Kozo Narita

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.

Amino acid sequence of α-bungarotoxin from the venom of bungarus multicinctus

Summary The primary structure of α-bungarotoxin isolated from the venom of Bungarus multicinctus was determined. It composes of 74 amino acid residues including ten half-cystines. Comparing the sequence with those of cobras and sea snake species, striking similarities can be found, especially between α-bungarotoxin and Naja nivea α-toxin, indicating 50 % sequence homology.

The N-terminal residue of bovine rhodopsin is acetylmethionine☆

By affinity chromatography on a concanavalin A-Sepharose column and gel filtration on a Sephadex G-25 column, a glycopeptide of 16 amino acid residues has been separated from a tryptic digest of reduced and carboxymethylated bovine rhodopsin. The glycopeptide was treated with cyanogen bromide and products were subjected to high-voltage paper electrophoresis. N-Blocked homoserine separated was reacted first with anhydrous hydrazine and then with dansyl chloride. The product was identified by thin-layer chromatography to be 1-acetyl-2-dansyl hydrazine, thus showing that the N-terminal blocking group was an acetyl group. The remaining peptide after cyanogen bromide treatment was partially sequenced by the Edman dansylation method. The present results and previously reported findings on the binding site of the sugar moiety, taken together, indicate that the N-terminal heptapeptide has the following structure: acetylMet-Asn(carbohydrate)-Gly-Thr-Glx-Gly-Pro.

[46] Acylamino acid-releasing enzyme from rat liver

Publisher Summary This chapter discusses an enzyme that releases acylamino acids from acylated amino-terminal positions of a number of peptides and proteins. Hydrolysis rates depend on nature of acyl groups, terminal amino acid sequences, and tertiary structure of acyl protein substrates. The enzyme may be useful for the removal of the N-terminal acylamino acid, from some of the N-terminally blocked peptides and proteins in amino acid sequence analysis. A similar enzyme has been found also in rabbit reticulocytes and acylamino acid-releasing activity exists in many animal tissues. This chapter discusses the enzyme isolated from rat liver. It elaborates on the enzyme activity is assayed by the ninhydrin method using a synthetic substrate, N-acetyl-L-methionyl-L-threonine (Ac-L-Met-L-Thr). The chapter explains the fractionation of the enzyme, its properties such as purity, stability, pH optimum and Km value, Isoelectric point, molecular weight and subunit structure, and activators and inhibitors. The use of Acylamino Acid-Releasing Enzyme for the Sequence Determination of an N-Acetylated Peptide has been discussed.

Proton magnetic resonance studies of ribonuclease T1. Assignment of histidine-40 peak and analysis of the active site

Abstract Nuclear magnetic resonances of the C-2 protons of the three histidine residues in ribonuclease T 1 have been studied at 360 MHz as a function of pH to discuss the structure of the active site. Comparison of the order of deuterium exchange of the histidine peaks with tritium incorporation rates into individual histidines of the enzyme leads to the unambigous assignment of one of the C-2 proton peaks to histidne-40. It has been concluded that histidine-40 is in the active site, interacting with a charged group of pK 4.1, which is replaced by the phosphate group of guanosine-3′-monophosphate in the enzyme-inhibitor complex. Histidine-92 is most likely a binding site for the complex, where the existence of a hydrogen bond between N-7 of the inhibitor and the ring NH proton of the histidine is suggested on the basis of NMR data.

Improved Tritium-Labeling for Quantitative C-Terminal Analysis

The carboxyl-terminal amino acid residues of proteins are selectively tritiated through racemization via the oxazolone intermediate by the action of acetic anhydride in a medium containing 3H2O and pyridine. The characterization of the tritium- labeled C-terminal amino acid, after acid hydrolysis of the tritiated protein, can afford a method for C-terminal analysis [824, 825]. This method has successfully been applied to the qualitative C-terminal determination of proteins [828, 901]. Accumulating data reveals that there still remain several problems to be improved involving reaction conditions or characterization of the tritiated amino acids. The mechanistic features of this reaction are examined below under the improved reaction conditions together with the scope and limitations of this method. Other improvements in the characterization procedure of the radioactive C-terminal amino acid have recently been proposed.

Crystallization of ribonuclease St.

Abstract The crystals of ribonuclease St, the extracellular ribonuclease from Streptomyces erythreus , have been obtained from (NH 4 ) 2 SO 4 solution with acetate buffer (pH 4.1). The crystals belong to a monoclinic space group C 2 with dimensions a = 88.4 A , b = 33.0 A , c = 69.0 A , β = 98.4 ° . There are two protein molecules per asymmetric unit. The crystals diffract beyond 2.0 A resolution.

[36] Identification of the N-terminal residue of rhodopsin

Publisher Summary This chapter discusses the identification of the N -terminal residue of rhodopsin. The N -terminal group is methionine with the substitute amino group. After reduction and carboxymethylation of the sulfhydryl groups, modified opsin is digested by trypsin and the sugar-containing N -terminal peptide is isolated by affinity chromatography on concanavalin A. Treatment of the peptide or carboxymethylated rhodopsin with cyanogen bromide gives rise to N -blocked homoserine. The blocking group is identified as an acetyl group by hydrazinolysis of the compound. Reduced carboxymethylated opsin is incubated overnight at 37° with 1.0 mg tosylphenylalanine chloromethyl ketone-treated trypsin and centrifuged at 1,100 g for .20 min. In this system methionine-1 is shown to be the initiator methionine residue. Therefore, the N -acetylation of opsin must occur as a posttranscriptional process. Jornval has suggested that acetylation may stabilize the amino terminal region, which might otherwise be susceptible to proteolysis. Opsin is synthesized on membranes of the rough endoplasmic reticulum in the inner segment of the rod cell and is then transported via the Golgi apparatus to the site of disk membrane assembly in the outer segment.