Hiroko Toda

The amino acid sequence of the Tetrahymena calmodulin which specifically interacts with guanylate cyclase

Abstract The amino acid sequence of the Tetrahymena calmodulin was determined. The protein is composed of 147 amino acids and the amino-terminal is acetylated. Compared to bovine brain calmodulin, there were eleven substitutions and one deletion of amino acid residues. The substitutions and deletion were concentrated in the carboxyl-terminal half of the molecule. Among the substitutions, those at positions 86 (Arg → Ile), 135 (Gln → His) and 143 (Gln → Arg) may introduce the functional difference. The deletion occurred near the carboxyl-terminal, this region being assumed to be exposed to the surface area ( R.H. Kretsinger and C.D. Barry (1975) ). The change in the sequence at this terminal region may be attributable to the specific activation of guanylate cyclase.

Characterization of structural unit of phospholamban by amino acid sequencing and electrophoretic analysis

The partial amino acid sequence of phospholamban from canine cardiac sarcoplasmic reticulum was determined by sequence analysis of the peptides obtained from the protein cleaved by cyanogen bromide and with TPCK-trypsin. The sequence determined initiated with N alpha-acetylated methionine followed by 44 amino acid residues intervening two unidentified residues. This polypeptide would represent a structural unit (protomer) of phospholamban. Analysis of temperature-dependent conversion of phospholamban from 26 kDa to lower molecular weight form (6 kDa) suggested that phospholamban holoprotein is composed of five identical protomers.

8 Bacterial and Mold Amylases

Publisher Summary Bacterial and mold amylases have been the objects of organic chemical and physicochemical studies as they are available in large quantities and can be easily purified and crystallized. A large number of bacterial and mold amylases have been isolated in crystalline form from various sources. Bacillus subtilis α -amylase was the first to be crystallized, which was later followed by B. subtilis , Aspergillus oryzae , B. coagulans , A. candidus , Pseudomonas saccharophila , B. polymyxa , B. macerans , A. niger , B. amyloliquefaciens , and B. stearothermophilus . Crystallization of amylases requires the presence of divalent cations, especially calcium ions. When crystallization is repeated, it becomes increasingly difficult as loss of the cations results in apparent high solubility of the enzymes. Most of the crystalline amylases have been shown to be homogeneous in sedimentation and electrophoretic analysis. It was found, however, that even after repeated crystallization, crystalline Taka-amylase A is still contaminated by traces of proteases that can only be removed by chromatography. Polyacrylamide gel electrophoresis and analytical ion exchange chromatography are used to examine the homogeneity of an amylase preparation. It has been shown by polyacrylamide gel electrophoresis that crystalline amylases from B. macerans , B. amyloliquefaciens , B. stearothermophilus , and A. oryzae are homogeneous.

Isolation and characterization of extracellular myeloperoxidase precursor in HL-60 cell cultures☆

An extracellular myeloperoxidase precursor of HL-60 cells was purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose chromatography, and monoclonal antibody affinity chromatography. The purified protein was a glycoprotein of approximately 89 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino-terminal amino acid sequence of the protein began at amino acid residue 49 of the 745-amino acid sequence deduced from a myeloperoxidase cDNA, suggesting that the protein consisted of 697 amino acid residues. The implications of the precursor in the processing of myeloperoxidase are discussed.

Isolation and characterization of Taka-amylase a apoprotein deglucosylated by digestion with almond glycopeptidase immobilized on sepharose

Taka-amylase A (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1), which contains a single asparagine-linked oligosaccharide unit, was digested with almond glycopeptidase immobilized on Sepharose 6B at 20 degrees C for 4 h. A maximum of 10% of the parent protein was isolated as apoprotein by column chromatography on Con-A Sepharose. The characteristics of the apoprotein were compared to those of the native Taka-amylase A. The removal of the sugar chain from Taka-amylase. A caused no change in the pH-activity profile or in kinetic parameters of the hydrolysis of soluble starch. The stability of the apoprotein toward changing pH and digestion by proteases did not show any appreciable difference from that of the native Taka-amylase. These results suggest that the carbohydrate moiety of Taka-amylase A is not an essential participant in the catalysis.

Tryptophan 65 is essential for hemolytic activity of the thermostable direct hemolysin from Vibrio parahaemolyticus

The effect of modification of aromatic and ionizable amino acid residues on the hemolytic activity of a thermostable direct hemolysin from Vibrio parahaemolyticus was examined. Tryptophan 65, one of the two tryptophan residues per subunit, was specifically modified with N-bromosuccinimide, resulting in complete loss of hemolytic activity. However, neither nitration with tetranitromethane of one of the nine tyrosine residues nor Nlm-ethoxyformylation of two of the four histidine residues caused any change in hemolytic activity. The hemolysin was fully active upon amidation of two reactive carboxyl group. On the other hand, acetylation of amino groups and the modification of one of the three arginine residues with 1,2-cyclohexanedione resulted in a partial loss of the hemolytic activity. The results suggest that Trp65 is essential for the hemolytic activity of V. parahaemolyticus hemolysin.