Comparison of two serologically distinct ribonucleic acid bacteriophages. II. Properties of the nucleic acids and coat proteins

Overby, L. R. (University of Illinois, Urbana), G. H. Barlow, R. H. Doi, Monique Jacob, and S. Spiegelman. Comparison of two serologically distinct ribonucleic acid bacteriophages. II. Properties of the nucleic acids and coat proteins. J. Bacteriol. 92:739-745. 1966.-The ribonucleic acid (RNA) molecules and coat proteins of two RNA coliphages, MS-2 and Qbeta, have been characterized. MS-2 RNA shows an S(20,w) of 25.8 and a molecular weight by light scattering of 10(6). The corresponding parameters for Qbeta-RNA were 28.9 and 0.9 x 10(6). A difference in base composition was reflected in the adenine-uracil ratio, which was 0.95 for MS-2 and 0.75 for Qbeta. The two RNA preparations are readily separated by chromatography on columns of methylated albumin. Both gave identical bouyant densities in cesium sulfate of 1.64 g/ml. The coat protein subunits were of similar molecular weights: 15,500 (Qbeta) and 14,000 (MS-2). They differed, however, in that the Qbeta-protein lacked tryptophan and histidine, whereas the MS-2 protein lacked only histidine.     

Purification and immunochemical properties of a protein antigen from serotype g Streptococcus mutans

A proteinaceous antigen (PAg) was purified from the culture supernatant of Streptococcus mutans 6715 (serotype g) by ultrafiltration, ammonium sulfate precipitation, DEAE-Sephacel ion-exchange chromatography, Phenyl-Sepharose CL-4B hydrophobic chromatography, and subsequent Sephacryl S-300 gel filtration. A yield of 0.1 mg of PAg was obtained from a liter of culture supernatant. The isoelectric point and molecular weight of PAg were pH 4.6 and 210,000, respectively. It contained 35% sugar, which was identified as glucose by gas-liquid chromatography. Amino acid analysis revealed that PAg contains 28% acidic and 11% basic amino acid residues. PAg retained its antigenicity after heating at 80 C for 10 min in deionized water, or after treatment with 0.1 M HC1 or 0.1 M NaOH at 37 C for 1 hr. Immunodiffusion and immunoelectrophoresis analyses revealed that PAg is serologically distinct from other cell-surface antigens such as serotype-specific polysaccharide and lipoteichoic acid. A cross-reaction between PAg and a protein antigen similarly prepared from serotype c S. mutans was observed in immunodiffusion tests.     

Bovine alpha-fetoprotein. Isolation and characterization.

Bovine AFP was purified by ion exchange chromatograph on C.M. cellulose and DEAE Sephadex A-50, gel filtration and immunosorbent technique. AFP was homogeneous when studied by gel electrophoresis under non denaturing and denaturing conditions, by ultracentrifugation and by immunological methods. The following molecular data were obtained: 1. Sedimentation equilibrium indicated a molecular weight of 66,500 and sedimentation velocity gave s degrees 20, w = 4.71 S. A partial specific volume v = 0.737 ml g-1 was derived from density measurements. 2. From these data, a Stokes radius of 3.26 nm, a diffusion coefficient D20 w = 6.61 10(-7) cm2 sec-1 and a frictional ratio f/fo = 1.21 were calculated. 2. Sodium dodecylsulphate disc electrophoresis suggests a molecular weight of 67,000. 3. Gel filtration pointed to a molecular weight of 75,000. 4. Microheterogeneity of AFP was demonstrated by isoelectric focusing. The isoelectric point of the major component is 4.6. 5. The chemical composition was determined. AFP is a glycoprotein containing 7 per cent carbohydrate including 1.67 per cent hexoses, 2.38 per cent N-acetyl glucosamine and 1.8 per cent N-acetyl neuraminic acid.     

Characterization and properties of Pholas luciferase as a metalloglycoprotein.

The luciferase of the bioluminescent boring mollusc, Pholas dactylus, has been purified by a new method which includes centrifugation in cesium chloride gradients. Homogeneous preparations have been obtained and molecular weight determinations and subunit analysis support the idea that this preparation is an oxyluciferin-luciferase complex. The preparation catalyzes oxidation of ascorbic acid in presence of H2O2, and this peroxidase activitity has been used for characterization (thermal and pH stabilities, activity as a function of pH, isoelectric point, turnover number). The existence of two atoms of copper has been established and their involvement in the peroxidase activity indicated. Chemical analyses have shown that Pholad luciferase is a glycoprotein and the existence of glucosamine, fucose, mannose, and galactose residues has been demonstrated. The apparent buoyant dentisty (1.340), the sedimentation coefficient (10.7 S), the Stoke's radius (83 A), the partial specific volum (0.707), and the molecular weight (350,000) have been determined. The frictional ratio (flf0 = 1.8) derived from the Stoke's radius indicates that the molecule is asymmetric. The quaternary structure has been examined. Subunits of molecular weight 150,000 and 46,000 have been observed. The latter has electrophoretic properties identical with luciferin or oxyluciferin.     

Structural and compositional changes associated with chlorine inactivation of polioviruses.

Chlorine inactivation of polioviruses resulted in the loss of viral ribonucleic acid, converting the viruses from 156S particles to 80S particles. However, it was found that virus inactivation occurred before the ribonucleic acid was released from the virions. Extraction of ribonucleic acid from partially inactivated virus suspensions indicated that chlorine inactivation was due to degradation of the ribonucleic acid before release and that ribonucleic acid loss was a secondary event. The empty 80S capsids had the same isoelectric point and ability to attach to host cells as infective virions. Thus, no major capsid conformational changes occurred during chlorine inactivation.     

Single-stranded fraction of deoxyribonucleic acid from Bacillus subtilis.

About 13% of the deoxyribonucleic acid (DNA) of various strains of Bacillus subtilis, independent of the stage of growth or competence for transformation, was rendered acid soluble by endonuclease S1. In a pH 11.2 CsCl gradient, 4% of the untreated DNA banded at the density typical for single-stranded molecules, whereas 9% of the remaining DNA (main band) was sensitive to endonuclease S1. Selective inhibition of DNA polymerase III, or of DNA-dependent ribonucleic acid polymerase, did not increase or abolish single-strandedness. The DNA purification procedure did affect the level of single-stranded DNA, indicating its binding to cell constituents containing ribonucleic acid, protein, and membranous material. The molecular weight of the single-stranded fraction resembled that of total denatured DNA, and its buoyant density in an alkaline CsCl gradient was centered partially at a density of 1.772 g/cm3 and partially at a density of 7.759 g/cm3. Incubation of DNA under conditions leading to renaturation of its single-stranded fraction led to an increase in transforming activity for the purA16+ marker (close to the origin of replication) relative to leu-8+ and metC3+ markers (located in the middle of the chromosome), indicating this region is the main source of the single-stranded fraction.     

Structural and compositional changes associated with chlorine inactivation of polioviruses.

Chlorine inactivation of polioviruses resulted in the loss of viral ribonucleic acid, converting the viruses from 156S particles to 80S particles. However, it was found that virus inactivation occurred before the ribonucleic acid was released from the virions. Extraction of ribonucleic acid from partially inactivated virus suspensions indicated that chlorine inactivation was due to degradation of the ribonucleic acid before release and that ribonucleic acid loss was a secondary event. The empty 80S capsids had the same isoelectric point and ability to attach to host cells as infective virions. Thus, no major capsid conformational changes occurred during chlorine inactivation.     

Two migration inhibitory factors with different chromatographic behavior and isoelectric points.

Migration inhibitory factor (MIF), produced by stimulation of guinea pig lymph node cells with concanavalin A, was fractionated by Sephadex G-100 gel filtration, sucrose density gradient electrophoresis, and isoelectrofocussing. Two distinct species were identified and separated. One, pH 3-MIF, has an isoelectric point of 3.0 to 4.5 and elutes from Sephadex G-75 columns with molecules having an apparent m.w. of 65,000 (Kd of 0.05 to 0.12). The other, pH 5-MIF, has an isoelectric point of 5.0 to 5.5 and elutes with molecules having an apparent m.w. between 25,000 and 43,000 (Kd of 0.15 to 0.23).     

Physicochemical properties of a lipase from Pseudomonas fluorescens.

The molecular weight of traicylglycerol lipase (EC 3.1.1.3) from Pseudomonas fluorescens is estimated to be approx. 33 000 by sodium dodecyl sulfate electrophoresis and Sephadex G-75 gel filtration. The lipase appears to be a single-chain protein and contains neither sugar nor lipid. The enzyme has a sedimentation coefficient (S20,w) of 3.06, an intrinsic viscosity of 3.0 g/ml and a partial specific volume of 0.730 g/ml, with an isoelectric point of pH 4.46. Amino acid analysis showed that the enzyme contained few sulfur-containing amino acid residues with no disulfide links. The N-terminal residue of the enzyme was found to be alanine and optical rotation dispersion analysis showed that about 20% of the enzyme structure was in a helicla configuration.     

Isolation, characterization and oxygen equilibrium of an extracellular haemoglobin from Eunice aphroditois (Passas).

The extracellular haemoglobin from the polychaeta,Eunice aphroditois, existed as a mixture of a heavy major component (so20, w = 56.96 +/- 0.125) and a light minor component (so20, w = 10.00 +/- 0.13S), the latter probably being a dissociation product of the former. The molecular weight of the purified heavier component, as detetermined by sedimentation equilibrium, was 3.44 x 10(6) +/- 0.04x10(6). The molecule had the electron-microscopic appearance typical of annelid haemoglobins, consisting of a stack of two hexagonal plates, with dimensions 26.32 +/- 0.27 nm across the flats of the hexagon, height of stack 17.86 +/- 0.34 nm. The sugar composition is reported, and the isoelectric point was approx. pH7.8. The haem content was 2.31 +/- 0.01%, corresponding to a minimal mol.wt. of 26700. Detergent/gel electrophoresis revealed the presence of at least four bands with molecular weights in the range 14600-31000. Five N-terminal amino acids were found. In addition to the 10S component, which co-existed with the 57S component at all pH values in the range 4.0-10.6, at low pH values (less than pH.5.0) A 16S and a 1.9 S component were found. The absorption and circular-dichroic spectra are reported, and the alpha-helical content, calculated from the ellipticity at 222 nm, was about 40%. The molecule bound O2 co-operatively with a maximum value of the Hill coefficient, h, of 3.9. Over the pH range 7.0-8.0 there was a positive Bohr effect.     

Studies of the bulbo-urethral (Cowper''s)-gland mucin and seminal gel of the boar

1. Moving-boundary electrophoresis of the mucin from the Cowper's gland of the boar revealed a sharp single peak at pH values from 1.1 to 9.0 and an isoelectric point of 1.1. 2. Neuraminidase treatment of the mucin, which removed at least 96% of the sialic acid groups, decreased the electrophoretic mobility at pH4 from -7.4x10(-5) (for the mucin) to -0.64x10(-5)cm(2)V(-1)s(-1). 3. Ultracentrifugal sedimentation values of s(20,w) showed a marked dependence on concentration. A hyperfine peak, similar to that given by ovine submaxillary secretion, persisted throughout the run at higher concentrations. Ultracentrifugal studies further showed a very low value for the diffusion coefficient (D(20,w) -1.57x10(-8)cm(2)/s). 4. Calculation of the approximate molecular weight from comparable s(20,w) and D(20,w) values gave a provisional value of 6.5x10(6). 5. Two proteins present in the boar vesicular secretion known as protein A and protein H (the haemagglutinating protein) were shown to promote the swelling of the mucin to form the characteristic rigid elastic gel of boar semen. It is suggested that protein A molecules particularly (mol.wt. 2.8x10(4)) cross-link with the long molecules of the mucin to form the seminal gel.     

Refined molecular weights for phage, viral and ribosomal RNA.

The RNAs of the Escherichia coli bacteriophages MS2 and Qbeta as well as E. coli 16S ribosomal RNA were examined under identical conditions by electron microscopy using the protein-free benzyldimethylalkylammonium chloride (BAC) spreading technique. From the contour length ratios of the RNAs and the known number of nucleotides for MS2, the chain lengths for Qbeta RNA and 16S RNA were found to be 4790 +/- 150 and 1645 +/- 55 nucleotides. Correcting for the base composition of Qbeta RNA the molecular weight of the Na salt of this RNA is (1.64 +/- 0.06) . 10(6) daltons. Since published values on the relative lengths of Qbeta RNA and several other homogeneous RNAs (E. coli 23S rRNA, E. Coli bacteriophage R17 and f2 RNAs, Pseudomonas aeruginosa phage PP7 RNA and Newcastle disease virus RNA) are available, we are able to calculate the approximate number of nucleotides for these useful standards.     

Production of a homogeneous Cl. botulinum type B neurotoxin

The method for obtaining the neurotoxin, or alpha-fraction of the toxin, of Cl. botulinum, type B, is described. In accordance with this method, the toxin was precipitated three times with hydrochloric acid in the isoelectric zone with subsequent extraction with phosphate (pH 6.8) and citrate-phosphate (pH 5.6) buffers, then fractionated in columns with DEAE cellulose (pH 5.6), DEAE Sephadex A-50 (pH 7.2) and Sephadex G-200 (pH 7.2). The homogeneous neurotoxin preparations with molecular weights ranging from 145,000 to 160,000 and having the isoelectric point at pH 5.5 and toxicity 5.0--10.0 x 10(7) Dlm per 1 mg protein were obtained.     

Guinea-pig liver leukotriene A4 hydrolase. Purification, characterization and structural properties

Leukotriene A4 hydrolase from perfused guinea-pig liver was purified 1200-fold to near homogeneity with a yield of about 20%. Apparent values of Km and Vmax at 37 degrees C (27 microM and 68 mumol x mg-1 x min-1), turnover number, and activation energy for the conversion of leukotriene A4 into leukotriene B4 were estimated from kinetic data obtained at -10 degrees C, 0 degree C and +10 degrees C (Arrhenius plots). Physical properties including Mr (67,000-71,000), pH optimum, isoelectric point and Stokes' radius were determined. The amino acid composition and N-terminal amino acid sequence were established after carboxymethylation of the enzyme. Unlike liver cytosolic epoxide hydrolase, the purified enzyme did not catalyze the conversion of leukotriene A4 into (5S,6R)-5,6-dihydroxy-7,9-trans-11,14-cis-icosatetraenoic acid.     

Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver. Purification, characterization and subcellular localization

1. Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver was purified to homogeneity. 2. The purified enzyme moves as a single electrophoretic band at pH 8.3 in 7.5% acrylamide and was coincident with the enzyme activity. 3. Gel filtration on Sephadex G-200 gave an apparent molecular weight of 110,000 with two apparent identical subunits of 54,000-56,000 as determined by sodium dodecyl sulphate gel electrophoresis. 4. The maximum of enzyme activity was obtained in the presence of 3-5 mM ZnCl2 at pH 6-6.2, however, higher concentrations of metal are inhibitory. The enzyme hydrolyses p-nitrophenylphosphate, o-carboxyphenylphosphate and phenylphosphate, was insensitive to NaF and was inhibited by phosphate and ATP. The Km for p-nitrophenylphosphate was 0.28 x 10(-3)M at pH 6 in 50 mM sodium acetate/100 mM NaCl. 5. Phosphate is a competitive inhibitor (Ki = 0.5 x 10(-3)M) whereas ATP seems to be a non-competitive inhibitor (Ki = 0.35 x 10(-3)M). The isoelectric point determined by isoelectric focusing on polyacrylamide gel is 7.5. 6. Cell fractionation studies indicate that the Zn2+-dependent acid p-nitrophenylphosphatase of chicken liver is a soluble enzyme form.     

Variant of Penicillinase Mediated by an R Factor in Escherichia coli

The penicillinase from an Escherichia coli strain harboring an R factor R(GN823) was purified and its properties were compared with those of a known type I penicillinase mediated by R factors. The molecular weight and S(20,w) of the enzyme were 22,600 and 2.42S, respectively. The isoelectric point of the enzyme was 6.9. These values are clearly different from those of type I penicillinase. The specific activity of the enzyme was 84,700 units per mg of the purified enzyme protein, which is about 20 times higher than that of the type I penicillinase. However, similarities were observed between the enzyme and the type I-penicillinase at optimal pH (6.5 to 7.0), optimal temperature (40 to 45C), substrate specificity, Michaelis constants for penicillins and cephaloridine, and effect of inhibitors. Furthermore, antiserum against type I penicillinase showed cross-reaction against this enzyme. The enzyme was named type Ib penicillinase, and the original type I penicillinase was renamed type Ia-penicillinase.     

Analysis of Euglena gracilis chloroplast deoxyribonucleic acid with a restriction endonuclease, EcoRI.

Cleavage of chloroplast deoxyribonucleic acid (DNA) of Euglena gracilis Z with restriction endonuclease RI from Escherichia coli (EcoRI) yielded 23 bands upon electrophoresis in gels of agarose. Four of the bands contained twice the stoichiometric amount of DNA. One of these bands contained two similarly sized fragments. The sum of the molecular weight of the 24 different fragments equaled the molecular weight of the circular molecule. The restriction fragments had different buoyant densities, with four having distinctly heavy densities in CsCl. Restriction fragments with a high buoyant density were preferentially lost when broken chloroplast DNA was purified by equilibrium density gradient centrifugation. Hybridization of chloroplast ribosomal ribonucleic acid to intact chloroplast DNA determined that there are two cistrons for 16S and 23S ribosomal ribonucleic acid. These two cistrons are located on six restriction fragments, all of which have buoyant densities greater than the intact molecule of chloroplast DNA.     

Isolation and characterization of tissue-specific isozymes of glucosephosphate isomerase from catfish and conger.

In teleosts glucosephosphate isomerase exists as two tissue-specific isozymes. Most tissues contain the more acidic liver-type isozyme, while white muscle contains the more basic isozyme; and a few tissues contain both the liver- and muscle-type isozymes as well as a hybird. The isozymes were isolated from catfish liver and muscle and from conger muscle and shown to be homogeneous by polyacrylamide gel electrophoresis, isoelectric focusing, analytical ultracentrifugation, and rechromatography. Both isozymes are of molecular weight 132,000 (S020,w = 7.0 S) and composed of two subunits of Mr approximately 65,000. The muscle and liver isozymes were shown to have distinct isoelectric points (catfish liver = 6.2; muscle = 7.0) and amino acid compositions. Tryptic peptide maps, after S-carboxymethylation and carbamylation, revealed several distinct differences in the primary structures of the isozymes. Although the isozymes could also be distinguished on the basis of their stabilities, most of their basic catalytic properties were found to be similar. A conger was obtained which was heterozygous for the variant allele at the muscle-glucosephosphate isomerase locus. A comparison of the variant conger muscle isozyme with the wild type revealed a single altered peptide, suggesting a point mutation. The structure-function studies, as well as the genetic studies, clearly establish that the two types of isozymes are of independent genetic origin.     

Physical studies of the hemocyanin of the marine gastropod, Kelletia kelleti (Forbes).

1. The hemocyanin of the Californian whelk, Kelletia kelleti, investigated at pH and ionic conditions close to physiological, has a molecular weight close to 9.0 x 10(6) and a sedimentation constant of 114S, characteristic of the di-decameric structure of molluscan hemocyanins. Light-scattering measurements at pH 8.0, 0.05 M Mg2+, 0.01 M Ca2+ gave a molecular weight of 9.0 +/- 0.6 x 10(6), and scanning transmission electron microscopy produced nearly the same particle mass of 9.22 +/- 0.50 x 10(6) daltons (Da). 2. Light-scattering measurements on the fully dissociated monomers in the presence of 8.0 M urea and at pHs 10.6 and 11.0 gave molecular weights of 4.50 x 10(5)-4.91 x 10(5), that are close to one-twentieth of the mass of the parent di-decameric hemocyanin assembly. 3. Changes in pH produced a bell-shaped molecular weight profile, with molecular weights close to 9.0 x 10(6) in the pH region of about 5.5-8.0, and progressive dissociation to 4.5 x 10(5) Da monomers in the region below pH 4.0 and above pH 9.0 or 10, depending on the absence or presence of stabilizing Mg2+ ions (0.01 M). 4. In the absence of divalent ions some aggregation of hemocyanin was found at pHs close to 5.0, with observed molecular weights above 10 x 10(6) (investigated at a hemocyanin concentration of 0.10 g/l). The early studies of Condie and Langer (Science 144, 1138-1140, 1964) had shown that Kelletia kelleti hemocynanin aggregates at acidic pHs close to the isoelectric point, forming linear polymers of the hemocyanin di-decamers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical and physical properties of the carboxypeptidase Y-inhibitor from Baker's yeast.

The purification as well as some characteristics of the carboxypeptidase Y-inhibitor from baker's yeast have been described in a previous report (Matern, H., Hoffmann, M. and Holzer, H. (1974) Proc. Natl. Acad. Sci. U.S. 71, 4874-4878). In this paper, chemical and physical properties of the purified inhibitor are presented. The molecular weight was estimated at 23 400--24 000 and appears to be a monomeric unit. Amino acid analysis and carbohydrate studies are given, showing the existence of three disulfide bonds and one sulfhydryl group per molecule and the absence of carbohydrate residues. The N-terminal amino acid is blocked by an acetyl group. The C-terminal amino acid is lysine. The isoelectric point (pI) is 6.6 and the inhibitor-enzyme complex is stable (at 25 degrees C) betwen pH 5 and 9. The apparent Ki value was calculated as 2.5.10(-9)M.