Identification and quantitation of levels of protein synthesis initiation factors in crude HeLa cell lysates by two-dimensional polyacrylamide gel electrophoresis

Protein synthesis initiation factors in purified preparations and in crude lysates of HeLa cells were fractionated by two-dimensional polyacrylamide gel electrophoresis in order to characterize their molecular forms. Specific spots in the complex cytoplasmic protein gel pattern which corresponded to the initiation factor proteins were identified by co-migration of purified initiation factors with 35S-labeled cell lysates, partial proteolytic digestion mapping, and immunoblotting analysis using antisera or affinity-purified antibodies to the initiation factors. Spots identified as eukaryotic initiation factor (eIF) 2 alpha, eIF-2 beta, eIF-2 gamma, eIF-4A, and four eIF-3 proteins of less than 50,000 Da corresponded to moderately abundant lysate proteins. Minor isoelectric variant forms of eIF-2 beta, eIF-2 gamma, and eIF-4A were detected by immunoblot analysis of lysate proteins, suggesting either covalent modification of these factor proteins or contaminating antibodies. eIF-2 beta and eIF-4B were present in at least two isoelectric forms, confirming covalent modification of these proteins. The cellular levels of the initiation factor proteins were measured by excising and counting radioactivity in gel-resolved spots corresponding to factors in lysates labeled in vivo. The individual factor protein abundancies span nearly a 10-fold range, from 1.1 to 9.8 million molecules/cell. The factor to ribosome ratio for eIF-2 was 0.8, for the average eIF-3 protein about 0.6, and for eIF-4A it was significantly higher at 3.0.     

Purification of a glycoprotein excreted by Trypanosoma cruzi to increase the permeability of the host-cell membrane

During invasion of the prospective host cell, metacyclic forms of Trypanosoma cruzi render the membrane of HeLa cells permeable to the alpha-sarcin toxin, by excreting a glycoprotein with N-acetyl-D-glucosamine residues. The molecular weight of the glycoprotein is 64,000 dalton and its isoelectric point is 4.8.     

Evidence for two functionally distinct forms of the human Ah receptor

The Ah receptor (AhR) was visualized using monoclonal antibody Rpt 1 on protein blots of HeLa cell cytosol; two bands were detected at 104 and 106 kDa. The photoaffinity ligand, 2-azido-3-[¹²⁵I]iodo-7,8-dibromodibenzo-p-dioxin, was added to HeLa cells in culture, and after 1 hour the cells were UV irradiated. Cytosolic and high salt nuclear preparations were isolated and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer of the protein to membrane. The AhR was visualized on the membrane, revealing two bands. Alignment of an autoradiogram with the membrane revealed that only the 106 kDa (upper) band was photoaffinity labeled. The nuclear fraction contained only the photoaffinity-labeled 106 kDa form of the AhR. The 104 kDa AhR does not appear to be a proteolytic product of the 106 kDa form. Cyanogen bromide fragmentation revealed that both forms contain the same size N-terminal fragment. Sucrose density gradient analysis of HeLa cell cytosol indicated that both forms cosedimented at 9 S. Both the 106 and 104 kDa AhR bands were detected in four different human cell lines. Together, these results would indicate that the AhR in human cell lines exists in two distinct forms.     

Regulated phosphorylation and low abundance of HeLa cell initiation factor eIF-4F suggest a role in translational control. Heat shock effects on eIF-4F

Initiation factor eIF-4F, a multiprotein cap binding protein complex, was purified from HeLa cells by m7G affinity chromatography and independently by phosphocellulose column chromatography. The m7G affinity-purified sample contains three major proteins, p220, eIF-4A, and p28 (also known as CBP-I or eIF-4E). The abundancies of these proteins are roughly 2, 10, and 0.8 X 10(6) molecules/cell, respectively. Two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the eIF-4F samples shows that p28 comprises two isoelectric variants, one of which labels with phosphate and disappears when samples are treated with alkaline phosphatase. The 45,000-dalton protein in eIF-4F appears to be identical to eIF-4A. The p220 subunit rarely produces discrete spots on two-dimensional gel electrophoresis; in purified samples it usually forms 3 closely spaced streaks. eIF-4F fractionated by phosphocellulose chromatography separates into forms containing either phosphorylated or unphosphorylated p28. However, both fractions possess similar specific activities in in vitro translation assays for eIF-4F activity. The phosphorylation of p28 decreases upon heat shock when protein synthesis is repressed. The correlation of dephosphorylation of p28 with the inhibition of protein synthesis and the relatively low abundance of the eIF-4F complex suggest that eIF-4F plays a role in the translational control of mRNA binding. Limitations of the in vitro assay system may account for the failure to detect phosphorylation-dependent activity differences.     

Purification of host DNA synthesis-suppressing factor (DSF) produced by infection with measles virus.

Host DNA synthesis-suppressing factor (DSF) produced into culture fluid of cloned HeLa cells (HeLa C-9) infected with a small plaque variant of Toyoshima strain of measles virus was purified by precipitation with ammonium sulfate, chromatography on CM-cellulose and DEAE-cellulose, and gel-filtration on Sephadex G-100 and G-200. The specific activity of the finally purified DSF was 302 units/mg of protein representing approximately 300-fold purification. The molecular weight of DSF was estimated to be about 55 000. By isoelectric focusing, two kinds of DSF having isoelectric points of 4.24 and 5.24 were detectable. The purified DSF was able to suppress host DNA synthesis of HeLa cells, continuous human lymphoid cells (NC-37), mouse L cells and Meth-A cells derived from an ascitic tumor of the mouse. The activity of the purified DSF was inactivated by heating at 56 C for 30 min or by treatment with trypsin.     

Purification of Host DNA Synthesis-Suppressing Factor (DSF) Produced by Infection with Measles Virus

Host DNA synthesis-suppressing factor (DSF) produced into culture fluid of cloned HeLa cells (HeLa C-9) infected with a small plaque variant of Toyoshima strain of measles virus was purified by precipitation with ammonium sulfate, chromatography on CM-cellulose and DEAE-cellulose, and gel-filtration on Sephadex G-100 and G-200. The specific activity of the finally purified DSF was 302 units/mg of protein representing approximately 300-fold purification. The molecular weight of DSF was estimated to be about 55 000. By isoelectric focusing, two kinds of DSF having isoelectric points of 4.24 and 5.24 were detectable. The purified DSF was able to suppress host DNA synthesis of HeLa cells, continuous human lymphoid cells (NC-37), mouse L cells and Meth-A cells derived from an ascitic tumor of the mouse. The activity of the purified DSF was inactivated by heating at 56 C for 30 min or by treatment with trypsin.     

Trypanosoma cruzi: amastigotes and trypomastigotes interact with different structures on the surface of HeLa cells

It is generally accepted that Trypanosoma cruzi trypomastigotes represent the infective forms of the etiological agent of Chagas' disease. However, the invasive capacity of amastigotes and their ability to sustain a complete infective cycle in mammalian cultured cells and hosts has been recently demonstrated. In order to compare the process of cell invasion by these different infective forms, I examined the interactions of trypomastigotes and amastigotes with HeLa cells using a new and simple method that improves parasite-cell interactions and significantly reduces incubation periods. T. cruzi forms were centrifuged onto HeLa cells grown on coverslips and parasite-cell interactions were examined by fluorescence and scanning electron microscopy. As expected, it was observed that all parasite forms attach and eventually enter the cells. However, whereas trypomastigotes preferentially invade HeLa cells at the edges, as has recently been demonstrated for other cell types, the initial steps of amastigote-HeLa cell interaction involve binding and entangling of the parasite to surface microvilli. Thus, different T. cruzi infective forms interact with different cell surface structures that could express different receptors at the HeLa cell membrane.     

A novel 53 kDa actin binding protein from porcine brain--further biochemical and immunological characterization

We have previously described some of the characteristics of an actin binding protein, 53 K protein, purified from porcine brains. The purification procedure was revised in order to investigate of this actin binding protein further. A Scatchard plot analysis showed that the association constant between actin and the 53 K protein has around the same value as those reported for the fascin-actin and for the filamin-actin interactions. The binding experiments also demonstrated the occurrence of competitive binding with other actin binding proteins such as filamin, alpha-actinin, caldesmon and tropomyosin for the actin filament. Antibody was produced against brain 53 K protein and further purified on an affinity column. Immunoblot analysis using the antibody showed that this protein is localized in both the soluble and membraneous fraction of the brain. Other tissues such as liver and lung also contain 53 K protein. The immunoblot analysis also revealed that the gelation product of rat brain extract described by Palmer et al. contains immunoreactive polypeptides having slightly lower molecular weights and more basic isoelectric points than porcine brain 53 K protein. Immunological localization of the 53 K protein within HeLa and BS-C-1 cells showed that this protein is distributed throughout the cell in small granules, and in some regions of the cell, these granules were aggregated into much larger granules.     

Purification of and production of an antibody against a 63,000 Mr stimulus-sensitive phosphoprotein in Paramecium

In vivo labeling of Paramecium cells with 32Pi most heavily labels a minor 63-kDa protein that undergoes a rapid, Ca2+-dependent dephosphorylation when the cell is stimulated to release. This stimulus-sensitive phosphoprotein was isolated and purified to apparent homogeneity. A polyclonal affinity purified antibody made against the purified protein recognizes both the phosphorylated and dephosphorylated forms of the protein. The phosphorylated 63-kDa protein is found in the cytosolic fraction; it is slightly acidic with two isoelectric forms at pI 5.8 and 6.2 and probably exists as a monomeric 60-65-kDa polypeptide in the native state. The labeled phosphoamino acid of the protein is phosphoserine. The affinity purified antibody recognizes a third isoelectric form at pI 6.3 that appears unlabeled. The specificity of the antibody was confirmed by showing that it immunoprecipitates the correct protein, i.e. the stimulus-sensitive 63-kDa phosphoprotein. The availability of purified 63-kDa protein as well as an antibody against it will now allow molecular, biochemical, and immunocytochemical studies into the role of this protein in the mechanism of exocytosis.     

Scanning isoelectric focusing in small density-gradient columns: I. Use of a Standard Spectrophotometer Cuvette for Focusing. Chemical Modification of Proteins by Migrating Reactive Ions

By the method of isoelectric focusing in a sucrose density gradient, small protein samples (less than 100 μg) have been separated and analyzed within 2 hr, using as electrolysis column a commercial standard quartz spectrophotometer cuvette, equipped with platinum electrodes and placed in an optical scanning device. Preparation of the cuvette prior to an isoelectric focusing experiment required about half an hour with no external apparatus, as the density gradient was created spontaneously in the cell by free interdiffusion of sucrose solutions. The cuvette temperature could be controlled by circulating water. The optical detection device permitted repeated scanning of the cuvette during the electrolysis process, thus providing information about the events occurring to a protein during focusing or prolonged electrolysis. By scanning with wavelength at the positions where the proteins have focused, their absorption spectra were obtained. the isoelectric points of separated proteins were estimated by fractionation of the cell contents and subsequent pH measurements on the fractions.The present paper also describes how individual Ampholine components, or groups of components, in their focused states gave rise to easily detected refractive-index gradients within the cell. The optical scanning device has been built in such a way that interference of these gradients with absorption measurements was abolished.Application of the technique to the isoelectric separation of commercial sperm whale myoglobin is reported. Ferrous or ferric forms of the focused myoglobin components were obtained by migration of reducing or oxidizing agents through the zones.     

S layer protein of Clostridium tetani: purification and properties.

S layer protein of Clostridium tetani strain AO 174, a nontoxigenic derivative of strain Harvard A 47, was prepared from the cell walls by 4 M urea extraction and purified by DEAE-Sepharose CL-6B chromatography followed by a combination of anion-exchange chromatography and reverse-phase chromatography using an HPLC system. The molecular weight of the S layer protein was estimated to be 140 kilodaltons (kDa) by SDS-PAGE. The amino acid composition of the 140 kDa protein was very similar to those of S layer proteins from the other bacterial species: it was rich in acidic amino acid and lacked cysteine. Also, the protein was unique in its extremely low content of proline (0.02 to 0.03 mol%). Multiple isoelectric forms ranging from pH 4.0 to 4.5 were observed in the purified preparation. Immunodiffusion analysis showed that the 140 kDa protein was a common antigen to the three strains of C. tetani tested.     

Host cell invasion mediated by Trypanosoma cruzi surface molecule gp82 is associated with F-actin disassembly and is inhibited by enteroinvasive Escherichia coli

The target cell F-actin disassembly, induced by a Ca2+-signaling Trypanosoma cruzi factor of unknown molecular identity, has been reported to promote parasite invasion. We investigated whether the metacyclic trypomastigote stage-specific surface molecule gp82, a Ca2+-signal-inducing molecule implicated in host cell invasion, displayed the ability to induce actin cytoskeleton disruption, using a recombinant protein (J18) containing the full-length gp82 sequence fused to GST. J18, but not GST, induced F-actin disassembly in HeLa cells, significantly reducing the number as well as the length of stress fibers. The number of cells with typical stress fibers scored approximately 70% in untreated and GST-treated cells, as opposed to approximately 30% in J18-treated samples, which also showed decreased F-actin content. J18, but not GST, inhibited approximately 6-fold the HeLa cell entry of enteroinvasive Escherichia coli (EIEC), which depends on actin cytoskeleton. Not only were fewer cells infected with bacteria in the presence of J18, there were also fewer bacteria per cell. The inhibitory activity of J18 was Ca2+ dependent. In co-infection experiments, preincubation of HeLa cells with EIEC drastically reduced gp82-dependent internalization of T. cruzi metacyclic forms. All these data, plus the finding that gp82-mediated penetration of metacyclic forms was associated with disrupted HeLa cell cytoskeletal architecture, indicate that gp82 promotes parasite invasion by disassembling the cortical actin cytoskeleton.     

Cellulase occurs in multiple active forms in ripe avocado fruit mesocarp

The existence of multiple forms of avocado (Persea americana Mill. cv Hass) cellulase in crude protein extracts of ripe avocado fruit is reported. Cellulase was separated into at least 11 multiple forms by native isoelectric focusing in the pH range between 4 and 7 and visualized by both activity staining using Congo red and immunostaining. The enzyme components were acidic proteins with isoelectric points in the range of pH 5.10 to 6.80, the predominant forms having isoelectric points of 5.60, 5.80, 5.95, and 6.20. All 11 forms were immunologically related with molecular masses of 54 kilodaltons.     

Intracellular distribution and degradation of immunoglobulin G and immunoglobulin G fragments injected into HeLa cells

Intact rabbit immunoglobulin G molecules (IgGs) and their papain or pepsin fragments were radio-iodinated and injected into HeLa cells. Whole IgGs, Fab2, and Fc fragments were degraded with half-lives of 60- 90 h, whereas half-lives of Fab fragments were 110 h. These results indicate that proteolytic cleavage in the hinge region of the IgG molecule is not the rate-limiting step in its intracellular degradation. The hingeless human myeloma protein, Mcg, was degraded at the same rate as bulk human IgG, providing further evidence that the proteolytically susceptible hinge region is not important for intracellular degradation of IgG molecules. SDS acrylamide gel analysis of injected rabbit IgG molecules revealed that heavy and light chains were degraded at the same rate. Injected rabbit IgGs and rabbit IgG fragments were also examined on isoelectric focusing gels. Fab, Fab2, and Fc fragments were degraded without any correlation with respect to isoelectric point. Positively charged rabbit IgGs disappeared more rapidly than their negative counterparts, contrary to the trend reported for normal intracellular proteins. The isoelectric points of two mouse monoclonal antibodies were essentially unchanged after injection into HeLa cells, suggesting that the altered isoelectric profile observed for intact rabbit IgG resulted from degradation and not protein modification. The intracellular distributions of IgG fragments and intact rabbit IgG molecules were determined by autoradiography of thin sections through injected cells. Intact IgG molecules were excluded from HeLa nuclei whereas both Fab and Fc fragments readily entered them. Thus, for some proteins, entry into the nuclear compartment is determined primarily by size.     

Isoelectric-focusing behavior of acid hydrolases in rat kidney lysosomes. Effects of the pH gradient, autolysis and neuraminidase.

Isoelectric focusing was used to study the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase in lysosomes isolated from rat kidney. The isoelectric points of the main protein and hydrolase peaks were 1-1.5 units lower when electrofocusing was done in a pH 3-10 gradient than in a pH 10-3 gradient, apparently because the lysosomal constituents aggregated strongly at their isoelectric points and tended to settle somewhat in the gradient due to gravity. In the extended pH gradient the acidic form of each hydrolase occurred as asingle, relatively discrete peak. However, when pooled acidic fractions were refocused in a restricted pH gradient (pH 6-3 or 3-5) multiple acidic enzyme and protein components were resolved with isoelectric points between 2.7 and 5.1. When autolysis was minimized by extracting lysosomal fractions at alkaline pH (0.2% Triton X-100, 0.1%p-nitrophenyloxamic acid, 0.1 M glycine buffer, pH9) and including 0.1%p-NITROPHENYLOXAMIC ACID, AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND CATHEPSIN D, in the pH gradient, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in two forms, an acidic form with an isoelectric point of about 4.4, and a basic form with an isoelectric point close to 6.2, 6.7 and 8.0, respectively. Acid phosphatase occurred in three forms with isoelectric points of 4.1, 5.6 and 7.4. When some autolytic digestion was permitted by extracting lysosomal fractions in an acidic medium (0.2% Triton X-100, 0.1 M sodium acetate buffer, pH 5.2) AT 0-4DEGREES C and omitting p-nitrophenyloxamic acid from the gradient, the acidic form of beta-glucuronidase and the intermediate form of acid phosphatase were lost, the isoelectric points of the acidic forms of acid phosphatase, arylsulfatase and beta-N-acetylhexosaminidase were increased 0.6-1.2 units, and the isoelectric point of the basic forms of acid phosphatase, arylsulfatase and beta-glucuronidase was increased 0.5 unit. When lysosomal extracts were incubated with bacterial neuraminidase before electrofocusing, the acidic forms of acid phosphatase, arylsulfatase and beta-glucuronidase were largely lost, the isoelectric point of the acidic form of beta-N-acetylhexosaminidase was increased from 4.5 to 6.4, and the isoelectric points of the basic forms of all four hydrolases were increased 0.5-1.5 units. Autoincubation of lysosomal extracts in vitro at pH 5.2 PRODUCED SIMILAR, THOUGH LESS MARKED, effects. cont'd     

A novel, highly phosphorylated protein, of the high-mobility group type, present in a variety of proliferating and non-proliferating mammalian cells

The present work describes a perchloric-acid-soluble high-mobility-group (HMG)-like protein present in HeLa and Ehrlich ascites cells, rat and calf liver. The protein is designated P1 and has, depending on the source, a molecular mass 48-53 kDa and an amino acid composition which, like the HMG proteins, is characterized by a high content of acidic and basic residues and of proline. The protein contains about 10 mol serine/100 mol amino acid residues, is highly phosphorylated and has, in contrast to the known HMG proteins, an acidic isoelectric point of 5.0. An estimate suggests that protein P1 in HeLa interphase cells contains 25-30 residues of phosphate. Like HMG 1 and 2 it is distributed between the nucleus and the cytoplasm. In HeLa metaphase cells P1 is further modified, resulting in an increase in apparent molecular mass from 53 kDa to 56 kDa.     

Electrophoretic Differences in the Capsid Proteins of Simian Virus 40 Plaque Mutants

The structural proteins of three mutants of simian virus 40 (SV40) which differ in plaque size, temperature sensitivity, oncogenicity, host cell restriction, and immunological properties were studied. The polypeptide components of these SV40 strains could not be distinguished by their polyacrylamide gel electrophoretic patterns. When the dissociated virions of two of the mutants were analyzed by the isoelectric focusing technique in a urea gradient, the capsid protein peaks were found to differ significantly in their isoelectric points. The capsid protein of the small-plaque mutant had an isoelectric point of pH 6.51 as compared with pH 6.28 for the large-plaque strain. Isoelectric focusing of the isolated capsid protein revealed three components, a single major subunit and two minor forms. The coat proteins of two of the mutants, small-plaque and minute-plaque strains, were indistinguishable by this technique. The capsid protein peaks obtained by isoelectric focusing were further analyzed by polyacryalmide gel electrophoresis.     

Hepatitis C virus core protein activates nuclear factor kappa B-dependent signaling through tumor necrosis factor receptor-associated factor

Hepatitis C virus (HCV) core protein, a viral nucleocapsid, has been shown to affect various intracellular events including the nuclear factor kappaB (NF-kappaB) signaling supposedly associated with inflammatory response, cell proliferation, and apoptosis. In order to elucidate the effect of HCV core protein on the NF-kappaB signaling in HeLa and HepG2 cells, a reporter assay was utilized. HCV core protein significantly activated NF-kappaB signaling in a dose-dependent manner not only in HeLa and HepG2 cells transiently transfected with core protein expression plasmid, but also in HeLa cells induced to express core protein under the control of doxycycline. HCV core protein increased the DNA binding affinity of NF-kappaB in the electrophoretic mobility shift assay. Acetyl salicylic acid, an IKKbeta-specific inhibitor, and dominant negative form of IKKbeta significantly blocked NF-kappaB activation by HCV core protein, suggesting HCV core protein activates the NF-kappaB pathway mainly through IKKbeta. Moreover, the dominant negative forms of TRAF2/6 significantly blocked activation of the pathway by HCV core protein, suggesting HCV core protein mimics proinflammatory cytokine activation of the NF-kappaB pathway through TRAF2/6. In fact, HCV core protein activated interleukin-1beta promoter mainly through NF-kappaB pathway. Therefore, this function of HCV core protein may play an important role in the inflammatory reaction induced by this hepatotropic virus.