Blot Overlay Assays Research Articles

The relationship between perlecan and dystroglycan and its implication in the formation of the neuromuscular junction.

Perlecan is a major heparan-sulfate proteoglycan (HSPG) within the basement membrane surrounding skeletal muscle fibers. The C-terminus of its core protein contains three globular domain modules which are also found in laminin and agrin, two proteins that bind to dystroglycan (DG, cranin) on the muscle surface with these modules. In this study, we examined whether perlecan can also bind to DG and is involved in signaling the formation of the neuromuscular junction (NMJ). By labeling cultured muscle cells with a polyclonal anti-perlecan antibody, this protein is found both within the extracellular matrix in a fibrillar network and at the cell surface in a punctate pattern. In Xenopus muscle cells, the cell-surface perlecan is precisely colocalized with DG. Both perlecan and DG are clustered at ACh receptor clusters induced by spinal neurons or by beads coated with HB-GAM, a heparin-binding growth factor. Blot overlay assays have shown that perlecan binds alpha-DG in a calcium and heparin-sensitive manner. Furthermore, perlecan is present in muscle lysate immunoprecipitated with an anti-DG antibody. Immunolabeling also showed colocalization between HB-GAM and perlecan and between HB-GAM and DG. These data suggest that perlecan is anchored to muscle surface via DG-dystrophin complex. Since DG is also a site of agrin binding, the neural agrin secreted by motoneurons during NMJ formation may compete with the pre-existing perlecan for cell surface binding. This competition may result in the presentation of perlecan-bound growth factors such as HB-GAM to effect synaptic induction.

Characterization of a 30-kDa Peripheral Nerve Glycoprotein That Binds Laminin and Heparin

We have shown previously that a bovine peripheral nerve protein with a molecular mass of about 30 kDa binds laminin in blot overlay assay. In this paper, we have characterized this 30-kDa laminin-binding protein (LBP30). LBP30 was extracted from the crude bovine peripheral nerve membranes at pH 12 or by 0.5 M NaCl but not by 2% Triton X-100. LBP30 bound to heparin-Sepharose in the presence of 0.5 M NaCl. The results of lectin staining indicated that LBP30 contained both terminally sialylated and nonsialylated Ser/Thr-linked oligosaccharides. LBP30 bound laminin-2 as well as laminin-1 but not fibronectin or collagen type IV. When immobilized LBP30 was incubated with the crude peripheral nerve membrane extracts, all of the endogenous peripheral nerve laminin chain isoforms, the alpha1, alpha2, beta1, beta2, and gamma1 chains, were detected bound to LBP30. The binding of LBP30 to laminin was inhibited by heparin, heparan sulfate, dextran sulfate, or NaCl but was not affected significantly by chondroitin sulfate, dextran, or EDTA. Although LBP30 bound to laminin-1 denatured with SDS in a nonreducing condition, the binding was reduced drastically when laminin-1 was denatured with SDS in a reducing condition, suggesting that the binding of LBP30 is somewhat dependent on the high order structure of laminin-1. Immunohistochemical analysis demonstrated the broad distribution of LBP30 in the perineurium and endoneurium of bovine peripheral nerve. These results indicate that LBP30 is a laminin- and heparin-binding glycoprotein localized in the perineurium and endoneurium of bovine peripheral nerve.

The PDZ Domain of Human Erythrocyte p55 Mediates Its Binding to the Cytoplasmic Carboxyl Terminus of Glycophorin C

The PDZ domain, also known as the GLGF repeat/DHR domain, is an approximately 90-amino acid motif discovered in a recently identified family of proteins termed MAGUKs (membrane-associated guanylate kinase homologues). Sequence comparison analysis has since identified PDZ domains in over 50 proteins. Like SH2 and SH3 domains, the PDZ domains mediate specific protein-protein interactions, whose specificities appear to be dictated by the primary structure of the PDZ domain as well as its binding target. Using recombinant fusion proteins and a blot overlay assay, we show that a single copy of the PDZ domain in human erythrocyte p55 binds to the carboxyl terminus of the cytoplasmic domain of human erythroid glycophorin C. Deletion mutagenesis of 21 amino acids at the amino terminus of the p55 PDZ domain completely abrogates its binding activity for glycophorin C. Using an alanine scan and surface plasmon resonance technique, we identify residues in the cytoplasmic domain of glycophorin C that are critical for its interaction with the PDZ domain. The recognition specificity of the p55 PDZ domain appears to be unique, since the three PDZ domains of hDlg (human lymphocyte homologue of the Drosophila discs large tumor suppressor) do not bind the cytoplasmic domain of glycophorin C. Taken together with our previous studies, these results complete the identification of interacting domains in the ternary complex between p55, glycophorin C, and protein 4.1. Implications of these findings are discussed in terms of binding specificity and the regulation of cytoskeleton-membrane interactions.

In vitro expressed dystrophin fragments do not associate with each other

Dystrophin, a component of the muscle membrane cytoskeleton, is the protein altered in Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD). Dystrophin shares significant homology with other cytoskeletal proteins, such as α-actinin and spectrin. On the basis of its sequence similarity with α-actinin and spectrin, dystrophin has been proposed to function as dimer. However, the existence of both dimers and monomers have been observed by electron microscopy. To address this apparent discrepancy, we expressed dystrophin fragments composed of different domains in an in vitro translation system. The expressed fragments were tested for their ability to interact with each other and full-length dystrophin by both immunoprecipitation and blot overlay assays. These assays were successfully used to demonstrate the dimerization of α-actinin and spectrin, yet failed to detect any interaction between dystrophin fragments. Although these in vitro results do not prove that dystrophin is not a dimer in vivo, they do indicate that this interaction is not like that of the α-actinin and spectrin.

Analysis of Small GTP-Binding Proteins of the Lens by GTP Overlay Assay Reveals the Presence of Unique GTP-Binding Proteins Associated with Fiber Cells

Low molecular weight GTP-binding proteins are molecular switches which are thought to play pivotal roles in cell growth, differentiation, cytoskeletal organization and vesicular trafficking. In this study, members of this family of proteins have been identified and characterized in the eye lens, for the first time. [α-32P]GTP blot overlay assays of monkey and human lens water soluble and membranous insoluble fractions revealed the presence of specific GTP-binding proteins in the range of 20–30 kDa (small GTPases) in both fractions, with much higher amounts in the membranous insoluble fraction. In the insoluble fraction, in addition to 20–30 kDa GTPases, there are three distinct GTP-binding proteins, ranging from 33–45 kDa. The small GTPases (20–30 kDa) were present throughout the lens in epithelium, cortex and nucleus, while the 33–45 kDA GTP-binding protein bands were exclusively associated with the cortex and nucleus (fiber cells). Analysis of lens fractions by two-dimensional electrophoresis, immunoprecipitation using monoclonal and sequence specific polyclonal antibodies and C3 exoenzyme mediated ADP-ribosylation demonstrated the presence of Ras, Rap, Rho, Rac, Rab and several other small GTPases. The 33–45 kDa GTP-binding proteins that are associated with lens fiber cells appear to be distinct from the small GTPases and from heterotrimeric GTPases, and were not detected in brain or heart tissue. The presence of different complements of GTP-binding proteins in lens fibers and epithelial cells suggests their involvement in important regulatory functions, possibly related to cell growth, differentiation and organization of the cytoskeleton.

Pathogenesis of Acanthamoeba keratitis: carbohydrate-mediated host-parasite interactions.

Acanthamoeba keratitis is a sight-threatening corneal infection. In a recent study, the saccharide mannose has been shown to inhibit the binding of Acanthamoeba organisms to the epithelium of the cornea (L. D. Morton, G. L. McLaughlin, and H. E. Whiteley, Infect. Immun. 59:3819-3822, 1991). In an attempt to determine the molecular mechanism by which acanthamoebae adhere to the surface of the cornea, the present study was designed to determine whether Acanthamoeba castellanii derived from an infected human cornea (i) binds to mannose-containing glycoproteins (mannose-GPs) of corneal epithelium and (ii) expresses one or more mannose-binding proteins. Mannose-GPs of primary cell cultures of rabbit corneal epithelium were isolated by using three different agarose-conjugated, mannose-specific lectins. By electrophoresis blot-overlay assays, 35S-labeled acanthamoebae were shown to bind to mannose-GPs of corneal epithelium and to a neoglycoprotein, mannose-bovine serum albumin (mannose-BSA). 35S-labeled acanthamoebae also bound to microtiter wells coated with mannose-BSA in a concentration-dependent manner. The binding of amoebae to mannose-GPs was blocked by free methyl-alpha-D-mannopyranoside. The parasites did not bind to galactose-BSA or to many other proteins lacking mannose residues. A membrane-associated mannose-binding protein (136 kDa) of A. castellanii was isolated by affinity chromatography of detergent extracts of unlabeled parasites and of cell surface biotin-labeled parasites on a p-aminophenyl alpha-D-mannopyranoside-agarose column. The affinity-purified protein of the amoeba was shown to bind specifically to mannose-BSA. In summary, a mannose-binding protein is present on the surface membranes of Acanthamoeba, and corneal epithelial cells express Acanthamoeba-reactive GPs. One of the mechanisms of Acanthamoeba adhesion to the corneal surface may involve interactions between the mannose-binding protein of Acanthamoeba and mannose-GPs on the surface of corneal epithelium.

Identification of hydrolase binding activities of the acrosomal matrix of hamster spermatozoa.

The interior of the mammalian sperm acrosome contains a structural framework, the acrosomal matrix, that may regulate both the distribution of hydrolases within the acrosome and their release during the acrosome reaction. To define the biochemical basis of this interaction, we examined the binding of two acrosomal hydrolase, proacrosin and N-acetylglucosaminidase (NAGA), to a purified acrosomal matrix fraction of hamster spermatozoa. Proacrosin-acrosin was chromatographically purified from acid extracts of hamster spermatozoa and consisted of four size variants of 50 kDa, 49 kDa, 45 kDa, and 43 kDa. Each of the four isoforms exhibited the same N-terminal amino acid sequence through 16 residues, suggesting that they may be modified by cleavage at the C-terminus. Polyclonal antiserum against the proacrosin isoforms specifically binds the acrosomal cap as shown by immunofluorescence microscopy. Neither proacrosin nor NAGA were solubilized when sperm were permeabilized with Triton X-100 under low ionic strength conditions; however, both hydrolases were releases by extraction with Triton X-100 containing 0.5 M NaCl. An acrosomal matrix fraction isolated under low ionic strength conditions retained bound proacrosin-acrosin and NAGA, and both hydrolases were released from the matrix by subsequent high-salt extraction. After high-salt treatment, the acrosomal matrix retained specific binding sites for both proacrosin and NAGA. In a blot overlay assay, a set of acrosomal matrix polypeptides between 29 kDa and 24 kDa specifically bound proacrosin. These data suggest that specific interactions between acrosomal matrix polypeptides and hydrolases represent a mechanism to sequester hydrolases within the acrosome and to regulate their release during the acrosome reaction.

Adherence to lipids and intestinal mucin by a recently recognized human pathogen, Campylobacter upsaliensis.

Campylobacter upsaliensis is a recently recognized human enteric pathogen associated with enteritis, colitis, bacteremia, and sepsis. Very little is known about the mechanisms of pathogenesis of this organism. The goals of this study were to determine whether C. upsaliensis binds to epithelial cells and whether there are specific lipid molecules that might serve as cell membrane receptors. In addition, we also explored C. upsaliensis binding to purified human small-intestinal mucin, since the mucus gel overlying the epithelium provides an initial contact surface for the bacteria and must be penetrated for the organisms to reach their cell receptors. Binding of C. upsaliensis to model epithelial cells was shown by microscopy adhesion assays, and binding to lipids was detected by thin-layer chromatography-overlay assays. Bacteria bound to phosphatidylethanolamine (PE), gangliotetraosylceramide (Gg4), and, more weakly, to phosphatidylserine (PS). There was no binding to ceramide, cholesterol, phosphatidylcholine, and globosides. Using receptor-based microtiter well immunoassays, we observed binding to be equal, specific, and saturable for PE and Gg 4 but low and nonspecific for PS. At least five bacterial surface proteins (50 to 90 kDa) capable of PE binding were identified by a lipid-silica affinity column technique. In slot blot overlay assays, biotin-labeled C. upsaliensis also bound in a concentration-dependent fashion to purified human small-intestinal mucin, implying that these microorganisms also express an adhesin(s) recognizing a specific mucin epitope(s). We speculate that binding to mucins may influence access of the bacteria to cell membrane receptors and thereby influence host resistance to infection.

N-tropomodulin: a novel isoform of tropomodulin identified as the major binding protein to brain tropomyosin.

We have identified and characterized two proteins in rat brain that bind to the neuron-specific tropomyosin isoform, TMBr3. The two proteins were identified by blot overlay assay, in which the proteins immobilized on the membrane were probed by epitope-tagged TMBr3, followed by detection with anti-epitope antibody. We have purified these proteins using a TMBr3 affinity column. Peptide sequencing as well as immunoblotting showed that one of the two proteins is identical to tropomodulin, a tropomyosin-binding protein originally identified in erythrocytes. The cDNA for the other protein was cloned from an adult rat brain cDNA library using degenerate oligonucleotides that we designed based on the peptide sequences. Sequence analysis of the cDNA clone revealed this protein to be a novel isoform of tropomodulin which is the product of a distinct gene, and is herein referred to as N-tropomodulin. Recombinant N-tropomodulin bound to TMBr3 as well as to other low molecular mass tropomyosins (TM5a or TM5), but not to high molecular mass tropomyosins (TM2 or TMBr1). Northern blotting and RNase protection assays as well as immunoblotting showed that N-tropomodulin is expressed predominantly in brain. Furthermore, RNase protection assays revealed no alternatively spliced regions within the coding sequence. Developmentally, N-tropomodulin was detected in rat brain as early as embryonic day 14 and reaches the adult level before birth. Immunofluorescence of primary frontal cortex cell cultures showed that N-tropomodulin is specifically expressed in neurons. The neuron-specific expression of N-tropomodulin strongly suggests specialized roles of this TM-binding protein in neurons.

Interaction of Kinesin Motor Domains with α- and β-Tubulin Subunits at a Tau-independent Binding Site: REGULATION BY POLYGLUTAMYLATION

Interaction of rat kinesin and Drosophila nonclaret disjunctional motor domains with tubulin was studied by a blot overlay assay. Either plus-end or minus-end-directed motor domain binds at the same extent to both alpha- and beta-tubulin subunits, suggesting that kinesin binding is an intrinsic property of each tubulin subunit and that motor directionality cannot be related to a preferential interaction with a given tubulin subunit. Binding features of dimeric versus monomeric rat kinesin heads suggest that dimerization could drive conformational changes to enhance binding to tubulin. Competition experiments have indicated that kinesin interacts with tubulin at a Tau-independent binding site. Complementary experiments have shown that kinesin does not interact with the same efficiency with the different tubulin isoforms. Masking the polyglutamyl chains with a specific monoclonal antibody leads to a complete inhibition of kinesin binding. These results are consistent with a model in which polyglutamylation of tubulin regulates kinesin binding through progressive conformational changes of the whole carboxyl-terminal domain of tubulin as a function of the polyglutamyl chain length, thus modulating the affinity of tubulin for kinesin and Tau as well. These results indicate that microtubules, through tubulin polymorphism, do have the ability to control microtubule-associated protein binding.

Coimmunoprecipitation of a 24-kDa protein with NHE1, the ubiquitous isoform of the Na+/H+ exchanger.

Ancillary proteins have been proposed to account for phosphorylation-independent regulation of the Na+/H+ exchanger (NHE), but such putative proteins have not been identified. Here we describe the specific association of NHE1 with a protein of approximately 24 kDa (p24). Immunoprecipitation of NHE1 from lysates of [35S] cysteine- and/or methionine-labeled cells with the use of an anti-NHE1 antibody demonstrated specific coimmunoprecipitation of p24 with NHE1. The stoichiometry of p24 relative to NHE1, assessed by their radiolabel content, was consistent between experiments and among cell types. Immunoblotting demonstrated that p24 is not a proteolytic product of NHE1. Internal deletion mutants and chimeras of NHE1/NHE3 suggest that p24 binds to residues 515-566 or 695-815 of NHE1 or to the transmembrane region of both NHE1 and NHE3. Protein p24 is not constitutively phosphorylated nor could phosphorylation be induced by serum or phorbol ester treatment. Binding of p24 to NHE1 is Ca2+ independent. Protein p24 failed to bind [gamma-32P]GTP in a blot-overlay assay, suggesting that it is not a low-molecular-weight GTP-binding protein. Identification of the p24:NHE1 interaction may contribute to our understanding of antiporter regulation.

Cytoplasmic dynein binds dynactin through a direct interaction between the intermediate chains and p150Glued.

Cytoplasmic dynein is a retrograde microtubule motor thought to participate in organelle transport and some aspects of minus end-directed chromosome movement. The mechanism of binding to organelles and kinetochores is unknown. Based on homology with the Chlamydomonas flagellar outer arm dynein intermediate chains (ICs), we proposed a role for the cytoplasmic dynein ICs in linking the motor protein to organelles and kinetochores. In this study two different IC isoforms were used in blot overlay and immunoprecipitation assays to identify IC-binding partners. In overlays of complex protein samples, the ICs bound specifically to polypeptides of 150 and 135 kD, identified as the p150Glued doublet of the dynactin complex. In reciprocal overlay assays, p150Glued specifically recognized the ICs. Immunoprecipitations from total Rat2 cell extracts, rat brain cytosol, and rat brain membranes further identified the dynactin complex as a specific target for IC binding. using truncation mutants, the sites of interaction were mapped to amino acids 1-123 of IC-1A and amino acids 200-811 of p150Glued. While cytoplasmic dynein and dynactin have been implicated in a common pathway by genetic analysis, our findings identify a direct interaction between two specific component polypeptides and support a role for dynactin as a dynein "receptor". Our data also suggest, however, that this interaction must be highly regulated.

Purification of developmentally regulated avian 400-kDa intermediate filament associated protein. Molecular interactions with intermediate filament proteins and other cytoskeleton components.

IFAPa-400, a 400-kDa developmentally regulated protein thought to be associated with intermediate filaments, has been purified from chick embryo hearts to investigate its interaction with vimentin and other IF proteins and to identify other cellular components to which this cytoskeletal protein associates. Previous studies suggested that this protein was associated with the vimentin-containing intermediate filament lattice of myoblasts and neuroblasts before their terminal differentiation, providing these cells with a particular intermediate filament cytoskeleton that could satisfy specific mechanical requirements during their intense morphogenetic activities. Although IFAPa-400 partially reassociated with vimentin and desmin in disassembly-reassembly experiments using crude IF preparations from chick embryo hearts, in vitro recombination of purified IFAPa-400 with vimentin and desmin failed to demonstrate any direct association. When purified IFAPa-400 was used as a probe in blot overlay assays, however, specific binding to vimentin and desmin was observed, providing the first evidence of a physical association between IFAPa-400 and intermediate filament proteins. The blot overlay experiments also demonstrated that IFAPa-400 binds to two unidentified polypeptides of 19 and 32 kDa. These results are thus consistent with the hypothesis that a structural lattice requiring a vimentin-IFAPa-400 combination constitutes the intermediate filament system of myogenic and neurogenic cells.

Identification of a band-3 binding site near the N-terminus of erythrocyte membrane protein 4.2.

Protein 4.2 (P4.2) is a major component of the erythrocyte plasma membrane accounting for approx. 5% of total membrane protein. The major membrane binding site for P4.2 is contained within the cytoplasmic domain of band 3 (cdb3), although the precise location of the cdb3 binding site is not known. To identify the cdb3 binding site, we used synthetic P4.2 peptides (15-mers) that spanned the entire 721-amino-acid large isoform of P4.2, and determined the binding of these peptides to cdb3 in an in vitro binding assay. One peptide, P8 (L61FVRRGQPFTIILYF), bound strongly to cdb3 and four others bound less strongly (P22, L271LNKRRGSVPILRQW; P27, G346EGQRGRIWIFQTST; P41, L556WRKKLHLTLSANLE; P48, I661HRERSYRFRSVWPE). These peptides have in common a cluster of two or three basic amino acid residues (arginine or lysine), in a region without nearby acidic residues. Cdb3 bound saturably to P8 with a Kd of 0.16 microM and a capacity of 0.56 mol of cdb3 monomer/mol of P8. Use of overlapping synthetic peptides further defined the cdb3 site as being contained within V63RRGQPFTIILYF. Replacement of R64R with R64G, G64R or G64G almost completely abolished cdb3 binding, suggesting that R64R is essential for cdb3 binding. P8 competitively inhibited binding of purified human erythrocyte P4.2 to cdb3. In blot overlay assays, cdb3 bound to a 23 kDa N-terminal P4.2 tryptic peptide containing V63RRGQPFTIILYF but not to other P4.2 tryptic peptides lacking this site. The V63RRGQPFTIILYF site is highly conserved in mouse and human erythrocyte P4.2 as well as between P4.2 and transglutaminase proteins, which are evolutionarily related to P4.2.

Down-regulation of a 67-kDa YIGSR-binding Protein upon Differentiation of Human Neuroblastoma Cells

Differentiated human neuroblastoma LA-N1 cells that were exposed to dibutyryl adenosine 3',5'-cyclic monophosphate for 5 days (primed cells) showed increased adhesion to laminin-, fibronectin-, and collagen type I-coated plates as compared to unprimed cells. Moreover, primed cells seemed to adhere best to laminin. The binding site in laminin, mediating cell attachment, was identified as containing the YIGSR sequence, a known cell binding motif, located in the short arm of the B1 chain of laminin. The synthetic peptide amide, C(YIGSR)3-NH2, containing a repeat of this binding motif, inhibited the attachment of neuroblastoma cells to laminin in a competitive manner, and its inhibitory activity was inversely dependent on laminin concentrations. Affinity chromatography of membrane-extracted proteins over an Affi-Gel 10 column conjugated to C(YIGSR)3-NH2, revealed a major YIGSR-binding protein with an apparent molecular mass of 67 kDa. The 67-kDa surface membrane protein was specifically eluted from the column with the soluble C(YIGSR)3-NH2 peptide, but not with an unrelated peptide. Furthermore, no 67-kDa laminin-binding protein was recovered from an unrelated peptide matrix with the free C(YIGSR)3-NH2 peptide. Ligand blot overlay assays with biotin-labeled C(YIGSR)3-NH2 peptide demonstrated that the 67-kDa receptor is indeed a YIGSR-binding protein. This 67-kDa laminin-binding protein appeared to be down-regulated upon differentiation of LA-N1 cells, as indicated by the level of this protein and its mRNA.

Macromolecular interactions in the nucleoporin p62 complex of rat nuclear pores: binding of nucleoporin p54 to the rod domain of p62.

Nuclear pore complexes are constructed from a large number of different proteins, called collectively nucleoporins. One of these nucleoporins, p62, has an alpha-helical coiled-coil COOH-terminal rod domain linked to an NH2-terminal domain that contains a series of degenerate pentapeptide repeats. In nuclear pores p62 forms a tight complex with at least two other proteins, p58 and p54, which can be extracted from isolated rat liver nuclei (Finlay, D. R., E. Meier, P. Bradley, J. Horecka, and D. J. Forbes. 1991. J. Cell Biol. 114:169-183). We have used a range of methods to demonstrate a strong binding between p62 and p54 in this complex and show that the rod domain of p62 appears to constitute the principal binding site for p54. Whole p62 and its rod domain expressed in Escherichia coli both bind strongly to p54 in blot-overlay assays. Most of the epitopes on the p62 rod recognized by polyclonal antisera are masked in the complex, whereas epitopes on the NH2-terminal domain of p62 are still exposed, both in the isolated complex and also in nuclear pores stained in situ by immunofluorescence in isolated rat nuclei. Moreover, it has been possible to exchange recombinant p62 rod for some of the native p62 in complexes partially dissociated by 4 M urea. Overall these results suggest a key role for the p62 rod domain in maintaining the structural integrity of the complex and also suggest a molecular model for the complex. This model is consistent with data that indicate that the analogous coiled-coil region of yeast nucleoporin NSP1 may function in a similar way.

The Drosophila lethal(2)giant larvae tumor suppressor protein forms homo-oligomers and is associated with nonmuscle myosin II heavy chain.

Inactivation of the Drosophila lethal(2)giant larvae (l(2)gl) gene causes malignant tumors in the brain and the imaginal discs and produces developmental abnormalities in other tissues, including the germline, the ring gland and the salivary glands. Our investigations into the l(2)gl function have revealed that the gene product, or p127 protein, acts as a cytoskeletal protein distributed in both the cytoplasm and on the inner face of lateral cell membranes in a number of tissues throughout development. To determine whether p127 can form oligomers or can stably interact with other proteins we have analyzed the structure of the cytosolic form of p127. Using gel filtration and immunoaffinity chromatography we found that p127 is consistently recovered as high molecular weight complexes that contain predominantly p127 and at least ten additional proteins. Blot overlay assays indicated that p127 can form homo-oligomers and the use of a series of chimaeric proteins made of segments of p127 fused to protein A, which alone behaves as a monomer, showed that p127 contains at least three distinct domains contributing to its homo-oligomerization. Among the proteins separated from the immuno-purified p127 complexes or isolated by virtue of their affinity to p127, we identified one of the proteins by microsequencing as nonmuscle myosin II heavy chain. Further blot overlay assay showed that p127 can directly interact with nonmuscle myosin II. These findings confirm that p127 is a component of a cytoskeletal network including myosin and suggest that the neoplastic transformation resulting from l(2)gl gene inactivation may be caused by the partial disruption of this network.

Characterization of the prenylated protein methyltransferase in human endometrial carcinoma

The processing or ras and of other GTP-binding proteins includes a final reversible step in which the carboxy terminal prenylated cysteine is methylated by the enzyme prenylated protein methyltransferase (PPMTase). The significance of this modification and of the role of PPMTase in human tumors has yet to be fully elucidated. Here we characterize the PPMTase of human endometrical carcinomas (tumors in which the frequency of ras gene mutations is relatively high) and compare it to the PPMTase of the normal endometrium. Our results show that in both types of tissues the enzyme is bound to the membranes. It can utilize synthetic substrate such as N- acetyl-S-farnesyl- l-cysteine (K m = 18–20 μ M) and is blocked by the PPMTase inhibitor S-farnesylthioacetic acid ( K i = 2 μM). In vitro methylation assays and [ α- 32P]GTP blot-overlay assays showed that the major endogenous PPMTase substrates are small GTP-binding proteins. Methylation of these proteins in vitro is blocked by farnesylthioacetic acid. The kinetic properties of PPMTase from the carcinomas and the normal tissues are very similar. however, levels of PPMTase activity (but not of its endogenous substrates) are higher in the carcinomatous endometrium than in the normal one. The elevated enzyme activity is restricted to the crude mitochondrial fraction (8.0 ± 0.4 vs. 5.4 ± 0.1 pmol N-acetyl farnesylcysteine methyl ester formed/min/mg protein by the carcinoma and by the normal endometrial preparations, respectively). As this fraction is enriched in plasma membranes, it appears that the elevated enzyme activity could be related to ras protein methylation; if so, selective methylation blockers might inhibit the growth of endometrial carcinomas.

A reverse gyrase with an unusual structure. A type I DNA topoisomerase from the hyperthermophile Methanopyrus kandleri is a two-subunit protein

Reverse gyrase, an ATP-dependent topoisomerase that positively supercoils DNA, has been purified to near-homogeneity from the hyperthermophile Methanopyrus kandleri. It migrates on SDS-polyacrylamide gel electrophoresis as two principal bands with apparent molecular masses of 150 and 50 kDa. Both proteins remain associated throughout all chromatographic steps. Transfer of a radioactive phosphate from DNA to the 50-kDa protein and gel retardation experiments indicate that this protein forms the covalent complex with DNA. A blot overlay assay identifies the 150-kDa protein as the potential ATPase. This is the first evidence that a reverse gyrase can be a topoisomerase consisting of two protomers. In analogy with the DNA gyrase A subunit (DNA breakage and reunion activity) and the B subunit (ATPase), the 50- and 150-kDa components of Mka reverse gyrase have been designated the A and B subunits, respectively. Methanopyrus reverse gyrase changes DNA linking number in steps of one and its A subunit covalently binds to the 5'-DNA phosphoryl group. It nicks DNA at sites that predominantly have a cytosine at the -4-position. The same rule was derived previously for monomeric reverse gyrase from sulfur-metabolizing hyperthermophiles and for topoisomerase I from mesophilic bacteria. Based on these results, Mka reverse gyrase is classified as belonging to group A of type I topoisomerases. The structural diversity of type I group A topoisomerases parallels the diversity of type II enzymes and suggests the evolution of an essential function by gene fusion.

Protein Kinase C Domains Involved in Interactions with Other Proteins

We have used a blot overlay assay to detect protein kinase C (PKC) interactions with other proteins. In many cases, the PKC binding proteins are also PKC substrates [Chapline et al. (1993) J. Biol. Chem. 268, 6858]. The purpose of the current studies was to characterize the PKC domains involved in the interactions with other proteins. alpha, beta, and epsilon isoforms of PKC interact with the same binding proteins in fibroblast cell extracts. These results indicate that constant rather than isozyme-specific (variable) regions are the major determinants of the interactions studied. PKC binding required phosphatidylserine (PS), indicating that the PS binding regulatory domain of PKC is involved in the interactions. The PKC pseudosubstrate peptide sequence, which is contained within the regulatory domain, also showed PS-dependent binding to the PKC binding proteins. To further investigate the role of the pseudosubstrate peptide in promoting PKC-protein interactions, an N-terminal truncation mutant lacking the pseudosubstrate sequence was prepared. Binding of the mutant alpha-PKC was diminished compared to wild-type alpha-PKC, although some binding was still apparent. These results indicate that the pseudosubstrate sequence contributes to, but is not the sole determinant of, PKC binding activity.