Purification and Characterization of Colicin D

Colicin D-CA23, obtained by sonic treatment of mitomycin C-induced cells of Escherichia coli K-12 W1485 (colD), was purified by ammonium sulfate precipitation, gel filtration on Sephadex G200, ion-exchange chromatography on diethylaminoethyl cellulose, and isoelectrofocusing. Polyacrylamide-gel electrophoresis, sedimentation velocity analysis, and antigenic analysis indicated that the preparation was homogeneous. Colicin D is composed entirely of amino acids and hence is a simple protein uncomplexed with lipid or lipopolysaccharide. It contains six residues of cysteine per molecule. The molecular weight of colicin D is approximately 92,000, as determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis and gel filtration on Sephadex G200. Its sedimentation coefficient is 4.41S. The behavior of colicin D in solutions of sodium dodecyl sulfate and 2-mercaptoethanol indicates that it does not consist of subunits and exists as a single polypeptide chain. Its high molecular weight and presence of six cysteine residues per molecule distinguish colicin D from all colicins previously described. Although colicins D and E3 have similar modes of action, their gross molecular properties are entirely different.     

Structure, subunit composition, and molecular weight of RD-114 RNA.

The properties and subunit composition of the RNA extracted from RD-114 virions have been studied. The RNA extracted from the virion has a sedimentation coefficient of 52S in a nondenaturing aqueous electrolyte. The estimated molecular weight by sedimentation in nondenaturing and weakly denaturing media is in the range 5.7 X 10(6) to 7.0 X 10(6). By electron microscopy, under moderately denaturing conditions, the 52S molecule is seen to be an extended single strand with a contour length of about 4.0 mum corresponding to a molecular weight of 5.74 X 10(6). It contains two characteristic secondary structure features: (i) a central Y- or T-shaped structure (the rabbit ears) with a molecular weight of 0.3 X 10(6), (ii) two symmetreically disposed loops on each side of and at equal distance from the center. The 52S molecule consists of two half-size molecules, with molecular weight 2.8 X 10(6), joined together within the central rabbit ears feature. Melting of the rabbit ears with concomitant dissociation of the 52S molecule into subunits, has been caused by either one of two strongly denaturing treatments: incubation in a mixture of CH3HgOH and glyoxal at room temperature, or thermal dissociation in a urea-formamide solvent. When half-size molecules are quenched from denaturing temperatures, a new off-center secondary structure feature termed the branch-like structure is seen. The dissociation behavior of the 52S complex and the molecular weight of the subunits have been confirmed by gel electrophoresis studies. The loop structures melt at fairly low temperatures; the dissociation of the 52S molecule into its two subunits occurs at a higher temperature corresponding to a base composition of about 63% guanosine plus cytosine. Polyadenylic acid mapping by electron microscopy shows that the 52S molecule contains two polyadenylic acid segments, one at each end. It thus appears that 52S RD-114 RNA consists of two 2.8 X 10(6) dalton subunits, each with a characteristic secondary structure loop, and joined at the 5' ends to form the rabbit ears secondary structure feature. The observations are consistent with but do not require the conclusion that the two 2.8 X 10(6) dalton subunits of 52S RD-114 RNA are identical.     

Characteristics of a new bacteriophage, Psp231a, infecting Pseudomonas phaseolicola HB10Y.

Bacteriophage Psp231a infects Pseudomonas phaseolicola, strain HB10Y, which is the host cell for the enveloped bacteriophage phi 6. This paper describes the biophysical characteristics of Psp231a and the physical properties of its nucleic acid. In electron micrographs the virion appears as an icosahedral structure, approximately 55 nm in diameter, with a short tail. The virion density is 1.48 g/cm3 in CsCl, and the sedimentation coefficient is approximately 407S. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of 12 polypeptides ranging in molecular weight from 5,000 to 117,000. The nucleic acid of Psp231a is linear, double-stranded DNA of molecular weight 28 X 10(6). Its density in CsCl is 1.716 g/cm3, and its sedimentation coefficient in 3 M CsCl is 20.0S, corresponding to an S020,W of 34S.     

Molecular size and shape of beta-connectin, an elastic protein of striated muscle

Connectin is an elastic protein of vertebrate striated muscle, and consists of doublet components, alpha and beta (also called titins 1 and 2). In the present study, beta-connectin isolated in the native state was investigated in order to characterize its molecular size and shape. The molecular weight was approximately 2.1 X 10(6) (SDS gel electrophoresis) or 2.7 X 10(6) (sedimentation equilibrium). The sedimentation coefficient (SO20, w) was 17S in 0.1 M phosphate buffer, pH 7.0. The intrinsic viscosity measured in an Ostwald-type viscometer was 1.8 dl/g. However, the viscosity was greatly dependent on the velocity gradient, and at a very low velocity gradient of 0.0007 s-1, a solution of connectin (0.3 mg/ml) showed a viscosity value of 17,000 cp. Flow birefringence measurements suggested a length distribution ranging from 300 to 450 nm. Electron microscopic observations revealed that connectin is a long flexible filament and the peaks of frequency of length distribution were at 150, 300, 450, and 600 nm. It was tentatively assumed that the connectin molecule is 300-400 nm long and 34-38 nm wide. It is likely that beta-connectin is derived from alpha-connectin, which has an apparent molecular weight of 2.8 X 10(6).     

Characterization of the extracellular haemoglobin of Haemopsis sanguisuga (L.).

The haemoglobin from the blood of the horseleech, Haemopsis sanguisuga (L.), had a sedimentation coefficient, SO20, w, of 59.11 +/- 0.55 S, and a molecular weight as determined by sedimentation equilibrium of 3.71 X 10(6)+/-9904 X 10(6). In the electron microscope the molecule appeared to be made up of two hexagonal plates, as is found with other worm haemoglobins, with dimensions 24.4+/-2.0 nm (across the hexagon) and 15.2+/-1.4 nm (height). The amino acid composition and spectrum were closely similar to those of the haemoglobins of other annelids (e.g. Lumbricus). The alpha-helical content, calculated from circular-dichroism measurements in the far-u.v. region, was 56-63%. The haem content was 2.49%, corresponding to a minimum molecular weight per haem group of 24 800, but detergent-gel electrophoresis indicated the presence of polypeptide chains of mol.wts. 12 600, 14 800, 15 500 and 25 100. The pH-induced dissociation of the native molecule yielded compotosol of Soya-bean root nodules.     

Caldesmon. Molecular weight and subunit composition by analytical ultracentrifugation

A wide range of values has been reported for the subunit and molecular weights of smooth muscle caldesmon. There have also been conflicting reports concerning whether caldesmon is a monomer or dimer. We attempted to resolve these uncertainties by determining the molecular weight of chicken gizzard smooth muscle caldesmon using the technique of sedimentation equilibrium in the analytical ultracentrifuge. Unlike previous methods that have been used to estimate the molecular weight of caldesmon, the molecular weight determined by equilibrium sedimentation does not depend upon assumptions about the shape of the molecule. We concluded that caldesmon in solution is monomeric with a molecular mass of 93 +/- 4 kDa, a value that is much less than those previously reported in the literature. This new value, in conjunction with sedimentation velocity experiments, led to the conclusion that caldesmon is a highly asymmetric molecule with an apparent length of 740 A in solution. The mass of a cyanogen bromide fragment, with an apparent mass of 37 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was determined to be 25.1 +/- 0.6 kDa using sedimentation equilibrium. These results imply that the reported molecular weights of other fragment(s) of caldesmon have also been overestimated. We have determined an optical extinction coefficient for caldesmon (E1%(280 nm) = 3.3) by determining its concentration from its refractive index which was measured in the analytical ultracentrifuge. From the above values of the molecular weight and the extinction coefficient, we redetermined that the caldesmon molecule has two cysteines and recalculated the stoichiometric molar ratio of actin/tropomyosin/caldesmon in the smooth muscle thin filament to be 28:4:1.     

Studies on erythrocruorin. VII. Reconstitution of earthworm erythrocruorin from the apoprotein.

Apoerythrocruorin prepared from the giant respiratory hemoprotein of the earthworm (60 S, Mr = 3 X 10(-6)) is an electrophoretically homogeneous molecule which sediments as a single peak of low molecular weight (3.5 S) and has a lower alpha-helical content (approx. 30%) than the native protein. Titration of globin with ferric heme indicates the presence of different binding sites; however, after purification by ion exchange chromatography, the reconstitution product contains 1 haem/23 000 g of protein as the native molecule. Reconstituted ferric erythrocruorin is a low molecular weight hemichrome with the same optical and physicochemical properties of the hemichrome formed by natural ferric erythrocruorin. Reconstituted ferrous erythrocruorin reacquires the alpha-helical content and the quaternary structure of the native molecule. Reassociation into 10-S speices (1/12 of the whole molecules) is fast and easy, while that into whole molecules is slow and somewhat erratic. The functional properties of reconstituted ferrous erythrocruorin (oxygen affinity, cooperativity in oxygen binding, magnitude of Bohr effect) are very similar to those of the "stable" low cooperativity form of the undissociated protein.     

Purification properties and subunit composition of pig heart lipoate acetyltransferase.

Lipoate acetyltransferase [acetyl-CoA: dihydrolipoate S-acetyl-transferase, EC 2.3.1.12], the core enzyme of the pyruvate dehydrogenase complex, has been highly purified by gel chromatography on Sepharose 6B and sucrose density gradient centrifugation in the presence of potassium iodide. The native enzyme has a sedimentation coefficient (S020,W) of 26.7S and a diffusion coefficient (D020,W) of 1.25 x 10(-7) cm2.-sec-1. The weight-average molecular weight was estimated to be 1.8 million from the sedimentation equilibrium data. The content of right-handed alpha helix in the enzyme molecule was estimated to be about 25% by optical rotatory dispersion and about 22% from the circular dichroism spectra. The enzyme was found to contain about 23 moles of protein-bound lipoic acid per mole of enzyme; some other properties are also reported. Lipoate acetyltransferase dissociated to yield a single subunit with a molecular weight of 74,000 as estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and by gel filtration on Bio-Gel in 6 M guanidine-HCl. The molecular weight was also estimated to be 74,000 from sedimentation equilibrium data in 6 M guanidine-HCl] containing 0.1 M 2-mercaptoethanol. Evidence is presented that 1 molecule of lipoate acetyltransferase apparently consists of 24 very similar subunits, each of which contains NH2-terminal alanine. Each subunit contains 1 molecule of covalently bound lipoic acid.     

A protein kinase copurified with chick oviduct progesterone receptor

A magnesium-dependent protein kinase activity was copurified with both the molybdate-stabilized 8S form of the chick oviduct progesterone receptor (PR) and its B subunit. In each case, purification was performed by hormonal affinity chromatography followed by ion-exchange chromatography. The Km(app) values of the phosphorylation reaction for [gamma-32P]ATP and calf thymus histones were approximately 1.3 X 10(-5) M and approximately 1.6 X 10(-5) M, respectively, and only phosphorylated serine residues were found in protein substrates, including PR B subunit. Physicochemical parameters of the enzyme [pI approximately 5.3, Stokes radius approximately 7.2 nm, sedimentation coefficient (S20,w) approximately 5.6 S, and Mr approximately 200,000] were compared to those of purified forms of PR (B subunit, pI approximately 5.3, Stokes radius approximately 6.1 nm, and Mr approximately 110,000; 8S form, Stokes radius approximately 7.7 nm and Mr approximately 240,000). The results suggest that most of the protein kinase activity copurified with both oligomeric and monomeric forms of PR belongs to an enzyme distinct from currently known receptor components. Its physiological significance remains unknown.     

A high molecular weight protease in the cytosol of rat liver. II. Properties of the purified enzyme

The properties of a soluble endoprotease from rat liver were studied. The enzyme was purified in a latent form. It sedimented as a single component with a sedimentation coefficient (S(0)20,w) of 19.8 S. Measurement by quasi-elastic light scattering gave a diffusion coefficient (D(0)20,w) of 2.5 X 10(-7) cm2 X s-1 and an effective hydrodynamic radius of 85 A. The enzyme had an unusually high molecular weight, estimated as 743,000 by sedimentation equilibrium and 722,000 by sedimentation velocity and diffusion measurements and as 760,000 by a recently developed low-angle laser light scattering method. Judging from electron microscopic observation and the calculated frictional and axial ratios, the enzyme molecule is disc-shaped. Analysis of the far-ultraviolet circular dichroic spectrum showed that the enzyme contains 50% alpha-helical, 25% beta-sheet, and 15% unordered structures with 10% beta-turns. The isoelectric point of the enzyme is 5.0. These properties indicate that the purified enzyme is a homogeneous molecule. In addition, the enzyme is a simple protein since it contains no measurable amounts of nucleic acid carbohydrate or lipid.     

Juvenile-hormone-binding protein from the hemolymph of Galleria mellonella (L). Isolation and characterization

A juvenile-hormone-binding protein (JHBP) has been isolated from Galleria mellonella hemolymph by gel filtration, phosphocellulose chromatography, and by chromatofocusing. The isolated protein is homogeneous as judged by column chromatography and gel electrophoresis in the presence and absence of denaturing agent. It has a relative molecular mass of 32,000, Stokes radius 2.4 nm, sedimentation coefficient of 2.3 S, molar absorption coefficient at 280 nm epsilon = 2.34 X 10(4) M-1 cm-1, and is composed of a single polypeptide chain. Chromatofocusing analysis (pI 8.6) and isoelectric focusing (pI 8.1) indicate that the JHBP is an alkaline protein. Its amino acid composition and fluorescence absorption spectra indicate that the protein does not contain tryptophan residues. The protein exhibits one class of binding sites for juvenile hormone (JH), 0.8 per molecule, with the following dissociation constants: JH I, 8.5 X 10(-8) M; JH II, 7.2 X 10(-8) M; JH III, 47 X 10(-8) M. The JHBP binds (10R, 11S)-JH II enantiomer with 2.3-times higher affinity then (10S, 11R)-JH II enantiomer. The pH optimum of binding is 7.0.     

Sedimentation Coefficient and Fragility under Hydrodynamic Shear as Measures of Molecular Weight of the DNA of Phage T5

T5 DNA molecules resemble fragments of T2 DNA of molecular weight 84 × 10⁶ with respect to sedimentation coefficient and susceptibility to breakage under hydrodynamic shear. The sedimentation coefficient falls by the same factor when either T2 or T5 DNA is broken at its characteristic critical shear rate. At a given high rate of shear, both DNA's are broken into fragments exhibiting the same sedimentation coefficient. It follows that 84 × 10⁶ is a proper estimate of the molecular weight of T5 DNA, and that particles of phage T5, like those of T2, contain a single DNA molecule.     

Thiol reduction of human α2-macroglobulin. The subunit structure

1. Human alpha(2)-macroglobulin was prepared from a fraction obtained during the large-scale separation of normal human plasma proteins for clinical use. 2. Sedimentation-equilibrium measurements indicated a molecular weight of 725000. A value of 18.1S was obtained for s(0) (20,w). 3. The dissociation that occurs in the pH range 4.5-2.5 and in the region of neutrality in urea-containing solutions is consistent with a dimeric structure of the molecule. 4. The effects of the thiol reagents mercaptoethanol, mercaptoethylamine and N-acetylcysteine were investigated over a range of experimental conditions. Distinct components having sedimentation coefficients of 15, 12 and 8.5S were identified. 5. Conditions were found under which limited reduction with thiol liberated a subunit with a molecular weight approximately one-quarter of that of the intact molecule. This subunit retains the serological specificity of the whole molecule.     

Subunits of the extracellular hemoglobin of Tubifex tubifex.

The molecular weight of the extracellular hemoglobin of Tubifex tubifex determined by equilibrium sedimentation is 3.0 +/- 0.2 . 10(6). Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that the hemoglobin dissociated into four subunits: 13 000 (subunit 1), 21 000 (subunit 2), 23 000 (subunit 3) and 47 000 (subunit 4); in the presence of mercaptoethanol two subunits were observed, 13 000 +/- 1000 (subunit I) accounting for 70--80% of the whole molecule, and 26 000 (subunit II). Electrophoresis of the subunits obtained in the absence of mercaptoethanol showed that subunit I originated from subunits 1 and 4, while subunit II originated from subunits 2 and 3. These relationships were supported by N-terminal group determinations. Gel filtration in 6 M guanidine hydrochloride showed that the molecular weight of subunit I is 17 500 and that of subunit II, 36 000. Tubifex hemoglobin appears to consist of at least seven polypeptide chains.     

Subunits of Tamm–Horsfall glycoprotein

1. The effect of 6m-guanidine hydrochloride on the urinary glycoprotein described by Tamm & Horsfall (1952) is to produce a homogeneous subunit of molecular weight approx. 100000. 2. Complete reduction of the disulphide bonds of this subunit does not decrease the molecular weight, suggesting that all disulphide bonds are intrachain. 3. Comparison of sedimentation and viscometric behaviour of unreduced and reduced material in 6m-guanidine hydrochloride is consistent with reduction causing an opening-up of intrachain disulphide bonds to give a more asymmetric molecule.     

Erythrocruorin from the water-flea Daphnia magna. Quaternary structure and arrangement of subunits.

The subunit structure of erythrocruorin from the cladoceran Daphnia magna was studied. The native protein was found to have a sedimentation coefficient (S2(20), w) of 17.9 +/- 0.2 S and a molecular weight, as determined by sedimentation equilibrium, of 494 000 +/- 33 000. Iron and haem determinations gave 0.312 +/- 0.011% and 3.84 +/- 0.04%, corresponding to minimal molecular weights of 17900 +/- 600 and 16 100 +/- 200 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave one band with mobility corresponding to a molecular weight of 31 000 +/- 1 500. The molecular weight of the polypeptide chain determined by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol is 31 100 +/- 1300. On a molecular-weight basis, Daphnia erythrocruorin is composed of 16 identical polypeptide chains carrying two haem groups each. The native structure is stable between pH5 and 8.5. At alkaline and acidic pH, a gradual decrease in the sedimentation coefficient down to 9.8S occurs. Above pH 10 and below pH4, a slow component with S20, w between 2.7S and 4.0S is observed. The 2.7S, 4.0S and 9.8S species are identified as single-chain subunits, subunit dimers and half-molecules respectively. We propose a model for the molecule composed of 16 2.7S subunits grouped in two layers stacked in an eclipsed orientation, the eight subunits of each layer occupying the vertices of a regular eight-sided polygon. Support for this arrangement is provided from electron microscopy and from analysis of the pH-dissociation pattern.     

Isolation and characterization of a new bacteriophage, Cp-1, infecting Streptococcus pneumoniae.

Several pneumococcal phages showing a morphology completely different from those of all other previously found pneumococcal bacteriophages have been isolated. Bacteriophage Cp-1, one of the phages isolated, showed an irregular hexagonal structure and a short tail of 20 nm. The virion density was 1.46 g/cm3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of nine polypeptides. The polypeptide showing a molecular weight of 39,000 accounted for more than the 90% of the total protein. The nucleic acid of Cp-1 was linear, double-stranded DNA with a mean length of 6.3 microns and a guanine-plus-cytosine content of 41%; its buoyant density was 1.699 and 1.422 g/cm3 in CsCl and CS2SO4, respectively. Its sedimentation coefficient (S20,w) was 19S. Cp-1 DNA showed a remarkable resistance to a large number of restriction endonucleases. A total of 12 fragments, ranging in molecular weight from 1.3 X 10(6) to 0.09 X 10(6), were produced by AluI, two fragments (molecular weight, 5.5 X 10(6) and 0.9 X 10(6)) were generated by HindIII, and two fragments (molecular weight, 6.0 X 10(6) and 5.7 X 10(6)) were produced by HaeIII. The easy visualization of th plaques produced by Cp-1, the small size of Cp-1 DNA (12 X 10(6) daltons), and other biological and physiochemical properties make this phage potentially useful for genetic studies.     

Radiation-induced double-strand scission of the DNA of mammalian metaphase chromosomes

The molecular weight distribution of double-stranded mammalian (hamster) DNA was determined by ultracentrifugation of isolated metaphase chromosomes previously layered onto sucrose gradients containing high salt concentrations to dissociate the protein and nucleic acid components. In untreated controls the distribution (as determined by counting the incorporated radioactivity in the resultant fractions) exhibited a peak at 225 X 10(6) daltons. Inclusion of mercaptoethanol and hydroxylamine into the gradients produced no significant change of these sedimentation patterns. Gamma-radiation-induced reduction in the number and weight average molecular weights was used to calculate a value of 1.05 X 10(11) double-strand breaks/gram rad, equivalent to about 600 eV/break. No significant difference was observed for chromosomes irradiated either before or after isolation from intact mitotic cells. Irradiation in the presence of cystamine resulted in at least a sevenfold reduction in the apparent double-strand scission. The observed sedimentation patterns were compared with those generated by a theoretical computer simulation of radiation-induced degradation which assumed random selection and breakage of molecules. These results suggested that at least 80 to 90 % of the isolated DNA was distributed approximately normally with a mean molecular weight of about 200 X 10(6) daltons and a standard deviation of about 50 X 10(6) daltons.     

Purification and properties of the light-activated cyclic nucleotide phosphodiesterase of rod outer segments.

Frog (Rana catesbiana) rod outer segment disc membranes contain a cyclic nucleotide phosphodiesterase (EC 3.1.4.17) which is activated by light in the presence of ATP. This enzyme is firmly bound to the disc membrane, but can be eluted from the membrane with 10 mM Tris-HCl buffer, pH 7.4 and 2 mM EDTA. The eluted phosphodiesterase has reduced activity, but can be activated approximately 10-fold by polycations such as protamine and polylysine. The eluted phosphodiesterase can no longer be activated by light in the presence of ATP, that is, activation by light apparently depends on the native orientation of phosphodiesterase in relationship to other disc membrane components. The eluted phosphodiesterase was purified to homogeneity as judged by analytical polyacrylamide gel electrophoresis and polyacrylamide gel isoelectric focusing. The over-all purification from intact retina was approximately 925-fold. The purification of phosphodiesterase from the isolated rod outer segment preparation was about 185-fold with a 28% yield. Phosphodiesterase accounts for approximately 0.5% of the disc membrane protein. The eluted phosphodiesterase (inactive form) has a sedimentation coefficient of 12.4 S corresponding to an approximate molecular weight of 240,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis separates the purified phosphodiesterase into two subunits of 120,000 and 110,000 daltons. With cyclic 3':5'-GMP (cGMP) as substrate the Km for the purified phosphodiesterase is 70 muM. Protamine increases the Vmax without changing the Km for cGMP. The isoelectric point (pI) of the native dimer is 5.7. Limited exposure of the eluted phosphodiesterase (inactive form) to trypsin produces a somewhat greater activation than is obtained with 0.5 mg/ml of protamine. The trypsin-activated phosphodiesterase has a sedimentation coefficient of 7.8 S corresponding to an approximate molecular weight of 170,000. The 110,000-dalton subunit is much less sensitive to trypsin hydrolysis and the 120,000-dalton subunit is rapidly replaced by smaller fragments. On the basis of the molecular weight of the purified phosphodiesterase (240,000) and the concentrations of phosphodiesterase and rhodopsin in the rod outer segment, it is estimated that the molar ratio ophosphodiesterase to rhodopsin in the rod outer segment is approximately 1:900. Since all of the disc phosphodiesterase molecules are activated when 0.1% of the rhodopsins are bleached, we conclude that in the presence of ATP 1 molecule of bleached rhodopsin can activate 1 molecule of phosphodiesterase.     

Characterization of mycoplasmavirus MVL2 DNA.

The size and molecular configuration of mycoplasmavirus MVL2 DNA are presented. The DNA was shown to be a covalently closed circular duplex molecule with a molecular weight of 7.4 X 10(6). In sucrose gradients at neutral pH, the form I and form II DNA molecules have sedimentation values of approximately 29S and 23S, respectively. Under alkaline conditions, the form I and form II have S values of 70 and 20, respectively.