Ivan Kluh

Amino acid sequence of hog pancreatic α-amylase isoenzyme I

Crystalline cll-amylase (EC 3.2.1 .l) from hog pancreas contains two a-amylase enzyme forms designated isoenzyme I and isoenzyme II with respect to their different electrophoretic mobilities [ 1,2]. We have characterized both isoenzymes by determination of relative molecular mass (M,) and amino acid composition; the C-terminal amino acid [3] and N-terminal amino acid sequence PCA-Tyr @‘CA, pyrrolid-2-oneScarboxylic acid) are identical in both isoenzymes. The MI and amino acid composition are likewise identical within the range of experimental error. So far partial sequential data have been published on isoenzyme II obtained by the analysis of its tryptic digest [ 51 and partial or complete data on certain cyanogen bromide fragments of isoenzyme I [6]. Here the primary structure of isoenzyme I comprising 496 amino acid residues is reported. The primary structure of isoenzyme I of pancreatic a-amylase can be compared with the structure of cu-amylase precursors from mouse pancreas, salivary glands and liver. The structures of these precursors have been derived from the nucleotide sequences of the corresponding mRNAs [7,8]. The comparison together with the data on the N-terminal sequence of cw-mylase from mouse pancreas and salivary glands [9] point to the possibility of identical post-translational cleavage of aamylase precursors in 2 animal species. excess of CNBr and separated by gel permeation chromatography on Sephadex G-100 in 0.4% formic acid. The individual fragments were isolated by rechromatography under identical conditions or by ion-exchange chromatography on SE-Sephadex G-25 in 8 M urea. The amino acid sequence of the N-terminal parts of the fragments isolated was determined by automatic sequential analysis based on the phenylisothiocyanate method in a Beckman model 890 C amino acid sequenator. The sequences thus obtained were checked and completed by sequential analysis of peptides from tryptic and chymotryptic digests of the corresponding fragments. These analyses were effected by manual degradation using phenylisothiocyanate method [ 111 which was replaced by the 4-NJdimethylaminoazobenzene4’-isothiocyanate/phenylisothiocyanate double coupling method [ 121 during later stages of the work.

Primary structure of N-terminal part of molecule of dolphin myoglobin

The partial primary structure of Black Sea dolphin (Dolphinus delphis) myoglobin has been determined by KaradZova and coworkers [l] by classical methods. The sequences of amino acids at positions 17-3 1, 60, and 84-85 have not been established. The difficulties encountered in the sequential investigation of the N-terminal part of the molecule of myoglobins can be ascribed to the low solubility of tryptic fragments arising from this part of the molecule. Another approach to the solution of this problem the stepwise degradation of amino acid residues from the native molecule of the globin by the phenylisothiocyanate technique has been proposed by Edman [2]. The automation of the procedure in the amino acid sequenator improved the yield to 98% and thus enabled the amino acid sequence of the 60-residue N-terminal fragment of the molecule of humpback whale myoglobin to be determined. This paper has followed the same line of approach to fill the existing gap in the amino acid sequence at position 17-3 1 in the molecule of Black Sea dolphin myoglobin and at the same time served as a practical test of the amino acid sequenator built in this laboratory according to Edman’s model [2]. 2. Material

Amino acid sequence of the N-terminal region of human hemopexin

Cyanogen bromide digestion of hemopexin at its 6 methionine residues results in 7 fragments (CB1–CB7) partially connected by disulfide bridges. By sequence studies of fragments CB1‐CB4 and peptides prepared by their enzyme cleavage, a continuous amino acid sequence of the N‐terminal region of human hemopexin, comprising 220 amino acid residues, was determined. The presence of intramolecular disulfide bonds, connecting half‐cystine residues and , was proved in fragments CB2 and CB3. Fragments CB1–CB4 include 5 sites, where hexosamine oligosaccharides are attached (positions 1,41,164, 217 and probably 223). In the sequenced region two sites sensitive to acid hydrolysis ‐ bonds ⋯ Asp‐Pro ⋯ in positions and were found. In spite of the fact that pooled material of many donors was studied, no sequence heterogeneity was discovered.