Masaru Funatsu

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.

Identification and isolation of endogenous insect phenoloxidase inhibitors

Inhibitors of phenoloxidase were identified in pupae of the housefly, Musca domestica L. The phenoloxidase inhibitors were purified from final instar pupae of the housefly by a combination of ammonium sulfate fractionation, ion-exchange chromatography, gel filtration and reverse-phase high performance liquid chromatography. The potent phenoloxidase inhibitors were heat-stable low molecular weight peptides with an inhibition constant of nM range. To the best of our knowledge, this is the first time that endogenous phenoloxidase inhibitors have been identified among the insects, and probably also among the invertebrates. It is likely that the inhibitors play a central role in regulating the action of active phenoloxidases and will also serve as important tools for understanding the structures and functions of phenoloxidases, as well as their role in insect metamorphosis.

Isolation of latent phenoloxidase from prepupae of the housefly, Musca domestica

Abstract Latent phenoloxidase was purified from prepupae of the housefly, Musca domestica vicina Maquart. The purification procedures included DEAE-cellulose column chromatography, sucrose density gradient centrifugation adn second sucrose density gradient centrifugation. The final preparations appear to be homogeneous based on results obtained from polyacrylamide gel electrophoresis in the presence of EDTA. Electrophoresis in the absence of EDTA caused spontaneous activation of latent phenoloxidase. While latent phenoloxidase was fairly stable over the range of temperatures between 0 and 40°C, it was quite sensitive to changes in pH, being stable only around pH 6.0. The molecular weight of latent phenoloxidase was estimated to be 178,000, as determined by gel filtration and sucrose density gradient centrifugation. Furthermore, phenoloxidase formed by the activation of latent phenoloxidase indicated a higher molecular weight (340,000) than that of latent phenoloxidase. Thus, it appears that the mechanism of the activation of latent phenoloxidase involves the association and disassociation system.

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.

Purification and some properties of prothoracicotropic hormone in the silkworm, Bombyx mori

Prothoracicotropic hormone was purified from the heads of Bombyx mori so that the nature of the hormone could be elucidated. The hormone extracted from acetone powder with 2% NaCl at pH 6.8 was partially purified by heat treatment, followed successively by ammonium sulphate precipitation, acetone fractionation and picric acid precipitation. Further purification was carried out by successive column chromatography on DEAE-cellulose, CM-Sephadex C-50, hydroxylapatite and Bio-Gel P-60. Finally, the hormone was isolated by preparative polyacrylamide gel disc-electrophoresis. These purification steps effected a 1700-fold increase in specific activity with a 4% yield of hormone preparation. From 1.5 × 106 heads, 5.3 mg of the hormone with a specific activity of 100,000 units per mg was isolated. The hormone labelled with 125I appeared as a single band with an apparent molecular weight of 18,000 on SDS-polyacrylamide gel electrophoresis. No loss of hormonal activity occurred in 6 M urea and 6 M guanidine hydrochloride solution. The isolated hormone was capable of inducing adult development of a debrained, dormant Bombyx pupa at a dose of 10ng, and was evidently different from the prothoracicotropic hormone (mol. wt 4400) obtained from the adult heads of Bombyx mori by Nagasawa et al. (1979) [Devl. Growth Differ.21, 29–38], which induced adult development of a brainless Samia pupa alone.

Isolation and chemical properties of a ricin variant from castor bean

Abstract M. Ishiguro , M. Tomi , G. Funatsu and M. Funatsu . Isolation and chemical properties of a ricin variant from castor bean. Toxicon 14, 157–165, 1976.—A ricin variant was isolated and purified from defatted castor bean meal by extraction with water at pH 3·8, precipitation with sodium chloride, fractionation with ammonium sulfate followed by gel filtration through Sephadex G-75 and chromatography on DEAE-cellulose. The preparation thus obtained appeared homogeneous on gel disc electrophoresis (pH 4·0 and 8·3), electrofocusing and ultracentrifugation. The ricin variant which was isoelectric at pH 7·25, proved to be a glycoprotein of 54,000 dalton. When injected intraperitoneally, it was found to have an ld 50 in mice of 0·031 μg of protein per g of body weight. The amino acid composition as well as the amino terminal amino acids (isoleucine and alanine) of this protein were determined. After reduction and carboxymethylation, the ricin variant was shown by gel disc electrophoresis to consist of two dissimilar subunits. Isolation and partial characterization of these subunits revealed that the A chain of the ricin variant and that of ricin D are very similar, whereas their B chains differed considerably in their chemical composition.

Studies on Tyrosinase in Housefly:Part I. Protyrosinase in the Pupae of Housefly and Its Activation

Occurrence of protyrosinase in the prepupae of housefly, Musca vicina Maquart, and its activation were described. The prepupae possessing no appreciable tyrosinase activity could be separated from the other aged pupae by putting them into water. Homogenate prepared from the prepupae contains protyrosinase and has no activation system for the proenzyme. As has been reported by Ohnishi2) a certain activator occurred naturally in the aged pupae apparently activates the protyrosinase in vitro. However, contrary to Ohnishi’s results3) it was found that this protyrosinase can be activated by the treatment with sodium dodecyl sulfate in vitro.

Studies on Xylanase from Trichoderma viride: Part I. Isolation and Some Properties of Crystalline Xylanase

The crude enzyme preparation obtained by isopropanol-precipitation of a water-extract of wheat bran culture of Trichoderma viride was first fractionated with ammonium sulfate precipitation, followed by DEAE-Sephadex A-25 column chromatography. Two active fractions, F-l and F-4, were obtained. Fraction F-4 was futher purified by column chromatography with CM-Sephadex C-50 and was finally crystallized by the addition of ammoniun sulfate to 35% saturation. This purified xylanase A was homogeneous as judged by ultracentrifugation and discelectrophoresis. Maximum conditions for the activity of crystalline xylanase were pH 3.5 and 50°C. The enzyme was stable in a concentrated solution below 40°C and between pH 2 and 7, but at low enzyme concentration, i.e. 9.7 μg per ml, it was easily inactivated under these same conditions.

In vitro action of prothoracicotropic hormone on RNA-synthesis in the prothoracic gland of the silkworm, Bombyx mori

Abstract Prothoracic glands of dauer pupae of the silkworm, Bombyx mori , were maintained in organ culture, and in vitro activation of prothoracic glands with prothoracicotropic hormone (PTTH) was studied. The PTTH purified from adult heads of Bombyx mori induced a significant stimulation of uridine incorporation into the RNA of cultured prothoracic glands. This stimulating effect showed an apparent dose-response relationship. On the basis of the inhibitory effect of actinomycin D on this RNA synthesis, it became apparent that the increase in uridine incorporation induced by PTTH action depends on a true stimulation of the RNA synthesis in prothoracic glands. The PTTH exerted no effect on the in vitro uridine incorporation into the RNA of both salivary glands and suboesophageal ganglia of dauer pupae of Bombyx mori . The RNA pattern of prothoracic glands stimulated with the PTTH as judged by polyacrylamide gel electrophoresis demonstrated the predominant increase in the synthesis of ribosomal RNA.

Primary structure of protein L14 isolated from Escherichia coli ribosomes.

Protein L14 is a component of the large subunit of Escherichia coli ribosomes [ 11. As shown by immune electron microscopy, at least a portion of this protein is located in the ‘seat’ of the 50 S subunit [2]. It can be crosslinked by bifunctional reagents to proteins L4 and Ll 1 [3], and this finding agrees with the immune electron microscopic result. L14 is among those proteins which rebind to the 18 S RNA fragment of 23 S RNA [4]. It has been more precisely shown that the binding site of L14 is between nucleotides 1300 and 2000 of the 23 S RNA [5]. Several mutants in which protein L14 is altered have been found by two-dimensional gel electrophoresis [6-91. The gene of protein L14 is located near the promotor of one of the operons within the spc-str region of the E. coli chromosome [lo]. It will be interesting to determine the nucleotide sequence of the gene for protein L14 and to compare it with the ammo acid sequence of this protein reported here.