87 (a p97-like molecule), defined by the monoclonal antibody (MoAb) 140.240 has been purified to homogeneity from the spent medium of cultured melanoma cells by a two-step immunoadsorbent procedure. The first immunoadsorbent step using glutaraldehyde-insolubilized MoAb 140.240 (ascites fluid) resulted in a 13-fold enrichment with 93% recovery in the bound material. In the second immunoadsorbent step constructed by the purified IgG2a of MoAb 140.240 (culture fluid) coupled to CNBr-activated Sepharose 4B the bound material from the first step was further purified resulting in a 330-fold purification with 90% recovery. SDS-polyacrylamide gel electrophoretic analysis of the final purified material revealed a single band migrating as a polypeptide with an approximate molecular weight of 87 Kd, consistent with the size of the molecule immunoprecipitated by MoAb 140.240 from lysates of radiolabelled melanoma cells. Preliminary amino acid analysis indicates a particularly high proportion of phenylalanine in gp87. We have also compared gp87 with two well defined antigens, HLA-A,B,C (integral membrane protein) and "94K" melanoma/carcinoma-associated antigen (peripheral membrane protein) with respect to antigen extractability from melanoma cells using phosphate-buffered saline, 0.1 M urea, 3 M NaCl, or nonionic detergent (NP-40). The results showed that whereas 94K antigen was extractable by each of the four different solutions, gp87, similar to HLA-A,B,C antigens, could only be extracted with NP-40, strongly suggesting that gp87 is an integral melanoma cell component.