Keiichi Enomoto

Bioactive Pigments from Marine Bacteria: Applications and Physiological Roles

Research into natural products from the marine environment, including microorganisms, has rapidly increased over the past two decades. Despite the enormous difficulty in isolating and harvesting marine bacteria, microbial metabolites are increasingly attractive to science because of their broad-ranging pharmacological activities, especially those with unique color pigments. This current review paper gives an overview of the pigmented natural compounds isolated from bacteria of marine origin, based on accumulated data in the literature. We review the biological activities of marine compounds, including recent advances in the study of pharmacological effects and other commercial applications, in addition to the biosynthesis and physiological roles of associated pigments. Chemical structures of the bioactive compounds discussed are also presented.

Incorporation in vitro of purified cytochrome b5 into liver microsomal membranes

Incubation of liver microsomes with cytochrome b5, purified after solubilization with detergents, caused an effective incorporation of the cytochrome into the microsomal membranes. The incorporated cytochrome was reducible by NADH and could not be removed by repeated washing with 0.3 M KCl or 10 mM EDTA. The incorporation was much more efficient at 37°C than at 0°C. Trypsin-solubilized cytochrome b5, which lacks the hydrophobic tail of the native protein, could not be inserted into the membranes. These findings confirm the view that the hydrophobic tail of the cytochrome molecule is responsible for its tight binding to the microsomal membranes.

Denaturation of cytochrome b5 by guanidine hydrochloride: Evidence for independent folding of the hydrophilic and hydrophobic moieties of the cytochrome molecule

Abstract Hepatic microsomal cytochrome b5 is an amphipathic protein consisting of a hydrophilic (heme-containing) moiety and a Hydrophobic (membrane-binding) segment and exists in aqueous media as a micelle. Circular dichroism studies indicated that denaturation of cytochrome b5 by guanidine hydrochloride is a two-stage process, the first transition occurring at the denaturant concentration of about 2.6 m and the second one at 5.0–5.5 m . A hydrophilic fragment of the cytochrome lacking the hydrophobic segment, on the other hand, underwent one-stage denaturation at a guanidine hydrochloride concentration of about 2.9 m . Detachment of the heme from the cytochrome and the fragment, measured by optical absorption, was effected at the denaturant concentrations of 2.6 and 2.9 m , respectively. Gel chromatography experiments showed that dissociation of the cytochrome micelle into the monomers took place concomitant with the second stage of denaturation. It is suggested that the two moieties of the cytochrome molecule exist as relatively independent domains undergoing unfolding separately and the hydrophobic domain is much more resistant to denaturation. It was further found that removal of the heme from the cytochrome by acid-acetone treatment rendered the hydrophilic domain unstable but did not affect the structure of the hydrophobic domain.

Inhibition of catalytic unit of adenylate cyclase and activation of GTPase of Ni protein by βγ-subunits of GTP-binding proteins

A protein factor which inhibited adenylate cyclase was purified to apparent homogeneity from rat brain and identified as the βγ‐subunits of the GTP‐binding regulatory proteins of adenylate cyclase. (i) The βγ‐subunits (protein factor) inhibited the partially purified catalytic unit of adenylate cyclase in the presence of an activator, forskolin or the stimulative regulatory protein (Ns), to 60 and 40% of the control, respectively; inhibition of the catalytic unit in the presence of forskolin required no guanine nucleotides. (ii) The subunits enhanced the GTPase activity of the purified α‐subunit of the inhibitory regulatory protein (Niα) 3.8‐fold, (iii) The subunits stimulated ADP‐ribosylation of niα catalyzed by islet‐activating protein (pertussis toxin). ADP‐ribosylation had no effect on the GTPase activity of Niα in the presence of the βγ‐subunits. The results suggest that direct inhibition of the catalytic unit by the βγ‐subunits liberated from Ni is essential for the receptor‐mediated inhibition of adenylate cyclase.

Quorum Sensing Signaling Molecules Involved in the Production of Violacein by Pseudoalteromonas

The production of violacein by Pseudoalteromonas sp. 520P1 has many features of quorum sensing. Signaling molecules were extracted from bacterial culture and subsequently identified as N-(3-oxooctanoyl)-homoserine lactone and N-tetradecanoyl-homoserine lactone. The former but not the latter induced the production of violacein in strain 520P1. We conclude that N-(3-oxooctanoyl)-homoserine lactone is a signaling molecule involved in the production of violacein.

Asymmetric binding of cytochrome b5 to the membrane of human erythrocyte ghosts.

The intact, amphipatic form of cytochrome b5 could bind to unsealed ghosts, but not to resealed ghosts, suggesting that the cytochrome could bind only to the inner (cytoplasmic) surface of the ghost membrane. This was further confirmed by the finding that the cytochrome could bind to closed, inside-out vesicles prepared from the ghosts. This asymmetric binding was not due to the exclusive localization of sialic acid and sugar chains on the outer surface of the ghosts membrane, because the cytochrome could not bind to ghosts even after enzymatic removal of these components. Although liposomes consisting of phosphatidylcholine or both phosphatidylcholine and sphingomyelin could effectively bind the cytochrome, this binding capacity was progressively decreased as increasing amount of cholesterol was included in the composition of phosphatidylcholine liposomes. Removal of cholesterol from resealed ghosts by incubation with egg phosphatidylcholine liposomes resulted in the binding of cytochrome b5 to the outer surface of the treated ghosts. The possibility is discussed that the asymmetric binding is due to preferential localization of cholesterol in the outer leaflet of the lipid bilayer that constitutes the ghost membrane.

Cytotoxic Prodigiosin Family Pigments from Pseudoalteromonas sp. 1020R Isolated from the Pacific Coast of Japan

Pseudoalteromonas sp. 1020R, isolated from the Pacific coast of Japan, produces prodigiosin family pigments. Structural analysis indicated that these are prodigiosin (2-methyl-3-pentyl-prodiginine) and three other prodigiosin congeners which differ only in the lengths of the alkyl side chains. These compounds exhibited different extents of cytotoxicity against U937 leukemia cells, and cell death was accompanied by typical features of apoptosis.

Evidence for the Presence of a GTP‐Dependent Regulatory Component of Adenylate Cyclase in Myelin from Rat Brain

Abstract: A GTP‐dependent regulatory component of adenylate cyclase was found in myelin from rat brain. The fraction solubilized from myelin contained a component that reconstituted guanine nucleotide‐responsive adenylate cyclase activity when combined with the catalytic unit of adenylate cyclase prepared from rat brain. Purified myelin demonstrated little adenylate cyclase activity, even in the presence of F− or Mn2+. The reconstituted activity was dependent on the amount of the solubilized myelin fraction and required the presence of 5′‐guanylylimidodiphosphate, a hydrolysis‐resistant analog of GTP. The elution pattern of the component solubilized from myelin in gel filtration was very similar to that of a GTP‐dependent regulatory component from synaptic plasma membranes. The content of the regulatory component‐like activity in myelin was estimated to be 50–60% of that in synaptic plasma membranes. Cholera toxin ADP‐ribosylated proteins having molecular weights of 48,000, 38,000, 23,000, 20,000 and 15,000 and other minor peptides in myelin, some of which were also present in synaptic plasma membranes. We conclude that myelin contained a GTP‐dependent regulatory component of adenylate cyclase despite the apparent lack of adenylate cylcase activity in myelin.

GTPase activity associates with the inhibitory GTP-binding regulatory component of adenylate cyclase purified from rat brain.

The inhibitory regulatory component of adenylate cyclase (Ni) was highly purified from rat brain synaptic membranes. A low K m GTPase activity was always associated with Ni through the purification, and the recovery of GTPase activity correlated well with that of Ni. Purified Ni was hardly ADP‐ribosylated by islet‐activating protein (IAP). A heat‐labile factor in the fraction of the stimulative regulatory component (Ns) restored ADP‐ribosylation and also activated the GTPase about 2‐fold. NaF which was reported to interact with Ni markedly reduced GTPase activity. The purified Ni fraction inhibited adenylate cyclase only in the presence of a heat‐stable factor found in the partially purified regulatory component. GTPase and inhibitory activities were weak in myelin which contained only a small amount of Ni. These findings support the view that GTPase activity is an intrinsic activity of Ni and some factors are necessary for the function of Ni.

Surface display of recombinant protein on the cell surface of Bacillus subtilis by the CotB anchor protein

We developed a novel surface display system based on the CotB anchoring motif in order to express foreign protein on the surface of vegetative Bacillus subtilis cells. CotB is a protein in the B. subtilis spore coat. In this system, three repeats of the immunodominant ovalbumin T-cell epitope (OVA323–339) were linked with the cholera toxin B subunit (CTB) to construct a fusion protein, CTB-OVA epi, which was then fused to the C-terminal of the CotB protein so that CTB-OVA epi was expressed in vegetatively-growing B. subtilis. The expression and localization of the CTB-OVA epi protein was confirmed by western blotting, immunofluorescence microscopy, and flow cytometry. The results indicated that a CotB-based surface display system was successfully used to express the CTB-OVA epi protein on the surface of vegetative B. subtilis cells.