Ethylenediaminetetraacetic Acid Extraction Research Articles

Characterization of a 28-kDa collagenous protein extracted with EDTA from adult rabbit alveolar bone

Bone proteins in mandibular alveolar bone from young adult rabbits (3-month-old) were extracted with 4 M guanidine hydrochloride (GuHCl), followed by 0.5M ethylenediaminetetraacetic acid (EDTA). The proteins in the EDTA extract were fractionated on Sepharose CL-6B in the presence of 4 M GuHCl, followed by HPLC using hydroxyapatite and then ‘Mono Q’ resin in the presence of 7 M urea, and a 28 kDa protein was isolated. The purified 28-kDa protein showed intense staining with silver following SDS-PAGE separation under reducing conditions. When the protein was digested with bacterial collagenase, a 19-kDa fragment was produced. However, the protein was not susceptible to cyanogen bromide. On SDS-PAGE under non-reducing conditions, the protein migrated as two bands; a new band at about 85-kDa, and the original 28-kDa band. The amino acid composition of the protein was similar to that of α1-pN-propeptide of type I procollagen from other tissues. Moreover, the characteristics of the purified 28-kDa protein were similar to those of a 28-kDa protein synthesized by rabbit alveolar bone-derived cellsin vitro.

Release of the component of Streptococcus faecalis Na +-ATPase from the membranes

The Na +-stimulated ATPase activity of Streptococcus faecalis was lost by washing the membranes with ethylenediaminetetraacetic acid (EDTA). ATPase activities of both the EDTA extract and the stripped membranes did not show any stimulation by Na + ions. However, the Na +-stimulated ATPase was readily reconstituted by an incubation of these fractions combined. It was only reconstituted from the fractions prepared under the condition that the Na +-ATPase is amplified, and not from those boiled or digested by trypsin. Thus, the component of Na +-ATPase of this organism is capable of being released from the membranes.

Some features of the Streptococcus faecalis Na +-ATPase resemble thoseof the vacuolar-type ATPases

In the ethylenediaminetetraacetic acid (EDTA) extract prepared from the membranes of Streptococcus faecalis, we found the 330-kDa protein that was coordinately increased with the induction of Na +-ATPase. It was missed in the EDTA extract of Nakl, a mutant defective in the Na +-ATPase, but restored in that of its revertant, NaklR. The 330-kDa protein showed the ATP hydrolytic activity by active staining, and mainly consisted of the polypeptides of 73 kDa, 52 kDa and possibly 38 kDa. In addition, the Na +-stimulated ATPase of the membranes was sensitive to both nitrate and N-ethylmaleimide, inhibitors for the vacuolar H +-ATPase. Thus, the Na +-ATPase of this organism has a structure similar to vacuolar H +-ATPase.

Effects of different extraction media on the electrophoretic pattern of proteins from partly mineralized bovine enamel.

Five different extraction solutions were used to isolate matrix proteins from immature bovine enamel, to evaluate the effect of this procedure on the pattern obtained after electrophoresis. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the dominating protein fraction in the ethylenediaminetetraacetic acid extract had a molecular weight of 67,000 daltons. The acetic acid and phosphate buffer extracts contained mostly low molecular weight proteins. Isoelectric focusing showed that most of the enamel proteins had isoelectric points below pH 7.0.

Proteoglycans in different mineralization stages of bovine alveolar bone.

A density fractionation procedure has been used to study the characteristic proteoglycans of bovine alveolar bone at different degrees of mineralization. Bone powder fractions were extracted with 4 M guanidium chloride (GdmCl) and subsequently demineralized with 0.5 M ethylenediamine tetraacetic acid (EDTA) containing protease inhibitors. Alveolar bone powder was distributed into density fractions from 1.8 to 2.1 g/cm3. The amount of non-collagenous proteins markedly decreased with increasing density. Hexuronic acid content was also decreased with increasing density. Chromatographic analysis of the proteoglycans indicated three species of proteoglycans in these extracts. A large proteoglycan, present only in GdmCl extract (corresponds to non-mineralized region), diminished with increasing density. A relatively small proteoglycan was present in both GdmCl and EDTA extract (mineralized region). The amino acid composition and immuno reactivity of each small proteoglycan was similar. Yet, all three proteoglycans were cross-reactive with anti-small proteoglycan. These data suggest that the decrease of proteoglycan and the change of its size is related to the mineralization process in bone.

Effect of ethylenediaminetetraacetic acid, Triton X-100, and lysozyme on the morphology and chemical composition of isolate cell walls of Escherichia coli.

Extraction of a partially purified preparation of cell walls from Escherichia coli with the nonionic detergent Triton X-100 removed all cytoplasmic membrane contamination but did not affect the normal morphology of the cell wall. This Triton-treated preparation, termed the "Triton-insoluble cell wall," contained all of the protein of the cell wall but only about half of the lipopolysaccharide and one-third of the phospholipid of the cell wall. This Triton-insoluble cell wall preparation was used as a starting material in an investigation of several further treatments. Reextraction of the Triton-insoluble cell wall with either Triton X-100 or ethylenediaminetetraacetic acid (EDTA) caused no further solubilization of protein. However, when the Triton-insoluble cell wall was extracted with a combination of Triton X-100 and EDTA, about half of the protein and all of the remaining lipopolysaccharide and phospholipid were solubilized. The material which remained insoluble after this combined Triton and EDTA extraction still retained some of the morphological features of the intact cell wall. Treatment of the Triton-insoluble cell wall with lysozyme resulted in a destruction of the peptidoglycan layer as seen in the electron microscope and in a release of diaminopimelic acid from the cell wall but did not solubilize any cell wall protein. Extraction of this lysozyme-treated preparation with a combination of Triton X-100 and EDTA again solubilized about half of the cell wall protein but resulted in a drastic change in the morphology of the Triton-EDTA-insoluble material. After this treatment, the insoluble material formed lamellar structures. These results are interpreted in terms of the types of noncovalent bonds involved in maintaining the organized structure of the cell wall and suggest that the main forces involved are hydrophobic protein-protein interactions between the cell wall proteins and to a lesser degree a stabilization of protein-protein and protein-lipopolysaccharide interactions by divalent cations. A model for the structure of the E. coli cell wall is presented.