Shuichiro Kubo

ADP-ribosylation of specific membrane proteins in pheochromocytoma and primary-cultured brain cells by botulinum neurotoxins type C and D

Type C1 and D toxins produced by Clostridium botulinum caused ADP‐ribosylation of a protein of 24 kDa in membrane preparations of rat clonal pheochromocytoma cells (PC12) and of proteins of 25 and 26 kDa in neuron‐rich culture of fetal rat brain cells. The ADP‐ribosylation reaction was dependent on the presence of MgCl2, GTP and GTPγS. The results obtained suggested that the ADP‐ribosylation reaction is responsible for the development of the biological activity of the botulinum neurotoxins and that the target of this reaction may be novel GTP‐binding proteins localized on cell membranes.

Light chains of chicken embryonic gizzard myosin.

1. Myosin from gizzards of 15-day-old chicken embryos was highly purified by ammonium sulfate fractionation in the presence of ATP and MgCl2, ultra-centrifugation and Sepharose 4B chromatography. 2. The myosin composed of heavy and three light chains as determined by sodium dodecyl sulfate (SDS) gel electrophoresis. The molecular weights of the light chains were 23,000 (L23), 20,000 (L20), and 17,000 (L17), respectively. The amount of L23 light chain decreased and disappeared, and the L17 light chain increased steadily in the course of development. The amount of L20 light chain did not change. 3. ATPase activity of the embryonic myosin was essentially the same as that of adult myosin. The change in the light chain pattern in the course of development did not correlate to the ATPase activity. 4. Antigenicity of the heavy chains in the embryonic myosin was the same as that of the adult heavy chains. However, antibodies to light chains were not detected in the antibodies to either the embryonic or adult myosins.

Protein kinase C phosphorylation of protamine is Ca2+ independent, but the addition of DNA renders it Ca2+ dependent

Protamine is a unique substrate of protein kinase C for its Ca2+-independent phosphorylation. The interaction between protein kinase C and protamine and the effect of DNA on the interaction was studied. Protein kinase C was retained in a protamine-immobilized Sepharose 4B column, even in the absence of Ca2+ and was eluted with ammonium sulfate or L-arginine. The eluted enzyme was fully activated by phosphatidylserine alone, when protamine was used as substrate. When DNA was included in the assay system, the activity elicited by phosphatidylserine alone was inhibited. The DNA effect on the activity in the presence of both Ca2+ and phosphatidylserine was much lower than on the activity elicited by phosphatidylserine alone, thereby demonstrating the Ca2+ sensitivity of protamine phosphorylation.

Clostridium botulinum type C toxin: a sketch of the molecule.

SummaryThe purification and crystallization of type C botulinum toxin along with its physical characteristics are described. The shape of Clostridium botulinum type C toxin molecule is globular like a pressed ball with a 7.4 nm diameter and a 4.3 urn thickness. The molecular volume is approximately 185 nl and the molecular weight is 141 000. The toxin molecule is composed of two parts, which are separable under appropriate conditions. These parts have some differences in the electrophoretic properties, amino acid distribution, immunological, and functional characteristics.The toxin molecule can be reconstituted by association of S-S bond between the two chains. The expression of the toxicity requires that the fragments of the polypeptide chain carrying the necessary information be functionally organized for the proper development of the specific tertiary structure for active conformation.

Purification and properties of cyclic nucleotide-independent phosvitin kinase from pig testis.

Cyclic nucleotide-independent and Ca2+-independent phosvitin kinase was purified from pig testis to apparent homogeneity by DEAE-cellulose, Sephadex G-200 chromatography, followed by subsequent DEAE-cellulose chromatography and phosvitin-Sepharose 4B affinity chromatography. The purified enzyme was homogeneous by analytical polyacrylamide gel electrophoresis, and it consisted of two polypeptides with molecular weights of 92000 (alpha) and 84000 (beta), which were present in the ratio of 1:2. Molecular weight of the native enzyme was estimated at 240000 by gel chromatography on Sepharose 6B, suggesting that the enzyme consists of alpha beta 2. The enzyme had maximal activity with phosvitin as the substrate. Casein was less active than phosvitin. Histone, protamine and myosin light-chains were practically ineffective. The enzyme possessed no ability to autophosphorylate. The apparent Km values were 7.4 microM for phosvitin, 65 microM for ATP and 0.6 mM for Mg2+. Vmax was 2.16 mumol/min per mg. The enzyme was inhibited by ammonium sulfate and heparin, and it was not affected by the addition of cyclic nucleotides and Ca2+ with calmodulin or phospholipid.

8 – Analysis of Antigenic Structure of Clostridium botulinum Type C1 and D Toxins by Monoclonal Antibodies

Publisher Summary This chapter presents an analysis of antigenic structure of Clostridium botulinum type C 1 and D toxins by monoclonal antibodies. Clostridium botulinum produces one of the most powerful neuroparalytic poisons that affect a great variety of animal species in all parts of the world. Clostridium botulinum cultures can be divided into four groups: (1) proteolytic, which produce toxin types A, B, or F; (2) non-proteolytic, which produce toxin types B, E, or F; (3) type C α , C β , and D cultures; and (4) proteolytic but non-saccharolytic type G. Hybridomas producing antibodies to toxins are screened by enzyme-linked immunosorbent assay. The production of a major toxin in certain type C and D cultures is governed by specific bacteriophages. Curing these cultures of their prophages results in concomitant loss of the dominant toxin production, and the non-toxigenic cultures are reconverted to toxin producers by the infection with phages.

Immunological evidence for the existence of two kinds of L2 components in myosin light chains.

Abstract 5,5′-dithiobis-(2-nitrobenzoic acid) treatment of myosin removed about 60% of the L2 components. In immunodiffusion, 5,5′-dithiobis-(2-nitrobenzoic acid) light chain produced one line with antibodies to the light chains, while L2 components fractionated by SP-Sephadex C-50 chromatography produced two lines. The line produced by the 5,5′-dithiobis-(2-nitrobenzoic acid) light chain fused with one line of the two lines of L2 components. The line of the 5,5′-dithiobis-(2-nitrobenzoic acid) light chain was fused with one line produced by chloromercuribenzoate-sensitive g2. These results indicated that L2 components contained 5,5′-dithiobis-(2-nitrobenzoic acid) light chain and the other L2 component, which were immunologically different, and that 5,5′-dithiobis-(2-nitrobenzoic acid) light chain was identical to chloromercuribenzoate-sensitive g2.