Bacillus megaterium, KM beta-galactosidase: purification by affinity chromatography and characterization of the active species

The enzyme β-galactosidase from Bacillus megaterium, strain KM has been purified by affinity chromatography. The enzyme was found to have a dimeric subunit structure, with the monomer having a molecular weight of 120,000. The K_eq of the monomer-dimer equilibrium was strongly shifted towards dissociation in the isolated state. Inclusion of 5% sucrose in the buffer (and maintenance of the temperature at 5 °) minimized this dissociation. Molecularly homogeneous monomer and dimer could be prepared on sucrose gradients. The dimer was determined to have an S_20,w of 8, while the monomer had an S_20,w of 3. The amino acid composition was found to be similar to that of the E. coli β-galactosidase although significant differences occur. The activity of the monomer was studied by both urea-denaturation experiments and by immobilization of the monomer on Sepharose-4B. The monomer, bound to Sepharose-4B, was found to be inactive but still capable of binding the inhibitor thio-methyl galactoside. Activity was reconstituted by adding free monomer, in 8 M urea, to the Sepharose-bound monomer, followed by removal of the urea by dialysis. In addition, free monomers from E. coli β-galactosidase were found to form active hybrids with Sepharose-bound B. megaterium β-galactosidase monomers. We conclude on the basis of these studies that the free monomer is inactive, and that the dimer is the active species, in marked contrast to E. coli β-galactosidase where only the tetrameric form is active.     

Studies on the structure of the acetylcholine receptor from Torpedo marmorata

The purified acetylcholine receptor of Torpedo marmorata has been characterized by sedimentation velocity measurements on dilute solutions using an ultracentrifuge and scanner. Several preparations were studied and all exhibited sedimentation coefficients in the vicinity of 24S. In a number of experiments the receptor could be resolved into two sedimenting boundaries of 18S and 26S, corresponding to minimum molecular weights of about 5 × 10⁵ and 10⁶, respectively. Additions of sodium dodecyl sulfate or Triton X-100 resulted in marked decreases in sedimentation coefficient, while treatment with Lubrol-WX had only a slight effect on the S values. Small changes in S_20,w were produced by guanidine hydrochloride alone, although addition of dithiothreitol with 6 M guanidine hydrochloride resulted in an 8.8S component. Electrophoresis in sodium dodecyl sulfate gave one principal band with a molecular weight of 46,000.     

Further evidence and biological significance for non-random localization of the centromere on mammalian chromosomes

A further quantitative analysis of the localization of the centromere on chromosomes was made using 16,817 individual chromosomes obtained from 723 mammalian species. Centromeric position was expressed quantitatively by the size of short arms as per cent weight (S_w) relative to the X-containing haploid set. When the class interval of S_w was 0·1 instead of the previous 0·2 (Imai, 1975), the frequency distribution of S_w showed an uneven (W-shaped) pattern with two distinct antimodes lying at S_w 0·6 (reconfirmation) and 0·1 (new finding). Two hypotheses, that are not mutually exclusive, are proposed to explain the non-random distribution of centromere position. One is that there are three structurally different short arms consisting of (1) centromere, (2) constitutive heterochromatin (as determined by C-banding), and (3) euchromatin, each arm-type being approximately characterized by the size of short arms (S_w) as S_w < 0·1, 0·1 ? S_w ? 0·6 and S_w > 0·6. The other possibility is concerned with an “orthogenetical” change of chromosome morphologies. When the chromosomes with S_w < 0·1, 0·1 ? S_w ? 0·6 and S_w > 0·6 are denoted as telocentric (T), acrocentric (A), and meta-, submeta- and subtelocentric (M, SM & ST), it was suggested that the chromosome morphologies tend to change orthogenetically (in a statistical sense) from T to M, SM & ST via A-chromosomes by rearrangements such as tandem growth of constitutive heterochromatic, pericentric inversions, and centric fusions.     

Comparison of molecular weight determination of sodium alginate by sedimentation-diffusion and light scattering

The weight average molecular weight, M̄_w, of sodium alginates were determined by the sedimentation-diffusion technique using photon correlation spectroscopy rather than boundary spreading in the analytical ultracentrifuge to determine the translational diffusion coefficients. This enables the diffusion coefficients to be determined easily and accurately. Excellent correlation is found between the observed zero concentration translational diffusion coefficient, D_O and the M̄_W values in a emphirical power law. The M̄_W obtained by sedimentation-diffusion and laser light scattering compare very favourably. The concentration dependence of the photon correlation spectroscopy data allowed determination of the coil overlap concentration, c*. The inverse proportionality of c* to both M̄_W and [η] is demonstrated.     

The Molecular Weight and Other Biophysical Properties of Bromegrass Mosaic Virus

Data are presented which show that bromegrass mosaic virus has a particularly low molecular weight and nucleic acid content. A molecular weight of 4.6 × 10⁶ was calculated from the sedimentation coefficient, S°_20,w = 86.2S, the diffusion coefficient, D_20,w = 1.55 × 10^-7 cm²/sec., and an assumed partial specific volume, [UNK] = 0.708 ml/gm. The virus has a ribonucleic acid content of 1.0 × 10⁶ atomic mass units. Electrophoresis experiments showed that the virus is stable in 0.10 ionic strength buffers in the pH range 3-6. Breakdown of the virus was observed outside this pH range. Some characteristics of the breakdown products are described.     

Effects of HgCl2 on principal parameters of exudation from maize detached root system

To elucidate the role of aquaporins in the control of the root pressure, we tested the effects of HgCl₂ (aquaporin blocker) at concentrations from 10^?8 to 10^?2 M on the exudation rate (J _w). Experiments were performed with detached roots of 5–7-day-old etiolated maize (Zea mays L.) seedlings. HgCl₂ suppressed exudation by 50–70% at the concentration of 2 × 10^?5 M. At the concentration of 2.5 × 10^?4 M, HgCl₂ reduced J _w during first 20–40 min, but in 2 h, it activated exudation by ten and more times. In this case, the root and osmotic pressures of the exudates increased by 1.5 times. The hydraulic conductance reduced approximately by 30% during first 30 min and increased severalfold in 2 h. The temperature coefficient (Q₁₀) of J _w attained 14 in 2 h. At the concentration of 10^?2 M, HgCl₂-induced acceleration of exudation was replaced by its inhibition essentially immediately. We suggested that a driving force for HgCl₂-induced stimulation of the J _w might be an increase in the osmotic component of the root pressure or the involvement of its nonosmotic (so-called metabolic) component. The results allow a supposition that aquaporins are involved in the control of water transport in the root.     

A note on osmotic pressure measured with ionophore treated red blood cells

A method is described by which the osmotic pressure of macromolecules or many low molecular weight substances can be measured relative to the known osmotic pressure of a reference substance. Measurements can also be made in the presence of univalent electrolytes. The method involves the use of ionophore treated mammalian red blood cells as osmometers. Details are given for the establishment of the isosmotic identity line for dextran M_w = 9 400, M_n = 5 500 and sucrose using nystatin treated human red blood cells. The sucrose concentrations used were from 20 to 33 mOsm (50–80 kPa).     

Physicochemical features of hemoglobin of the crustacean, Triops longicaudatus

Hemoglobin from the notostracan, Triops longicaudatus, was purified and characterized physicochemically. Stoke's radius (77.7 Å) and the sedimentation coefficient (S_20,w = 17.1) were estimated by gel filtration chromatography and density gradient centrifugation, respectively and then used to calculate the molecular weight (600,400 daltons), the frictional ratio (1.33), and the diffusion coefficient (2.70). These physical parameters, along with data on the amino acid composition, are compared with similar data on other crustacean hemoglobins.     

The stabilizing influence of divalent ions and Na+ on the di-decameric structure of Yoldia limatula hemocyanin

The stabilizing influence of Ca²⁺, Mg²⁺, Ba²⁺ and Na⁺ on the di-decameric structure of the hemocyanin of the bivalve, Yoldia limatula has been investigated by light-scattering molecular weight measurements and by analytical ultracentrifugation. The molecular weight (M_w) data, examined as a function of decreasing divalent ion and sodium ion concentrations at pH 8.0 and at a constant hemocyanin concentration of 0.10 g·l⁻¹, show biphasic transition profiles, with a sharp initial decline in M_w as the concentration of the stabilizing cations is reduced. The analysis of the molecular weight data is best described in terms of the four-species, di-decamer-decamer-dimer-monomer scheme of association-dissociation equilibria. About 25 to 35 bound divalent ions and about 10 bound Na⁺ ions per half-molecule or decamer are required in order to account for the initial step of the observed transitions. The subsequent transitions representing the decamer to dimer and the dimer to nonomer steps of the reaction account for the additional binding of three to four and two to four cations per dimer and per monomer, respectively. The relatively large number of divalent ions per decamer suggests strong ionic stabilization of the decamer to decamer contacts within the parent di-decameric assembly of Yoldia hemocyanin. This is consistent with earlier observations showing relatively few hydrophobic groups at the decamer to decamer contact areas.     

Mammary glucose 6-phosphate dehydrogenase. Molecular weight studies

Glucose 6-phosphate dehydrogenase was isolated from lactating rat mammary glands by a procedure extended and modified from one previously described. The sedimentation coefficient, S_20,W, was 10.3 in 0.01 m potassium phosphate, pH 6.9, containing 0.1 m NaCl at three protein concentrations between 0.51 and 1.45 mg/ml. The partial specific volume, v?, was 0.735 ml/g as determined by equilibrium sedimentation centrifugation in H₂O and D₂O containing buffers at pH(D) 6.5 containing 0.01 m potassium phosphate and 0.1 m NaCl. In the same buffer, but with 2.0 m NaCl, the apparent partial specific volume, φ′, was 0.756 ml/g. Equilibrium sedimentation of the enzyme at an initial concentration of 0.8 mg/ml was performed in 0.01 m potassium phosphate, pH 6.5, containing 1.0 mm EDTA, 7.0 mm mercaptoethanol, and various concentrations of NaCl between 0 and 2.0 m and with or without 0.1 mm NADP⁺. Weight-average and Z-average molecular weights were calculated and, from these values, the molecular weights of the monomer and dimer were derived. Under these conditions, the enzyme existed principally as a dimer, of molecular weight approximately 235,000, at low salt concentration, and as a monomer, of molecular weight approximately 120,000 in 1.0 m and 2.0 m NaCl. The subunit molecular weight was found to be 64,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Equilibrium sedimentation in 6 m guanidine hydrochloride gave a subunit molecular weight of 62,000 (assuming v? was unaltered) or 58,000 or 54,000 (assuming v? is decreased by 0.01 or 0.02, respectively, in 6 m guanidine). We conclude that rat mammary glucose 6-phosphate dehydrogenase has a molecular weight similar to that of glucose 6-phosphate dehydrogenases isolated from various other mammalian sources with the notable exception of human erythrocyte glucose 6-phosphate dehydrogenase which, like the microbial glucose 6-phosphate dehydrogenases thus far examined, has a significantly lower molecular weight.     

Characterization of Ribosomes from Neurospora crassa

Ribosomes isolated from growing hyphae of Neurospora crassa contain 53 per cent protein and 47 per cent RNA and have a sedimentation coefficient of 81S at 20°C and infinite dilution. These ribosomes are stable at pH 7.4 in the presence of 0.01 M and 0.002 M MgCl₂ but undergo a dissociation into smaller particles if the MgCl₂ concentration is lowered to 0.0001 M. Two types of RNA with sedimentation coefficients of 19S₂₀⁵⁰ and 13S₂₀⁵⁰ have been extracted from the 81S particles.     

Purification and properties of acetate kinase from Clostridium thermoaceticum

Acetate kinase (ATP: acetate phosphotransferase EC 2.7.2.1) has been purified from Clostridium thermoaceticum. The enzyme of a specific activity of 282 μmoles min^-1 mg^-1 appeared homogeneous as judged from Sephadex chromatography and sedimentation velocity. Polyacrylamide gel electrophoretic patterns at pH 9.0 and 9.5 showed heterogeneity. Velocity curves obtained with varying amount of acetate were of the Michaelis-Menten type with an apparent K _m of 0.135 M. With varying amounts of ATP sigmoidal kinetic was observed (S_0.5=1.64 mM), suggesting cooperative binding of this substrate. The enzyme had only moderate thermal stability with a temperature optimum of about 60°C and exhibited a broken line in an Arrhenius graph. From gel filtration a molecular weight of about 60 000 daltons was estimated for the enzyme. The S_20w value was 6.0 S.     

Molecular weight of cytochrome oxidase

The molecular weight of bovine heart mitochondrial cytochrome oxidase in 2% or 3% deoxycholate was determined by the sedimentation velocity method to be 228,000 daltons from an S_20,w=8.44×10^–13 sec, a D_20,w=3.21×10^–7 cm² sec^–1, and the reported by others. The S_20,w value was only slightly concentration-dependent. When the deoxycholate in our preparation was replaced with Tween 80 the S value increased to between 16 and 17. When one preparation in Tween 80 was allowed to stand at room temperature, the S value increased in successive determinations to reach 51.5 at the end of approximately 7 h. The minimum molecular weight of the enzyme as calculated from the heme content (determined from the absorbance at 603 nm) and total protein content (determined from total nitrogen) was 110,000. An amino acid analysis when related to the heme content yielded a minimum molecular weight of 85,000.Deceased.     

Characterization of T-Even Bacteriophage Substructures: II. Tail Plates

Tail plates obtained from T4D amber mutants were examined with respect to sedimentation behavior, subunit molecular weights, amino acid composition, isoelectric points, and morphology. Intact plates had an S_20,w of 77S from pH 5 to 9. The only conformational change noted was that below pH 5 tail plates readily dimerized yielding vis-à-vis dimers with an S_20,w of 124S. Dissociated plates consisted of three major proteins with molecular weights of 53 K ± 5, 31 K ± 3, and 17 K ± 2 daltons. The amino acid analyses indicated that plates had a composition distinct from fibers and tubes and were relatively rich in tryptophan. Degradation studies with dimethyl sulfoxide (DMSO) indicated that tail plates had a unique biological structure. After treatment with DMSO, and to some extent without DMSO, or from lysates of defective mutants, tetrad structures were observed in the electron microscope. These structures had an amino acid content and relative amounts of types of subunits similar but not identical to intact plates. It was proposed that plates were composed of nine such tetrads giving rise to a structure with six- and threefold symmetry.     

Bacillus thuringiensis parasporal crystal toxin: Dissociation into toxic low molecular weight peptides

Parasporal crystals of Bacillus thuringiensis can be dissociated into low molecular weight peptides (< 5000 daltons) by dissolving them in 0.1 M N-morpholinopropane sulfonic acid buffer pH 7.8 containing 0.05 M dithiothreitol and 2M–4M KSCN, or by performic acid oxidation. The peptides obtained by dissolving in KSCN were still toxic to silkworm larvae.     

Low temperature solution behaviour of Methylophilus methylotrophus electron transferring flavoprotein: a study by analytical ultracentrifugation

The solution behaviour of electron transferring flavoprotein (ETF) from Methylophilus methylotrophus was investigated at low temperature (4 °C) by analytical ultracentrifugation. The concentration dependence of the apparent weight average molecular weight, M_w,app, established the existence of the protein in heterodimeric state (M = 63,700 Da), but also signified the possible dissociation of the heterodimer at lower concentrations into its constituent subunits (M = 28,900 Da and 33,700 Da, together with FAD and AMP cofactors of collective M = 1120 Da). This similarity in subunit size allows approximate quantification of the dissociation in terms of expressions for a monomer-dimer equilibrium. The dissociative behaviour was confirmed by determination of the point average molecular weight, M_w,app(r), as a function of the ETF concentration, c(r), throughout the sedimentation equilibrium distributions obtained with loading concentrations of 0.4 and 0.7 mg/ml. By means of the recently formulated ``psi' procedure for direct analysis of solute self-association a value of (1.5 ± 0.1) μM has been obtained for the dissociation constant K_d. Sedimentation velocity experiments yielded an estimate of the heterodimer sedimentation coefficient, s⁰ _20,w, of (4.5 ± 0.2) S which for M = 63,700 Da suggests a globular structure. Received: 29 November 1996 / Accepted: 2 December 1996     

Molecular weight and symmetry of the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The molecular weight and polypeptide chain stoichiometry of the native pyruvate dehydrogenase multienzyme complex from Escherichia coli were determined by independent techniques. The translational diffusion coefficient (D^o_20,w) of the complex was measured by laser light intensity fluctuation spectroscopy and found to be 0.90 (±0.02) × 10^?11m²/s. When this was combined in the Svedberg equation with the measured sedimentation coefficient (s^o_20,w = 60.2 (±0.4) S) and partial specific volume ($\text{v}\text{?}\text{= 0.735 (±0.01)}\text{ml/g}$), the molecular weight of the intact native complex was calculated to be 6.1 (±0.3) × 10⁶. The polypeptide chain stoichiometry (pyruvate decarboxylase: lipoate acetyltransferase: lipoamide dehydrogenase) of the same sample of pyruvate dehydrogenase complex was measured by the radioamidination technique of Bates et al. (1975) and found to be 1.56:1.0:0.78.From this stoichiometry and the published polypeptide chain molecular weights estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, a minimum chemical molecular weight of 283,000 was calculated. This structure must therefore be repeated approximately 22 times to make up the native complex, a number which is in good agreement with the expected repeat of 24 times if the lipoate acetyltransferase core component has octahedral symmetry. It is consistent with what appears in the electron microscope to be trimer-clustering of the lipoate acetyltransferase chains at the corners of a cube. It rules out any structure based on 16 lipoate acetyltransferase chains comprising the enzyme core.The preparation of pyruvate dehydrogenase complex was polydisperse: in addition to the major component, two minor components with sedimentation coefficients (s^o_20,w) of 90.3 (±0.9) S and 19.8 (±0.3) S were observed. Together they comprised about 17% of the total protein in the enzyme sample. Both were in slowly reversible equilibrium with the major 60.2 S component but appeared to be enzymically active in the whole complex reaction. The faster-sedimenting species is probably a dimer of the complex, whereas the slower-sedimenting species has the properties of an incomplete aggregate of the component enzymes of the complex based on a trimer of the lipoate acetyltransferase chain.     

Properties of a discrete high molecular weight poly(A) containing mitochondrial RNA

Pulse-labeled mitochondrial RNA from hamster cells contains a number of discrete high molecular weight poly[A(+)] and poly[A(?)] RNAs. Characterization of the largest and most plentiful of the poly[A(+)] RNAs, the “20S_E RNA,” showed it to be a labile, unmethylated component with a molecular weight ～- 730,000. Hybridization of the 20S_E RNA to mtDNA was 60–70% inhibited in the presence of excess 17S rRNA, suggesting a significant degree of primary sequence homology between these RNA species. In vitro treatment with RNAse III converted the 20S_E RNA to a poly[A(?)] “17S” product, while similar treatment of mitochondrial 17S rRNA or a poly[A(+)] 12S_E RNA had no effect on these RNAs. These data support the proposition that the 20S_E RNA is a precursor to the 17S rRNA.     

Crystallization and amino acid composition of a serine protease from rat skeletal muscle

A serine protease from rat skeletal muscle was crystallized in good yield, and the homogeneity of the preparation was proved by ultracentrifugical analysis and polyacrylamide disc electrophoresis. The S_{20, w} value of the enzyme was 2.2 S and the molecular weight was calculated to be 22,000–24,000 from the results of sedimentation equilibrium analysis. Analysis showed 87% coincidence in the amino acid composition with that of a serine protease from the small intestine. The apparent Km and k_cat(sec^?1) values for N-acetyl-L-tyrosine ethyl ester were 1.1 × 10^?3 M and 9.0, respectively.     

Evidence for an intermediate in the denaturation and assembly of phosphoglucose isomerase

The denaturation of dimeric rabbit muscle phosphoglucose isomerase in guanidine hydrochloride occurs in two discrete steps consisting of partial unfolding followed by subunit dissociation. In 3.5 to 4.5 m guanidine hydrochloride the enzyme forms a stable denaturation intermediate. Formation of this intermediate abolishes catalytic activity, shifts the protein fluorescence emission maximum from 332 to 345 nm, exposes all of the unavailable sulfhydryl groups, and decreases the s_20,w from 6.8 to 4.6 S. The intermediate dissociates into fully unfolded polypeptide chains with further increases in the concentration of the denaturant. The fluorescence maximum shifts to 352 nm and the s_20,w of the denatured monomer is 1.6 S. From the equilibrium constant for subunit association, 3 × 10⁴M^?1, in 4.7 m guanidine hydrochloride, the apparent free energy of association is estimated to be ?6 kcal mol^?1. Reconstitution of the enzyme protein takes place by the reversal of the steps observed upon denaturation. The denatured monomers refold and associate to reform the dimeric intermediate which then anneals to yield the intact enzyme molecule.