John Bridgen

Evolutionary relationships in superoxide dismutase

Two distinct types of superoxide dismutase have been isolated from aerobic organisms. A blue-green Cu-Zn protein with a mol. wt of 32 000 and comprising two identical subunits has been obtained from a wide range of eukaryotes (e.g. [3,4]) while a pinkish-purple Mncontaining enzyme, also composed of identical subunits, has been isolated from eukaryotic mitochondria [S] and from prokaryotes [6,7]. In addition, an Fecontaining superoxide dismutase, similar to its Mncontaining counterpart, has been obtained from,!?. coli [8]. The prokaryotic Mn and Fe-containing enzymes are dimers with mol. wts of 40 000 and the mitochondrial Mn-enzyme, with a mol. wt of 80 000, is a tetramer. The subunits of the Mn and Fe-containing dismutases are of the same chain length, comprising approx. 185 to 190 residues [6-81. In addition they possess similar amino acid compositions and N-terminal sequences [9]. The Cu-Zn enzyme from bovine erythrocytes on the other band possesses a smaller subunit (of 150 residues [ 1 O]), with a different amino acid composition [ 111 and N-terminal sequence [9]. These results led Steinman and Hill [9] to suggest that the two classes of dismutase are probably composed of entirely different proteins of independent evolutionary origin. Insofar as the sequence comparisons of the respective protein chains were confined to the N-terminal segments of 25 to 30 residues, the possibility

Solid phase sequencing: A new support for the high sensitivity degradation of peptides and proteins

Since the introduction by Laursen [l] of the automated solid-phase Edman degradation, numerous modifications and improvements have been made to the original method. Of particular value have been the introduction of methods for coupling peptides containing lysine residues [2] and for coupling peptides derived from cyanogen bromide digests [3]. Recently, the use of derivatised porous glass for determining the N-terminal sequence of cytochrome c by a solid-phase procedure has been described [4]. I would like to describe an extension of this method using a new support, N-(2-aminoethyl)3aminopropyl glass, which contains activated amino groups suitable for coupling peptides and proteins by the diisothiocyanate method, as well as for coupling large and small cyanogen bromide fragments. The use of this method for the degradation of very small quantities of peptides and proteins is also described.

Amino acid sequence homology in alcohol dehydrogenase.

Thiol groups have been implicated in the catalytic activity of a number of dehydrogenases (for recent review see [ 11). Moreover, in the case of glyceraldehyde-3-phosphate dehydrogenase there is good evidence to show [2] that one cysteine residue per subunit (Cys-149 in the primary structure [3]) is directly involved in the catalytic reaction. The enzyme is inhibited by reaction of this cysteine with thiol group reagents and the amino acid sequence around the reactive cysteine is virtually identical in enzymes from a number a widely different species [4]. Alcohol dehydrogenase are also inhibited by reagents that react with thiol groups [5-71. In the horse liver enzyme inhibition by iodoacetate has been shown to be due to its selective reaction with one cysteine residue [8] identified as cysteine-46 in the enzyme [9]. Alcohol dehydrogenase from yeast is inhibited in a similar manner and although there is some similarity in the amino acid sequence around the reactive cysteines in the two proteins [6] it is not as extensive as that found between the corresponding glyceraldehyde-3phosphate dehydrogenases [4]. Moreover, differences in sequence close to the reactive cysteine in alcohol dehydrogenase have been shown to occur [ 10, 1 l] even within mammals. Alcohol dehydrogenase from yeast and liver differ in several respects. For example, the latter is a dimer with a subunit consisting of 374 amino acids [8,9] while the former is a tetrameric protein with a smaller subunit consisting of about 330 amino acids [6, 121. Although there is a similarity in the amino acid sequence around the respective reactive cysteines [6] comparison of the compositions of other tryptic peptides

Superoxide dismutase from Bacillus stearothermophilus: Crystallization and preliminary X-ray diffraction studies

Superoxide dismutase from Bacillus stearothermophilus is a dimeric manganese-containing enzyme with a molecular weight of 40,000. It has been crystallised in the monoclinic space group P21 with unit cell dimensions of a = 50 A, b = 70 A, c = 69 A and β = 111 ° 10′. The asymmetric unit appears to be the dimer.

A polyamide support for solid-phase protein sequencing

The solid-phase sequencing procedure of Laursen [l] requires the covalent attachment of a peptide or protein to an inert support. The essential properties of the support are that it should be capable of being highly substituted with functional groups, that these functional groups should be accessible for chemical reaction in a variety of aqueous and organic media, and that it should be stable under the reaction conditions used for the Edman degradation. An ideal support should also be capable of coupling efficiently all sizes of polypeptides including proteins and subsequently allow these to be degraded with a repetitive yield sufficient to obtain at least 30 residues of amino acid sequence. The present supports polystyrene [ 1 ] and porous glass [2] do not fulfill all of these criteria. Polystyrene derivatives [ 1,3] do not swell in aqueous media necessitating the performance of the coupling reactions in solvent systems where the peptide may be poorly soluble. Also, attachment yields for large peptides or proteins are often very low and once attached, these molecules may only be degraded with relatively low efficiency. With porous glass derivatives [2,4] the reverse is true and small peptides are attached only in low yield. The repetitive yield for the solid phase Edman degradation on these supports to be 90-93s [5] allowing only the first 20-25 residues of amino acid sequence to be determined. A polydimethylacrylamide-based resin has recently been shown [6] to have significant advantages for solid-phase peptide synthesis over conventional polystyrene matrices. Since this resin is already functionalised with (protected) amino groups and appeared to

Molecular heterogeneity of alkaline phosphatase

Alkaline phosphatase (EC 3. I .3.1) from E. coli is a dimeric enzyme whose subunits are coded by the same gene [ 11. Molecular heterogeneity of the subunits has been ascribed to differential modification of the N-terminus of each monomer after translation [2, 31. In an attempt to verify this hypothesis we have looked for heterogeneity in the N-terminal sequence of the enzyme using an automatic sequenator [4].

Isolation, characterization and partial sequence of cyanogen bromide fragments and thiol peptides from pig kidney D-amino-acid oxidase.

A partial characterization of the primary structure of D-amino-acid oxidase (D-Amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3.) from hog kidney has been achieved by a CNBr cleavage of the 14C-carboxymethylated protein. Four fragments have been isolated and purified and their alignment made possible by overlapping with methionine-containing peptides derived from tryptic digestion of the 14C-carboxymethylated protein. A partial sequencing of the CNBr fragments has been carried out by the automated Edman procedure and by manual sequence analysis. Chymotryptic peptides containing the 5 alkylated thiols of the monomer enzyme (Curti, B., Ronchi, S., branzoli, U., Ferri, G. and Williams, Jr., C. H. (1973) Biochim. Biophys. Acta 327, 266-273) have been isolated and their sequence determined. The present results do not show any significant homologies with the known sequences of other flavoproteins.