Brian Martin

The complete amino acid sequence of copper, zinc superoxide dismutase from Saccharomyces cerevisiae

The amino acid sequence of the copper zinc superoxide dismutase from Saccharomyces cerevisiae has been determined by automated Edman degradation. Peptides were obtained from cyanogen bromide cleavage, Staphylococcus aureus V8 protease digestion, tryptic and chymotryptic digests of the citraconylated reduced and carboxymethylated enzyme, and by further fragmentation of selected peptides with trypsin. From the alignment of these peptides and the previously published sequence of the first 54 amino terminal residues (24) the complete sequence was deduced by direct sequence identification of all 153 amino acid residues and of all peptide overlaps. The amino acid sequence corresponds to a molecular weight of 15,950 for each of the two identical subunits in the native enzyme. The primary structure of yeast copper, zinc superoxide dismutase is 55% identical with the sequence of the copper, zinc enzyme from bovine erythrocytes. Importantly, all the copper and zinc ligands, six histidine residues and one aspartate residue from the bovine enzyme, are conserved in the yeast enzyme. The high overall sequence homology and conservation of important metal binding active site amino acid residues suggest that the three-dimensional structure and in particular the active site geometry is virtually the same for the bovine and yeast enzyme. In contrast no sequence homology is apparent by comparison with the manganese or iron class of superoxide dismutases indicating that the two classes have not evolved from a common ancestor.

CHARACTERISTICS OF HIPROLY BARLEY III. AMINO ACID SEQUENCES OF TWO LYSINE-RICH PROTEINS

Two lysine-rich components isolated from «Hiproly» barley have been subjected to sequence determination. The complete sequence of the smallest component, designated SP II B, has been determined. This protein consists of 72 amino acid residues and has a molecular weight of 8.072. The largest component, designated SP II A, was blocked to N-terminal Edman degradation, but sequence analyses of cyanogen bromide- and tryptic fragments showed that it was identical to the smallest component, except for the eight to eleven residues of which SP II A is longer in the N-terminus. Of these residues seven have been sequenced, and the following total sequence is reported (Glx, Val, Ser, Ser)-Lys-Lys-Pro-Glu-Gly-Val-Asn-Thr-Gly-Ala-Gly-Asp-Arg-His-Asn-Leu-Lys-Thr-Glu-Trp-Pro-Glu-Leu-Val-Gly-Lys-Ser-Val-Glu-Glu-Ala-Lys-Lys-Val-Ile-Leu-Gln-Asp-Lys-Pro-Glu-Ala-Gln-Ile-Ile-Val-Leu-Pro-Val-Gly-Thr-Ile-Val-Thr-Met-Glu-Tyr-Arg-Ile-Asp-Arg-Val-Arg-Leu-Phe-Val-Asp-Lys-Leu-Asp-Asn-Ile-Ala-Gln-Val-Pro-Arg-Val-Gly.

Isolation and characterization of the folate-binding protein from cow's milk

The folate-binding protein from cows milk has been purified in milligramme scale by combination of ion-exchange chromatography and affinity chromatography. The molecular weight has been determined by sedimentation equilibrium ultracentrifugation and found to be 30,000±2,000. The amino acid composition is compatible with this value. The molecule contains six disulphide bridges and no free SH-groups. The three per cent carbohydrate content was accounted for by six glucosamine residues per mole of protein. About 50 per cent of the amino acid sequence has been delineated, including the N-terminal sequence and the C-terminal sequence. Isoelectric focusing gave rise to four major peaks with isoelectric points ranging from 8.5 to 7.6, but no heterogeneity was observed in the sequence.

Use of carboxypeptidase Y for carboxy-terminal sequence determination in proteins

Treatment of several proteins-native pancreatic ribonuclease, human carbonic anhydrase B and human carbonic anhydrase C- with carboxypeptidase Y in the presence of 0.5% sodium dodecyl sulphate resulted in the sequential release of amino acid residues from the carboxyl terminus of the polypeptides. This provides a convenient method to study the carboxy-terminal sequence of proteins whose carboxyl termini are inaccessible in the native state to cleavage by carboxypeptidase.

The complete amino acid sequence of manganese-superoxide dismutase from Saccharomyces cerevisiae

The complete amino acid sequence of manganese superoxide dismutase isolated from Saccharomyces cerevisiae has been determined by automated Edman degradation. Peptides for sequence analysis were produced by cleavage with cyanogen bromide, hydroxylamine and S. aureus protease V8. The native enzyme consists of four identical polypeptide chains 203 residues long each containing a single sulfhydryl group and no disulfide bridges. A subunit molecular weight of 22,690 was calculated from the complete sequence. The sequence exhibits a significant degree of homology with the managanese superoxide dismutase from E. coli and B. stearothermophilus.

Amino acid sequence of carboxypeptidase Y. II. Peptides from enzymatic cleavages

Carboxypeptidase Y (E.C.3.4.12.1) with the lysine side-chains blocked by citraconylation has been digested with trypsin and the resulting peptides purified by gel filtration followed by ion-exchange chromatography on DE 52 cellulose. Eight of the expected ten peptides have been completely or partially sequenced in a liquid phase automatic sequencer. The remaining two peptides were sequenced as part of a CNBr peptide (see preceeding paper). Selected peptides obtained from enzymatic cleavages with A. mellea protease, chymotrypsin, post-proline cleaving enzyme, S. aureus V8 protease, and subtilisin have upon sequencing provided overlaps which have made the reconstruction of 96% of the complete sequence possible together with a tentative assignment of the carbohydrate attachment sites. No homology with sequences of known proteases has been observed.

Partial amino acid sequence of the large subunit of ribulosebisphosphate carboxylase from barley

The large subunit of D-ribulose-1,5-bisphosphate carboxylase (E.C. 4.1.1.39) from barley has been subjected to partial amino acid sequencing. The amino-terminal sequence comprising 46 residues of the reduced and carboxymethylated large subunit polypeptide has been determined. The carboxy-terminal end contained the sequence-Leu-Ala-Val · COOH. Eight of the ten cyanogen bromide fragments have been partially sequenced from the N-terminus. A ninth fragment was found to be blocked to N-terminal sequencing due to cyclization of a glutamine residue after cyanogen bromide cleavage. The eight sequences which have been determined comprise 210 of the approximate 490 residues in the large subunit. The sequences have been compared to five sequences of the large subunit from spinach. Among 37 residues which could be compared four differences were encountered.

Amino acid sequence of carboxypeptidase Y. I. Peptides from cleavage with cyanogen bromide

The partial amino acid sequence of carboxypeptidase Y from Saccharomyces cerevisiae has been determined by automatedEdman degradation. Peptides were obtained by cyanogen bromide cleavage of the native enzyme and of the carboxymethyl-diisopropylphosphoryl enzyme and by further fragmentation of selected cyanogen bromide peptides after reduction and alkylation, by trypsin, A. mellea protease and S.aureus V8 protease. Together with the peptides presented in the following paper (26) 416 of approximately 430 amino acid residues can be placed in their sequential order.

THE AMINO ACID SEQUENCE OF PROTEINASE A INHIBITOR 3 FROM BAKER'S YEAST

The amino acid sequence of yeast proteinase A inhibitor 3 (IA3) was deter mined to be Ac-Met-Asn-Thr-Asp-Gln-Gln-Lys-Val-Ser-Glu-Ile-Phe-Gln-Ser-Ser-Lys-Glu-Lys-Leu-Gln-Gly-Asp-Ala-Lys-Val-Val-Ser-Asp-Ala-Phe-Lys-Lys-Met-Ala-Ser-Gln-Asp-Lys-Asp-Gly-Lys-Thr-Thr-Asp-Ala-Asp-Glu-Ser-Glu-Lys-His-Asn-Tyr-Gln-Glu-Gln-Tyr-Asn-Lys-Leu-Lys-Gly-Ala-Gly-His-Lys-Lys-Glu.This structure was established by automated Edman degradation of peptides derived from cyanogen bromide fragmentation and enzymatic digestion with S. aureus V8 protease and chymotrypsin. High pressure liquid chromatography was used to separate the peptides from the different cleavage reactions. The cyanogen fragment residues 2–33 contained the inhibitory activity.

The amino terminal sequence of superoxide dismutase from Saccharomyces cerevisiae

The amino-terminal amino acid sequence of fifty-four residues was determined for the Cu,Zn-superoxide dismutase isolated from Saccharomyces cerevisiae. The sequence shows a high degree of homology to that of the Cu,Zn-enzyme isolated from bovine erythrocytes, but the enzyme appears to be distinctly different from the family of Mn or Fe superoxide dismutases.