Gunki Funatsu

The complete amino acid sequence of ribonuclease from the seeds of bitter gourd (Momordica charantia)

The complete amino acid sequence of ribonuclease (RNase MC) from the seeds of bitter gourd (Momordica charantia) has been determined. This has been achieved by the sequence analysis of peptides derived by enzymatic digestion with trypsin, lysylendopeptidase, and chymotrypsin, as well as by chemical cleavage with cyanogen bromide. The protein contains 191 amino acid residues and has a calculated molecular mass or 21 259 Da. Comparison of this sequence with sequences of the fungal RNases, RNase T2, and RNase Rh, revealed that there are highly conserved residues at positions 32–38 (TXHGLWP) and 81–92 (FWXHEWXKHGTC). Furthermore, the sequence of RNase MC was found to be homologous to those of Nicotiana alata S‐glycoproteins involved in self‐incompatibility sharing 41% identical residues.

Conserved amino acid residues in ribosome-inactivating proteins from plants

The amino acid sequences of eleven RIPs sequenced to date have been compared in the expectation that this would be useful in the location of functionally and/or structurally important sites of these molecules. In addition to several highly conserved hydrophobic amino acids, thirteen absolutely conserved residues have been found in ricin A-chain: Tyr21, Phe24, Arg29, Tyr80, Tyr123, Gly140, Ala165, Glu177, Ala178, Arg180, Glu208, Asn209 and Trp211. The role of these residues as well as of the C-terminal region have been discussed based on the results of chemical and enzymatic modifications, site-directed mutagenesis, and deletion studies.

The complete amino acid sequence of the B-chain of ricin E isolated from small-grain castor bean seeds. Ricin E is a gene recombination product of ricin D and ricinus communis agglutinin

The complete amino acid sequence of the B-chain of ricin E has been determined. The reduced and carboxymethylated B-chain was digested with trypsin, followed by separation and purification of the resulting peptides using reverse-phase HPLC. The amino acid sequence of each tryptic peptide was determined employing the DABITC/PITC double-coupling method. The B-chain of ricin E proved to consist of 262 amino acid residues. By comparing the amino acid sequence of the B-chain of ricin E with those of ricin D and of Ricinus communis agglutinin, it was found that the B-chain of ricin E was composed of the N-terminal half of ricin D and C-terminal half R. communis agglutinin. This result suggested that the gene recombination probably occurred at the center region of two B-chain genes of ricin D and R. communis agglutinin.

Isolation and Characterization of Ricin E from Castor Beans

A toxic protein, distinct from ricin D, was isolated from castor beans produced in Japan and referred to as ricin E. Ricin E was extracted from defatted meal of castor beans and purified by gel filtration through Sephadex G-75 column followed by DEAE- and CM-cellulose column chromatographies. Ricin E was found to be a glycoprotein and had two N-terminal amino acids, Ile and Ala, which were identical to those of ricin D. The molecular weight of ricin E was 64,000 by SDS-polyacrylamide gel electrophoresis and its sedimentation coefficient was 4.45 S. The isoelectric point of ricin E, estimated to be 8.8 by ampholine electrophoresis, was higher than that of ricin D. Ricin E had equal toxicity for mice and equal cytoagglutinating activity for Sarcoma 180 ascites tumor cells to those of ricin D, and this cytoagglutination was inhibited by galactose.

Chinese Hamster Cell Variants Resistant to the A Chain of Ricin Carry Altered Ribosome Function

Ricin, a toxic lectin from Ricinus communis, is composed of two different polypeptide chains, A and B, and the ricin A chain (RA) blocks protein synthesis. We studied cell lines resistant to cytotoxic action of RA. One low-RA-resistant cell line, AR10, isolated from Chinese hamster ovary (CHO) cells, was resistant to a low dose of RA (1 microgram/ml) and showed a 10-fold-higher resistance to RA and ricin than that of CHO. We further mutagenized AR10 to isolate high-RA-resistant cell lines AR100-6, AR100-9, and AR100-13, which were resistant to higher doses of RA and ricin (100- to 1,000-fold) than CHO was. The binding of [125I]ricin to AR10, AR100-6, AR100-9, and AR100-13 cells was decreased to about 30% of that of CHO. The internalization of [125I]ricin in AR10 cells and in the high-RA-resistant clones was the same. Polyuridylate-dependent polyphenylalanine synthesis, using S-30 extracts from either AR100-9 or AR100-13, was about 100-fold more resistant to the inhibitory action of RA than when CHO, AR10, and AR100-6 cells extracts were used. The protein synthesis with ribosomes (80S) from AR100-9 or AR100-13 was 10- to 100-fold more resistant to RA than it was with parental ribosomes when combined with the S-100 fraction of CHO cells. The polyphenylalanine synthesis assay using the ribosomes constituted from the 60S subunit of AR100-9 and the 40S subunit of CHO indicated that the resistant phenotype of AR100-9 cells is due to an alteration of the 60S ribosomal subunit.

Crystal structure of a ribonuclease from the seeds of bitter gourd (Momordica charantia) at 1.75 Å resolution

The ribonuclease MC1 (RNase MC1) from seeds of bitter gourd (Momordica charantia) consists of 190 amino acid residues with four disulfide bridges and belongs to the RNase T(2) family, including fungal RNases typified by RNase Rh from Rhizopus niveus and RNase T(2) from Aspergillus oryzae. The crystal structure of RNase MC1 has been determined at 1.75 A resolution with an R-factor of 19.7% using the single isomorphous replacement method. RNase MC1 structurally belongs to the (alpha+beta) class of proteins, having ten helices (six alpha-helices and four 3(10)-helices) and eight beta-strands. When the structures of RNase MC1 and RNase Rh are superposed, the close agreement between the alpha-carbon positions for the total structure is obvious: the root mean square deviations calculated only for structurally related 151 alpha-carbon atoms of RNase MC1 and RNase Rh molecules was 1.76 A. Furthermore, the conformation of the catalytic residues His-46, Glu-105, and His-109 in RNase Rh can be easily superposed with that of the possible catalytic residues His-34, Glu-84, and His-88 in RNase MC1. This observation strongly indicates that RNase MC1 from a plant origin catalyzes RNA degradation in a similar manner as fungal RNases.

The complete amino acid sequence of chitinase-c from the seeds of rye (Secale cereal).

The complete amino acid sequence of rye seed chitinase-c (RSC-c) has been analyzed. This was done by first sequencing the tryptic peptides from RCm-RSC-c and then connecting them by analyzing the peptides produced by digestions with lysylendopeptidase and Staphylococcus aureus V8 protease of RCm-RSC-c, and by chymotryptic digestion and formic acid cleavage of S. aureus V8 protease peptides. RSC-c consists of 243 amino acid residues and has a molecular mass of 26,093, and has 92% sequence homology with barley seed basic chitinase which lacks a Cys-rich domain. Cys204 is free and six cysteine residues are linked by disulfide bonds between Cys23 and Cys85, Cys97 and Cys105, and Cys223 and Cys236.

Primary structure of the 16S rRNA binding protein S15 from Escherichia coli ribosomes

Protein S15 from the 30s ribosomal subunit of E. coli is one of the 16s rRNA binding proteins. It binds at about the same region of the 16s rRNA as protein S8, namely approximately 600-750 nucleotides from the 3’-end (for a review see [ 11). Determination of the primary structure of protein S15 is necessary for a closer investigation of the molecular mechanism of the interaction between this protein and 16s rRNA. Protein S1.5 which is a very basic protein [2], consists of 87 amino acid residues and its N-terminal sequence has been determined using a liquid phase sequenator [3] f In this paper, the complete determination of the primary structure of this protein is reported.

Primary structure of protein S12 from the smallEscherichia coli ribosomal subunit

Protein S12 is a very basic protein of the E. coli 30 S ribsomal subunit [ 1,2]. It controls the fidelity of translation [3] and plays an important role in the initiation of natural messenger RNA translation [4]. Protein S12 is the product of gene strA [S] and mutation in this protein confers resistance to [6] and dependence on streptomycin [7]. The amino acid replacements in mutants with altered S12 proteins are clustered in two regions: tryptic peptide T6, or T15 [S-l 11. Some mutants resistant to neamine have two altered ribosomal proteins, S12 and S.5 [ 121, and the amino acid replacements in S12 are also located in peptide T 15 [13]. The tryptic peptides of protein S12 have been isolated and their amino acid compositions reported [8,10]. The amino acid sequence of the N-terminal region of S12 has been determined [ 14,151. Protein S12 consists of 123 amino acids and this report summarizes the determination of the amino acid sequence.

Interaction of ricin and its constituent polypeptides with dipalmitoylphosphatidylcholine vesicles

The interaction of ricin and of its constituent polypeptides, the A- and B-chain, with dipalmitoylphosphatidylcholine (DPPC) vesicles was investigated. The A- and B-chain were individually associated with DPPC vesicles, although the intact ricin was not associated. The maximum binding and association constants were evaluated to be 154 micrograms per mg of DPPC and Ka = 2.30 X 10(5) M-1 for the A-chain, and 87 micrograms per mg of DPPC and Ka = 14.5 X 10(5) M-1 for the B-chain, respectively. The A-chain could induce the phase transition release of carboxyfluorescein from DPPC vesicles to a greater extent than the B-chain, whereas the release induced by the intact ricin was negligible. The evidence indicated that the hydrophobic regions on the A-chain and on the B-chain were buried inside when the two chains constituted the intact ricin molecule through one interchain disulfide bond, and that the A-chain caused perturbation of the DPPC bilayer at the phase transition temperature with consequent leakage of carboxyfluorescein.