Yuhei Morita

Structure of rice ferricytochrome c at 2·0 Å resolution

The crystal structure of ferricytochrome c from rice embryos has been solved by X-ray diffraction to a resolution of 2.0 A, applying a single isomorphous replacement method with anomalous scattering effects. The initial molecular model was built on a graphics display system and was refined by the Hendrickson and Konnert method. The R factor was reduced to 0.25. Rice cytochrome c consists of III amino acid residues. In comparison with animal cytochromes c, the peptide chain extends for eight residues at the N-terminal end, which is characteristic for plant cytochromes c. These additional residues display a collagen-like conformation and an irregular reverse turn, and are located around the C-terminal alpha-helix on the surface or the rear side of the molecule. Two hydrogen bonds between the carbonyl oxygen of the N-terminal acetyl group and O eta of Tyr65, and between the peptide carbonyl oxygen of Pro-1 and O epsilon 1 of Gln89, are involved in holding these eight residues on the molecular surface, where Tyr65 and Gln89 are invariant in plant cytochromes c. Except for the extra eight residues, the main-chain conformations of both rice and tuna cytochromes c are essentially identical, though small local conformational differences are found at residues 24, 25, 56 and 57.

Purification, stabilization and characterization of nitrite reductase from barley roots

SummaryNitrite reductase (NiR) isolated from barley (Hordeum vulgare L.) roots was stabilized in a buffer solution containing a sulfhydryl-reducing reagent and glycerol. The enzyme was purified 340fold by ammonium sulfate fractionation and chromatography on DEAE-Sephadex A-50, Sephadex G-200 and DEAE-cellulose. Purified NiR had a specific activity of 28 μmol NO2- reduced min-1 mg-1 of protein. The purified preparation was reddishbrown having absorption maxima at 282, 388 and 577 nm. The barley-root enzyme was almost identical with spinach-leaf NiR with respect to molecular weight, isoelectric point, pH stability, pH optimum, affinity for substrate, behavior toward inhibitors. It is concluded that NiR is the same enzymatic entity regardless of its localization in photosynthetic or nonchlorophyllous tissues. The electron-transport system for NiR in root tissue is discussed in comparison with that in leaf tissue.

Ferredoxin-sepharose affinity chromatography for the purification of assimilatory nitrite reductase

Assimilatory nitrate reduction in higher plants and algae proceeds in two steps: the first stage is catalyzed by nitrate reductase (NAD(P)H-nitrate oxidoreductase, EC 1.6.6.2.) and yields nitrate, and the subsequent step is catalyzed by nitrite reductase (ferredoxin-nitrite oxidoreductase, EC 1.6.6.4) to yield ammonia with the use of six electrons [1,2]. Recently nitrate reductase has been obtained in a highly purified state by the use of affinity chromatography in which FAD [3], Blue dextran [4], NADH [5], and p-nitroaniline, respectively [6] were used as specific or general ligands coupled to Sepharose gels. To date no attempts have been made to purify nitrite reductase (NiR) by affinity chromatography, although NiR has been prepared in a highly purified form by conventional purification procedures such as ion exchange chromatography, gel filtration and preparative gel electrophoresis [7], the yields of the enzyme were, however, rather low and the procedures were time-consuming. In this communication, we describe a simple and efficient method for the purification of NiR from a higher plant by application of ferredoxin-Sepharose affinity chromatography and some properties of the purified preparation.

Elucidation of the role of sugar chains in glucoamylase using endo-β-N-acetylglucosaminidase from Flavobacterium sp.

Abstract Endo-β-N-acetylglucosaminidase (glycopeptide- d -mannosyl-N4-(N-acetyl- d -glucosaminyl)2-asparagine 1,4-N-acetyl-β-glucosaminohydrolase, EC 3.2.1.96), homogenized from the culture filtrate of Flavobacterium sp., could liberate about 50% of the sugar chains from the glucoamylase of Rhizopus niveus. The native and carbohydrate-depleted glucoamylases were compared in their various enzymatic properties. It was found that they were identical in their catalytic activities. However, the carbohydrate-depleted glucoamylase was less thermally stable than the native glucoamylase. Moreover, the carbohydrate-depleted glucoamylase was more sensitive to proteinases such as pronase, subtilisin and trypsin. These results suggest that the sugar chains of the glucoamylase contribute to the high stability of the enzyme. However, circular dichroism spectra of the native and carbohydrate-depleted glucoamylase were found to be identical.

Resonance Raman study of plant tissue peroxidases Common characteristics in iron coordination environments

Abstract Resonance Raman spectra of isozymes 2, 3, 9, 15, and 16 of Japanese radish peroxidase and isozymes 1, 3, and 7 of turnip peroxidase were measured and compared with those of horseradish peroxidase. All the resting isozymes gave the ν 10 band around 1629–1631 cm −1 , indicating the five-coordinate high-spin structure. The reduced form of all these isozymes provided the iron-histidine stretching Raman line at distinctly higher frequencies in comparison with those of hemoglobin and exhibited a clear pH-dependent frequency shift at neutral pH in accord with the results for horseradish peroxidase. Therefore, we conclude that strong hydrogen bonding of the proximal histidine and a small structural change of the proximal histidine at neutral pH without breakage of the hydrogen bond are the common characteristics of plant tissue peroxidases (EC 1.11.1.7) which contrast with those of oxygen-carrier hemoproteins.

Purification and complete amino acid sequence of a major prolamin of rice endosperm

The complete amino acid sequence of a major prolamin from rice endosperm was determined by protein sequencing. The prolamin fraction showed five bands on isoelectric focusing, having pIs of 5·6, 7·1, 7·3, 7·6 and 8·0. The pI 7·1 component was the major prolamin, it was purified by ion exchange chromatography and RP-HPLC. The N-terminal amino acid residue of this protein was not detected by Edman degradation analysis. The blocked N-terminal peptide was isolated from the acidic fraction of a peptic digest of the protein. The N-terminal amino acid was identified as pyroglutamic acid after digestion of the peptide with carboxypeptidase A. The complete primary structure of the prolamin was established by sequencing its tryptic (T1–T7) and peptic (P5–P35) peptides. The N-terminal sequence of the protein was found to be pGx-Phe-Asp-Val-Leu-Gly-Gln-Ser-Tyr-Arg after the blocked T2 peptide was unblocked with pyroglutamate amino peptidase. The protein consists of one polypeptide of 131 residues, and has a molecular weight of 14930. The protein was rich in glutamine (22%), leucine (13%) and alanine residues (7%). Lysine, cysteine and methionine residues were not detected, however.

Mössbauer effect in peroxidase

Mossbauer spectroscopy provides valuable information on the electronic configuration of heme-iron and the surrounding ligand field strength in heme-proteins complementary to that derived from magnetic susceptometry and electron spin resonance (ESR) spectroscopy. Quite recently, the method has been applied on some biological materials containing iron atom, and some investigations have been reported on heme-proteins such as hemoglobin, myoglobin, catalase and cytochrome c Gonser et al. 1964; Bearden et al. 1965; Lang and Marshall 1966; Caughey et al. 1966, but not yet on peroxidase. This communication presents an outline of the experimental results on Mossbauer effect in Japanese-radish peroxidase a (JRP-a) and some of its derivatives enriched artificially with 57Fe in order to obtain any information complementary to that derived from the ESR study of this enzyme Morita and Mason 1965.

A preliminary crystallographic investigation of rice cytochrome c.

Abstract Cytochrome c of rice embryos was crystallized from a concentrated solution of ammonium sulphate. The crystals belong to the hexagonal space group P61 or P65 with unit cell dimensions a = 43·8 A and c = 110·0 A. Considerations of cell volume and protein molecular weight indicate one molecule of cytochrome c in the asymmetric unit.

Studies on Amylases of Aspergillus oryzae Cultured on Rice Part I:Isolation and Purification of Glucoamylase

Four fractions of glucoamylase, different chromatographically from one another, were isolated and purified from a culture of Aspergillus oryzae on steamed rice. All these fractions were found to be homogeneous in sedimentation analysis, and had almost the same enzymatic activities in the hydrolysis of soluble starch. The limit of hydrolysis of starch was about 60per thousand, which was very similar to those of Taka-amylase B of Okazaki and Taka-amylase B 2 of Sawasaki.

Preliminary crystallographic study of ω-amino acid: Pyruvate aminotransferase from Pseudomonas sp. F-126

Abstract Large single crystals of ω-amino acid: pyruvate aminotransferase, were prepared by dialysis of the enzyme solution against 2.2 m -ammonium sulphate solution at pH 7.8. X-ray diffraction patterns show that the crystals belong to the orthorhombic space group I222 or I212121 with unit cell dimensions a = 124.1 A , b = 137.9 A , and c = 61.2 A . The asymmetric unit consists of one monomer of molecular weight 43,000.