SDS-containing Polyacrylamide Gels Research Articles

The Plastid-encoded ccsA Gene Is Required for Heme Attachment to Chloroplast c-type Cytochromes

A chloroplast gene, ycf5, which displays limited sequence identity to bacterial genes (ccl1/cycK) required for the biogenesis of c-type cytochromes, was tested for its function in chloroplast cytochrome biogenesis in Chlamydomonas reinhardtii. Targeted inactivation of the ycf5 gene results in a non-photosynthetic phenotype attributable to the absence of c-type cytochromes. The cloned ycf5 gene also complements the phototrophic growth deficiency in strain B6 of C. reinhardtii. B6 is unable to synthesize functional forms of cytochromes f and c6 owing to a chloroplast genome mutation that prevents heme attachment. The selected (phototrophic growth) as well as the unselected (holocytochrome c6 accumulation) phenotypes were restored in complemented strains. The complementing gene, renamed ccsA (for c-type cytochrome synthesis), is expressed in wild-type and B6 cells but is non-functional in B6 owing to an early frameshift mutation. Antibodies raised against the ccsA gene product recognize a 29-kDa protein in C. reinhardtii. The 29-kDa protein is absent in strain B6 but is restored in a spontaneous revertant (B6R) isolated from a culture of B6. Sequence analysis of the ccsA gene in strain B6R indicates that it is a true revertant. We conclude that the ccsA gene is expressed and that it encodes a protein required for heme attachment to c-type cytochromes.

Antigenic structure of histone H1�

In order to study the antigenic structure of histone H1(0) the purified protein from mouse liver was subjected to different chemical and enzymatic treatments (CNBr, acetic acid, trypsin, chymotrypsin). The resulting peptides were fractionated in SDS-containing or acid-urea polyacrylamide gels, transferred by electroblotting onto nitrocellulose paper and probed with specific rabbit anti-H1(0) antiserum. The C-terminal fragments 99-193 (obtained following acetic acid hydrolysis) and 107-193 (obtained by chymotrypsin digestion) also exhibited strong immunoreactivity. Fragment 1-30 (CNBr cleavage) contained antigenic determinants while the shorter fragments 1-22 and 1-28 (acetic acid hydrolysis) failed to show any detectable reactivity. It was concluded that, in contrast to histone H5 whose reactivity is mainly concentrated to the globular domain of the molecule, the antigenic determinants in histone H1(0) are more or less evenly distributed along the polypeptide chain with the possible exception of the short unstructured N-nose.

Large‐scale preparation of T4 endonuclease VII from over‐expressing bacteria

Endonuclease VII is the product of gene 49 of phage T4 and was the first enzyme shown to resolve Holliday structures in vitro [Mizuuchi, K. et al. (1982) Cell 29, 357-365]. Low amounts of the enzyme were originally purified from phage-infected cells [Kemper, B. & Garabett, M. (1981) Eur. J. Biochem. 115, 123-131]. We now report a purification procedure for milligram amounts of cloned endonuclease VII expressed in Escherichia coli with gene 49 under the control of a temperature-inducible promoter on a plasmid system [Tomaschewski, J. (1988) PhD Thesis, University of Bochum, FRG]. The protein was purified 500-fold from crude extracts in five steps with a recovery of 15%. The steps include (a) poly(ethyleneglycol)/dextran two-phase separation; (b) DEAE-cellulose; (c) single-stranded DNA-agarose; (d) Mono-Q and (e) Mono-S chromatography. The final protein was more than 98% pure as estimated from SDS/PAGE analysis. The protein has an apparent molecular mass of 17.8 kDa on SDS-containing polyacrylamide gels and 36 kDa when determined by gel filtration or sedimentation through sucrose gradients in the presence of high salt (600 mM NaCl). In the absence of additional salt, the enzyme has a tendency to aggregate and products of molecular masses differing in steps of about 18 kDa appear on SDS-containing polyacrylamide gels.

Electrophoretic and Immunochemical Characteristics of H1 Histone Variants in Peas ( Pisum sativum L.)

The heterogeneity of the pea HI histone family was studied in plumula and epicotyl of Pisum sativum at three stages of germination. By electrophoretic fractionation in urea-acetic acid or SDS-containing polyacrylamide gels, four major subfractions of H1 histone were identified. Specific quantitative ratios of H1 histone variants in two stem parts, characterized by different degrees of cell differentiation at three stages of germination, were established. The monospecific polyclonal anti-pea H1a serum raised in rabbits reacts with the four H1 variants in pea plumula and epicotyl, and immunologically «recognizes» two additional subfractions from the total histone protein. It was shown that the H1 histone of primary pea roots presents six subfractions analogous to their stem counterparts, and the lack of strong organ specificity of plant H1 histones was suggested. The results from the immunochemical testing of H1 variants of evolutionary distant plants — peas, beans and maize — confirm the existence of species-specific H1 families in plants.

Properties of purified actin depolymerizing factor from chick brain.

Actin depolymerizing factor (ADF) from 19-day embryonic chick brains has been purified to greater than 98% homogeneity with a yield of 7.2 mg/100 g of brain. Quantitative immunoblotting with a monospecific antibody to ADF indicated that ADF comprises 0.3% of the total brain protein, resulting in an actual purification yield of about 20%. Brain ADF migrates as a single polypeptide of 19,000 kDa on SDS-containing polyacrylamide gels. The molecular weight of the native protein determined from sedimentation equilibrium in buffers containing from 50 to 200 mM KCl is 20,000. The secondary structure of ADF calculated from the circular dichroic spectrum consists of about 22% alpha-helix, 24% beta-sheet, and 18% beta-turn. ADF contains a blocked N-terminus, a single tryptophan residue located about one-third of the way from one end of the protein, and six cysteine residues (all in reduced form in the native protein). All six cysteine residues could be chemically modified with eosinylmaleimide under nondenaturing conditions; however, ADF activity was lost when more than one cysteine residue was modified. ADF microheterogeneity has been observed upon nonequilibrium pH gradient electrophoresis in polyacrylamide gels containing 9 M urea, the major isoform having a pI of congruent to 7.9-8.0. ADF can interact with either monomeric or filamentous actin to give a complex which can be isolated by gel filtration chromatography. Both major and minor isoforms of the ADF are found in the complex. Assembly-competent actin and active ADF can both be recovered from the complex by chromatography on ATP-saturated DEAE-cellulose.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Interferon on Secretion of Proteins by Various Murine Cell Lines

The interferons (IFNs) have been shown to be antagonistic to the growth stimulatory effects of mitogens on cultured cells. A report of the interactions of IFN-beta and platelet-derived growth factor on BALB/c-3T3 mouse cells established that IFN itself induced the secretion of a limited number of proteins from this cell line. The present work was undertaken to determine if other murine cell lines treated with homologous IFN-beta also secreted new or additional protein(s) in response to this agent and if this response correlated with other phenotypic properties of the cells. The cell lines examined included L929 cells and two derivatives of this line (GM347 and WDIFN), CAK-TK-, Swiss-3T3, and BALB/c-3T3. Each line was exposed to [35S]methionine in the absence and in the presence of IFN-beta, the supernatant fluids collected, and the radioactive, secreted proteins examined by fluorography after electrophoresis through SDS-containing polyacrylamide gels. Two cell lines (GM347 and Swiss-3T3) did not appear to secrete new or additional proteins after IFN treatment. However, four lines (L929, WDIFN, CAK-TK-, and BALB/c-3T3) did secrete new or additional proteins in response to IFN. Thus IFN-induced secretion of protein appeared to be a common but not universal phenomenon. In addition, although the number and apparent size(s) of the IFN-induced, secreted proteins were different in these various lines, one protein (Mr = 89-90,000) appeared to be secreted by each of them. In this respect it was unique. Moreover the IFN-induced secretion of protein did not appear to correlate with the antiviral or antiproliferative effects of IFN.

Expression of μ and γ 1 membrane forms of immunoglobulin segregate in somatic cell hybrids

In the present investigation, we have utilized the somatic cell hybridization technique to generate an experimental model for studying the differential expression of membrane (mIg) and secreted (sIg) forms of immunoglobulin that characterize different stages of B cell development. We describe here that fusion of the dextran-binding myeloma, MOPC 104E (μ,λ 1) and the phthalate-binding B cell hybridoma, 2C3E1 (γ 1, kappa) results in the formation of antigen-specific, double hybrids (tribrids) that coexpress both parental secreted forms of Ig but express only one of the two possible membrane forms of immunoglobulin (Ig). This segregated expression of membrane Ig is a new and unexpected finding that has been substantiated here by both immunological and biochemical methods. Analysis by SDS-containing polyacrylamide gels (SDS-PAGE) reveals distinct and characteristic migration patterns for each of the four Ig heavy chains in the tribrids (μ membrane, μ. secreted, γ 1, membrane and γ 1 secreted). Immunochemical analysis of the immunoglobulin from the tribrids confirms the coexpression of both secreted forms of immunoglobulin in most of the tribrid lines tested and indicates that about 30% of the tribrids express only phthalate-specific γ 1, membrane Ig, while 38% express only dextran-binding μ. membrane Ig. About 30% of the tribrids secrete both antibodies but express no membrane form and less than 1% are non-secretors. Approximately 2% initially express both membrane forms of Ig, as determined by immunoeytoadherence assay using appropriate target cells but subsequently express only one membrane form during propagation in vitro. SDS-PAGE analysis of surface labeled tribrids confirms that in tribrids expressing membrane Ig, only a single mIg is synthesized. These results suggest that the expression of the secreted and membrane forms of immunoglobulin are separately regulated and the tribrids represent a model with which to study the mechanisms involved in the regulation of each structurally distinct immunoglobulin form.

Spectral and potentiometric analysis of cytochromes from Bacillus subtilis.

Bacillus subtilis cytoplasmic membranes contain several cytochromes which are linked to the respiratory chain. At least six different cytochromes have been separated and identified by ammonium sulphate fractionation and ion-exchange chromatography. They include two terminal oxidases with CO-binding properties and cyanide sensitivity. One of these is an aa3-type cytochrome c oxidase which has characteristic absorption maxima in the reduced-oxidized difference spectrum at 601 nm in the alpha-band and at 443 nm in the Soret band regions. In the alpha-band two separate electron transitions with Em = +205 mV and Em = +335 mV can be discriminated by redox potentiometric titration. The other CO-binding cytochrome c oxidase contains two cytochrome b components with alpha-band maxima at 556 nm and 559 nm. Cytochrome b556 can be reduced by ascorbate and has an Em + +215 mV, whereas cytochrome b559 has an Em = +140 mV. Furthermore a complex consisting of a cytochrome b564 (Em = +140 mV) associated with a cytochrome c554 (Em = +250 mV) was found. This cytochrome c554, which can be reduced by ascorbate, appears to have an asymmetrical alpha-peak and stains for heme-catalyzed peroxidase activity on SDS-containing polyacrylamide gels. A protein with a molecular mass of about 30 kDa is responsible for this activity. A cytochrome b559 (Em = +65 mV) appears to be an essential part of succinate dehydrogenase. Finally a cytochrome c550 component with an apparent mid-point potential of Em = +195 mV has been detected.

Analyses of protein extracts of human breast cancers: changes in glycoprotein content linked to the malignant phenotype

The protein and glycoprotein composition of Triton X-100 extracts of breast biopsies from 17 women with benign breast disease and from 11 women with invasive breast carcinoma were investigated using electrophoresis in SDS-containing gradient polyacrylamide gels, followed by Coomassie Blue (CB) staining and the binding of radio-iodinated wheat germ agglutinin (WGA). Patterns were analysed after the CB-step for differences in protein composition, and after the WGA-step for differences in glycoprotein composition. Tissue extracts from patients with benign breast disease have less CB stained bands than similar extracts from the cancer patients. A particular consistent change was the appearance of an extra band at 58 Kdaltons in the cancer extracts. In contrast to the CB results, WGA detected less major bands, in the 40-60 Kd region, in the cancer extracts than at similar locations in benign extracts. Analysis of blood sera using the above techniques suggested that certain serum proteins could account for some of the WGA changes, but not the changes after CB staining. However, residual contamination of the specimens by blood proteins seemed unlikely because of the washing procedure used, unless these components were very strongly associated with the tissue. Differential synthesis of serum proteins by benign and malignant breast tissue may also explain some of our findings. Examination of the histopathology adjacent to the extracted tissue suggested that the degree of reduction in WGA-binding may be related to the extent of local invasiveness. Other animal and human studies suggest that reduced glycosylation of tumour-associated proteins may be linked to increased malignancy. The current findings may reflect a general pattern of change in tumour glycoprotein composition linked to malignant expression.

Absence of the α and γ subunits of 7S nerve growth factor in denervated rodent iris: Immunocytochemical studies

Immunocytochemical studies were performed to determine if denervated rodent iris produces nerve growth factor (NGF) in a form chemically similar to that of the 7S NGF complex in mouse submandibular glands. Antisera to the α, β, and γ subunits of 7S NGF were raised in rabbits and characterized on immunoblots of SDS-containing polyacrylamide gels. Antisera were applied to stretch preparations of rat and mouse irides that were cultured for periods of 2 to 6 days or sympathetically denervated by superior cervical ganglionectomy and left in situ 4 days. Antibody binding was visualized by indirect immunofluorescence. In control studies done on plastic sections of mouse submandibular glands, antisera co-localized the three subunits of 7S NGF within secretory granules of granular tubule cells. In denervated rat iris, β NGF immunoreactivity was evident in a cellular plexus that resembled in distribution and morphology nerve fibers in the normal iris, in agreement with a previous study ( R. A. Rush (1984). Nature (London) 312, 364–367 ). Identical staining patterns were observed in mouse iris. In neither rat nor mouse, however, did the nerve-like processes stain with antibodies to the α or γ subunits nor was any other structure in the iris detectably immunoreactive for these proteins. This evidence suggests that the NGF-like protein in denervated rodent iris is not synthesized as part of the 7S NGF complex. Iris also did not react with antibodies to epidermal growth factor, a protein co-localized with NGF in mouse submandibular glands and in guinea pig prostate.

On the origin of the helper component of tobacco vein mottling virus: translational initiation near the 5′ terminus of the viral RNA and termination by UAG codons

The nature of the polypeptide products encoded by the 5'-terminal region of the RNA of the potyvirus, tobacco vein mottling virus (TVMV), was investigated. Single-stranded DNA probes complementary to either nucleotides 1100-2100 or 2100-2820 from the 5' terminus of the RNA were prepared by subcloning recombinant plasmids in bacteriophage M13. These were hybridized to TVMV RNA, the DNA:RNA hybrids translated in a reticulocyte lysate cell-free translation system (hybrid-arrested translation), and the products analyzed by electrophoresis on SDS-containing polyacrylamide gels. The hybrids produced altered patterns of polypeptides which indicated that the major product, P75, was translated from the 5' terminus of the RNA. The N-terminal portion (molecular weight approximately 35,000) of P75 did not react with antisera to any of the five known potyviral proteins, suggesting the existence of a previously unidentified cistron at the 5' terminus. Translation of additional RNA sequences produced a polypeptide with a molecular weight of 68,000 which was immunoprecipitable by antiserum to TVMV helper component, establishing the coding region for helper component near the 5' terminus. Limited DNA sequence analysis of the region encoding the C terminus of P75 revealed an open reading frame of 369 nucleotides followed by a pair of UAG Codons. Pulse-chase experiments demonstrated that in the first 10 min of incubation, the only polypeptide initiated was P75, suggesting that products downstream from P75 arose from in vitro fragmentation of TVMV RNA and translation of the RNA fragments.

Protein counterparts of human and simian cytomegaloviruses

Cytomegalovirus (CMV) proteins from isolates of both human (HCMV) and Simian (SCMV) origin have been compared. Three classes were analyzed: the immediate-early (IE) proteins, other infected-cell-specific proteins not present in virus particles, and the proteins that constitute the mature extracellular virion. Comparisons were based on one-and two-dimensional (charge-size) separations in denaturing polyacrylamide gels, and on the selectivity of biosynthetic radiolabeling with [ 32P)orthophosphate and [ 3H]glucosamine. Results indicate that most, if not all, of the HCMV and SCMV proteins recognized, have counterparts in strain Colburn. As a group, the simian strains exhibit protein similarities that distinguish them from the human strains. Among the most diagnostic of these are the 205K and 145K virion proteins, each of which is about 7K smaller than its HCMV counterpart, and the predominant IE proteins, which are 10K to 20K (depending upon the strain) larger than their HCMV counterparts. The proteins of strain Colburn are shown to be more like those of the simian isolates than the human, and more like those of a vervet strain than rhesus. Leads provided by experiments using strain Colburn have aided in the identification of a previously unrecognized, abundant virion protein that is a principal phosphate acceptor, both in vivo and in vitro, Three additional phosphorylated proteins are identified in HCMV virions, as well as three glycoproteins. Only two HCMV strain-specific protein differences were detected by comparisons based on separation in SDS-containing polyacrylamide gels—one in the IE protein of strains Towne and Davis; the other in a virus capsid protein of strain AD169.

In vitro labeling of proteins by reductive methylation: Application to proteins involved in supramolecular structures

Actin and tropomyosin, purified from both muscle and brain, and alpha-actinin, purified from muscle, have been labeled in vitro by reductive methylation to specific activities of greater than 10(5) dpm/micrograms protein. Actin so modified bound DNase I and polymerized identically to unmodified actin. Furthermore, the spectral properties of actin did not change after labeling. The interactions of labeled tropomyosin and alpha-actinin with F-actin were nearly identical to those of the unmodified proteins. These modified proteins comigrated with their unmodified counterparts in both SDS-containing polyacrylamide gels and isoelectric focusing gels. The labeled actin was quantitatively extracted from SDS-containing polyacrylamide gels (yield greater than 98% of radioactivity applied demonstrating that all of the radioactivity was protein bound. The reductive methylation procedure worked well at pH 8.0-8.5 in either pyrophosphate buffer or Bicine buffer using formaldehyde with [3H]-sodium borohydride as the reducing agent. The procedure could also be performed at pH 7.0 in phosphate buffer using (14C]-formaldehyde with sodium cyanoborohydride as the reducing agent. Proteins so labeled are ideal for use in quantitative experiments involving protein-protein interactions.

Cytoskeletal and contractile structures in bovine lens cell differentiation

Bovine lenses from animals of different ages were separated into two epithelial sections, a cortical region and the lens nucleus. Both the 10000 g supernatant fraction and pellet of these sections were analysed by electrophoresis in SDS-containing polyacrylamide gels. When comparing total protein patterns of the cytoskeletal preparations from the different parts of lenses of different ages a decrease in the amount of vimentin, the protein subunit of lens intermediate-sized filaments (IF), was observed upon lens cell differentiation and aging. Amounts of monomeric (G) and filamentous (F) actin in the different stages of lens cell differentiation were quantitated using the DNase I inhibition technique. A significant increase in the relative amount of F-actin was observed upon fibre cell formation. A slight, but significant increase in the total amount of actin relative to the total amount of cellular protein was observed when passing from the central part of the lens epithelium to the epithelial cells in the elongation zone. In the fibre cells the amount of total actin decreased from cortex to nucleus. A possible function of microfilament-assembly in the process of lens cell differentiation is suggested.

Synthesis in vitro of urokinase-like material using polyA (+)RNA from human kidney and from cultured human embryonic kidney cells

Total RNA was extracted from human kidney or human embryonic kidney cells in culture, then passed through oligo-d(T) cellulose to obtain total polyA(+)RNAs. Messenger activity of the preparations was checked in vitro using the rabbit reticulocyte lysate system and peptides synthesized invitro were separated on SDS-containing polyacrylamide gels. Identification of urokinase-like material was achieved by immuno-precipitation with antibodies raised against pure commercial urokinase and by radioimmunoassays where urokinase synthesized in vitro was competed out with unlabelled urokinase. The data show that both polyA(+)RNAs code for urokinase-like material and that the apparent Mw of the in vitro translations product is roughly 47 K daltons (in reducing conditions).

Synthesis of rat prostatic binding protein in xenopus oocytes and in wheat germ

Prostatic binding protein (PBP) is a quantitatively important steroid-binding protein present in rat ventral prostate. Electrophoresis on SDS-containing polyacrylamide gels shows that PBP is composed of two subunits, F and S having molecular weights of 16,000 and 18,000. Upon reduction these subunits dissociate further into smaller components. Translation of mRNA from rat ventral prostate in a wheat germ cell-free system or in Xenopus oocytes results in the formation of polypeptides immunoprecipitable with an anti-PBP antiserum. However, as opposed to the wheat germ system, only the oocytes synthesize polypeptides, that are electrophoretically identical to those of native cytosolic PBP.

Comparison of the Capsid Polypeptides of Various Bluetongue Virus Serotypes

The molecular sizes of the capsid polypeptides of different serotypes of bluetongue virus (BTV) have been compared to those of serotype 10A, after electrophoretic fractionation on SDS-containing polyacrylamide gels. Computer-programmed dis criminant analyses of the polypeptides’ migration distances indicated significant differences between serotypes. The results obtained are in agreement with those of molecular hybridization studies of the viral genome segments, suggesting that the main determinants of its serological specificity reside in the two polypeptides forming the outer coat of the virus.

Outer membrane proteins of Escherichia coli: II. Heterogeneity of major outer membrane polypeptides

When E. coli outer membrane protein is dissolved in sodium dodecyl sulfate (SDS) solution and boiled briefly, a single major peak (peak B) with a molecular weight of 42,000 daltons is observed on SDS-containing polyacrylamide gels. If the protein is dissolved in SDS solution at 37 °C and applied to gels without further treatment, peak B disappears and two other major peaks appear: Peak A, which is composed of aggregates and migrates more slowly than peak B, and peak C which is composed of monomeric protein not fully reacted with SDS and which migrates faster than peak B. When cyanogen bromide peptides of protein from peak A and peak C were compared, it was evident that peak A and peak C contained entirely different polypeptides. This was further confirmed by differential labeling studies with methionine and leucine. The cyanogen bromide peptide profiles of protein from peak A suggested that this peak was composed of two polypeptides, and this was confirmed by electrophoresis in an alkaline gel system which resolves peak B into three subcomponents. Two of these were derived from peak A and the third was derived from peak C. These results indicate that the outer membrane of E. coli contains at least three nonidentical major polypeptides, each of which has a nearly identical molecular weight of about 42,000 daltons. These polypeptides are present in identical proportions in the soluble and insoluble fractions obtained when the outer membrane is treated with Triton X-100 plus EDTA.

An investigation of the subunit structure and AMP-deaminases from rabbit and chicken muscle

Native rabbit and chicken muscle AMP-deaminases have been compared by gel filtration chromatography. These studies, along with previously-reported ultracentrifugal analyses, suggest that these enzymes are virtually identical in size and shape. Chemical and physical evidence is presented which shows that: (a) each enzyme has four subunits; (b) the molecular weights of these subunits are indistinguishable (69,000 Daltons and 73,000 Daltons, as determined by gel filtration chromatography in guanidine hydrochloride and electrophoresis in SDS-containing polyacrylamide gels, respectively; and (c) the native enzymes are not stabilized by disulfiede bridges. Although previous studies suggest that these enzymes share certain structural features, the results presented here provide direct evidence, at the molecular level, that the adenylate deaminases from these sources have similar quaternary structures.