Identification of a proacrosin precursor in the cell-free translation of boar testicular poly(A)(+)-mRNA.

A cell-free translation system was used to determine the molecular mass of the protein component of precursor(s) to boar proacrosin. Poly(A)(+)-mRNA was extracted from freshly excised boar testis into phenol/chloroform, precipitated in chilled (-20 degrees C) ethanol, then translated in a cell-free, reticulocyte lysate system with Tran 35S-label. Analysis of the resulting products by SDS-PAGE followed by autoradiography demonstrated multiple bands of translated proteins. Both Western blotting and immunoprecipitation with a specific polyclonal antibody to boar proacrosin yielded a single major band with a relative molecular weight of approximately 64,000. These results suggest that proacrosin (Mr = 53,000-55,000), which contains both protein and carbohydrate moieties, results from the cellular processing of a proacrosin precursor molecule.     

Identification and purification of a kidney membrane protein which specifically binds the amino-terminal domain of native parathyroid hormone

Nitrocellulose blots of bovine kidney membrane proteins were prepared from denaturing polyacrylamide gels. Strips of the blots were incubated with parathyroid hormone (PTH), washed, and then incubated with antisera against the hormone. Exposure to horseradish peroxidase-linked second antibody led to staining of a 51-kDa protein. No staining was observed in blots not incubated with PTH. Fragments 35-84 and 19-84 of PTH reacted strongly with the antisera, but did not lead to staining of the 51-kDa protein on the blots. Staining was visible, but greatly reduced, when fragment 9-84 was used. Oxidation of the native hormone at positions 8 and 18 led to reductions in staining of the band which were quantitatively similar to the reductions in biological activity induced by such oxidations. These properties suggested that the 51-kDa protein recognizes the amino-terminal portions of PTH, which is the segment of the molecule required for its biological activities. Several micrograms of the 51-kDa protein were purified to homogeneity by selective extraction from the membranes with detergent and by elution from multiple two-dimensional gels. The purified protein retained its PTH-dependent staining and specificity. This protein may be a PTH receptor or a fragment of a PTH receptor from kidney.     

Biosynthesis of rat insulin-like growth factor II. I. Immunochemical demonstration of a approximately 20-kilodalton biosynthetic precursor of rat insulin-like growth factor II in metabolically labeled BRL-3A rat liver cells

BRL-3A rat liver cells synthesize mature 7484-dalton rat insulin-like growth factor II (rIGF-II) as a approximately 22-kDa precursor, presumably prepro-rIGF-II. In the present study, we have biosynthetically labeled intact BRL-3A cells with [35S]cysteine and immunoprecipitated cell lysates and media with antisera to rIGF-II. A approximately 20-kDa protein was identified in immunoprecipitates of cell lysates having properties consistent with pro-rIGF-II. The approximately 20-kDa protein is precipitated by immune sera but not by nonimmune serum. Its immunoprecipitation is specifically inhibited by unlabeled rIGF-II but not by insulin. It is not precipitated from labeled lysates of a subclone of BRL-3A cells (BRL-3A2) that does not synthesize rIGF-II. The approximately 20-kDa protein is rapidly labeled intracellularly (10 min) but is not detected in BRL-3A media. In pulse-chase experiments, radioactivity in the approximately 20-kDa protein disappears during the chase and appears, at later times, in specifically immunoprecipitated approximately 19-, approximately 10-, approximately 8-, and approximately 7-kDa proteins in media and, to a limited extent, intracellularly. A protein with electrophoretic mobility identical to that of the approximately 20-kDa protein observed in cell lysates is immunoprecipitated from 35S-proteins whose synthesis is directed by BRL-3A RNA in a reticulocyte lysate cell-free translation system supplemented with microsomal membranes, and presumably arises by cotranslational removal of the signal peptide from approximately 22-kDa prepro-rIGF-II. Processing of the approximately 20-kDa protein in intact BRL-3A cells to intermediate and mature rIGF-II species appears to occur at the time of secretion and/or shortly thereafter, with the different forms appearing at approximately the same time.     

Ligand-induced association of surface immunoglobulin with the detergent insoluble cytoskeleton may involve alpha-actinin

B cell surface immunoglobulin (SIg) plays an important role in antigen recognition and cellular activation. Cross-linking of SIg by bivalent antibody converts it into a detergent insoluble state. The resultant SIg may be partially solubilized by incubating the detergent insoluble cytoskeleton in buffers that convert F actin to G actin. Immunoprecipitation of SIg from the detergent soluble fraction of [35S]methionine-labeled B cells results in the co-isolation of 112 kDa, 42 kDa, (actin), and three additional proteins in the 70- to 73-kDa molecular mass range, isoelectric point 4.8 to 5.6. Analysis of anti-Ig immunoprecipitates made after preclearing with anti-alpha-actinin showed complete depletion of the 112-kDa protein, suggesting that the 112-kDa protein is immunologically related to alpha-actinin. These immunoprecipitates also showed partial depletion of 70- to 73-kDa proteins and mu and delta heavy chains. After treatment of a rat B cells with anti-Ig, much of the Ig-associated 112-kDa protein and 70- to 73-kDa proteins became detergent insoluble, concomitant with most of the SIg. The migration of the SIg-associated 112-kDa and 70- to 73-kDa proteins from the detergent soluble fraction to the detergent insoluble fraction after ligand treatment, suggests that these proteins might be involved in linking SIg to the underlying cytoskeleton and could be involved in the transmission of mitogenic signals.     

A novel 49-kilodalton protein from Artemia cross-links microtubules in vitro.

A 49 kilodalton (kDa) protein, previously proposed to cross-link microtubules, was purified to apparent homogeneity from cell-free extracts of the brine shrimp Artemia. When incubated with tubulin under assembly conditions, the purified 49-kDa protein cross-linked the resulting microtubules. Preformed microtubules were also cross-linked when incubated with the 49-kDa protein. Upon centrifugation through sucrose cushions the 49-kDa protein cosedimented with microtubules, suggesting a stable association between the cross-linking protein and tubulin. Such microtubules were interconnected by particles which were circular, bilobed, or elongated in shape. Disruption of microtubule cross-linking and dissociation of the 49-kDa protein from microtubules occurred in the presence of ATP and 5'-adenylyl-imidodiphosphate (AMP-PNP), a nonhydrolyzable analogue of ATP. The 49-kDa protein was moderately resistant to heat, it did not stimulate tubulin assembly, and it did not react with antibodies to neural microtubule-associated proteins (MAPs) and kinesin. These observations indicate that the 49-kDa protein is different from many known MAPs, a conclusion strengthened by the inability of antibodies raised to the 49-kDa protein to recognize these proteins. The amino terminal 15 amino acid residues of the 49-kDa protein were determined by Edman digestion and an antibody raised to this peptide reacted with the 49-kDa protein on Western blots. Microtubule cross-linking was unaffected by the synthetic amino-terminal peptide, even when it was present at a fivefold molar excess over the 49-kDa protein. A search of three protein databanks revealed that the amino terminus of the 49-kDa protein is unique among published sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tropomyosin shares immunologic epitopes with group A streptococcal M proteins

Tropomyosin is an alpha-helical coiled-coil protein with structural similarities to the streptococcal M protein. In order to show serologic cross-reactivity between streptococcal M proteins and tropomyosin, we selected from a panel of murine mAb those which reacted with M proteins and tropomyosins in the ELISA. Western blots were used to study the reactions of each mAb with human and rabbit cardiac and rabbit skeletal tropomyosins. The antibodies were further characterized for their reactions with the additional autoantigens myosin, actin, keratin, and DNA. Five mAb were found which reacted with either PepM5 or ColiM6 protein and tropomyosin in Western blots or ELISA. Two of the tropomyosin positive mAb were also antinuclear antibodies and were inhibited with DNA. In Western blots of cardiac tropomyosins, the mAb reacted with either the 70-kDa dimer of tropomyosin, the 35-kDa monomer, or both. Some differences were observed in the reactions of the mAb with the different tropomyosins in Western blots. The heart cross-reactive epitopes shared between M proteins and tropomyosin were in most instances shared with cardiac myosin. Differences were observed among the reactions of the mAb with the different tropomyosins. This report constitutes the first evidence of serologic cross-reactivity between streptococcal M proteins and tropomyosins.     

Nucleotide sequence and expression of a cDNA encoding chick brain actin depolymerizing factor

Chick brain actin depolymerizing factor (ADF) is a 19-kDa protein that severs actin filaments and binds actin monomers. We have obtained a cDNA encoding ADF by screening a chick embryo lambda gt11 cDNA library with both a rabbit anti-ADF antiserum and two oligonucleotide probes. Several non-full-length clones of 636 bases and one full-length clone of 1886 bases were isolated and sequenced. The full-length cDNA encodes a protein of 165 amino acids with a calculated molecular weight of 18,520. The deduced amino acid sequence shows 73% identity with the porcine brain actin binding protein cofilin. The coding region of the ADF cDNA has been placed in an expression vector, and the resulting protein shows immunoreactivity with an anti-ADF antiserum but not with an anti-cofilin antibody. The expressed ADF has been purified and has an actin depolymerizing activity identical with that of brain ADF. Like cofilin, ADF contains a sequence similar to the nuclear transport signal sequence of the SV40 large T antigen and a calcium/calmodulin-dependent protein kinase II phosphorylation consensus sequence. Northern blots of both embryonic chick brain and muscle RNA revealed two ADF mRNAs of length 2.1 and 0.9 kilobases. Southern blots suggest that the ADF gene is present in a single copy within the chicken genome. ADF contains regions of homology with other actin binding proteins including tropomyosin, gelsolin, and depactin.     

Monocyte-to-macrophage differentiation: synthesis and secretion of a complex extracellular matrix

Although monocyte- and macrophage-derived molecules are known to promote extracellular matrix (ECM) disruption and destabilization, it is less appreciated that they also synthesize molecules contributing to ECM formation, stabilization, and function. We have identified and characterized the synthesis of proteoglycans and related proteins, some not previously known to be associated with macrophages. Proteoglycan extracts of [(35)S]sulfate- and (35)S-trans amino acid-radiolabeled culture media from THP-1 monocytes induced to differentiate by treatment with phorbol myristate acetate revealed three major proteins of ~25, 90, and 100 kDa following chondroitin ABC lyase digestion. The 25-kDa protein was predominant for monocytes, whereas the 90- and 100-kDa proteins were predominant for macrophages. Tandem mass spectrometry identified (i) the 25-kDa core protein as serglycin, (ii) the 90-kDa core protein as inter-α-inhibitor heavy chain 2 (IαIHC2), and (iii) the 100-kDa core as amyloid precursor-like protein 2 (APLP2). Differentiation was also associated with (i) a >500-fold increase in mRNA for TNF-stimulated gene-6, an essential cofactor for heavy chain-mediated matrix stabilization; (ii) a >800-fold increase in mRNA for HAS2, which is responsible for hyaluronan synthesis; and (iii) a 3-fold increase in mRNA for versican, which interacts with hyaluronan. Biochemical evidence is also presented for an IαIHC2-APLP2 complex, and immunohistochemical staining of human atherosclerotic lesions demonstrates similar staining patterns for APLP2 and IαIHC2 with macrophages, whereas serglycin localizes to the underlying glycosaminoglycan-rich region. These findings indicate that macrophages synthesize many of the molecules participating in ECM formation and function, suggesting a novel role for these molecules in the differentiation of macrophages in the development of atherosclerosis.     

Production of a monoclonal antibody to an attachment protein derived from human cementum.

Cementum is the mineralized structure that covers the surface of the roots of teeth; it serves as the attachment site for collagen fibers of adjacent soft connective tissues. Very little is known about how cementum formation is regulated or how it affects other periodontal structures. We have raised a monoclonal antibody that may aid in studies to determine the biology and function of cementum. Mice were immunized with a 55-kDa attachment protein partially purified from human cementum and a monoclonal antibody, H166, was produced. Incubation of tissue sections with this antibody and fluorescein isothiocyanate-conjugated secondary antibody revealed that it immunostains cementum but not dentin, gingiva, or periodontal ligament. Alveolar bone did not bind the antibody, although a few paravascular cells were positive. Long bones, kidney, liver, skin, and several other tissues were negative. Protein fractions separated from cementum extracts by binding to immobilized H166 column contained 55-, 49-, 39-, 29- to 31-, and 23- to 26-kDa components that cross-reacted with the antibody in Western blots; these components were previously shown to be derived from a common precursor. We conclude that the antibody recognizes a group of proteins related to 55-kDa attachment protein in cementum. Our data show that the antibody could serve as a marker for cementum.     

Binary insecticidal crystal protein from Bacillus thuringiensis,strain PS149B1: effects of individual protein components and mixtures in laboratory bioassays

A family of novel binary insecticidal crystal proteins, with activity against western corn rootworm, Diabrotica virgifera virgifera LeConte, was identified from Bacillus thuringiensis Berliner. A binary insecticidal crystal protein (bICP) from B. thuringiensis strain PS149B1 is composed of a 14-kDa protein (Cry34Abl) and a 44-kDaprotein (Cry35Ab1). These proteins have been co-expressed in transgenic maize plants, Zea mays L., and effectively control western corn rootworm larvae under field conditions. Laboratory experiments were conducted to better understand the contribution of each component protein to the in vivo activity of the bICP. The 14-kDa protein is active alone against southern corn rootworm, Diabrotica undecimpunctata howardi Barber, and was synergized by the 44-kDa protein. In mixtures, the concentration of the 14-kDa protein had a greater impact on efficacy than the 44-kDa component. Although both proteins are clearly required for maximal insecticidal activity, laboratory results did not support the formation of a stable, fixed-ratio complex of the two component proteins.     

Actin and tubulin synthesis in monolayer and suspension cultures of murine L cells

The rates of total protein, actin and tubulin synthesis were studied for monolayer (L-929) and suspension (LS) cultures of mouse L cell. Data on pulse 34S-label incorporation into the cellular protein pool show that LS characterized by a short cell cycle have, comparatively to L-929, higher rates of protein synthesis and phosphorylation. According to PAGE data, the level of actin and tubulin synthesis in suspension line exceeds that in monolayer one. The correlation between growth conditions, biosynthetic parameters and dynamics of cytoskeleton is discussed.     

Molecular cloning, bacterial expression and properties of Rab31 and Rab32.

GTP-binding proteins of the Rab family were cloned from human platelets using RT-PCR. Clones corresponding to two novel Rab proteins, Rab31 and Rab32, and to Rab11A, which had not been detected in platelets previously, were isolated. The coding sequence of Rab31 (GenBank accession no. U59877) corresponded to a 194 amino-acid protein of 21.6 kDa. The Rab32 sequence was extended to 1000 nucleotides including 630 nucleotides of coding sequence (GenBank accession no. U59878) but the 5' coding sequence was only completed later by others (GenBank accession no. U71127). Human Rab32 cDNA encodes a 225 amino-acid protein of 25.0 kDa with the unusual GTP-binding sequence DIAGQE in place of DTAGQE. Northern blots for Rab31 and Rab32 identified 4.4 kb and 1.35 kb mRNA species, respectively, in some human tissues and in human erythroleukemia (HEL) cells. Rabbit polyclonal anti-peptide antibodies to Rab31, Rab32 and Rab11A detected platelet proteins of 22 kDa, 28 kDa and 26 kDa, respectively. Human platelets were highly enriched in Rab11A (0.85 microg x mg of platelet protein(-1)) and contained substantial amounts of Rab32 (0.11 microg x mg protein(-1)). Little Rab31 was present (0.005 microg x mg protein(-1)). All three Rab proteins were found in both granule and membrane fractions from platelets. In rat platelets, the 28-kDa Rab32 was replaced by a 52-kDa immunoreactive protein. Rab31 and Rab32, expressed as glutathione S-transferase (GST)-fusion proteins, did not bind [alpha-(32)P]GTP on nitrocellulose blots but did bind [(35)S]GTP[S] in a Mg(2+)-dependent manner. Binding of [(35)S]GTP[S] was optimal with 5 microm Mg(2+)(free) and was markedly inhibited by higher Mg(2+) concentrations in the case of GST-Rab31 but not GST-Rab32. Both proteins displayed low steady-state GTPase activities, which were not inhibited by mutations (Rab31(Q64L) and Rab32(Q85L)) that abolish the GTPase activities of most low-M(r) GTP-binding proteins.     

Studies on Trypanosomatid Actin I. Immunochemical and Biochemical Identification

In this study, the presence of actin in cultured trypanosomatids was investigated using polyclonal antibodies to heterologous actin. Polyclonal antisera to rabbit muscle actin and a monospecific anti-actin antibody react with a 43-kDa polypeptide in extracts of Trypanosoma cruzi, Herpetomonas samuelpessoai and Leishmania mexicana amazonensis on protein immunoblots. The 43-kDa polypeptide co-migrates with skeletal muscle actin and is retained within trypanosomatid cytoskeletons. Attempts to isolate H. samuelpessoai actin through DNase I affinity chromatography showed that the 43-kDa polypeptide did not bind to the column. Instead, low yields of a 47-kDa polypeptide were obtained indicating that the trypanosomatid actin displays unusual DNase I binding behavior when compared to actins from higher eukaryotes. Immunofluorescence studies confirmed that cytoskeletons retain the actin-like protein. In H. samuelpessoai , actin is localized in the region close to the flagellum, whereas in T. cruzi it is more homogeneously distributed. The data presented here show that trypanosomatid actin displays biochemical characteristics similar to actins of other protozoa.     

A 50-kDa cytosolic protein complexed with the 90-kDa heat shock protein (hsp90) is the same protein complexed with pp60v-src hsp90 in cells transformed by the Rous sarcoma virus.

We have previously shown that a 50-kDa protein is one component of a heteromeric complex immunoprecipitated by the 90-kDa heat shock protein (hsp90) monoclonal antibodies 8D3 and 3G3 (Perdew, G. H., and Whitelaw, M. L. (1991) J. Biol. Chem. 266, 6708-6713). In this report, we compare the 50-kDa protein with that found in pp60v-src-hsp90-p50 complexes immunoprecipitated from Rous sarcoma virus-transformed cells with antibodies to pp60v-src. 35S- and 32P-labeled p50 proteins from each system were identical in their mobilities by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. The profile of N-chlorosuccinimide cleavage products derived from each 32P-labeled p50 protein were also identical when resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have developed a mouse monoclonal antibody, 3M/1B5p50, capable of detecting p50 on Western blots. This antibody detected the 50-kDa protein which co-purified with the pa104 pp60v-src mutant of the avian sarcoma virus oncoprotein in 44A rat fibroblasts. We did not detect p50 in association with native glucocorticoid receptor in L cells or with the overexpressed glucocorticoid receptor in Chinese hamster ovary cells. Two experiments utilizing immunochemical staining implied that essentially all cytosolic p50 is associated with hsp90. Firstly, immunoprecipitating hsp90 from Hepa 1 cytosol with monoclonal antibody 3G3 left the cytosol depleted of p50. Secondly, cytosol fractionated by sucrose gradient revealed that p50 cosedimented with hsp90, confirming the existence of p50 only in association with hsp90.     

Human trophoblast glycoproteins defined by monoclonal antibody 1D2

A cell surface antigen has been defined by a monoclonal antibody 1D2, raised following immunisation with lectin-purified syncytiotrophoblast glycoproteins. 1D2 was nonreactive with any one of 8 common trophoblast proteins in immunodot. Analysis of nonreduced western blots of syncytiotrophoblast microvillous plasma membrane (StMPM) protein indicated that mAb 1D2 was reactive with a series of sialylated proteins with molecular weights of 16-22 kilodaltons. Immunoprecipitates of radiolabelled StMPM protein contained molecules that co-migrated with placental alkaline phosphatase in addition to those identified by western blotting. This set of human trophoblast molecules has not been previously identified by monoclonal antibodies; the antigenicity is widely distributed in human tissues.     

Stretch-induced contractile differentiation of vascular smooth muscle: sensitivity to actin polymerization inhibitors

Signaling mechanisms forstretch-dependent growth and differentiation of vascular smooth musclewere investigated in mechanically loaded rat portal veins in organculture. Stretch-dependent protein synthesis was found to depend onendogenous release of angiotensin II. Autoradiography after[³⁵S]methionine incorporation revealed stretch-dependentsynthesis of several proteins, of which SM22 and actin wereparticularly prominent. Inhibition of RhoA activity by cell-permeant C3toxin increased tissue mechanical compliance and reducedstretch-dependent extracellular signal-regulated kinase (ERK)1/2activation, growth, and synthesis of actin and SM22, suggesting a roleof the actin cytoskeleton. In contrast, inhibition of Rho-associatedkinase by Y-27632 did not reduce ERK1/2 phosphorylation or actin and SM22 synthesis and did not affect tissue mechanical compliance butstill inhibited overall growth. The actin polymerization inhibitors latrunculin B and cytochalasin D both inhibited growth and caused increased tissue compliance. Whereas latrunculin Bconcentration-dependently reduced actin and SM22 synthesis,cytochalasin D did so at low (10⁸ M) but not at high(10⁶ M) concentration. The results show that stretchstabilizes the contractile smooth muscle phenotype. Stretch-dependentdifferentiation marker expression requires an intact cytoskeleton forstretch sensing, control of protein expression via the level ofunpolymerized G-actin, or both.