95 kd sperm proteins bind ZP3 and serve as tyrosine kinase substrates in response to zona binding

In the mouse, the zona pellucida (ZP) glycoprotein ZP3 both binds intact sperm and induces acrosomal exocytosis. The subsequent signaling pathway(s) is still uncertain, but Gi-like proteins have been implicated. By analogy with other signal transduction mechanisms, we examined anti-phosphotyrosine antibody reactivity in mouse sperm. Antibodies reacted with three proteins of 52, 75, and 95 kd. Indirect immunofluorescence localized reactivity to the acrosomal region of the sperm head. The 52 kd and 75 kd phosphoproteins are detected only in capacitated sperm, whereas the 95 kd protein is detected in both fresh and capacitated sperm. For the 95 kd protein, the level of immunoreactivity is not related to sperm motility but is enhanced by both capacitation and sperm interaction with solubilized ZP proteins. In addition, binding of radiolabeled whole ZP or purified ZP3 to blots of separated sperm proteins identified two ZP binding proteins of 95 kd and 42 kd. 95 kd sperm proteins that bind to ZP3 also react with anti-phosphotyrosine antibodies (in a ZP concentration-dependent manner), supporting the idea that the same 95 kd sperm protein serves as a ZP3 receptor and as a tyrosine kinase substrate. These findings and our evidence on acrosome reaction triggering via sperm receptor aggregation suggest that a 95 kd protein in the sperm plasma membrane is aggregated by ZP3, which stimulates tyrosine kinase activity leading to acrosomal exocytosis.     

Purification of the major UsnRNPs from broad bean nuclear extracts and characterization of their protein constituents.

Small nuclear ribonucleoprotein particles containing the five major nucleoplasmic snRNAs U1, U2, U4, U5 and U6 as well as two smaller sized snRNAs were purified from broad bean nuclear extracts by anti-m3G, monoclonal antibody, immunoaffinity chromatography. We have so far defined 13 polypeptides of approximate mol. wts. of 11 kd, 11.5 kd, 12.5 kd, 16 kd, 17 kd, 17.5 kd, 18.5 kd, 25 kd (double band), 30 kd, 31 kd, 35 kd, 36 kd and 54 kd. Upon fractionation of the UsnRNPs by anion exchange chromatography, essentially pure U5 snRNPs were obtained, containing the 11 kd, 11.5 kd, 12.5 kd, 16 kd, 17 kd, 17.5 kd, 35 kd and 36 kd polypeptides. These may therefore represent the common snRNP polypeptides and which may also be present in the other snRNPs. By immunoblotting studies, using anti-Sm sera and mouse monoclonal antibodies we show that the 35 kd and 36 kd proteins are immunologically related to the mammalian common B/B' proteins. The broad bean 16 kd and 17 kd proteins appear to share structural elements with the mammalian D protein. The three proteins of mol. wts. 11 kd, 11.5 kd and 12.5 kd probably represent the broad bean polypeptides E, F, and G. Cross-reactivity of proteins of mol. wts of 30 kd and 31 kd with Anti-(U1/U2)RNP antibodies suggests that they may represent the broad bean A and B" polypeptides. The 54 kd protein and the 18.5 kd protein could be candidates for the U1 specific 70 k and C polypeptides. Our results demonstrate a strong similarity between the overall structure of broad bean and mammalian snRNPs.     

Membrane organization of the dystrophin-glycoprotein complex

The stoichiometry, cellular location, glycosylation, and hydrophobic properties of the components in the dystrophin-glycoprotein complex were examined. The 156, 59, 50, 43, and 35 kd dystrophin-associated proteins each possess unique antigenic determinants, enrich quantitatively with dystrophin, and were localized to the skeletal muscle sarcolemma. The 156, 50, 43, and 35 kd dystrophin-associated proteins contained Asn-linked oligosaccharides. The 156 kd dystrophin-associated glycoprotein contained terminally sialylated Ser/Thr-linked oligosaccharides. Dystrophin, the 156 kd, and the 59 kd dystrophin-associated proteins were found to be peripheral membrane proteins, while the 50 kd, 43 kd, and 35 kd dystrophin-associated glycoproteins and the 25 kd dystrophin-associated protein were confirmed as integral membrane proteins. These results demonstrate that dystrophin and its 59 kd associated protein are cytoskeletal elements that are tightly linked to a 156 kd extracellular glycoprotein by way of a complex of transmembrane proteins.     

Developmental expression of the protein product of Vg1, a localized maternal mRNA in the frog Xenopus laevis.

Vg1 is a maternal mRNA localized in the vegetal cortex of Xenopus laevis oocytes, that encodes a protein homologous to the mammalian growth factor TGF-beta. Using a polyclonal antibody to a T7-Vg1 fusion protein, we have identified the native protein. We find that a single protein of Mr 40 kd is immunoprecipitated following in vitro translation of oocyte poly(A)+ RNA, whilst two proteins of Mr 45 and 43.5 kd are immunoprecipitated from oocyte and embryo extracts. Synthesis of at least the 40 kd, in vitro, and 45 kd, in vivo, proteins is specifically inhibited following treatment of the respective systems with antisense Vg1 (but not histone H4) oligodeoxynucleotides. Tunicamycin treatment reveals the in vivo proteins to be glycosylated versions of a 40 kd protein, modified by the addition of either two or three N-linked oligosaccharide side chains. Both proteins are sensitive to digestion by the enzyme endoglycosidase-H, and are segregated within a membrane fraction from which they can be released by high pH treatment. Their synthesis is first detectable in stage IV oocytes and continues throughout early embryogenesis until the late gastrula. During embryogenesis the relative proportions of the two proteins change, the 45 kd protein being predominant in early embryogenesis and the 43.5 kd protein in late embryogenesis. Synthesis only occurs in the vegetal hemisphere at all stages; however, in the large oocyte diffusion of both proteins into the animal hemisphere occurs.     

Specific proteolysis by fibrinolysin-coagulase from Yersinia pestis of Yersinia pseudotuberculosis outer membrane proteins coded by the Ca(2+)-dependence plasmid]

The pesticinogenicity 9.5 kb plasmid from Yersinia pestis strain EV76 has been marked by the kanamycin phosphotransferase gene inserted into PstI site and designated pP3. The obtained plasmid pP3 determines the synthesis of 45 kd pesticin, alpha and beta-forms of fibrinolysin coagulase (37 and 35 kd) and the 29, 19 and 13 kd proteins in Escherichia coli mini cells. When transferred into Yersinia pseudotuberculosis strain 6933 the plasmid causes the proteolysis of outer membrane proteins. The 150 kd protein is reduced to 138 kd, the 48.5 kd protein is reduced to 45 kd. The proteins secreted into the cultural medium (51 and 38 kd) are also cleaved. The proteolysis of the 150 kd protein was found to occur at the stage of secretion via the inner membrane. The purified fibrinolysin coagulase from Escherichia coli strain JM83 harbouring the plasmid pP3 induces the proteolysis in vitro of the isolated membrane proteins from Yersinia pseudotuberculosis strain 6953 similar to the proteolysis registered in vivo.     

Xenopus laevis sperm proteins, previously identified as surface proteins with egg coat binding capability, are indeed histone H4, histone H3, and sperm specific protein SP2.

Recently, four Xenopus sperm proteins thought to be involved in binding to the egg envelope were identified (Lindsay and Hedrick, J. Exp. Zool., 245:286-293, '88). We have studied the three more abundant ones of apparent molecular weight of 14, 19, and 25 kd in SDS-PAGE. We have shown that these proteins are indeed nuclear basic proteins: the 14 kd is the histone H4, the 19 kd is the histone H3, and the 25 kd is the sperm-specific protein SP2.     

Protein Phosphorylation Induced by Phorbol Esters and Cyclic AMP in Anterior Pituitary Cells: Possible Role in Adrenocorticotropin Release and Synthesis

Forskolin, an activator of adenylate cyclase, stimulates adrenocorticotropin (ACTH) release and increases proopiomelanocortin mRNA levels in anterior pituitary cells by enhancing cyclic AMP (cAMP)-dependent protein kinase activity. The phorbol ester phorbol 12-myristate 13-acetate (PMA) evokes these same responses from anterior pituitary cells by activating protein kinase C. Both protein kinases most likely induce their cellular effects by catalyzing the phosphorylation of specific proteins. To elucidate the mechanisms by which cAMP-dependent protein kinase and protein kinase C promote ACTH secretion and synthesis, the phosphoproteins regulated by forskolin and PMA were identified in the cell line AtT-20, which consists of a homogeneous population of corticotrophs. Phosphoproteins were analyzed in different subcellular fractions by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Forskolin increased phosphate incorporation into two proteins in the cytoplasmic fraction of 24 kilodaltons (kd) (pI 6.8) and 40 kd (pI 5.8), two proteins in the plasma membrane fraction of 32 kd (pI 8.3) and 60 kd (pI 8), and one protein in the nuclear fraction of 20 kd (pI 8.7). Insertion of the inhibitor of cAMP-dependent protein kinase into the AtT-20 cells, using a liposome technique, blocked the rise in phosphate incorporation induced by forskolin. PMA also stimulated phosphate incorporation into proteins in AtT-20 cells. PMA increased the phosphorylation of three cytoplasmic proteins of 25 kd (pI 7.6), 40 kd (pI 5.8), and 40 kd (pI 8.1) as well as two membrane proteins of 32 kd (pI 8.3) and 60 kd (pI 8) and one nuclear protein of 20 kd (pI 6.3).(ABSTRACT TRUNCATED AT 250 WORDS)     

The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species.

Uvomorulin belongs to the group of Ca2+-dependent cell adhesion molecules, which are integral membrane proteins with several structural features in common. In particular, the cytoplasmic part of these proteins is highly conserved in different species, suggesting a common biological function. To test this assumption we transfected a uvomorulin full-length cDNA into uvomorulin-negative mouse NIH 3T3 and L cells. Immunoprecipitations with anti-uvomorulin antibodies detected, in addition to uvomorulin, three independent proteins of 102, 88 and 80 kd which are of host origin and which form complexes with uvomorulin. Using cDNA constructs coding for uvomorulin with cytoplasmic or extracellular deletions it is shown that the 102, 88 and 80 kd proteins complex with the cytoplasmic domain of uvomorulin. Peptide pattern analysis revealed that these three proteins are identical in different mouse cells. When uvomorulin cDNA was introduced into cell lines from other species, such as human HeLa and avian fibroblasts, the expressed uvomorulin was also associated with endogenous 102, 88 and 80 kd proteins and, moreover, each of these proteins showed structural similarities to the respective mouse molecule. A panel of antibodies specific for known cytoplasmic proteins of mol. wts similar to those of the three proteins did not react with any of the described components. This suggests that the 102, 88 and 80 kd proteins constitute a new group of proteins for which we propose the nomenclature of catenin alpha, beta and gamma respectively. The characterization of these proteins provides a first molecular basis for a possible cytoplasmic anchorage of uvomorulin to the cytoskeleton.     

Identification in chicken macrophages of a set of proteins related to, but distinct from, the chicken cellular c-ets-encoded protein p54c-ets.

Using an antiserum to a bacterially expressed polypeptide corresponding to 56 amino acids of v-ets, we previously identified in chicken tissues a protein of 54 kd (p54c-ets) which shares extensive sequence homology to the v-ets-encoded domain of the E26-transforming protein p135gag-myb-ets and is thus apparently encoded by the c-ets proto-oncogene. We report here that the anti-ets serum specifically identifies in chicken cells a second set of proteins of 60 kd (p60), 62 kd (p62) and 64 kd (p64) which appear to be highly related to each other but display only a limited domain of homology with p54c-ets and p135gag-myb-ets and are thus probably encoded by a gene(s) partially related to, but different from c-ets. In contrast to p54c-ets which is expressed at high levels in chicken lymphoid tissues, prominent syntheses of p62 and p64 were found in both normal and transformed chicken macrophages but not in avian cells corresponding to immature stages of the myeloid differentiation pathway. These observations together with the fact that differentiation of avian myeloblastosis virus-transformed myeloblasts into macrophage-like cells after treatment with 12-O-tetradecanoylphorbol-13-acetate is accompanied by the synthesis of p62 and p64 suggest a role for these proteins in chicken macrophage differentiation or function. Induction of differentiation of human leukemia cell lines HL60 and U937 into macrophages is also accompanied by the increased synthesis of c-ets-encoded 68 kd, 62 kd and 58 kd proteins.     

HMG-like chromosomal proteins in Trypanosoma cruzi.

HMG-like chromosomal proteins from Trypanosoma cruzi were studied. Four HMG-like proteins, designated HMG A, HMG-B, HMG-C, and HMG-E, were isolated and found to have molecular weights of 35.5 kd, 27.5 kd, 21.8 kd and 10.4 kd, respectively. Immunological relatedness was demonstrated between the mammalian HMG 1,2 and the HMG-A and HMG-B from T. cruzi. The relative amounts of HMG-C and HMG-E proteins vary in T. cruzi depending to the proliferative stage of the cells. HMG-E protein is increased in proliferating cells when compared to its level in non-proliferating cells. HMG-C is increased in the non-proliferating cells. Probably, the shifts observed in the relative amounts of HMG-like proteins are related to the proliferating cells of this flagellate. The results are consistent with those described for other lower eukaryotes where the HMG-like proteins isolated are similar but not identical to HMG proteins from vertebrates.     

Analysis of HPV-1 E4 gene expression using epitope-defined antibodies.

Six monoclonal antibodies (mAbs) have been raised against the E4 proteins of HPV-1. Five of these were found to recognize denaturation-resistant epitopes as determined by Western blotting--and their binding sites were identified by determining their reactivity against a panel of bacterial E4--beta-galactosidase fusion proteins which contained progressive deletions at the C-terminal end of the E4 region. The five mAbs were found to bind to four distinct sites. By using these epitope-defined mAbs, along with anti-peptide antibodies raised against putative N- and C-terminal E4 sequences, we have determined the relationships between the eight distinct polypeptides (mol. wt 10/11 kd, 16/17 kd, 21/23 kd and 32/34 kd) previously shown to be expressed from the E4 gene of HPV-1 in productively infected papillomas. The 17 kd E4 polypeptide appears to be the product of a spliced mRNA encoding five amino acids from open reading frame (ORF) E1 joined onto 120 from the E4 ORF. The 16 kd and 10/11 kd proteins, which may be derived from this, lack sequences (approximately 15 and 70 amino acids respectively) encoded by the 5' end of the E4 gene. The 32/34 kd proteins were detected by all antibodies which reacted with the 16/17 kd polypeptides, suggesting that they represent dimers of the latter species. The 21/23 kd polypeptides, however, do not appear to be simple dimers of the 10/11 kd protein as previously predicted, and reacted with antibodies whose epitopes mapped in the N-terminal half of the E4 protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

The E5 oncoprotein of bovine papillomavirus binds to a 16 kd cellular protein.

The E5 oncoprotein of bovine papillomavirus type 1 is the smallest known viral transforming protein. It is a 44 amino acid polypeptide asymmetrically oriented in Golgi and plasma membranes which appears to modify (either directly or indirectly) the internalization and phosphorylation of at least two growth factor receptors: EGF and CSF-1. To identify cellular proteins associated with E5, we have constructed two E5 fusion proteins, each of which contains a well-characterized epitope at the E5 amino terminus. These E5-epitope fusion proteins are biologically active, localize normally to cellular membranes and form dimers. Both monoclonal and polyclonal antibodies against the inserted epitopes specifically co-precipitate E5 and an associated 16 kd cellular protein. A transformation-defective E5 mutant containing a substitution within the hydrophobic portion of E5 is defective in its ability to bind the 16 kd protein. These findings suggest a role for E5/16 kd binding in the process of cellular transformation.     

Regulation of light-harvesting chlorophyll-binding protein (LHCP) mRNA accumulation during the cell cycle in Chlamydomonas reinhardi

Light-harvesting chlorophyll a/b protein (LHCP) synthesis is highly regulated during the cell cycle in light-dark synchronized C. reinhardi cells. LHCPs are a family of cytoplasmically synthesized proteins which are imported into the chloroplast. LHCPs are derived from at least two precursor proteins (32 kd and 30 kd) that are synthesized in vitro and immunoprecipitated by antiserum against chlorophyll-protein complex II proteins. A DNA copy of the mRNA encoding a 32 kd LHCP precursor was cloned from cDNA synthesized from poly(A) RNA obtained from mid-light-phase synchronous cells. Using cloned cDNA (pHS16) as a hybridization probe, we found that a single 1.2 kb RNA complementary to pHS16 accumulates in a wave-like manner during the mid-light phase of the 12 hr light-12 hr dark cycle and correlates with the pattern of chlorophyll synthesis. Light, during the light phase in the light-dark cycle, is required for accumulation of this RNA.     

腺苷转运体亲和层析分离

本文合成了一种腺苷亲和层析凝胶,并采用亲和层析法从牛脑细胞膜上分离出了几种膜上结合的腺苷结合蛋白质。这些蛋白质在ＳＤＳ－ＰＡＧＥ电泳凝胶上为单一或主要的蛋白带,分子量分别为６４ｋｄ,４５ｋｄ,３５ｋｄ。腺苷转运体抑制剂潘生丁和ＮＢＭＰＲ对６４ｋｄ蛋白与＾３ｈ－腺苷的结合抑制作用远强于腺苷受体的激动剂ＮＥＣＡ和Ｒ－ＰＩＡ;这表明６４ｋｄ蛋白为牛脑细胞膜上结合的腺苷转运体。     

Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase.

Affinity-purified bovine brain phosphatidylinositol 3-kinase (PI3-kinase) contains two major proteins of 85 and 110 kd. Amino acid sequence analysis and cDNA cloning reveals two related 85 kd proteins (p85 alpha and p85 beta), which both contain one SH3 and two SH2 regions (src homology regions). When expressed, these 85 kd proteins bind to and are substrates for tyrosine-phosphorylated receptor kinases and the polyoma virus middle-T antigen/pp60c-src complex, but lack PI3-kinase activity. However, an antiserum raised against p85 beta immunoprecipitates PI3-kinase activity. The active PI3-kinase complex containing p85 alpha or p85 beta and the 110 kd protein binds to PDGF but not EGF receptors. p85 alpha and p85 beta may mediate specific PI3-kinase interactions with a subset of tyrosine kinases.     

Identification of a developmentally regulated calcium-binding protein in Trypanosoma brucei

Calcium ions mediate cellular activity by binding to specific cellular proteins. The following study systematically examines the cellular complement of calcium-binding proteins in different cell fractions and life cycle stages of Trypanosoma brucei. Using a 45Ca-gel overlay procedure, eight calcium-binding proteins were consistently observed. The majority of proteins were cytosolic (84, 70, 64, 22, and 15 kd) while the remainder (55, 46, and 29 kd) were particulate. Although calmodulin was detected amongst the calcium-binding proteins, it did not represent the majority of calcium-binding activity. Of special interest was the 46 kd calcium-binding protein which was associated with 3-fold more calcium in cultured procyclic forms than in slender bloodstream forms. By contrast, promastigote forms of Leishmania mexicana did not contain the 46 kd calcium-binding protein. These data suggest that responsiveness to calcium signals may vary during the trypanosome life cycle as a result of changes in the cellular complement of calcium-binding proteins.     

Structure of the spinach chloroplast genes for the D2 and 44 kd reaction-centre proteins of photosystem II and for tRNASer (UGA)

We have determined the sequence of the spinach (Spinacia oleracea) chloroplast genes for the photosystem II proteins, D2 and the 44 kd reaction-centre, chlorophyll a-binding protein, and for tRNASer (UGA). The 3' end of the D2 gene overlaps the first 50 bp of the 5' end of the gene for the 44 kd protein. Northern RNA hybridization analysis indicates the two genes are cotranscribed into a single 3.5 kb RNA. The predicted molecular weight of the 353-residue D2 protein is 39536 and that of the 473-residue 44 kd protein is 51816. Both proteins are hydrophobic containing at least five possible membrane-spanning domains. D2 shows significant homology to the 32 kd herbicide-binding protein (Zurawski et al., (1982) Proc. Natl. Acad. Sci. USA 79, 7699-7703), and parts of the 44 kd protein show obvious similarities to parts of the 51 kd reaction-centre, chlorophyll a-binding protein of photosystem II (Morris and Herrmann (1984) Nucleic Acids Res. 12, 2837-2850). The gene for tRNASer (UGA) which is on the opposite strand to and transcribed towards the photosystem II genes is 72% homologous with the corresponding Escherichia coli tRNASer.     

The pim-1 oncogene encodes two related protein-serine/threonine kinases by alternative initiation at AUG and CUG.

The pim-1 gene is frequently found activated by proviral insertion in murine T cell lymphomas. Overexpression of pim-1 in lymphoid cells by transgenesis formally proved its oncogenic potential. The pim-1 cDNA sequence predicts that both murine and human pim-1 encode a 34 kd protein with homology to protein kinases. In this study, we show that the murine pim-1 gene encodes a 44 kd protein in addition to the predicted 34 kd protein. The 44 kd protein is an amino-terminal extension of the 34 kd protein and is synthesized by alternative translation initiation at an upstream CUG codon. Contrary to previous findings by others, we provide evidence that both murine and human pim-1 gene products are protein-serine/threonine kinases. Murine 44 kd and 34 kd pim-1 proteins exhibit comparable in vitro kinase activity and are both mainly cytoplasmic, but they differ in in vivo association state and half-life.