Selenium-containing peroxidases of germinating barley

Germinating barley grown on an artificial medium was exposed to⁷⁵Se-selenite for 8 d. Then the leaves were homogenized and proteins were separated by means of Sephadex G-150 filtration, followed by DEAE-Sepharose chromatography. Each fraction collected was assayed for total protein, radioactivity, and peroxidase activity. In barley leaves, three protein peaks (peaks no. I, II, and III) with peroxidase activity could be separated by Sephadex G 150 filtration. Each fraction was then further separated on DEAE-Sepharose chromatography. Thus, peaks I and II were resolved by DEAE-Sepharose into one major and two minor peaks of radioactivity. However, only the major peak showed peroxidase activity. Peak III was resolved from the gel filtration on the DEAE-sepharose into one major and four minor peaks of radioactivity. The major and three of the minor radioactivity peaks contained peroxidase activity. The protein fractions were separated by polyacrylamide gel electrophoresis. The molecular weights of separated proteins were estimated by means of molecular markers, and⁷⁵Se radioactivity was evaluated by autoradiography. Thus, gel filtration peak I contained four bands with mol wts of 128, 116, 100, and 89 kDa. Of these, the 89 kDa protein contained selenium. Peak II contained three protein bands, with mol wts 79.4, 59.6, and 59.9. The 59.6 band was a selenoprotein. Peak III contained four protein bands (and some very weak bands). The four major bands had mol wts of 38.6, 31.6, 30.2, and 29.2 kDa. The last mentioned band was a selenoprotein.     

Ribulose 1,5-bisphosphate carboxylase and polyhedral bodies of Chlorogloeopsis fritschii

Ribulose 1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) activity was approximately equally distributed between supernatant and pellet fractions produced by differential centrifugation of disrupted cells of Chlorogloeopsis fritschii. Low ionic strength buffer favoured the recovery of particulate RuBP carboxylase. Density gradient centrifugation of resuspended cell-free particulate material produced a single band of RuBP carboxylase activity, which was associated with the polyhedral body fraction, rather than with the thylakoids or other observable particles. Isolated polyhedral body stability was improved by density gradient centrifugation through gradients of Percoll plus sucrose in buffer, which yielded apparently intact polyhedral bodies. These were 100 to 150 nm in diameter and contained ring-shaped, 12 nm diameter particles. It is inferred that the C. fritschii polyhedral bodies are carboxysomes. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of SDS-dissociated polyhedral bodies revealed 8 major polypeptides. The most abundant, with molecular weights of 52,000 and 13,000, correspond with the large and small subunits, respectively, of RuBP carboxylase.Abbreviations RuBP ribulose 1,5-bisphosphate - Ru5P ribulose 5-phosphate - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - EDTA ethylenediamine tetraacetic acid - Tris tris (hydroxymethyl) methylamine - IB isolation buffer - TCA trichloroacetic acid     

Purification and characterization of a liver-specific antigen.

A liver-specific antigen (F-antigen) previously demonstrated in saline extracts of BALB/c mouse liver by double immunodiffusion was isolated and characterized. The antigen was found widely distributed among mammals but absent from avian and frog liver extracts. In immunoelectrophoresis it had an electrophoretic mobility similar to that of serum beta2-globulins, was relatively thermolabile, and was precipitated at 30 to 70% saturated ammonium sulfate concentrations. Evidence was presented that this antigen is a protein or a moiety closely associated with protein. Gel-filtration on Sephadex G-200 revealed liver-specific antigenicity in the second peak. Ion-exchange chromatography on DEAE-Sephadex A-50 revealed four peaks of which only the third one exhibited liver-specific antigenicity. This active peak contained 11 polypeptides on SDS polyacrylamide gel electrophoresis. After electrophoresis on acrylamide gel in the absence of SDS, antigenic activity was detected on one fast-moving band. Extraction of the protein band followed by SDS gel electrophoresis showed one major component of m.w. 75,000 and two major bands of m.w. 72,000 and 93,000, respectively.     

Preliminary evidence for a processing error in the biosynthesis of Gaucher activator in mucolipidosis disease types II and III.

Activator protein (AP), which stimulated fibroblast sphingomyelinase activity, was isolated from the spleen of a patient with Gaucher's disease type I by the combined techniques of heat and alcohol denaturation, DEAE-cellulose column chromatography, gel filtration, preparative polyacrylamide-gel electrophoresis and decyl-agarose chromatography. Urea/sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis showed two bands, one with an Mr of approx. 3,000 and the other with an Mr of 5,000-6,500. Similarly, SDS/polyacrylamide-gel electrophoresis performed in the absence of urea revealed the presence of two components, one of which adsorbed to a concanavalin A (Con A) column. Both components stimulated sphingomyelinase activity. On a non-denaturing polyacrylamide gel containing Triton X-100, four major components, two of which bound to Con A, were detected with the dye Stains-All. Cross-reacting material (CRM) to polyclonal Gaucher spleen AP antibodies was detected in normal fibroblasts and in fibroblasts from patients with sphingomyelinase and beta-glucocerebrosidase deficiency states (Niemann-Pick and Gaucher's diseases respectively). CRM in normal fibroblasts adsorbed to Con A columns and had the same mobility on SDS/polyacrylamide-gel electrophoresis as Con A-adsorbing Gaucher spleen AP. Normal AP was not observed in mucolipidosis type II (I-cell disease) fibroblasts; instead, extracts from these cells revealed the presence of two closely migrating bands with higher Mr values than normal fibroblast CRM. Furthermore, extracts of media from I-cell fibroblast cultures, but not from control or Gaucher fibroblast cultures, contained AP activity towards sphingomyelinase and beta-glucocerebrosidase. Fibroblasts from a patient with mucolipidosis type III (pseudo-Hurler polydystrophy) showed an intermediate pattern consisting of normal as well as the higher-Mr CRM. Our data provide evidence for the existence of AP in cultured skin fibroblasts and suggest that these proteins may be targetted to the lysosome by post-translational modification in a similar manner to that reported for lysosomal enzymes.     

New insights into the pneumococcal fratricide: relationship to clumping and identification of a novel immunity factor

In 1971, Tomasz and Zanati discovered that competent pneumococci have a tendency to form aggregates when pelleted by centrifugation and resuspended in 0.01 N HCl by brief vortexing. Interestingly, no clumping was observed with parallel cultures of non-competent cells treated in the same way. We set out to elucidate the mechanism behind this striking phenomenon, and were able to show that it depends on extracellular DNA that is presumably released by so-called competence-induced cell lysis. Competence-induced cell lysis, which was first described a few years ago, seems to rely on the concerted action of several murein hydrolases. Our results confirmed and extended previous findings by showing that competence-induced aggregation is abolished in a lytA-lytC double mutant, and absolutely requires CbpD and its N-terminal CHAP amidase domain. Furthermore, we discovered a novel competence stimulating peptide (CSP)-induced immunity protein, encoded by the early competence gene comM (spr1762), which protects competent pneumococci against their own lysins. Together, the murein hydrolases and the immunity protein constitutes a CSP-controlled mechanism that allows competent pneumococci to commit fratricide by killing non-competent pneumococci sharing the same ecological niche. Through such predatory behaviour, pneumococci can get access to transforming DNA and nutrients, promote the release of virulence factors, and at the same time get rid of competitors.     

Protein B23 (M.W./pI = 37 kD/5.1) is the only major protein extracted from HeLa nucleoli with 3M urea.

HeLa nucleoli were isolated using the NP-40 method and subsequently extracted with 3M urea. The extract was incubated at 60 degrees C for 30 min, and precipitated proteins were removed by centrifugation. The supernatant was analyzed by one- and two-dimensional SDS polyacrylamide gel electrophoresis (PAGE). Protein B23 was the only major protein extracted from HeLa nucleoli by this procedure. Using this procedure, 1 mg of protein B23 was obtained from 2 g of HeLa cells. The purity of the extracted protein B23 was 98%, as measured by one- and two-dimensional gel electrophoresis.     

X-ray study of competence development in Bacillus subtilis

Summary Pre-competent and competent cultures of Bacillus subtilis were x-irradiated before and after centrifugal separation of cells in a Renografin density gradient. Pre-competent cultures have no cells at the radiosensitivity of the cells in the bulk competent culture, but there is a substantial fraction of cells in a multi-target state, with heterogeneous target numbers. On reaching maximal competence, the survival becomes entirely linear in radiosensitivity. Irradiation of the separated competent cells shows that competence development correlates with disappearance of multi-target cells from the non-competent band of cells and the appearance of single-target cells in the competent band at a radiosensitivity equal to that of the bulk competent culture. Thus the multi-target state may be a required stage in the development of competence in this system.     

Purification and properties of nuclear and cytoplasmic DNA polymerases from JLS-V9 cells.

Four DNA polymerases, two enzymes from the nucleus and two from the cytoplasm, were purified 2000- to 7000-fold from continuous mouse cell-line (JLS-V9), by sequential column chromatography. Each of these polymerases require all the deoxynucleoside-5′-triphosphates in order to synthesize DNA, using activated DNA as a primer-template, and can copy the ribonucleotide strand of hybrid templates, but their rate of efficiency varies. The molecular weights of these DNA polymerases range from 35,000 to 160,000, as estimated by Sephadex column chromatography. Three out of the four DNA polymerases are probably a single polypeptide chain, since they have a single major band in polyacrylamide gel electrophoresis as well as one enzymatically active peak in guanidine hydrochloride gel filtration. The highly purified preparation of the high molecular weight cytoplasmic DNA polymerase contains two major bands in sodium dodecyl sulfate polyacrylamide gel electrophoresis and two enzymatically active peaks in guanidine hydrochloride gel filtration.     

Biological expressions of lymphocyte activation. V. Characterization of a soluble immune response suppressor (SIRS) produced by concanavalin A-activated spleen cells.

Supernatant fluids from murine spleen cell cultures incubated with concanavalin A for 48 hr contain a factor(s), soluble immune response suppressor (SIRS), which suppresses plaque-forming cell responses to sheep erythrocytes by murine spleen cells in vitro. In the present studies, some of the biochemical and biophysical properties of SIRS were investigated. SIRS was non-dialysable; the suppressive activity was stable at 56 degrees C for 30 min, but was destroyed by treatment at 70 degrees C for 30 min, 80 degrees C for 10 min, or at pH 2. The suppressive activity was not absorbed by the stimulating antigen, SRBC, or antisera against murine IgG or mu-chain, suggesting that SIRS does not contain immunoglobulin determinants. Murine spleen and thymus, but not kidney cells, however, absorbed SIRS activity. Enzyme treatments revealed that SIRS was resistant to DNase and RNase, but was destroyed by trypsin and chymotrypsin. In gel filtration with Sephadex G-100, SIRS activity eluted in the fraction corresponding to m.w. in the range between 48,000 and 67,000. With polyacrylamide gel electrophoresis, SIRS activity migrated in the region cathodal to albumin. Isopycnic centrifugation in a cesium chloride gradient suggested that SIRS is a glycoprotein. These supernatant fluids with SIRS activity were also found to contain macrophage migration inhibitory factor (MIF). In the experiments using gel filtration, polyacrylamide gel electrophoresis, and isopycnic centrifugation to fractionate supernatant fluids, SIRS and MIF activity were found in the same fractions, and to date we have been unable to dissociate definitively SIRS activity from MIF activity.     

Proteins of axonal transport: investigation of solubility characteristics and behaviour in gel filtration

Rapid axonal transport of proteins in retinal ganglion cells of the rabbit was studied following intraocular injections of labelled amino acids. Approximately 10% of the transported radioactivity was found in the supernatant following homogenization and high-speed centrifugation of the nerve terminal region. Relatively simple manipulations with ionic strength, pH and the presence of a chelating agent could solubilize an equivalent amount of radioactivity from the pellet. Lithium diiodosalicylate solubilized most rapidly transported membrane proteins. Gel filtration of readily soluble rapidly transported radioactivity gave a main macromolecular radioactive peak with an approximate mol. wt. of 500,000 dalton as determined on Sephadex G-200. However, gel filtration on Sepharose CL-6B gave a mol. wt. of about 160,000 for the same radioactive peak. SDS polyacrylamide gel electrophoresis of rapidly transported soluble proteins and fractions derived from these proteins via gel filtration and ion exchange chromatography revealed in all cases a very complex picture of labelled polypeptides. Thus rapid axonal transport of soluble proteins in this system seems to involve many different macromolecules.     

Purification of RNAase II by preparative polyacrylamide gel electrophoresis.

Purification of RNAase II to electrophoretic homogeneity is described. The exonuclease is activated by K+ and Mg2+ and hydrolyses poly(A) to 5'-AMP, exclusively as described by Nossal and Singer (1968, J. Biol. Chem. 243, 913--922). To separate RNAase II from ribosomes, DEAE-cellulose chromatography was used. Two additional chromatographic steps give a preparation that yields 10 bands after analytical polyacrylamide gel electrophoresis. Preparative polyacrylamide gel electrophoresis resulted in a final preparation which on analytical polyacrylamide gels gives a single band. A molecular weight of 76 000 +/- 4000 was obtained from Sephadex G-200 chromatography, with three bands from sodium dodecyl sulfate (SDS) denaturation and SDS gel electrophoresis. The subunits have a molecular weight of 40 000 +/- 2000, 33 000 +/- 2000, and 26 000 +/- 1000. The enzyme thus appears to consist of three dissimilar subunits.     

Subcellular distribution of enkephalin precursor proteins in rat dental pulp and the usefulness as a substrate for enkephalin-producing enzymes

The subcellular distribution of enkephalin (EK) precursor proteins was investigated to clarify the intracellular site of biosynthesis of EK in rat dental pulp tissue. The contents of met-EK-like peptides in nuclear, microsomal, and supernatant fractions of the pulp tissue were markedly increased after sequential digestion with trypsin and carboxypeptidase B, indicating the enrichment of the precursors in these fractions. Sephadex G-100 gel filtration showed a common peak of the precursor proteins in the homogenate and its microsomal and supernatant fractions, and the molecular weight was determined to be about 58,000 by SDS polyacrylamide gel electrophoresis. Both the partially purified precursor protein from the supernatant fraction and N alpha-benzoyl-DL-arginine-beta-naphthylamide (BANA) were used as substrates for a lysosomal enzyme separated by Sephadex G-75 gel filtration. The major peak of EK-producing activity of the enzyme was identical with that of BANA-degrading activity of the enzyme. These results demonstrate the EK-producing activity of lysosomal proteinase, and also indicate the usefulness of the two substances as substrates for the enzyme.     

Inhibition of the Development of Competence in Streptococcus sanguis (Wicky) by Reagents that Interact with Sulfhydryl Groups: Discernment of the Competence Process

Reagents that interact with sulfhydryl groups are shown to inhibit competence factor (CF)-induced competence development in Streptococcus sanguis (Wicky) strain WE4 (Wicky 4 Ery(R)). Inhibition is correlated with specific inhibition of either the function or biosynthesis of three competent cell-related proteins and is reversed by either 2-mercaptoethanol or dithiothreitol. Mercuric chloride (5 muM) or N-ethylmaleimide (NEM; 50 muM) inhibited (i) the function but not the biosynthesis or activation of the competent cell-associated autolysin; (ii) the biosynthesis of a competent cell-associated protein of unknown function, demonstrated by polyacrylamide gel electrophoresis of acidified phenol extracts; and (iii) the biosynthesis or activation of distinct deoxyribonucleic acid (DNA)-binding sites. Neither reagent at the indicated concentration interfered with the uptake of CF by cells or with the uptake and expression of DNA by competent cells. Neither reagent inactivated CF or genetic markers coded by the transforming DNA, nor did they inhibit cell growth or viability appreciably. The data reveal that either mercuric chloride or NEM can differentially inhibit induced protein synthesis and, in addition, conclusively show that some autolytic activity is essential for the onset of the competent state.     

Hormone-Carrier-Neurosecretory Granule Association in the Urophysis of the Goby Gillichthys mirabilis*

Nishioka, R. S., Bern, H. A., Gunther, R. L. 1980. Hormone-carrier-neurosecretory granule association in the urophysis of the goby Gillichthys mirabilis. (Department of Zoology and Cancer Research Laboratory, University of California, Berkeley, California, N.S.A.) — Acta zool. (Stockh.) 61(1): 29–38. The caudal neurosecretory system of the mudsucker, Gillichthys mirabilis, was investigated using continuous sucrose density gradient centrifugation, electron microscopy, polyacrylamide gel electrophoresis, and bioassay. A strong light-scattering band near the 41 % sucrose concentration was shown to contain neurosecretory granules by electron microscopy and urotensin II activity by bioassay. Aliquots of this band were run in polyacrylamide gel electrophoresis and the selected regions of the gel containing those bands unique to urophysial tissue were cut out and eluted after the gel had been sliced longitudinally. The eluate from the region of the unique band (rf 0.46) gave the highest urotensin II bioassay activity when compared to adjacent areas. The remaining half of the gel was stained to confirm the localization of the unique bands. Gels made from regions adjacent to the 41 % sucrose concentration showed less stainability of the bands, and eluate of regions from its unique bands contained correspondingly less bioactivity.     

A rapid purification method for human erythrocyte pyruvate kinase

Human erythrocyte pyruvate kinase (ATP: pyruvate phosphotransferase, E.C.2.7.1.40) is purified 30,000-fold, using a method which includes ammonium sulfate precipitation, Sephadex G-75 filtration, and Blue Dextran-Sepharose 4B chromatography. The enzyme is resolved into two peaks on Blue Dextran-Sepharose 4B. The first peak with sp act of 300 corresponds to the mature form (R4) whereas the second peak with sp act of 180 corresponds to R2R'2. Peaks I and II give one band on 10% polyacrylamide gel without SDS. Peak II gives two bands on 10% SDS gel with molecular weights 60,000 (R') and 57,500 (R). On the other hand, peak I gives only one band on 10% SDS gel having a molecular weight of 57,500. Both the R4 and R2R'2 forms of the enzyme have the same pH optimum of 7.2.     

Electroporation of Haemophilus influenzae is effective for transformation of plasmid but not chromosomal DNA.

Electroporation of plasmid and chromosomal DNAs were tested in Haemophilus influenzae because of an interest in introducing DNA into mutants that are deficient in competence for transformation. The initial experiments were designed to investigate and optimize conditions for electroporation of H. influenzae. Plasmid DNA was introduced into the competence proficient strain Rd and its competence-deficient uptake mutants com-52, com-59, and com-88, and the recombination deficient mutant rec1. Plasmid DNA could also be electroporated into the non-transforming strains Ra, Rc, Re and Rf. Plasmid DNA without sequences that are involved in tight binding (uptake) of DNA by competent cells of H. influenzae Rd was electroporated into both competent and non-competent cells. Competent cells were several orders of magnitude less efficient than non-competent cells for electroporation of plasmid DNAs. Electroporation of H. influenzae chromosomal DNA was not successful. Low levels of integration of chromosomal markers were observed following electroporation and these could be ascribed to transformation. The treatment of cells with DNasel following electroporation separated the effects due to electroporation from those due to transformation. The DNasel treatment did not affect the efficiency of plasmid incorporation, but severely restricted effects due to natural DNA transformation.     

Fractionation of individual, biologically active factor VIII multimers

We have designed an electrophoretic system for the fractionation of individual, biologically active multimers of factor VIII. Human factor VIII, purified by gel filtration on Sepharose CL-2B from plasma cryoprecipitate, was submitted to electrophoresis without SDS on 2.0% polyacrylamide gels in 0.04 M Tris/0.06 M Tes buffer, pH 7.5. Staining with Coomassie blue revealed a series of protein bands. Measurement of electrophoretic mobility showed constant size intervals between adjacent bands. Electrophoresis in a second dimension, in the presence of SDS, resulted in an identical order of mobilities, suggesting that the different migration rates of factor VIII proteins in the first electrophoretic system were size- and not charge-dependent. After electrophoresis in the absence of SDS both factor VIII coagulant and ristocetin cofactor activities as well as factor VIII-related antigen were recovered by elution from gel slices. The distribution of activity peaks resembled that of Coomassie-stained factor VIII proteins found in control gels. We thus demonstrate that an electrophoretic fractionation of factor VIII multimers is possible even at neutral pH where factor VIII activities are retained.     

Purification and characterization of spermidine N1-acetyltransferase from chick duodenum

We have reported that spermidine N1-acetyltransferase has a larger role than ornithine decarboxylase in putrescine synthesis in chick duodenum induced by 1 alpha,25-dihydroxycholecalciferol (calcitriol) [Shinki, T., Kadofuku, T., Sato, T. and Suda, T. (1986) J. Biol. Chem. 261, 11712-11716]. In the present study, spermidine N1-acetyltransferase was purified from the duodenal cytosol of calcitriol-treated chicks to homogeneity judged by SDS/polyacrylamide gel electrophoresis. The purified enzyme converted spermidine only to N1-acetyl-spermidine. The apparent molecular mass of the purified spermidine N1-acetyltransferase was found to be 36 kDa by gel filtration on Sephacryl S-200 and 18 kDa by SDS/polyacrylamide gel electrophoresis. When duodenal crude 105,000 x g extracts were directly applied to a Sephacryl S-200 column without prior purification, three peaks with spermidine N1-acetyltransferase activity appeared. The first peak was in the void volume, the second peak was in the fraction corresponding to an apparent molecular mass of 70 kDa, and the third peak was in the fraction corresponding to 36 kDa. These results suggest that spermidine N1-acetyltransferase exists as a dimer of the 18 kDa subunits and is stabilized in (a) form(s) bound to other components or proteins in intact cells.     

Mouse DNA methylase: methylation of native DNA.

An improved method of purification of DNA methylase from Krebs II ascites cells is reported. The enzyme sediments at 8.3 S on glycerol-gradients and a major band on SDS polyacrylamide gel electrophoresis has a molecular weight of 184 000. Aggregation occurs at low salt and this may interfere with enzymic activity. The preferred double stranded DNA substrate is that rendered partially unmethylated by an in vitro repair mechanism or by isolation from methionine starved cells. Methylation of native partially methylated DNA is favoured under conditions of low salt and high temperature; conditions which encourage 'breathing' of the DNA. Methylation of native, unmethylated DNA also involves breathing but results in formation of a salt resistant tight binding complex between the enzyme and the DNA.