C.Y. Lai

Studies on the structure of rabbit muscle aldolase: I. Cleavage with cyanogen bromide: an approach to the determination of the total primary structure

Abstract S -Carboxymethylated aldolase was treated with CNBr to cleave the methionyl bonds and the products were separated by chromatography on Sephadex G-75. Five fractions were obtained, designated CnI to CnV according to their order of elution. All fractions except CnI were homogeneous in both N -terminal and C -terminal analyses. CnI was present only in 10% yield and appeared to be an aggregate of peptides. CnII had the N -terminal sequence Pro-His-, and was derived from the N -terminus of aldolase. CnIII, containing C -terminal tyrosine, was the only fragment devoid of homoserine, and was derived from the C -terminus of the enzyme. CnIV contained the Schiff base-forming lysine residue. CnV, with N -terminal sequence Val-Thr-Pro-, represented the overlapping peptide at the COOH-terminal side of this lysine (9). The sequence of the four fragments in the aldolase molecule has thus been deduced as: (NH 2 ) CnII-CnIV-CnV-CnIII (OH). Since aldolase contains 12 methionine residues, the present results confirm the 4-subunit structure of the enzyme. Fraction CnIII has been shown to contain 2 peptides derived from the C -termini of the α and β chains, and may represent the only region in which the 2 chains differ. The procedure has provided a means to locate in the molecule the functional groups involved in the manifestation of aldolase activity. The significance of this approach to the determination of the total primary structure of the enzyme is discussed.

Primary structure of two COOH-terminal hexapeptides from rabbit muscle aldolase: A difference in the structure of the α and β subunits

Abstract Two COOH-terminal hexapeptides isolated from rabbit muscle aldolase have been shown to have the following primary structures: Ile-Ser- Asn -His-Ala-Tyr (α chain) and Ile-Ser- Asp -His-Ala-Tyr (β chain). This is the first structural difference identified for the two types of chains found in the crystalline aldolase preparations.

Mechanism of action of aldolases: XII. Primary structure around the substrate binding site of rabbit muscle aldolase

Abstract A tryptic peptide of 28 amino acid residues containing the substrate combining site was isolated from rabbit muscle FDP-aldolase. The complete amino acid sequence of the active site peptide was established as: Ala-Leu-Ser-AspN-His-His-Ile-Tyr-Leu-GluN-Gly-Thr-Leu-Leu-βGLys-AspN-Pro-Met-Val-Thr-Pro-Gly-His-Ala-Cys-Thr-GluN-Lys. The significance of this primary structure in relation to the mechanism of action of aldolase is discussed.

Subunit structure of rabbit muscle aldolase: Extent of homology of the α and β subunits and age-dependent changes in their ratio☆

Abstract The α and β subunits of rabbit muscle aldolase, dissociated in 8 m urea and separated by chromatography on DEAE-cellulose, were cleaved with cyanogen bromide. The corresponding cyanogen bromide peptides from the two subunits were found to be very similar in amino acid composition and in tryptic fingerprints, possibly differing in only a single peptide. In aldolase prepared from skeletal muscle of young rabbits the α subunit predominates, in contrast to preparations from adult rabbits which contain nearly equal quantities of the α and β subunits. The change in ratio occurs between 3 and 5 months of age. These results suggest that the two subunits have a common genetic origin, and that the β subunit may arise by a modification of one or more amino acids near the COOH-terminus of the molecule.

Organic phosphate groups in native and borohydride-reduced aldolase

Abstract The number of Schiff base-forming sites in rabbit muscle aldolase has been determined by analysis of organic phosphate incorporated into the molecule on reduction of the enzyme-substrate complex with sodium borohydride. On the basis of these results there is a minimum of three Schiff base-forming sites per mole of enzyme. Native aldolase was found to contain 0.5–1.0 equivalents of endogenous organic phosphate. The quantity of covalently linked phosphate could be increased by precipitating the enzyme with acid from solutions containing dihydroxyacetone phosphate. Hydrolysis of the 14 C-labeled product, obtained by precipitation of the enzyme-dihydroxyacetone phosphate mixtures with acid, yields neither dihydroxyacetone nor N 6 -β-glyceryllysine, but instead, an unidentified radioactive amino acid derivative. The properties of the bound phosphate and the requirements for its incorporation suggest that it is derived from the Schiff base intermediate, perhaps by transfer of the activated intermediate to another group in the protein.

Studies on the structure of rabbit muscle aldolase: III. Primary structure of the BrCN peptide containing the active site

Abstract The four peptide segments obtained from rabbit muscle aldolase by cleavage with BrCN and separation with gel-filtration chromatography (1) have been redesignated according to their positions in the molecule, N-A-B-C. The primary structure of segment A, containing 66 amino acid residues, including the Schiff base-forming lysine at the active site, has been elucidated by isolation and sequence analyses of the proteolytic subfragments. Preliminary separation of tryptic peptides containing 7–25 residues was achieved by chromatography on Sephadex G-25 which facilitated subsequent purification. For the study of the tryptic peptide of 25 residues further fragmentation with pepsin then subtilisin (Nagarse) was employed. Edman degradation directly after subtilisin cleavage of a peptide was found useful in avoiding deamidation of a glutamine NH 2 -terminus newly formed in the proteolysis. The sequence of 90 amino acids in the center region of the polypeptide chain of rabbit muscle aldolase has now been established.

Codfish muscle aldolase: Purification, properties, and primary structure around the substrate-binding site☆

Abstract FDP aldolase has been purified and crystallized from the muscle of codfish. The specific activity of the codfish enzyme was found to be two-thirds that of rabbit muscle aldolase. Other catalytic properties, the molecular weight, and the carboxyl-terminal structure were similar to those of rabbit muscle aldolase. A tryptic peptide containing the Schiff base-forming lysine (1) has been isolated and its primary structure determined as: Ala-Leu-Ser-Asp-His-His-Val-Tyr-Leu-Gln-Gly-Thr-Leu-Leu-βGlys-Pro-Asn-Met-Val-Thr-Ala-Gly-His-Ser-CMCys-Ser-His-Lys. Extensive homology observed in this region of the aldolase molecules isolated from biologically divergent sources indicates the importance of this structure for catalysis of the aldolase reaction.

Frog muscle aldolase: Purification of the enzyme and structure of the active site☆

Abstract Fructose 1,6-diphosphate aldolase has been crystallized from frog muscle. The preparations are homogeneous in ultracentrifugation and disc gel electrophoresis and dissociate in urea or sodium dodecyl sulfate to yield only one type of subunit. The molecular weight of frog muscle aldolase was estimated as 164,000 and the molecular weight of the subunit as 41,000. A comparison of the amino acid compositions of two peptides derived from the active site indicates a high degree of homology with the corresponding peptides from fish aldolases. However, unlike the aldolases from fish muscle, aldolase from frog muscle contains only a single methionine residue per subunit; this methionine residue is present in the active site peptide and appears to occupy the same position in all of the active site peptides thus far examined.

Studies on the structure of rabbit muscle aldolase: II. Primary structure of peptide CnV and chemical evidence for the four-subunit structure of aldolase☆

Abstract The primary structure of fragment CnV, the smallest of peptides obtained by CNBr cleavage of aldolase, has been determined and the known sequence of amino acids around the substrate binding site extended to include a total of 41 residues. CnV, an 18 amino acid peptide with homoserine at the C -terminus, has been shown to exist in 2 electrophoretically distinct forms, one having homoserine γ-lactone at the C -terminus. Four moles of CnV were obtained from one mole of aldolase by quantitative analysis, confirming the 4-subunit structure of the enzyme.

Lobster muscle aldolase: Isolation, properties and primary structure at the substrate-binding site☆

Abstract Fructose 1,6- P 2 aldolase has been isolated from lobster muscle. The crystalline enzyme is shown to be homogeneous by ultracentrifugation, polyacrylamide gel electrophoresis and gel filtration. The enzyme has a molecular weight of 160,000 based on sedimentation equilibrium, sedimentation velocity and gel filtration. The amino acid composition of the lobster muscle aldolase is similar to that of rabbit muscle aldolase, except for the content of half-cystine, methionine and aromatic amino acids. Tyrosine is found to be the COOH-terminal amino acid by carboxypeptidase A digestion and hydrazinolysis. The release of 4 moles of tyrosine per mole of enzyme by carboxypeptidase A digestion suggests a tetrameric structure of lobster muscle aldolase similar to that of other mammalian aldolases. The kinetic parameters are similar to those described for rabbit muscle aldolase, except that lobster muscle aldolase has a much lower rate of cleavage of fructose 1- P as compared with fructose 1,6- P 2 . The amino acid sequence of the active site peptide isolated from tryptic digests has been determined. It shows extensive homology with similar peptides isolated from vertebrate muscle and liver. A region containing 12 amino acids immediately adjacent to the lysine residue which forms the Schiff base intermediate is identical in all aldolases thus far examined.