The common 90-kd protein component of non-transformed ''8S'' steroid receptors is a heat-shock protein.

Non-transformed steroid receptors have an approximately 8S sedimentation coefficient that corresponds to an oligomeric structure of 250-300 kd which includes a non-hormone binding 90-kd protein. A monoclonal antibody BF4 raised against the purified, molybdate-stabilized, 8S progesterone receptor (8S-PR) from chick oviduct, recognizes 8S forms of all steroid hormone receptors. BF4 was found specific for a 90-kd protein present in great abundance in all chicken tissues, including that present in 8S-forms of steroid receptors. Here, using immunological and biochemical techniques, we demonstrate that this ubiquitous BF4-positive 90-kd protein is in fact the chicken 90 kd heat-shock protein (hsp 90): it increased in heat-shocked chick embryo fibroblasts, and displayed identical migration in two-dimensional gel electrophoresis and the same V8 peptide map as the already described hsp 90. We discuss the possibility that the interaction between hsp 90 and steroid hormone-binding subunits may play a role in keeping the receptor in an inactive form.     

Hyperphosphorylation of N-60, a protein structurally and immunologically related to nucleolin after tumour-promoter treatment.

Okadaic acid, a non-TPA-type tumour promoter, induces hyperphosphorylation of a 60-kd protein in primary human fibroblasts. Treatment with TPA-type tumour promoters (e.g. TPA and teleocidin) did not cause this hyperphosphorylation. Phosphorylation of this protein was not seen at times earlier than 90 min after the addition of 75 ng/ml okadaic acid to the proliferating cell cultures. The presence of inhibitors such as actinomycin D and cycloheximide, did not significantly influence the level of hyperphosphorylation induced by okadaic acid treatment. By immunoblotting using an antibody anti-nucleolin, the 60-kd protein was identified as a fragment of nucleolar protein, nucleolin. Similarly, antibodies against the 60-kd protein cross-reacted with nucleolin. Furthermore peptide mapping, using staphylococcal V8 protease, showed that the 60-kd protein phosphorylated by casein kinase II in vitro and the okadaic-acid-induced hyperphosphorylated 60-kd protein exhibited identical phosphopeptide maps, indicating that there is also structural relatedness between N-60 and nucleolin. Hyperphosphorylation of the nucleolin fragment (N-60) was suppressed by anti-tumour promoter retinoic acid.     

Light chains of sea urchin kinesin identified by immunoadsorption

Previous studies with monoclonal antibodies indicate that sea urchin kinesin contains two heavy chains arranged in parallel such that their N-terminal ends fold into globular mechanochemical heads attached to a thin stalk ending in a bipartite tail [Scholey et al., 1989]. In the present, complementary study, we have used the monoclonal antikinesin, SUK4, to probe the quaternary structure of sea urchin (Strongylocentrotus purpuratus) kinesin. Kinesin prepared from sea urchin cytosol sedimented at 9.6 S on sucrose density gradients and consisted of 130-kd heavy chains plus an 84-kd/78 kd doublet (1 mol heavy chain: 1 mol doublet determined by gel densitometry). Low levels of 110-kd and 90-kd polypeptides were sometimes present as well. The 84-kd/78 kd polypeptides are thought to be light chains because they were precipitated from the kinesin preparation at a stoichiometry of one mol doublet per 1 mol heavy chain using SUK4-Sepharose immunoaffinity resins. The 110-kd and 90-kd peptides, by contrast, were removed using this immunoadsorption method. SUK4-Sepharose immunoaffinity chromatography was also used to purify the 130-kd heavy chain and 84-kd/78-kd doublet (1 mol heavy chain: 1 mol doublet) directly from sea urchin egg cytosolic extracts, and from a MAP (microtubule-associated protein) fraction eluted by ATP from microtubules prepared in the presence of AMPPNP but not from microtubules prepared in ATP. The finding that sea urchin kinesin contains equimolar quantities of heavy and light chains, together with the aforementioned data on kinesin morphology, suggests that native sea urchin kinesin is a tetramer assembled from two light chains and two heavy chains.     

Protein import into yeast mitochondria is inhibited by antibodies raised against 45-kd proteins of the outer membrane.

Import of several precursor proteins into isolated yeast mitochondria is inhibited by rabbit antiserum raised against the total mitochondrial outer membrane or against electrophoretically purified 45-kd outer membrane proteins. Antisera against other outer membrane proteins are only marginally active or inactive. Inhibition by the antiserum against 45-kd proteins is only weak with untreated mitochondria, but reaches 80-90% with mitochondria that had been pretreated with 0.1 mg/ml trypsin. This trypsin pretreatment by itself inhibits precursor import only slightly (30-50%). Selective inhibition of import does not correlate with binding of the various IgGs to the mitochondrial surface and is also observed with the corresponding Fab fragments. Inhibition by antibodies against 45-kd outer membrane proteins strongly suggests the existence of a mitochondrial surface protein mediating protein import and offers a means of isolating this protein.     

Induction of resistance to alkylating agents in E. coli: the ada+ gene product serves both as a regulatory protein and as an enzyme for repair of mutagenic damage.

The expression of several inducible enzymes for repair of alkylated DNA in Escherichia coli is controlled by the ada+ gene. This regulatory gene has been cloned into a multicopy plasmid and shown to code for a 37-kd protein. Antibodies raised against homogeneous O6-methylguanine-DNA methyltransferase (the main repair activity for mutagenic damage in alkylated DNA) were found to cross-react with this 37-kd protein. Cell extracts from several independently derived ada mutants contain variable amounts of an altered 37-kd protein after an inducing alkylation treatment. In addition, an 18-kd protein identical with the previously isolated O6-methyl-guanine-DNA methyltransferase has been identified as a product of the ada+ gene. The smaller polypeptide is derived from the 37-kd protein by proteolytic processing.     

Effect of continuous heat stress on cell growth and protein synthesis in Aedes albopictus

Aedes albopictus (clone C6/36) cells, which normally grow at 28 degrees C, were maintained at a supraoptimal temperature of 37 degrees C. The effect of continuous heat stress (37 degrees C) on cell growth was analyzed as were the modifications occurring with protein synthesis during short- and long-term heat stress. We observed that cells in lag or exponential growth phase, present inhibition of cell growth, and cells in the lag phase showed more sensitivity to death than cells growing exponentially. During the first hour of exposing the cells to 37 degrees C, they synthesized two heat shock proteins (hsps) of 82 kd and 70 kd, respectively, concomitant with inhibition of normally produced proteins at control temperature (28 degrees C). However, for incubations longer than 2 hr at 37 degrees C, a shift to the normal pattern of protein synthesis occurred. During these transitions, two other hsps of 76 kd and 90 kd were synthesized. Pulse chase experiments showed that the 70-kd hsp is stable at least for 18 hr, when the cells are returned to 28 degrees C. However, if cells were incubated at 37 degrees C, the 70-kd hsp is stable for at least 48 hr. The 70-kd hsp was localized in the cytoplasmic and in the nuclear compartment. Our results indicate a possible role of hsp 70-kd protein in the regulation of cell proliferation.     

Biochemical characterization of HgCl2-inducible polypeptides encoded by the mer operon of plasmid R100.

Minicells carrying the subcloned mer operon from plasmid R100 were pulse-labeled with [35S]methionine, and the labeled polypeptides were analyzed at various subsequent times by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Hg(II) reductase monomer encoded by plasmid R100 occurred as two proteins of 69 and 66 kilodaltons (kd). The minor 66-kd protein is a modified form of the 69-kd protein. This modification occurs in vivo. Both of these mer proteins are found in the soluble fraction of the cell; however, the 66-kd protein appears to have a slight affinity for the cellular envelope. Both the 69- and 66-kd mer proteins have pI values greater (pI = 5.8) than that reported (pI = 5.3) for the analogous monomer encoded by plasmid R831. The 15.1- and 14-kd mer proteins are localized in the inner membrane and are probably elements of the mer-determined Hg(II) uptake system. These two mer membrane proteins, which are antigenically unrelated to the Hg(II) reductase monomer, are quite basic (pI values greater than 7.8). The 12-kd mer protein is also a basic polypeptide that is present in the soluble fraction of the cell. Unlike the two membrane-bound mer proteins, the 12-kd mer protein is processed from a 13-kd precursor.     

A new 185,000-dalton skeletal muscle protein detected by monoclonal antibodies

The M line, which transverses the center of the thick filament region of skeletal muscle sarcomeres, appears to be a complex array of multiple structural elements. To date, two proteins have definitely been shown to be associated with the M line. They are MM-CK, localized in the M 4,4' substriations, and a 165,000-dalton (164 kd) protein, referred to as both M-protein and myomesin. Here we report the positive identification of a third M-line protein of 185 kd. In the course of making monoclonal antibodies (mAbs) against a 165-kd fraction, we also obtained mAbs that bound to the M line of isolated myofibrils as detected by indirect immunofluorescence, but recognized a protein band of 185 kd in immunoblotting experiments with either the original immunogen or low ionic strength myofibril extracts as antigenic targets. The evidence that the 185- and 165-kd proteins are distinct protein species is based on the separation of the two proteins into discrete peaks by ion exchange chromatography, the distinctive patterns of their degradation products, and non-cross-reactivity of any of seven mAbs. These mAbs recognize three unique antigenic determinants on the 185-kd molecule and at least two and probably four sites on the 165-kd molecule as determined from competitive binding and immunofluorescence experiments. To resolve the problem of multiple nomenclature for the 165-kd protein, the 185-kd protein will be referred to as myomesin and the 165-kd protein as M-protein.     

B cell growth modulating and differentiating activity of recombinant human 26-kd protein (BSF-2, HuIFN-beta 2, HPGF).

The human "26-kd protein' is a secreted glycoprotein expressed, for example, in (blood) leukocytes, in epithelial cells treated with various inducers, but most strongly in interleukin-1 (IL-1)-treated fibroblasts. After finding it has antiviral and 2-5A synthetase-inducing activity, one group of authors called this protein IFN-beta 2. However, recently the full-length 26-kd cDNA sequence was shown to be identical with that of a B-cell-differentiating lymphokine called BSF-2, and another report suggested that the 26-kd protein could support the growth of some transformed murine B cell lines. To define its biological activities, we expressed the recombinant 26-kd protein by translating in Xenopus laevis oocytes a pure, synthetic chimeric mRNA containing the 26-kd protein coding region surrounded by Xenopus laevis beta-globin untranslated regions. A similar construction, but containing the HuIFN-beta cDNA coding region, was used to produce HuIFN-beta by the same procedure. Both recombinant glycoproteins were secreted, glycosylated, and their amounts were measured by [35S]methionine incorporation by the oocyte. Here we show that the recombinant 26-kd protein exhibits a high growth factor activity when assayed on an IL-HP1-dependent murine B cell hybridoma (sp. act. approximately 2 X 10(8) U/mg) as well as a potent differentiating activity on human CESS cells (sp. act. approximately 5 X 10(7) U/mg). While rHuIFN-beta was inactive in the latter two assays, it had the expected antiviral activity of 1-5 X 10(8) U/mg. The parallel recombinant 26-kd protein preparations had no detectable antiviral activity (i.e. a maximal specific activity of 1-3 X 10(2) U/mg, if any). The 26-kd protein is thus clearly an interleukin, and considering the confusing nomenclature now in use, this factor may better be renamed "interleukin 6'.     

A novel virulence-associated cell surface structure composed of 47-kd protein subunits in Yersinia enterocolitica.

A fresh human isolate of Yersinia enterocolitica serotype 03, and its derivative that had lost the virulence-associated 46-Md plasmid, were grown under defined conditions and compared for their outer membrane protein and cell surface structure. Under these conditions, the virulent strain grown at 37 degrees C expressed one major outer membrane protein (47 kd) not present in the plasmidless strain or in either strain grown at room temperature. A 200-kd protein also seen in the same preparations was shown to be an oligomer composed of the 47-kd protein subunits. Four different electron microscopic techniques showed tack-like projections covering the surface of those bacteria that expressed the 47-kd protein. These were specifically labeled with antibody to the 47-kd protein. This surface structure appeared to mediate aggregation (auto-agglutination) of the bacteria bringing their surfaces into unusually close apposition.     

Wheat germ agglutinin binds to the contact site A glycoprotein of Dictyostelium discoideum and inhibits EDTA-stable cell adhesion

Wheat germ agglutinin (WGA), a lectin that primarily reacts with N-acetylglucosamine residues, specifically inhibits the EDTA-stable type of intercellular adhesion of aggregation competent Dictyostelium discoideum cells. The major WGA-binding protein of these cells is a developmentally-regulated glycolipoprotein of 80 kd apparent mol. wt., designated as contact site A. This glycoprotein is a target site of antibody fragments that block the EDTA-stable cell adhesion, and is characterized by sulfated carbohydrate residues. WGA does not significantly bind to glycoproteins of a mutant, HL220, which produces a 68-kd component in place of the 80-kd glycoprotein. Inhibition of N-glycosylation by tunicamycin causes wild-type cells to produce a WGA-binding but unsulfated 66-kd component and a non-binding 53-kd component. These results indicate that the 80-kd glycoprotein contains two classes of carbohydrate residues, a WGA-binding one that is defective in HL220, and another, sulfated, one that is absent from the 66-kd wild-type product; both are missing in the 53-kd protein. WGA and a monoclonal antibody that is blocked by N-acetylglucosamine were further used to probe for glycoproteins in the multicellular slug stage that share carbohydrate structures - and possibly functions - with the contact site A glycoprotein. Glycoproteins in the 95-kd range have previously been implicated in cell-to-cell adhesion during the slug stage. We distinguished a 95-kd glycoprotein that binds WGA from another one that binds antibody.     

Synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein in the chloroplast membranes of peas and Chlamydomonas reinhardi

The synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein (HSP) in the chloroplast membranes was studied in pea plants and Chlamydomonas reinhardi. HSPs were detected in both systems by in vivo labeling and in vitro translation of poly(A)⁺RNA, using the wheat-germ and reticulocyte lysate systems. Heat-shock treatment of pea plants for 2 h at 42-45°C induces the expression of ˜10 nuclear coded proteins, among which several (18 kd, 19 kd, 22 kd) are predominant. A 22-kd protein is synthesized as a 26-kd precursor protein and is localized in a chloroplast membrane fraction in vivo. Following post-translational transport into intact chloroplasts in vitro of the 26-kd precursor, the protein is processed but the resulting 22-kd mature protein is localized in the chloroplast stroma. If, however, the in vitro transport is carried out with chloroplasts from heat-shocked plants, the 22-kd protein is preferentially transported to the chloroplast membrane fraction. In C. reinhardi the synthesis of poly(A)⁺RNAs coding for several HSPs is progressively and sequentially induced when raising the temperature for 1.5 h from 36°C to 42°C, while that of several preexisting RNAs is reduced. Various pre-existing poly(A)⁺RNAs endure in the cells at 42°C up to 5 h but are no longer translated in vivo, whereas some poly(A)⁻RNAs persist and are translated. As in pea, a poly(A)⁺RNA coded 22-kd HSP is localized in the chloroplast membranes in vivo, although it is translated as a 22-kd protein in vitro. The in vitro translated protein is not transported in isolated pea chloroplast which, however, processes and transports other nuclear coded chloroplast proteins of Chlamydomonas. The poly(A)⁺RNA coding for the 22-kd HSP appears after 1 h at 36°C. Its synthesis increases with the temperature of incubation up to 42°C, although it decreases after ˜2 h of heat treatment and the already synthesized RNA is rapidly degraded. The degradation is faster upon return of the cells to 26°C. None of the heat-induced proteins is identical to the light-inducible proteins of the chloroplast membranes.     

Post-translational glycosylation of the contact site A protein of Dictyostelium discoideum is important for stability but not for its function in cell adhesion

The functions of type 1 and 2 carbohydrates of the contact site A (csA) glycoprotein of Dictyostelium discoideum have been investigated using mutants lacking type 2 carbohydrate. In two mutant strains, HG220 and HG701, a 68-kd glycoprotein was synthesized as the final product of csA biosynthesis. This glycoprotein accumulated to a much lower extent on the surfaces of mutant cells than the mature 80-kd glycoprotein did in wild-type cells. There was also no accumulation of the 68-kd glycoprotein observed within the mutant cells nor was a precursor of lower molecular mass detected, in accordance with previous findings that indicated cotranslational linkage of type 1 carbohydrate by N-glycosylation. Pulse-chase labelling showed that a 50-kd glycopeptide was cleaved off from the mutant 68-kd glycoprotein and released into the medium, while the fully glycosylated 80-kd glycoprotein of the wild type was stable. These results assign a function to type 2 carbohydrate in protecting the cell-surface-exposed csA glycoprotein against proteolytic cleavage. HG220 cells were still capable of forming EDTA-stable contacts to a reduced extent, consistent with the low amounts of the 68-kd glycoprotein present on their surfaces. Thus type 1 rather than type 2 carbohydrate appears to be directly involved in intercellular adhesion that is mediated by the csA glycoprotein. Tunicamycin-treated wild-type and mutant cells produce a 53-kd protein that lacks both type 1 and 2 carbohydrates. While this protein is stable and not transported to the cell surface in the wild type, it is cleaved in the mutants and fragments of it are released into the extracellular medium. These results suggest that the primary defect in the two mutants studied is relief from a restriction in protein transport to the cell surface, and that the defect in type 2 glycosylation is secondary.     

Identification of tyrosine-phosphorylated colony-stimulating factor 1 (CSF-1) receptor and a 56-kilodalton protein phosphorylated in intact human cells in response to CSF-1

Colony-stimulating factor 1 (CSF-1) selectively supports the survival, proliferation, and maturation of hemopoietic cells of the monocyte/macrophage lineage. Although the cellular receptor for CSF-1, (the c-fms protein) is a protein-tyrosine kinase activated by the binding of CFS-1, the role of phosphorylation of cellular proteins in CSF-1 signal transduction is poorly understood. Therefore, we examined the CSF-1-stimulated phosphorylation of cellular proteins in human BeWo choriocarcinoma cell line (known to express the c-fms protein). BeWo cells were metabolically labeled with 32Pi, stimulated with recombinant human CSF-1, and extracted with detergent. Phosphotyrosyl proteins were isolated from detergent extracts by affinity chromatography on a highly specific antibody to phosphotyrosine. Rapid phosphorylation of 170-kd protein, followed closely by the phosphorylation of a 56-kd protein, was observed in response to CSF-1. The 170-kd phosphotyrosyl protein bound to wheat germ agglutinin and was secondarily immunoprecipitated with a specific anti-fms serum, consistent with its identity as the CSF-1 receptor. Although purified human macrophages that proliferate in culture in response to CSF-1 are not generally accessible, CSF-1 did stimulate the phosphorylation of a 56-kd protein in intact mononuclear leukocytes from human peripheral blood. Thus, the BeWo cell line may represent a good model for the study of CSF-1-stimulated cellular protein phosphorylation.     

51-kd protein, a component of microtubule-organizing granules in the mitotic apparatus involved in aster formation in vitro

Mitotic apparatuses (MAs) isolated from sea urchin metaphase eggs were chilled on ice to depolymerize microtubules, homogenized, and incubated with tubulin. This caused formation of many small asters with microtubules focusing on granules which were probably fragments of the centrosome. The aster-forming protein components of the granules in the homogenized MAs were solubilized in 0.5 M KCl containing 50% glycerol. After dialysis against low-ionic-strength buffer solution, proteins congregated to form granular assembly capable of initiating aster formation. Phosphocellulose column chromatography enabled the separation of the aster-forming protein fraction which contained a 51,000 molecular weight protein (51-kd protein) as a major component. The protein fraction possessing the aster-forming activity was also prepared from methaphase whole egg homogenate, and the elution profile of the 51-kd protein on phosphocellulose column also coincided with that of the aster-forming activity. The granular assembly reconstituted from the phosphocellulose fraction formed asters whose microtubules show the same growth rate and length distribution as those of asters reconstructed from the granules in the homogenized MAs. Anti-51-kd protein antibody that was raised in rabbit and affinity-purified stained the center of asters which were reconstructed either from the granules in the homogenized MAs or from the granular assembly reconstituted from the phosphocellulose fraction. These results suggest that the 51-kd protein is a component in the aster-forming activity of the centrosomal component in vitro.     

In vivo sulfation of the contact site A glycoprotein of Dictyostelium discoideum

During the development of Dictyostelium discoideum from the growth phase to the aggregation stage, a glycoprotein with an apparent mol. wt. of 80 kd is known to be expressed on the cell surface. This glycoprotein, referred to as contact site A, has been implicated in the formation of species-specific, EDTA-stable contacts of aggregating cells. When developing cells were labeled in vivo with [³⁵S]sulfate, the 80-kd glycoprotein was found to be the most prominently sulfated protein. Another strongly sulfated protein had an apparent mol. wt. of 130 kd and was, like the 80-kd glycoprotein, developmentally regulated and associated with the particulate fraction of the cells. The [³⁵S]sulfate incorporated into the 80-kd and 130-kd proteins was not present as tyrosine-O-sulfate, a modified amino acid found in many proteins of mammalian cells. D. discoideum cells incubated with [³⁵S]sulfate in the presence of tunicamycin, an inhibitor of N-glycosylation, produced a 66-kd protein that reacted with monoclonal antibodies raised against the 80-kd glycoprotein, but no longer contained [³⁵S]sulfate. These results suggest that sulfation of the 80-kd glycoprotein occurred on carbohydrate residues. The possible importance of sulfation for a role of the 80-kd glycoprotein in cell adhesion is discussed.     

Initiation of adenovirus DNA replication: detection of covalent complexes between nucleotide and the 80-kilodalton terminal protein

We have previously shown that the 5'-terminal deoxycytidine residue of each nascent adenovirus 5 DNA strand synthesized in vitro is covalently linked to the 80-kilodalton (kd) terminal protein precursor via a phosphodiester bond to a serine residue in the protein. When extracts prepared from adenovirus 5-infected cells are incubated with [alpha-33P]dCTP as the only added deoxynucleoside triphosphate, complexes consisting of nucleotide covalently linked to the 80-kd protein can be detected. The nucleotide moieties present in such complexes include d(pC) and d(pCpA), the 5'-terminal nucleotide and dinucleotide of adenovirus 5 DNA, respectively, as well as some longer oligonucleotides. The formation of these complexes requires the presence of adenovirus DNA containing the attached 55-kd terminal protein and ATP. Extracts from H5ts125-infected cells which are defective in DNA replication catalyze complex formation to the same extent as extracts prepared from wild-type infected cells; thus, the presence of the adenovirus-coded 72-kd DNA-binding protein is apparently not required. Most, if not all, of the 80-kd protein-nucleotide complexes that are formed are noncovalently bound to the input viral DNA. These observations are consistent with the protein-priming model for the initiation of adenovirus DNA replication.     

Molecular heterogeneity in human beta-galactosidase and neuraminidase deficiency

Human lysosomal beta-galactosidase and neuraminidase exist in a complex together with a 32-kilodalton (kd) glycoprotein. The latter protein was found to have a dual function: it is required for the aggregation of monomeric 64-kd beta-galactosidase into high molecular weight (600-700 kd) multimers and it is an essential subunit of neuraminidase together with a 76-kd polypeptide. The severe neurological disorder galactosialidosis, characterized by a coexistent deficiency of beta-galactosidase and neuraminidase, was found to be due to a genetic defect of the 32-kd protective protein. The molecular background of the clinical heterogeneity within this syndrome is described and will undoubtedly be further elucidated since we have recently isolated the gene coding for the protective protein. The sequence of normal and mutant (enzyme) proteins will also provide better insight into the characteristics of the beta-galactosidase-neuraminidase-protective protein complex. Another interesting model for the study of posttranslational processing is the defective phosphorylation of beta-galactosidase in cells from patients with GM1-gangliosidosis.