Protein migration from transplanted nuclei in Amoeba proteus. I. The relation to the cell cycle and RNA migration, as studied by autoradiography

Autoradiography has been used to examine the migration of proteins from a radioactively labelled amoeba nucleus following transplantation into an unlabelled homophasic amoeba. Nuclei were transferred at three times in the cell cycle coinciding with DNA synthesis (4 h post-division); a peak of RNA synthesis (25 h); and a relative lull in synthetic activity (43 h). Six amino acids were added individually to the culture medium to label the nuclear proteins. Migration of the proteins from the donor nucleus was found to be greatest following the transfer of [3H]aspartic acid-labelled nuclei and least with protein labelled with the basic amino acids. All amino acids exhibited the greatest extent of migration following the 25-h transfers, i.e., coinciding with a peak of RNA synthesis at 26-27.5 h. Actinomycin D (actD) inhibition of RNA synthesis reduced, but did not eliminate the extent of protein migration from the transplanted nucleus, thus indicating the existence of two classes of migratory proteins. Firstly, proteins, associated with RNA transport, which migrated mainly into the host cytoplasm. The second class migrated into the host nucleus from the transplanted nucleus, irrespective of RNa synthesis. The shuttling character of the latter class of proteins is consistent with a role of regulation of nuclear activity.     

Susceptibility of muscle soluble proteins to degradation by mast cell chymase

We investigated the in vitro susceptibility of muscle soluble proteins to the major alkaline proteinase (chymase) from skeletal muscle tissue, an enzyme originating from intramuscular mast cells, but also present in certain muscle fibers. Cytoplasmic proteins from rat skeletal muscle tissue were fractionated into four groups according to their different isoelectric points: fraction A (pI 9.5-7.0), B (pI 7.0-5.6), C (pI 5.5-4.5) and D (pI 5.3-3.5). Chromatography of these fractions on octyl-Sepharose CL-4B revealed the presence of a higher percentage of hydrophobic proteins in fraction C and D as compared to fraction A and B. In vitro degradation of these protein fractions by chymase, isolated from rat skeletal muscle tissue, was monitored (a) by measuring the ability of these proteins to bind Coomassie G-250, and (b) by analyzing the digestion mixture in isoelectric focusing gels. Both methods revealed fraction B proteins to be degraded very rapidly. While there was also a significant breakdown of fraction A proteins, fraction C and D proteins were degraded only very slowly, if at all. These differences in degradability are not due to the presence of a proteinase inhibitor in fraction C and D. The results suggest that mast cell chymase preferentially degrades those groups of muscle soluble proteins, the constituents of which have neutral to basic isoelectric points and a relatively low surface hydrophobicity.     

THE CYTONUCLEOPROTEINS OF AMEBAE : II. Some Aspects of Cytonucleoprotein Behavior and Synthesis

Radioactivity, apparently in cytonucleoproteins, from an amino acid-labeled nucleus implanted into a non-radioactive cell appeared in the host nucleus within 10 minutes, and the typical equilibrium ratio 70:30 donor nucleus radioactivity:host nucleus radioactivity was reached in 4 to 5 hours at 25°C. If such binucleates grew and divided, no localization of radioactivity was observable in cells fixed during mitosis, but the protein label remained concentrated in the daughter interphase nuclei for at least 4 generations. Continued migration of cytonucleoproteins was observed if these daughter nuclei were transplanted to other unlabeled cells. The Q₁₀ (19° to 29°C) of the migration rate of radioactive cytonucleoproteins was ca. 1.3, suggesting that passage through the cytoplasm occurred by diffusion. Both non-migratory nuclear proteins and cytonucleoproteins appear to be synthesized in the cytoplasm.     

Stigma proteins of the two loci self-incompatible grass Phalaris coerulescens

Summary Protein extracts from four self-incompatible genotypes of Phalaris coerulescens were subjected to analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and ultra-thin isoelectric focusing. A comparison between stigma, root and leaf extracts showed that there was no significant difference in electrophoretic or isoelectric focusing patterns between the genotypes for both root and leaf proteins. However, stigma protein patterns did vary between genotypes especially within the molecular weight region of 43 000–97 000 and within the pI range 5–7. The stigma-specific changes strongly suggest a link between the self-incompatible genotype and these stigma proteins. However, because there are two loci involved, it is not yet possible to precisely assign particular proteins to each S- or Z-allele.     

A method for three-dimensional protein characterization and its application to a complex plant (corn) extract

Knowledge of host protein properties is critical for developing purification methods for recombinant proteins from a specific host, or for choosing suitable hosts and targeted expression tissues for a specific recombinant protein. A method to obtain a three-dimensional (3D) map (surface hydrophobicity (SH), isoelectric point (pI), and molecular weight (MW)), of a host's aqueous soluble protein properties was developed. The method consists of hydrophobic partitioning in a PEG 3350 (15.7%)-Na(2)SO(4) (8.9%)-NaCl (3%) aqueous two-phase (ATP) system followed by quantitative, 2D-electrophoretic characterization of the proteins of each equilibrium phase and the original extract. The pI and MW of host proteins were obtained directly through 2D electrophoresis. The partition coefficients of individual proteins were obtained by quantitative matching of protein spots in the top and bottom phase gels and calculating the protein partition coefficients from this information. Correlation of the partition coefficient to a SH scale was established by partitioning several model proteins with known surface hydrophobicities in the same ATP system. The inclusion of the extract gel provided for a spot selection criterion based on satisfactory mass balance closure. The method is illustrated by application to a mixture of model proteins and to complex mixtures, that is, corn germ proteins extracted at pH 7 and pH 4.     

Characterization of proteins associated with nuclear ribonucleoprotein particles by two-dimensional polyacrylamide gel electrophoresis.

Rat liver nuclear ribonucleoprotein particles were prepared by two different methods and defined as 40S ribonucleoprotein (40S RNP) and heterogeneous nuclear ribonucleoprotein (HnRNP) particles. The RNP particles were either solubilized in 8 M urea--6 mM 2-mercaptoethanol--20 mM glycine--20 mM Tris--HCl (pH 8.4) or subjected to removal of RNA by phenol extraction prior to solubilizing the proteins in the urea buffer. The proteins associated with 40S RNP and HnRNP were heterogeneous and very similar in their electrophoretic patterns when analyzed by two-dimensional PAGE, except a protein with molecular weight of 62 000 and an isoelectric point (pI) of 6.2 was present only in HnRNP particles. At least 12 major and 22 minor components could be identified in both preparations. The major proteins were found at pI values varying from 6.0 to 8.5 and with molecular weights from 32 000 to 42 000, and a group of proteins with molecular weight approximately 65 000 were more prominent in HnRNP than in 40S RNP. The other components were found mainly at pI ranges from 5.0 to 6.5 with molecular weights from 43 000 to 65 000. The phenol method extracted essentially all proteins associated with either 40S RNP and HnRNP, but was less effective in extracting a group of proteins with pI values from 5.0 to 5.5 and more efficient for proteins with pI values from 7.5 to 8.5. When chromatin proteins isolated by phenol extraction were compared with HnRNP particle proteins isolated by the same method, the electrophoretic mobilities of the HnRNP particle proteins were found to be identical with a fraction nonhistone chromatin proteins. The 40S RNP particles were further purified by metrizamide isopycnic density gradient centrifugation. The electrophoretic patterns of these proteins were very similar to those prepared by sucrose density gradient centrifugation. Therefore, we concluded that the proteins of RNP particles constituted part of the chromatin proteins.     

中国武汉地区葎草花粉变应原蛋白质组分的双向电泳分析

应用蛋白质双向电泳的分析技术对武汉地区生长的主要过敏原———葎草(Humulus scandensLour.)花粉的蛋白质组分进行了分析。应用三氯乙酸法提取葎草花粉总蛋白质,通过等电聚焦和第二向SDS-聚丙烯酰胺凝胶电泳分析获得了完整的葎草花粉全蛋白质图谱。应用专业分析软件(Im ageM aster 2D)对电泳图谱分析表明:通过等电聚焦和第二向SDS-PAGE电泳,有122个不同的蛋白质组分被检测出来,并进一步确定每个蛋白质相应的分子量、等电点和相对含量。研究中获得的高分辨率的双向电泳图谱是我国葎草花粉变应原蛋白质第一张完整的蛋白质图谱,将为今后葎草花粉中致敏蛋白质的检测、分离、基因克隆和变应原的标准化奠定基础。     

Neurotoxins of Bungarus multicinctus vernom. Purification and partial characterization.

The purification to homogeneity of nine neurotoxic components of the venom of Bungarus multicinctus is described. The purified components include alpha-bungarotoxin and two other alpha-type synaptic toxins and beta-bungarotoxin and five other beta-type synaptic toxins. The purified toxins have been characterized by electrophoresis, isoelectric focusing, amino acid analysis, and N-terminal amino acid determination. The alpha-type synaptic neurotoxins constitute a discrete class with molecular weights of 7000-8500, isoelectric points (pI) of 9.0-9.2, and N-terminal isoleucine or methionine. The beta-type synaptic neurotoxins constitute a second group with molecular weights of 20 000-22 000 and pI = 8.8-9.7. Fractions 10 through 13 exhibit a chain structure consisting of a 6000-7000 light chain and a 11 000-15 000 heavy chain apparently covalently stabilized by interchain disulfides. Fractions 9A and 14 were single chains of 11 000-14 000 which resemble the sequenced beta-type synaptic neurotoxin notexin (Halpert, J., and Eaker, D. (1975), J. Biol. Chem. 250, 6990). All of the beta-type synaptic toxins have a single tryptophan and N-terminal aspartic acid or asparagine.     

The presence of actin in nuclei: a critical appraisal.

To assess the significance of actin associations with nuclei, we have examined Amoeba proteus nuclei for the presence of labeled actin under a variety of circumstances without (in most instances) isolating nuclei or breaking up cytoplasms prior to the extraction of proteins.We first established that: the 42,000 dalton proteins (presumed to be actin) present in cytoplasm and non-isolated nuclei are identical electrophoretically; the putative actin of amebas has the same size and almost the same isoelectric point as rat muscle actin; and the peptide “fingerprints” of putative ameba actin and rat actin are very similar after tryptic digestion. We therefore concluded that the 42,000 dalton protein of ameba is actin.We determined that: the concentrations of actin in the cytoplasm and nucleus of amebas are the same; actin is readily lost from nuclei that are released from lysed cells; shortly after a ³⁵S-labeled nucleus is transplanted into unlabeled cytoplasm, or an unlabeled nucleus is transplanted into ³⁵S-labeled cytoplasm, the concentration of ³⁵S-actin in nucleus and cytoplasm is the same; and when cells containing ³⁵S-actin are subjected to long chase periods on unlabeled food, the concentrations of ³⁵S-actin in nucleus and cytoplasm fall in parallel. These observations taken together suggest that actin is not tightly associated with nuclei. Rather, actin may associate with nuclei for the trivial reason that the nuclear envelope is no barrier to free movement of that protein between the two compartments.We conclude that the mere presence of actin in nuclei is insufficient grounds for assuming that it has any role in nuclear functions, such as, for example, chromosome condensation.     

Studies on the microheterogeneity of anterior pituitary follicle-stimulating hormone in the female rat. Isoelectric focusing pattern throughout the estrous cycle

Anterior pituitary (AP) glands were removed from adult female rats at different times throughout the estrous cycle, and the isohormones of follicle-stimulating hormone (FSH) present within them were separated by isoelectric focusing in polyacrylamide gels (PAGE-IEF; pH range 3.0-8.0). Gel eluents were analyzed for FSH content by radioimmunoassay (RIA) and radioreceptor assay (RRA). All AP samples exhibited several peaks of FSH immunoactivity within a pH range of 6.2-4.0; the major peak constantly exhibited an isoelectric point (pI) of 4.9-4.5. To quantify differences in the IEF pattern of AP-FSH between the pituitaries collected during the different days of the cycle, each PAGE-IEF profile was divided into 7 regions (pI 7.0-6.3 = Area 1, 6.2-5.5 = Area 2, 5.4-5.0 = Area 3, 4.9-4.5 = Area 4, 4.4-4.0 = Area 5, 3.9-3.5 = Area 6, and less than 3.5 = Area 7), and the amount of FSH present within each was determined. In all APs collected at 0900 h of diestrus 1 (D1) and 2 (D2), proestrus (P), and estrus (E); at 1300 h of D1, D2 and E; at 2200 h of P; and at 0200 h of E, the majority of FSH immunoactivity (37-55% of total FSH on gel) focused within Area 4, whereas Areas 2 and 3 contained minor amounts of FSH activity (11-26% and 14-24%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

THE CYTONUCLEOPROTEINS OF AMEBAE : I. Some Chemical Properties and Intracellular Distribution

Autoradiographs of whole Amoeba proteus host cells fixed after the implantation of single nuclei from A. proteus donors labeled with any one of 8 different radioactive amino acids showed that the label had become highly concentrated in the host cell nucleus as well as in the donor nucleus and that the cytoplasmic activity was relatively low. When these amebae were sectioned, the radioactivity was found to be homogeneously distributed throughout the nuclei. The effect of unlabeled amino acid "chaser," the solubility of the labeled material, and the long-term behavior of the labeled material gave evidence that the radioactivity was in protein. At equilibrium, the host cell nucleus contained approximately 30 per cent of the radioactivity distributed between the two nuclei. This unequal nuclear distribution is attributed to the presence of two classes of nuclear proteins: a non-migratory one that does not leave the nucleus during interphase, and a migratory one, called cytonucleoprotein, that shuttles between nucleus and cytoplasm in a non-random manner. It is estimated that between 12 per cent and 44 per cent of the cytonucleoproteins are present in the cytoplasm of a binucleate cell at any one moment. Nuclei of Chaos chaos host cells also concentrated label acquired from implanted radioactive A. proteus nuclei.     

Nuclear import and export of plant virus proteins and genomes

Nuclear import and export are crucial processes for any eukaryotic cell, as they govern substrate exchange between the nucleus and the cytoplasm. Proteins involved in the nuclear transport network are generally conserved among eukaryotes, from yeast and fungi to animals and plants. Various pathogens, including some plant viruses, need to enter the host nucleus to gain access to its replication machinery or to integrate their DNA into the host genome; the newly replicated viral genomes then need to exit the nucleus to spread between host cells. To gain the ability to enter and exit the nucleus, these pathogens encode proteins that recognize cellular nuclear transport receptors and utilize the host's nuclear import and export pathways. Here, we review and discuss our current knowledge about the molecular mechanisms by which plant viruses find their way into and out of the host cell nucleus.     

Analysis of total proteins in pollen of Humulus scandens Lour in Wuhan Region of China by two-dimensional electrophoresis

Total proteins in the pollen of Humulus scandens Lour,one of the most popular aeroallergens in China,were analyzed by two-dimensional electrophoresis in the current study.The proteins were extracted by Trichloracetic acid (TCA) method,and then separated by isoelectric focusing as the first dimension and SDS-PAGE as the second dimension.The spots of proteins were visualized by staining with Coomassie Brilliant Blue.After analysis with software (ImageMaster 2D),122 different proteins were detected;isoelectric point (pI),Molecular weight (MW) and relativevolume of each protein in the pollen were also discovered.This is the first high-resolution,two-dimensional protein map of the pollen ofHumulus scandens Lour in China.Our finding has built a solid foundation for identification,characterization,gene cloning and standardization of allergenic proteins in the pollen ofHumulus scandens Lour for further studies.     

Multiple antigens in the rat visceral yolk sac induce teratogenic antisera

Preparative isoelectric focusing was used to fractionate the supernatant from a homogenate of day 19 rat visceral yolk sac. Three fractions, of pI ranges 3.5-5.0, 5.0-7.0, and 7.0-9.0, were isolated and used to immunize rabbits, by four or six weekly injections, each containing 5 mg protein. The resulting antisera were all teratogenic when injected into rats on day 9 of gestation, but widely differing potencies were observed. The most potent antiserum was that against yolk sac components focusing in the pI 7.0-9.0 range: An optimum teratogenic dose of 50 mg protein per kg body weight was observed, and a dose of 100 mg/kg was shown to cause 100% embryonic resorption. Antiserum against the fraction focusing in the pI 3.5-5.0 range was the least teratogenic: A significant incidence of embryonic malformation and death was seen only at doses of 600 mg/kg and above. The two fractions that yielded the more teratogenic antisera were refocused over narrower pH ranges, yielding four subfractions in the pI 5.0-7.0 range and eight subfractions in the pI 7.0-9.0 range. Antisera against each of these 12 fractions were raised in rabbits; most of these antisera were shown to be teratogenic, although of differing potencies. It is concluded that the yolk sac contains many antigens that can elicit antibodies with teratogenic and yolk sac-localizing properties.     

Analysis and separation of synaptosomal membrane proteins

Synaptosomal membrane proteins solubilized with 8% CHAPS-8 M urea were analyzed with twodimensional electrophoresis (2DE). The membrane proteins were resolved up to 250 spots on a 2DE map, ranging in isoelectric points (pI) from 3.5 to 10.0 and molecular weights (MW) from 10 kDa to 200 kDa. Comparison of the mapped proteins of synaptosomal membranes with those of myelin and mitochondorial membranes revealed that synaptosomal membrane proteins were characteristic in the area of pI from 4.0 to 7.5 and MW from 20 kDa to 130 kDa, and that at least 30 spots were synaptosomal membrane-specific proteins. Most of these 30 proteins have not been previously described, named, and characterized Serial numbers (from SY1 to SY30) were assigned to the proteins on the map in order to investigate them systematically. A preliminary attempt to separate synaptosomal membrane proteins was carried out using a reversed-phase HPLC system. Several proteins could either be isolated or enriched. SY10 (pI 4.6; MW 56 kDa) was one of these proteins, and was of particular interest for its unusual behavior on the reversed-phase column, and for its binding to an immobilized protein A-gel.     

Peridinin-Chlorophyll a Proteins of the Dinoflagellate Amphidinium carterae (Plymouth 450)

The marine dinoflagellate Amphidinium carterae (Plymouth 450) releases several water-soluble peridinin-chlorophyll a proteins after freezethawing. These chromoproteins have a molecular weight of 39.2 × 10³ and are comprised of noncovalently bound peridinin and chlorophyll a and a nonoligomeric protein. They have distinct isoelectric points and may be resolved into six components by either isoelectric focusing on polyacrylamide gel or ion exchange chromatography. The predominant chromoprotein, which has a pI of 7.5, constitutes about 90% of the extractable peridinin-chlorophyll a protein. It consists of an alanine-rich apoprotein of molecular weight 31.8 × 10³ stoichiometrically associated with 9 peridinin and 2 chlorophyll a molecules. Additionally, the peridinin-chlorophyll a proteins with pI values of 7.6 and 6.4 were purified and found to have amino acid and chromophore composition essentially identical with the pI 7.5 protein. Peridinin-chlorophyll a protein, pI 7.5, after treatment at alkaline pH was transformed into several more acid pI forms of the protein, strongly suggesting that the naturally occurring proteins are deamidation products of a single protein. Fluorescence excitation and emission spectra demonstrate that light energy absorbed by peridinin induces chlorophyll a fluorescence presumably by intramolecular energy transfer. The peridinin-chlorophyll a proteins presumably function in vivo as photosynthetic light-harvesting pigments.     

Chicken erythrocyte membranes: comparison of nuclear and plasma membranes from adults and embryos

These experiments were designed to determine whether the migration of RNA molecules from an implanted nucleus to the host cytoplasm and from there into the host cell nucleus against a concentration gradient might reflect an artefact induced by the process of nuclear transplantation. That is, are RNA molecules, as previously shown for certain nuclear proteins, caused to artefactually leave a manipulated nucleus and then move into the host cell nucleus (as well as return to the grafted nucleus) during the recovery period?A variety of experiments involving different kinds of manipulative sequences and different numbers of nuclear transplantations suggest—but do not prove—that no artefact is involved in the migration of RNA from one nucleus to another but two experiments strongly support the view that the shuttling activity is a normal physiological process. One of the latter involved a determination of the rate of egress of ³H-RNA from an implanted nucleus and reveals that that rate, in contrast with the equivalent rate of egress for labeled proteins which is clearly abnormal after micromanipulation, is totally consonant with the rate of movement of RNA from nucleus to cytoplasm established from experiments that do not involve micromanipulation. The other experiment involves comparison of (1) the amount of radioactivity acquired by an unlabeled nucleus present in the cell at the time a labeled nucleus is implanted with (2) the amount of radioactivity acquired by an unlabeled nucleus implanted after a labeled nucleus had been implanted and had time to recover from any possible operation-induced trauma. With ³H-protein nuclei the host nuclei of (1) acquired much more label than the host nuclei of (2) because in (1) the host nuclei were able to acquire much of the artefactually-released ³H-protein. For the ³H-RNA experiments, however, little difference was found between (1) and (2) in the amount of label acquired by the host cell nuclei. It can be concluded that little, if any, of the non-random shuttling activity of RNA molecules can be a reflection of an artefact.     

Physicochemical characterization and phylogenetic comparison of fish lens proteins.

The composition of soluble eye lens proteins from four chondropterygiian and fourteen teleostean fishes were analyzed for heterogeneity in MW and pI. Lens proteins from all the fish species studies are distributed in the pI range 4.3-9.0 with polypeptides in the range 17,500-31,000 Da. Phylogenetic trees are constructed based on the observations.