Masachika Irie

Crystal and molecular structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus

The crystal structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus, has been determined at 2.5 Å resolution by the multiple isomorphous replacement method. The crystal structure was refined by simulated annealing with molecular dynamics. The current crystallographic R‐factor is 0.200 in the 10—2.5 Å resolution range. The molecular structure which is completely different from the known structures of RNase A and RNase T1 consists of six α‐helices and seven β‐strands, belonging to the α+β type structure. Two histidine and one glutamic acid residues which were predicted as the most probably functional residues by chemical modification studies are found to be clustered. The steric nature of the active site taken together with the relevant site‐directed mutagenesis experiments (Irie et al.) indicates that: (i) the two histidine residues are the general acid and base; and (ii) an aspartic acid residue plays a role of recognizing adenine moiety of the substrate.

Induction of metallothionein in a human astrocytoma cell line by interleukin-1 and heavy metals

The effects of cytokines and heavy metals on the expression and localization of metallothioneins (MTs) within U373MG astrocytoma cells were analyzed by using indirect immunofluorescence using a monoclonal anti‐MT antibody (MT45). IL‐1, CdCl2 (50μM) or ZnCI2, (500μM) remarkably augmented intracellular MT levels, whereas IL‐6 or 10 μM of ZnCl2 showed no inducing activity. From 24 to 48 h after the addition of CdCl2 or IL‐1, immunoreactive MTs were found in the cytoplasm and the nucleus. After 72 h, immunoreactive MTs accumulated in a granular form near the cell surfaces in the presence of CdCl2 (50 μM) or IL‐1 plus ZnCI2 (10 μM). However, this accumulation was not observed when only IL‐1 was added. Thus, Zn2+ facilitated the appearance of the granular form of immunoreactive MTs at a concentration where they do not induce MTs by themselves.

Isolation and characterization of a major allergenic component (gp55) of Aspergillus fumigatus

IgE class antibodies specific for antigens in a water-soluble extract of Aspergillus fumigatus (strain NHL-5759) were analyzed by immunoblotting with sera from patients with allergic bronchopulmonary aspergillosis. All the sera tested were reactive with a major 50 to 60 kd protein in the extract. This allergen, designated gp55, was purified by gel filtration and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigen was found to be present in the water-soluble extract in the form of a complex composed of approximately eight molecules of gp55. The carbohydrate and phosphate content of the purified antigen were 23.1% and 0.46%, respectively. The molar ratio of mannose to galactose residues was 2.76:1, and the protein was glycosylated predominantly with N-linked oligosaccharides. The serologic activity of the gp55 antigen was abolished by treatment with nonspecific protease (Pronase) but not by treatment with sodium metaperiodate or endoglycosidases. Thus the major antigenic site of the glycoprotein is located within its peptide moiety. The antigen itself displayed no chymotryptic or tryptic activity. The amino acid sequence of the 20 N-terminal residues of the antigen (ATPHEPVFFSWDAGAVTSFP) is different from that of any other protein previously reported.

Three-dimensional structure of ribonuclease Ms*3'-guanylic acid complex at 2.5 A resolution.

The crystal structure of ribonuclease Ms*3+‐guanylic acid complex has been determined by molecular replacement methods based on the known structure of ribonuclease T1. The pattern of hydrogen‐bonds between the enzyme and the guanine base is similar to that discovered by Arni et al. [(1988) J. Biol. Chem. 263, 15358–15368] in the crystal structure of ribonuclease T1*2′‐guanylic acid complex. As for the possible general base in the trans‐phosphorylation step of the catalysis, 0 c 1 of Glu17 is within the hydrogen‐bond distance (2.7 Å) of the 2′‐0 of the nucleotide while N c 2 of His14 is significantly more distant (3.4 Å) from the 2′‐0.

A monoclonal antibody to scopolamine and its use for competitive enzyme-linked immunosorbent assay.

A hybridoma clone producing a monoclonal antibody (SC78.H81) against scopolamine was established. The monoclonal antibody was an IgG1 (k) antibody with high affinity (1.6 x 10(9) M-1 for methylscopolamine). The monoclonal antibody was cross-reactive with methylscopolamine and butylscopolamine, and showed weak cross-reactivity with 6 beta- and 7 beta-hydroxyhyoscyamine. The cross-reaction with L-hyoscyamine, atropine, scopine and DL-tropic acid was very weak. A competitive enzyme-linked immunosorbent assay using SC78.H81 was established to quantify scopolamine. The sensitivity of the assay allowed detection of 20 pg assay-1 (0.2 ng ml-1) of scopolamine. The assay was applied to the estimation of scopolamine content in hairy root cultures of a Duboisia hybrid.

Amino Acid Sequence of an Unique Ribonuclease with a C-Terminus rich in O-Glycosylated Serine and Threonine from Culture Medium of Lentinus edodes

The mushroom Lentinus edodes produces three base-non-specific and acid ribonucleases, RNases Le2, Le37, and Le45. The latter two are excreted from mycelia into the medium. The primary structure of RNase Le37, which had a molecular mass of 37 kDa, was sequenced. It was a member of the RNase T2 family, as is RNase Le2. RNase Le37 was some 30 amino acid residues longer at the C-terminal end than RNase Le2. The C-terminal region of RNase Le37 was rich in O-glycosylated serine and threonine. In fungal glucoamylases and chitinases, which hydrolyze raw-starch and chitin, respectively, have structures resembling the structure of the C-terminal of RNase Le37.

Interaction of folic acid with ribonuclease A

Abstract The interaction of bovine pancreatic ribonuclease A (EC 3.1.4.22) with folic acid was studied by the Hummel and Dreyer gel-filtration method on Sephadex G-25 columns. The pH-dependence curve of the binding was bell-shaped, the optimum being near pH 5.0–5.5. The maximum number of the bound ligand was three per mol of enzyme. The first molecule of folic acid bound non-cooperatively and the binding of the second and the third obeyed the kinetics of positive cooperativity. p- Aminobenzoylglutamic acid , a constiuent of folic acid, bound to the enzyme with less affinity than the latter and with similar pH-dependence curves of binding. Studies on the effects of nucleotides on the interaction of the enzyme with folic acid suggested that the first binding site for folic acid was not identical to the binding site for pyrimidine nucleotides (B1 site) but located at or near the site for adenosine 5′-phosphate (B2 site). Both folic acid and p- aminobenzoylglutamic acid competitively inhibited the enzyme activity of RNAase A when cytidine 2′,3′-cyclic phosphate and cytidyl-3′,5′-uridine were used as substrates. The pterin moiety of folic acid was not essential for binding to RNAase A but increased the affinity for the enzyme and the modes of interaction of folic acid and p- aminobenzoylglutamic acid with the enzyme revealed to be essentially the same.

Enzymatic Properties of Phenylalanine101 Mutant Enzyme of Ribonuclease Rh from Rhizopus niveus

To investigate the role of Phe101, a component of a base recognition site (B2 site) of a base-nonspecific RNase Rh from Rhizopus niveus, we prepared several enzymes mutated at this position, F101W, F101L, F101I, F101A, F101Q, F101R, and F101K, and their enzymatic activities towards RNA, 16 dinucleoside phosphates, and 2′, 3′-cyclic pyrimidine nucleotides were measured. Enzymatic activity toward RNA of F101W, F101L, and F101I were about 7, 20, and 3.8% of the native enzyme, respectively, and those of the other mutants were less than 1% of the RNase Rh. Similar results were also obtained with GpG as substrate. Thus, it was concluded that Phe101 is a very important residue as a component of the B2 site of RNase Rh, and its role could be replaced by Leu, then Trp and Ile, though in less effectively. The results suggested that some kind of interaction between B2 base and the side chain of amino acid residue at the 101th position, such as π/π or CH/π interaction is very important for the enzymatic activity of RNase Rh. The mutation of Phe101 markedly affected the enzymatic activity toward dinucleoside phosphates and polymer substrates, but only moderately the rate of hydrolysis of cyclic nucleotides, indicating the presence of secondary effect of the mutation on B1 site.

Crystallization of a new class of microbial ribonuclease from Rhizopus niveus

Crystals of ribonuclease Rh, a new class of microbial ribonuclease from Rhizopus niveus, were obtained from polyethylene glycol 8000 solution by a vapour diffusion technique in the hanging drop mode. Two crystal forms, type I and type II, were obtained from the same droplet solution. Both forms belong to the space group P2(1)2(1)2(1), but their cell dimensions are markedly different: a = 68.3 A, b = 73.0 A, c = 50.0 A for type I and a = 67.5 A, b = 72.3 A, c = 44.2 A for type II. The type I crystals diffract beyond 2.0 A resolution and are suitable for X-ray structure analysis at high resolution.

Amino Acid Sequence of a Nuclease (Nuclease Le1) from Lentinus edodes

The fruit bodies of Lentinus edodes produce two acid nucleases, nucleases Le1 and Le3, both of which are thought to be candidates for the enzymes producing a tasty substance, 5′-GMP. To obtain the basic information on the mechanism of production of 5′-GMP, and structure-function relationship of these nucleases, the primary structure of nuclease Le1 was estimated by both protein chemistry and gene cloning. Nuclease Le1 is a glycoprotein and consists of 290 amino acid residues, and about 2 and 6 residues of hexosamine and neutral sugar, respectively. The nucleotide sequence of cDNA and genomic DNA encoding nuclease Le1 indicated the presence of 20 amino acid residues of a signal peptide. Nuclease Le1 has 115 and 108 residues of identical amino acid residues with nucleases P1 and S, respectively. The amino acid residues concerning the coordination with Zn2+ in nuclease P1 are all conserved in nuclease Le1. Nuclease Le1 contains 8 half-cystine residues and 4 of them are located at the same places as those of nucleases P1 and S.