Biochimica et Biophysica Acta (BBA) - Protein Structure最新文献

CD studies have been carried out on horse pancreatic colipases which contain a tryptophan in place of the phenylalanine found at position 52 in the pig protein. From the CD spectra of native proteins in the far ultraviolet region, it was estimated that the secondary structure consists of equal amounts of β-structure and aperiodic arrangements. Colipases contain no α-helical structure. The CD spectra of horse and pig colipases display a positive band at 226 nm the pH dependence of which is characteristic of aromatic chromophores. The near ultraviolet region of the CD spectra of horse and pig colipases contains well-resolved bands at 283–284 nm and 277–278 nm, which corresponds to the tyrosines, with one band at 266–268 nm and one shoulder at 261–263 nm reflecting the contribution of the phenylalanine residues. Comparative analysis of the contribution of sidechains in the aromatic region of the spectra is consistent with a lower solvent accessibility of the lone tryptophan of horse colipase B as compared to isocolipase A. Study of the near ultraviolet CD spectrum of colipase B in the presence of micellar concentration of sodium taurodeoxycholate reveals that the tryptophan (Trp₅₂) moves towards a more hydrophobic environment upon the formation of the colipase-bile salt complex. These results support the conclusion that this residue belongs to the previously identified hydrophobic domain of the colipase molecule which interacts with organized lipids (lipid binding site). The conservative substitution of this residue for a phenylalanine in the pig protein suggests that the aromatic structure might be of critical importance for the binding of colipase to the lipid-water interface.

An α^M-subunit of legumin from Pisum sativum has been purified from a genotype which showed no α^M-subunit heterogeneity on two-dimensional isofocusing/electrophoresis gels. N-terminal sequence analysis of the subunit showed it to be homologous to the acidic subunits of glycinin, from Glycine max, and particularly similar to glycinin subunit A₂. Amino acid analysis showed the α^M-subunit to contain three cysteine and three methionine residues per subunit, plus 14 lysine and 39 arginine. Fewer than the expected number of tryptic peptides from the α^M-subunit were resolved by two-dimensional peptide mapping, but the expected number could be detected by peptide mapping of α^M-subunits which had been hydrolysed with the arginine-specific protease from mouse submaxillary gland.

The fluorescence polarization of fluorescent derivatives of hemoglobin and myoglobin was measured as a function of the concentration of added polymers (PEG-6 000, PEG-20 000) and globular proteins (lysozyme, ribonuclease A, β-lactoglobulin). The results indicated that the effective size and shape of 1-anilino-9-naphthalene sulfonate myoglobin are unaltered in the presence of up to 25 g/dl poly(ethylene glycol), whereas they are significantly altered in the presence of comparable concentrations of other proteins. The results are consistent with the hypothesis that in the presence of high concentrations of added protein, 1-anilino-9-naphthalene sulfonate myoglobin self-associates to form a dimer similar in size and shape to 1-anilino-9-naphthalene sulfonate hemoglobin.

The binding of probe molecules such as fluorescein isothiocyanate, eosin isothiocyanate and erythrosin isothiocyanate to the Ca²⁺-ATPase of sarcoplasmic reticulum followed by illumination of the labelled protein causes substantial reductions of ATPase activity over a 1-h period. The degree of light-sensitivity induced by these probes is related to the triplet yield of these probe molecules. Consistent with this, the greatest effect is seen with erythrosin isothiocyanate and the least effect with fluorescein isothiocyanate. These reductions of ATPase activity associated with illumination are also associated with an aggregation of the protein molecules. This is indicated by laser flash photolysis measurements and also by polyacrylamide gel electrophoresis. A reduction in the number of thiol groups present on the ATPase molecule parallels the reduction of enzyme activity and changes in the protein mobility. The results are discussed in relation to the use of these probe molecules to study biological systems and also in terms of oxidative processes which may affect protein function in vivo.

No sarcoplasmic calcium-binding proteins reminiscent of those described in other arthropods could be detected in locust flight and leg muscle. Instead, these tissues are rich in calmodulin. The purification and functional properties of this protein, which was purified to electrophoretic homogeneity, are very similar to those of calmodulin from bovine brain.

A ‘Hollow Cylinder Protein’ (HCP) similar to the protein originally isolated by Harris from human erythrocyte membranes (Harris, J.R. (1968) Biochim. Biophys. Acta 150, 534–537) is present in the cytosol of erythrocytes at a concentration of more than 15 μg/ml packed erythrocytes. When negatively stained and examined in the electron microscope, cytosol HCP is similar in morphology to the HCP associated with erythrocyte ghost membranes. Cytosol HCP can be purified by isoelectric precipitation at pH 5.2 followed by repeated sucrose gradient centrifugation at alkaline pH. Negatively stained purified cytosol HCP appears as a hollow cylinder with apparent dimensions of 18.0 nm in length by 11.8 nm in diameter and contains a hollow core. Purified cytosol HCP migrates as a single band by non-denaturing polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis shows that it is composed of five peptides having apparent molecular weights 21 500, 23 500, 26 000, 27 500 and 29 000. Chymotryptic peptide maps of each of these bands indicate that each is a unique polypeptide chain. These results indicate that erythrocyte cytosol HCP is a macromolecular complex composed of multiple copies of five non-identical subunits arranged as a hollow cylinder.

The proalbumin hexapeptide extension was synthesized beginning from the C-terminal end by stepwise N-terminal peptide chain elongation starting from $\text{N-tert-}\text{butyloxycarbonyl}\text{-(N}{}^{\mathrm{g}}\text{-}\text{nitro)arginyl}\text{-(N}{}^{\mathrm{g}}\text{-}\text{nitro)arginine}$ 4-nitrobenzyl ester; $\text{[α]}{}_{365}{}^{20}\text{-12°}\text{C}$ ($\text{c = 1}$; dimethylformamide). The other amino acids were incorporated by excess mixed anhydrides of Ddz-amino acids (Ddz; 3,5-dimethoxyphenylisopropyloxycarbonyl) yielding the fully protected hexapeptide in crystalline quality. After removal of the protective groups by acid treatment and hydrogenation the peptide was purified by Dowex ion-exchange and Sephadex chromatography. The purity was confirmed by thin-layer chromatography and amino acid analysis.

Native and modified methemoglobin (β-93-SH groups blocked) were reduced by NADH-dependent methemoglobin reductase in the absence and the presence of organic phosphate (inositol hexaphosphate). These experiments were performed with dilute as well as concentrated methemoglobin solutions (physiological heme concentration). It is shown that: (a) in dilute solutions the blockage of β-93-SH groups lowers the rate of methemoglobin reduction in the absence of organic phosphate but the rates of native and modified methemoglobin reduction are similar in the presence of organic phosphate; (b) at physiological heme concentration the blockage of β-93-SH groups does not affect the rate of reduction but the organic phosphate stimulates the reduction of both native and modified methemoglobins in a similar fashion, as it does in dilute solutions. It is concluded that, although in dilute solutions the blockage of β-93-SH groups alters the reduction rate, at physiological heme concentration the presence of free β-93-SH groups does not have any significant effect on methemoglobin reduction. On the contrary, the organic phosphates do accelerate the rate of reduction at all ranges of heme concentration.

Surfactant apoproteins were prepared from detergent-solubilized rat surfactant by immunoaffinity chromatography. SDS-polyacrylamide gel electrophoresis of the apoproteins, without prior chemical reduction, revealed several bands of molecular weights 50 000–78 000, 140 000 and 160 000. Following treatment with dithiothreitol, the apoproteins were resolved into three bands of molecular weights 38 000, 32 000 and 26 000. Further analysis of the apoproteins by two-dimensional polyacrylamide gel electrophoresis showed that each of the proteins of molecular weights 38 000, 32 000 and 26 000 were crosslinked by disulfide bridges and formed homopolymers. Based on periodic acid-Schiff staining, the 38 000 dalton protein appeared to be the richest in carbohydrates, followed by the 32 000 and 26 000 dalton proteins. Partial proteolysis of the 38 000 and 32 000 dalton proteins showed similarity in the sizes of peptides generated. Surfactant-associated proteins from human and monkey lungs were also analyzed by polyacrylamide gel electrophoresis. A non-serum glycoprotein of molecular weight 38 000 was found. In different systems of polyacrylamide gel electrophoresis, this protein showed an electrophoretic mobility similar to that of the 38 000 dalton protein of rat surfactant. However, it formed polymers of molecular weight higher than those of polymers found in rat surfactant.

X-ray absorption near-edge spectroscopy (XANES) of Co(II) in three derivatives of superoxide dismutase, namely [Cu(II)-Co(II)], [Cu(I)-Co(II)] and […-Co(II)], suggests a tetrahedral coordination of the metal for all compounds. Significant differences, detected in the spectrum of the [Cu(II)-Co(II)] derivative as compared to the other species, indicate that a conformational change and/or a different charge of the imidazole bridging the two metal sites in superoxide dismutase occur in coincidence with the change of copper valence. The XANES spectra of the cobalt derivatives of alcohol dehydrogenase, carbonic anhydrase and stellacyanin show features that can be accounted for by an increasing degree of covalency in the metal first sphere of coordination, in the following order: alcohol dehydrogenase > stellacyanin > superoxide dismutase ⩾ carbonic anhydrase.