Molecular organization in bacterial cell membranes: IV. Isolation by preparative electrophoresis in sodium dodecylsulphate and properties of the two major polypeptide groups of a “soluble” fraction from Streptomyces albus membranes

Two polypeptide fractions have been purified from a “soluble” fraction of n-butanol-extracted Streptomyces albus membranes by preparative electrophoresis in sodium dodecylsulphate. They accounted for approx. 80% of the protein of the fraction (i.e. 20% of the total membrane protein). The ultraviolet spectrum of Group 1 (relative mobility 1.0) revealed the presence of nucleotide material, while that of Group 3 (relative mobility 0.65±0.05) showed the presence of a possibly aggregated protein-like material. About 100 and 30% of the protein contents (Lowry method) of Groups 3 and 1, respectively, were recovered as amino acid residues. These results confirm the protein nature of both fractions and suggest an overestimation of the protein value in Group 1. Both polypeptide groups can be classified as “extrinsic” membrane proteins on the basis of their similar amino acid composition (Vanderkooi, G. and Capaldi, R. A. (1972) Ann. N.Y. Acad. Sci. 195, 135–138). Three N-terminal amino acids were found in each fraction: one common (alanine), methionine, leucine or isoleucine (Group 3) and glutamic acid, lysine (Group 1). The sedimentation coefficients calculated were 2.46 S for Group 3 and 1.54 S for Group 1. Analysis of the isolated groups by gel electrophoresis under non-dissociating conditions or with Triton X-100, gave aggregate-like patterns.Sodium dodecylsulphate electrophoresis revealed an anomalous staining behaviour of Group 3 depending upon the dissociating conditions. The whole “soluble” fraction bound 0.40 mg dodecylsulphate /mg protein (0.55 mg detergent/mg protein corrected for overestimation). After dialysis, the fraction retained 10% of the bound dodecylsulphate. Circular dichroism of the isolated groups after exhaustive dialysis showed similar spectra, although of lower dichroism, to those obtained by other authors on soluble enzymes treated with sodium dodecylsulphate. Strong acid conditions were required to change the CD spectra of the polypeptides.     

Binding of cytochrome c to the cytochrome bc1 complex (complex III) and its subunits cytochrome c1 and b1.

Cytochrome $\text{c}$₁, the electron donor for cytochrome $\text{c}$, is a subunit of the mitochondrial cytochrome $\text{bc}$₁ complex (complex III, cytochrome $\text{c}$ reductase). To test if cytochrome $\text{c}$₁ is the cytochrome $\text{c}$-binding subunit of the $\text{bc}$₁ complex, binding of cytochrome $\text{c}$ to the complex and to isolated cytochrome $\text{c}$₁ was compared by a gel-filtration method under non-equilibrium conditions (a $\text{bc}$₁ complex lacking the Rieske ironsulfur protein was used; von Jagow et al. (1977) Biochim. Biophys. Acta $\text{462}$, 549–558). The approximate stoichiometries and binding affinities were found to be very similar. Binding of cytochrome $\text{c}$ to isolated cytochrome $\text{b}$ which is another subunit of the reductase was not detectable by the gel-filtration method. Further, the same lysine residues of cytochrome $\text{c}$ were shielded towards chemical acetylation in the complexes $\text{c}\text{:}\text{c}$₁ and $\text{c}\text{:}\text{bc}$₁. From this we conclude that the same surface area of cytochrome $\text{c}$ is in direct contact with cytochrome $\text{bc}$₁ and with cytochrome $\text{c}$₁ in the respective complexes and that therefore cytochrome $\text{c}$ is most probably the structural ligand for cytochrome $\text{c}$ in mitochondrial cytochrome $\text{c}$ reductase.     

Quantitative measurement of hypermodified adenosine ribonucleosides in plant tissues

A procedure for analyzing subnanomole levels of hypermodified ribonucleosides in small amounts of plant tissue is described. It involves sodium metaperiodate oxidation, tritiated borohydride reduction, and separation of hypermodified nucleosides by Sephadex LH-20 column in 35% ethanol followed by paper chromatography. Data for the identification of modified nucleosides by glc and paper chromatography are included.     

Motility of the N-terminal tail of phosphorylase b as revealed by crosslinking.

There are lysyl-ε-NH₂ groups within about 3.5 Å distance across the intersubunit contact area of rabbit muscle phosphorylase $\text{b}$, as shown by cross-linking with malonic diimidate. These include the lysines of N-terminal region as revealed by limited tryptic digestion, but the contribution of the tail lysines to overall formation of covalent dimers is small. The fine structure of dimer band on dodecylsulfate-gelelectrophoretograms of crosslinked phosphorylases suggests that the tail retains its freedom in the phosphorylase $\text{b}$-AMP complex. Amidination induces the dissociation of phosphorylase $\text{b}$ dimer, which is slow relative to crosslinking.     

The polypeptide composition and ultrastructure of nuclear ghosts isolated from mammalian cells

The polypeptide species of non-membranous nuclear ghosts from purified cell nuclei are conserved among a variety of human, hamster and mouse cell types studied, including HeLa, BHK, 3T6 and Hep-2 cell lines. The polypeptide species present in nuclear ghosts from HeLa cells synchronized in various stages of the cell cycle are largely the same with minor variations. The isolated nuclear ghosts are similar, in terms of polypeptide composition, to other residual nuclear structures isolated by independent techniques. The nuclear ghosts appear as flattened sac-like structures when viewed by scanning electron microscopy. Transmission electron microscopy of the nuclear ghosts reveals ring-like structures which may represent the nuclear pores. Also observed are novel rod-shaped structures approximately 260 nm in length and 50 nm in diameter. The latter images either arise by a rearrangement during isolation of the nuclear ghost macromolecules or are a heretofore undescribed structure of intact nuclei.     

Action of Solanum malacoxylon on calcium metabolism in the rat

The administration of an aqueous extract of the leaves from $\text{Solanum malacoxylon}$ to vitamin D-deficient rats fed a normal calcium, normal phosphorus diet markedly increased serum calcium concentration within 48 hours. The $\text{Solanum malacoxylon}$ extract also stimulated intestinal calcium transport in the vitamin D-deficient rat but was without effect on the mobilization of calcium from bone. The extract from 100 mg of dry $\text{Solanum malacoxylon}$ leaves was more effective than 25 units of vitamin D given daily to vitamin D-deficient rats in stimulating intestinal calcium transport but its effect was not additive to that of the vitamin D. The results demonstrate that the action of $\text{Solanum malacoxylon}$ is independent of vitamin D and, although it can substitute for vitamin D in the stimulation of intestinal calcium transport activity, it cannot substitute for vitamin D in the mobilization of calcium from bone.     

Synthesis of compositionally unique DNA by terminal deoxynucleotidyl transferase

Studies on the composition and characterization of DNA product(s) synthesized by calf thymus terminal deoxynucleotidyl transferase were performed using homopolymeric single-stranded, calf thymus double-stranded, and native DNA resident in calf thymus chromatin preparations as priming DNA species. Synthesis was carried out using equimolar concentrations of all four deoxynucleoside triphosphates as substrates and Mg²⁺ or Mn²⁺ as an effective divalent cation. Irrespective of the nature of the priming DNA or the divalent cation, the DNA product contained 60–70% dGMP residues, 10–15% each of the two pyrimidine residues, and 5–10% dAMP residues. The product synthesized using chromatin DNA as initiator was predominantly single-stranded and its synthesis was resistant to actinomycin D. The predilection of terminal deoxynucleotidyl transfease to synthesize dGMP-rich products on natural or homopolymeric DNA primers suggests that such products may represent biologically important recognition signal sequences.     

A calorimetric study of the binding of 2′-deoxyuridine-5′-phosphate and its analogs to thymidylate synthetase

The binding of dUMP, dTMP, UMP, and 5-fluoro-2′-deoxyuridylate (FdUMP) to Lactobacillus casei thymidylate synthetase (TSase) was examined by direct thermal titration. The binding of each ligand was examined in two different buffers, so that proton interactions could be observed. In agreement with an earlier study (N. V. Beaudette, N. Langerman, R. L. Kisliuk, and Y. Gaumont, 1977, Arch. Biochem. Biophys.179, 272–278), dUMP binding is driven predominantly by enthalpy changes at pH 7.4, with 0.77 ± 0.07 mol of protons binding along with the substrate. When the pH is decreased to 5.8, binding affinity increases, and a substantial increase in the entropic contribution to the binding is observed. In contrast to the binding of protons with substrate at pH 7.4, protons are released at pH 5.8. The proton effects suggest a model in which binding occurs through an electrostatic interaction between dianionic nucleotide and protonated enzyme residues. Binding of FdUMP at pH 7.4 involves the uptake of protons, and is also predominantly driven by changes in enthalpy. A good fit to the thermal data is obtained using the single-site binding constant, K = 9.5 × 10⁴m^?1. Our earlier interpretation (Arch. Biochem. Biophys., 1977, 179, 272–278) of the thermal data indicating two sites is in error. Preliminary date are presented which suggest that two-site binding of FdUMP occurs on prolonged incubation during equilibrium dialysis. Binding of the product dTMP shows different behavior. The reaction is entropically driven, suggesting that a significant hydrophobic interaction occurs between the protein and the 5-methyl group of the nucleotide. Only 0.48 ± 0.08 mol of protons are absorbed at pH 7.4. Binding of the nucleotide UMP could not be detected at pH 7.4.     

Proton nuclear magnetic resonance studies of the manganese (II) binding site of concanavalin A. Effect of saccharides on the solvent relaxation rates

The longitudinal relaxation rate ($\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$) of water protons was studied in solutions of Mn(II)-concanavalin A at a number of frequencies. These relaxation rates were lowered in the presence of a variety of saccharides which have affinities for concanavalin A which range over two orders of magnitude. A good correlation was found in which saccharides which bind tightly have the greatest effect and saccharides which bind weakly or not at all have little effect on the $\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ values. The temperature dependence of the proton relaxation rates showed that the lowering of these rates in the presence of saccharides was most likely due to a change in the exchange rate of solvent interacting with protein-bound Mn(II), $\text{1}\text{T}{}_{\mathrm{<mtext>m</mtext>}}$.An analysis of the temperature and frequency dependence of the $\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ and $\text{1}\text{T}{}_{\mathrm{<mtext>2</mtext><mtext>p</mtext>}}$ (transverse) solvent proton relaxation rates resulted in evaluation of a number of parameters for solvent water molecules interacting in the first coordination sphere of Mn(II) bound to concanavalin A. The ratio of the number of water molecules (q) to the Mn(II)-proton distance (r) obtained from a computer fit of the data over a limited temperature range is in accord with the findings of Koenig et al. ((1973) Proc. Nat. Acad. Sci.70, 475) and Meirovitch and Kalb ((1973) Biochim. Biophys. Acta303, 258). However, our studies of $\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ and $\text{1}\text{T}{}_{\mathrm{<mtext>2</mtext><mtext>p</mtext>}}$ of water over a more extensive temperature range are best fit with the following conclusions: at low temperatures (<20 °C), the data are consistent with an outer-sphere relaxation process. At higher temperatures (> 30 °C), the water molecule in the inner coordination sphere of the bound Mn(II) begins exchanging more rapidly and contributes to the relaxation processes ($\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ and $\text{1}\text{T}{}_{\mathrm{<mtext>2</mtext><mtext>p</mtext>}}$). The relaxation time of protons in the inner coordination shell, T_1M, contributes over the entire temperature range and produces a frequency dependence in the relaxivity data from 6 to 100 MH_z since the contributions to the correlation times are in the range 10^?9-10^?8 sec.     

Microviscosity of the lipid domains of normal and hypercholesterolemic very low density lipoprotein.

Very low density lipoprotein (VLDL) has been isolated from normal (n) and dietary-induced hypercholesterolemic (hc) rabbits. Incorporation of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene into the lipid domains of both n VLDL and hc VLDL allowed assessment of the fluidity characteristics of these particles, utilizing fluorescence polarization techniques. Over the temperature range of 5° – 45°, the lipid region of n VLDL consists of an invariant phase, characterized by a microviscosity, η, at 30° of 0.6 ± 0.2 poise and a fusion activation energy, ΔE, of 7.6 ± 1.5 kcal/mole. The lipid region of hc VLDL, over the same temperature range, also is invariant and is characterized by a value of η at 30° of 4.6 ± 0.3 poise, and a ΔE of 7.8 ± 1.5 kcal/mole. Thus, large differences in the fluidit of the lipid in n VLDL and hc VLDL are evident, most probably due to the greatly increased content of cholesterol esters in hc VLDL, compared to n VLDL.     

The enthalpy of oxidation of flavin mononucleotide

The oxidation enthalpy of reduced flavin mononucleotide at pH 7.0 in 0.2 m phosphate buffer has been studied by determining the heat associated with the reaction: FMNH₂ + 2 Fe(CN)^?3₆ ? FMN + 2 Fe(CN)^?4₆ + 2 H⁺. (a) (The quinone, semiquinone, and hydroquinone forms of FMN are represented as FMN, FMNH, and FMNH₂, respectively.) Calorimetric experiments were performed in a flow microcalorimeter which was modified to prevent sample contamination by oxygen. The enthalpy observed for reaction (a), after correction for dilution and buffer effects, was ?39.2 ± 0.4 kcal (mole FMNH₂)^?1 at 25 °C. The potential difference, ΔE′, developed by reaction (a) was determined potentiometrically and corresponded to a free energy change, ΔG′, of ?30.3 kcal (mole FMNH₂)^?1. The resulting entropy change, ΔS′, was thus calculated to be ?29.8 e.u. Reaction (a) was also studied at temperatures of 7 °C and 35.5 °C. ΔC_p′ for the reaction was calculated as ?155 ± 18 cal deg^?1 (mole FMNH₂)^?1 at 20 °C. ΔH′ for the reaction (b), FMNH₂ ? FMN + H₂, (b) was calculated as +14.2 ± 0.7 kcal mole^?1 at 25 °C, relative to the enthalpy of the hydrogen electrode being identically equal to zero at all values of pH and temperature. The free energy at pH 7.0 for reaction (b), calculated from the potential was found to be ?9.7 kcal mole^?1, which resulted in an entropy for reaction (b) of 80.2 e.u. A thermal titration of reaction (a) was used to calculate the thermodynamic parameters for the formation of semiquinone dimer according to the reaction FMNH₂ + FMN ? (·FMNH)₂. (c) The free energy, enthalpy, and entropy changes for reaction (c) were estimated to be ?6.1 kcal mole^?1, ?7 kcal mole^?1, and ?3 e.u., respectively.     

Multiple types of cell surface heparan sulfate are produced by primary cultures of embryonic mouse cells

The cell surface heparan sulfate produced by primary cultures of 12-day mouse embryo cells has been found to consist of at least two forms, designated I and II. These two forms can be distinguished by both ion-exchange chromatography on DEAE-cellulose and eletrophoresis at pH 1. However, no difference in molecular weight is observed when the two forms are compared by gel filtration on Bio-Gel A-15m. These data suggest that the two forms differ in their content of sulfate residues. Multiple types of cell surface heparan sulfate are also produced by primary cell cultures derived from various mouse embryonic organs, including heart, lung, kidney and liver. Type II, the minor form produced by the primary embryonic mouse cells, behaves on ion-exchange chromatography and electrophoresis at pH 1 as the heparan sulfate produced by several mouse cell lines that exhibit contact inhibition of growth. The predominant form, type I, behaves on ion-exchange chromatography as the heparan sulfate derived from either DNA or RNA virus-transformed cell lines which lack growth control. The cell surface heparan sulfate produced by chick myoblasts, human fibroblasts, and bovine endothelial cells behave as single types on ion-exchange chromatography. These data suggest that an individual cell type produces a single type of cell surface heparan sulfate and provide support for a model in which cell-cell interactions are mediated, in part, by the quantity and, possibly, arrangement of sulfate residues within the heparan sulfate polymer.     

The determination of 2'-O-methylnucleosides in RNA

A rapid and sensitive procedure is described for determining the 2′-O-methylnucleoside methylnucleoside composition of an RNA sample. The RNA is enzymatically hydrolyzed to nucleosides and the 2′-O-methylnucleoside fraction is isolated by DEAE-cellulose (borate) column chromatography. Boric acid is removed as its methyl ester and the 2′-O-methylnucleosides are resolved by liquid chromatography in the presence of ethylene glycol. The sensitivity of this method is sufficient to distinguish RNA samples which differ only 2–3% in 2′-O-methylnucleoside composition.