Akio Ide

INHIBITION OF NEUTROPHIL-SUPEROXIDE GENERATION BY ALPHA -TOCOPHEROL AND COENZYME Q

Effects of various derivatives of alpha-tocopherol (VE) and coenzyme Q (CoQ) on superoxide (O2.-) generation of neutrophils and protein kinase C (PKC) activity were examined. VE and CoQ8 inhibited O2.- generation of neutrophils stimulated by a protein kinase C mediated process monitored by cytochrome c reduction and spin trapping methods. The inhibitory action was observed not only with alpha-tocopherol, but also with beta-, gamma-, delta-tocopherols and with tocol which is a chemical similar to VE but lacking methyl groups on the chromanol ring structure and which is not a radical scavenger. By contrast, no inhibition was observed with 2-carboxy-2, 5, 7, 8-tetramethyl-6-chromanol (CTMC, trolox) or 2, 2, 5, 7, 8,-pentamethyl-6-chromanol (PMC) which are water soluble VE derivatives having radical scavenging activity. Compounds having a similar isoprenoid chain, such as CoQ, also have inhibitory activity on PKC-dependent O2.- generation of neutrophils. The inhibitory activity of CoQ derivatives is dependent on the length of the unsaturated isoprenoid chain. CoQ derivatives having 16, 24 and 32 carbon isoprenoid chains corresponding to CoQ4, 6, and 8 inhibited O2.- generation but 4 and 40 carbon isoprenoid chains corresponding to CoQ2 and 10 had no inhibitory activity on O2.- generation. Alpha-tocopherol and CoQ inhibited PKC activity but the ID50 for O2.- generation and PKC activity was different for each compound. However, no direct relationship between VE content and O2.- generation of neutrophils was observed. These results suggest that isoprenoids of VE and CoQ participate in the inhibition of the NADPH oxidase activation system through modulation of the neutrophil membrane probably by the inhibition of PKC.

Immunoblot analysis of chitinolytic enzymes in integument and molting fluid of the silkworm, Bombyx mori, and the tobacco hornworm, Manduca sexta

Abstract The occurrence of proteins related to chitinolytic enzymes in integument of the silkworm, Bombyx mori, and the tobacco hornworm, Manduca sexta, during the larval-pupal transformation was determined by immunoblot analysis using rabbit polyclonal antibodies for B. mori chitinase (EC 3.2.1.14) and β-N-acetylglucosaminidase (EC 3.2.1.30) as probes. Similar temporal patterns of appearance and immunoreactivities of chitinase-like and β-N-acetylglucosaminidase-like proteins were observed in the two species. Several chitinase-like proteins (MWapp 50–200 kDa) were resolved by denaturing electrophoresis. During the latter part of the fifth larval stadium, the major immunoreactive protein in B. mori integument and molting fluid had an apparent molecular mass of 88 kDa, which was previously observed (Koga et al., 1989). In M. sexta integument on days 6–8 of the fifth larval stadium and in molting fluid, the major immunoreactive protein had an apparent molecular mass of 97 kDa, which was 22 kDa larger than the 75 kDa chitinase detected in molting fluid (Koga et al., 1983). In integument on days 3–7 of the fifth larval stadium, another immunoreactive protein with an apparent molecular mass of 119 kDa was present. In contrast to multiple immunoreactive chitinase-like proteins, only a single major β-N-acetylglucosaminidase-immunoreactive protein was detected in integument and molting fluid from either species. The immunoreactive β-N-acetylglucosaminidase-like proteins had an apparent molecular mass of 67.5 kDa and a pI of 5.0, which are identical values to those of β-N-acetylglucosaminidases determined previously. The β-N-acetylglucosaminidases cleaved N-acetylchitooligosaccharides from the non-reducing end in an exo-fashion. The results of this study suggest that chitinases are synthesized in B. mori and M. sexta integuments as zymogens, which are activated by limited proteolysis whereas β-N-acetylglucosaminidases are not.

Appearance of chitinolytic enzymes in integument of Bombyx mori during the larval-pupal transformation. Evidence for zymogenic forms

Abstract The appearance of chitinolytic enzymes, chitinase and β-N-acetylglucosaminidase, involved in ecdysis of the silkworm, Bombyx mori, was investigated using integuments prepared from fifth instar larvae during and after spinning behavior just before the larval-pupal transformation. β-N-Acetylglucosaminidase activity appeared a day after the beginning of spinning (SP1) and gradually increased for 2 more days (SP3), while chitinase activity appeared later at the SP3 stage (1 day before the ecdysis). It was shown by immunoblotting that the changes in activity were due to increases in the amounts of enzymes present. A probable zymogenic form of chitinase, whose molecular weight was about 215 kDa, was detected during spinning period by immunoblotting using anti-65-kDa chitinase antibody. The zymogen was observed 2 days before the appearance of enzyme activity. High molecular proteins (120–190 kDa) related to β-N-acetylglucosaminidase were also observed throughout the spinning period by immunoblotting, but this appearance pattern was different from that of chitinase. The results support, at least in the case of chitinase the hypothesis, that insect chitinolytic enzymes are synthesized as inactive precursors which are activated by limited proteolysis.

Human Pro-Tumor Necrosis Factor: Molecular Determinants of Membrane Translocation, Sorting, and Maturation

Human pro-tumor necrosis factor (pro-TNF) is a type II transmembrane protein with a highly conserved 76-residue leader sequence. We have analyzed the behavior, both in a microsomal translocational system and by transfection, of a series of mutants with deletions from the cytoplasmic, transmembrane, and linking domains. Cytoplasmic deletions included the Arg doublet at -49 and -48 and/or the Lys doublet at -58 and -57; additional mutants included deletion of residues -73 to -55 and -73 to -55, -49, and -48. The transmembrane and linking domain mutants included deletions in the -42 to -35 region, combined with the deletion of residues -32 to -1. Two hybrid mutants combined the cytoplasmic deletions with the deletion of residues -32 to -1. All of the cytoplasmic deletion mutants were properly translocated, as were the transmembrane deletion mutants with deletions up to residues -36, -35, -32 to -1, although the last one exhibited reduced efficiency; further incremental deletions, including deletions of residues -38 to -35 and -32 to -1, completely blocked translocation. Both hybrid mutants were effectively translocated; furthermore, transfection analysis revealed competent expression and maturation of both the cytoplasmic and hybrid mutants. Thus, proper expression and maturation of human pro-TNF can be accomplished with as few as approximately 12 of the 26 residues of the native transmembrane domain and with a net negative charge in the cytoplasmic domain flanking the transmembrane region.

Effects of 20-hydroxyecdysone and KK-42 on chitinase and β-N-acetylglucosaminidase during the larval-pupal transformation of Bombyx mori

Abstract The appearance of chitinolytic enzymes, chitinase and β-N-acetylglucosaminidase, in integuments of fifth-larval instars of the silkworm, Bombyx mori, was investigated by injection of 20-hydroxyecdysone into the hemolymph of the ligated larvae, and by topical application of an imidazole compound (KK-42, 1-benzyl-5[(E)-2,6-dimethyl-1,5-heptadienyl] imidazole) along the dorsal vessel of the larvae at the beginning of spinning behavior. 20-Hydroxyecdysone induced both enzyme activities. However, the induction patterns were different between two types of chitinolytic enzymes. Chitinase was rapidly induced only by high hormone levels (30 μg/insect, 7.5 μg/g live wt) and soon decreased, while β-N-acetylglucosaminidase was gradually induced even by low hormone levels (6 μg/insect, 1.5 μg/g live wt). KK-42 suppressed both the larval-pupal transformation and appearance of chitinolytic enzymes. Application of KK-42 (50 μg/insect) caused 1-day delay in β-N-acetylglucosaminidase and 2-day delay in chitinase. It was shown by immunoblotting and activity staining that the appearance of the enzyme activities was associated with that of the respective enzyme molecules. The molecular species of β-N-acetylglucosaminidase appeared was mainly the 67.5 kDa subunit. In the case of chitinase, several molecular species including active forms (88 and 65 kDa) and zymogenic form (about 215 kDa) were observed. These results suggest that β-N-acetylglucosaminidase is induced in an active form by relatively low ecdysteroid levels, whereas chitinase is induced through activation of the zymogen by higher levels of hormone.

Requirement of protein association with membranes for phosphorylation by protein kinase C

To clarify the requirement of the association of substrate proteins with phospholipid membranes for phosphorylation by protein kinase C (PKC), we studied the relationship between membrane association of PKC-substrate proteins and their phosphorylation by PKC. In the presence of phosphatidylserine, 12-O-tetradecanoylphorbol-13-acetate induced PKC autophosphorylation in either the presence or the absence of Ca2+, and this phosphorylation was not inhibited by increasing salt concentration (up to 200 mM NaCl). Thus, Ca2+ and ionic strength did not markedly affect the enzymatic activity of PKC. Annexin I required Ca2+ for both its association with phospholipid membranes and phosphorylation by PKC, whereas histone and monomyristilated lysozyme (C14:0-lysozyme) did not. This result indicates that the membrane association of substrates closely correlates with their phosphorylation by PKC. Similar correlation was also observed in the effects of ionic strength on the membrane association of the substrates and their phosphorylation by PKC; increased ionic strength (200 mM NaCl) remarkably inhibited both the membrane association and the phosphorylation of histone and annexin I by PKC but C14:0-lysozyme was not markedly affected. These results suggest that the membrane association of PKC-substrate proteins is a prerequisite for their phosphorylation by PKC. This concept further conforms to the mechanisms of PKC inhibitors; some types of PKC inhibitors are mediated all or in part through inhibition of the substrate-membrane interaction.

Ricin A-chain induces fusion of small unilamellar vesicles at neutral pH

The interaction of ricin and its constituent polypeptides, the A‐ and B‐chain, with small unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC) or dimyristoylphosphatidylcholine (DMPC) was investigated by means of differential scanning calorimetry measurements. The A‐chain, at neutral pH, entirely shifted the endothermic peak of small unilamellar vesicles of DPPC from 37°C to 41°C at low protein/lipid ratios. The potency of either ricin or the B‐chain to induce the shift of endothermic peak was much less than that of the A‐chain. The A‐chain was also found to cause mixing of endothermic peaks of DMPC vesicles and DPPC vesicles. These data strongly suggest that the A‐chain has the ability to induce fusion of phospholipid vesicles.

Kinetics of a Chitinase from a Prawn, Penaeus japonicus

Kinetic analysis was done on a chitinase (EC 3.2.1.14) purified from the liver of a prawn, Penaeus japonicus, using N-acetylchitooligosaccharides (GlcNAcn, n = 2 to 6), p-nitrophenyl N-acetylchitooligosaccharides (pNp-GlcNAcn, n = 1 to 5), and colloidal chitin as the substrates. The enzyme hydrolyzed GlcNAc4 to two molecules of GlcNAc2, GlcNAc5 to GlcNAc2 plus GlcNAc3, and GlcNAc6 by two ways to GlcNAc2 plus GlcNAc4 (87%), and two molecules of GlcNAc3 (13%). Neither GlcNAc2 nor GlcNAc3 was hydrolyzed. The Km and kcat were 0.249 mm and 3.38 sec−1 for GlcNAc4,0.018 mm and 2.67 sec−1 for GlcNAc5, and 0.005 mm and 2.72 sec−1 for GlcNAc6, respectively. The cleavage patterns of p-nitrophenyl N-acetylchitooligosaccharides were different from those of the corresponding N-acetylchitooligosaccharides. The enzyme hydrolyzed colloidal chitin to produce mainly GlcNAc2 and a trace of GlcNAc3. Allosamidin inhibited prawn chitinase in a competitive way with a Ki of 0.1 μm and IC50 of 0.14 μm. These results suggest that p...

Association of a myristoylated protein with a biological membrane and its increased phosphorylation by protein kinase C

A hydrophilic enzyme, lysozyme, was myristoylated in vitro by the N‐hydroxysuccinimide ester of myristic acid, and the monomyristoylated lysozyme was isolated by CM‐cellulose cation‐exchange column chromatography. The monomyristoylated lysozyme associated with phospholipid vesicles, whereas the association of native lysozyme was negligible. The membrane‐associated monomyristoylated lysozyme was phosphorylated with partially purified rat brain Ca2+‐ and phospholipid‐dependent protein kinase (protein kinase C) in the presence of Ca2+, phosphatidylserine and phorbolmyristate acetate. Thus, the myristoylated lysozyme became a substrate of protein kinase C through its hydrophobic association with the membrane. The present results suggest that the myristoylation of cytoplasmic proteins may have an important role in signal transduction.

Growth and alkaloid production of callus culture induced from Securinega suffruticosa

Abstract The growth of, and production of alkaloids by, callus derived from budding stem explants of the germinated seeds of Securinega suffruticosa has been studied. The major alkaloids produced were securinine and allosecurinine with the latter being present in the greatest amount. The effects of pH, growth hormones, sucrose concentration and light and dark on callus growth and alkaloid production have been examined in detail. The pattern of alkaloid production in the callus culture appeared to be similar to that in the root of the securinega plant.