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.     

Cetyltrimethylammonium bromide polyacrylamide gel electrophoresis: estimation of protein subunit molecular weights using cationic detergents.

Polyacrylamide gel electrophoresis in the presence of the cationic detergent, cetyltrimethylammonium bromide (CTAB), has been previously used to obtain more accurate estimates of the molecular weight of certain highly charged and membrane protein subunits that exhibit anomalous electrophoretic behavior in the presence of sodium dodecyl sulfate (SDS). The improved method reported herein is comparable to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) method in simplicity, time, and quality of gels, but the CTAB-PAGE method appears to have a wider range of application for diverse types of proteins. The technique may also be used for verification of molecular weight data and thus detection of possible anomalous results obtained using the anionic SDS-PAGE method. The described method eliminates the precipitates formed between ammonium persulfate and cationic detergents during gel polymerization and between cationic detergents and the protein dyes during staining that have complicated previous methods. The reliability of the technique is indicated by the high correlation coefficient (?0.97) between R_f and molecular weight. Data are presented to indicate that the method can be used to estimate the subunit molecular weight of unknown proteins with a 95% level of confidence.     

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.     

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.     

Studies of oligosaccharide linkages by simultaneous determination of component aldehydes in dialdehyde fragments as (2,4-dinitrophenyl)hydrazones

The component aldehydes in dialdehyde fragments formed by periodate oxidation of oligosaccharides were converted quantitatively into the corresponding (2,4-dinitrophenyl)hydrazones by the simple procedure of treatment with excess (2,4-dinitrophenyl)hydrazine hydrochloride in 1,2-dimethoxyethane. Then, by chromatographic separation of the hydrazones on a small column of silica gel and subsequent spectrophotometric analysis, it was possible to determine the position of glycosidic substitution in μmolar amounts of various types of glucobioses, oligosaccharides of senega, and some synthetic (1→6)-β-D-gluco-oligosaccharides.     

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 mechanism of activation of rabbit plasminogen by urokinase. Lack of a preactivation peptide

We have obtained direct evidence which we interpret to prove that an amino terminal peptide need not be released from rabbit plasminogen prior to its conversion to plasmin by urokinase. The single chain plasminogen molecule possesses an amino terminal amino acid sequence of NH₂-glu-pro-leu-asp-asp. When this plasminogen is activated to plasmin by urokinase in the presence of the Kunitz bovine trypsin-plasmin-kallikrein inhibitor (BTI), a two chain disulfide linked molecule of plasmin is obtained. The heavy chain of this plasmin is directly derived from the original amino terminus of plasminogen since it possesses the identical amino terminal sequence as does native plasminogen. When the same plasminogen activation is carried out in the absence of BTI, the heavy chain of the plasmin obtained has a molecular weight of 6,000–8,000 less than the heavy chain of the plasmin obtained in the presence of this inhibitor. In addition, the heavy chain of this latter plasmin has an amino terminal sequence which differs from the original native plasminogen. These data show, in agreement with others, that the activation of plasminogen by urokinase is accompanied by the loss of an amino terminal peptide from plasminogen but also show, in contrast to the human plasminogen system, that cleavage of the internal peptide bond, leading to plasmin formation, can occur without cleavage of the amino terminal peptide.     

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.     

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.     

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.     

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.     

Specificity reversal in phospholipase A2 hydrolysis of lipid mixtures

The activity and specificity of phospholipase A₂ from cobra venom ($\text{Naja naja naja}$) toward binary mixtures of phosphatidylcholine and phosphatidylethanolamine in mixed micelles with the nonionic surfactant Triton X-100 were examined. In mixtures containing 5–50 mol % phosphatidylcholine, the rate for phosphatidylethanolamine hydrolysis was enhanced greatly over that for phosphatidylcholine. This is in marked contrast to previous studies with individual phospholipid species in mixed micelles where phosphatidylcholine was found to be the preferred substrate and phosphatidylethanolamine was found to be a very poor substrate. Possible explanations for this specificity reversal are considered.     

Vacuum ultraviolet circular dichroism spectrum of beta-turn in solution.

The vacuum ultraviolet circular dichroism spectrum of an isolated 4 → 1 hydrogen bonded β-turn is reported. The observed spectrum of N-acetyl-Pro-Gly-Leu-OH at ? 40°C in trifluoroethanol is in good agreement with the theoretically calculated CD spectrum of the β-turn conformation. This spectrum, particularly the presence of a strong negative band around 180 nm and a large ratio $\text{[θ]}{}_{201}\text{[θ]}{}_{225}$, can be taken as a characteristic feature of the isolated β-turn conformation. These CD spectral features can thus be used to distinguish the β-turn conformation from the β-structure in solution.     

The effect of in vivo treatment with EHDP and/or 1,25-DHCC on calcium uptake and release in isolated kidney mitochondria

Previous work on calcium transport (uptake and release) in isolated mitochondria, in vitro, has shown that addition of EHDP to the medium does not influence calcium uptake, but does delay calcium release. In vivo treatment of normal chicks with high doses of EHDP (10 mg P/kg body weight/day) has now also been found not to affect the in vitro calcium uptake in isolated chick kidney mitochondria, but to delay the subsequent release as compared with controls. The effect is not due to the decrease in 1,25-DHCC, since chronic administration of this metabolite did not correct the delay. In fact 1,25-DHCC in itself had a delaying effect on accumulated calcium release.