The helicase-like domain of plant potexvirus replicase participates in formation of RNA 5' cap structure by exhibiting RNA 5'-triphosphatase activity.

Open reading frame 1 (ORF1) of potexviruses encodes a viral replicase comprising three functional domains: a capping enzyme at the N terminus, a putative helicase in the middle, and a polymerase at the C terminus. To verify the enzymatic activities associated with the putative helicase domain, the corresponding cDNA fragment from bamboo mosaic virus (BaMV) was cloned into vector pET32 and the protein was expressed in Escherichia coli and purified by metal affinity chromatography. An activity assay confirmed that the putative helicase domain has nucleoside triphosphatase activity. We found that it also possesses an RNA 5'-triphosphatase activity that specifically removes the gamma phosphate from the 5' end of RNA. Both enzymatic activities were abolished by the mutation of the nucleoside triphosphate-binding motif (GKS), suggesting that they have a common catalytic site. A typical m(7)GpppG cap structure was formed at the 5' end of the RNA substrate when the substrate was treated sequentially with the putative helicase domain and the N-terminal capping enzyme, indicating that the putative helicase domain is truly involved in the process of cap formation by exhibiting its RNA 5'-triphosphatase activity.

Immunohistochemical Identification of Hyperglycemic Hormone- and Molt-Inhibiting Hormone-Producing Cells in the Eyestalk of the Kuruma Prawn, Penaeus japonicus.

This study deals with the localization of crustacean hyperglycemic hormone (CHH, Pej-SGPIII) and molt-inhibiting hormone (MIH, Pej-SGP-IV) in the eyestalk of the kuruma prawn Penaeus japonicus using immunohistochemistry. High-titer and highly specific antisera were raised in rabbits against synthetic Pej-SGP-III C-terminal peptide (Glu-Glu-His-Met-Ala-Ala-Met-Gln-Thr-Val-NH2) and Pej-SGP-IV C-terminal peptide (Val-Trp-Ile-Ser-Ile-Leu-Asn-Ala-Gly-Gln-OH), both of which were conjugated with bovine serum albumin by a cross linker. Eyestalks were removed from mature male prawns at the intermolt stage of the molting cycle and fixed in Bouin's solution. Serial sections stained immunohistochemically showed that neurosecretory cells of Pej-SGP-III and Pej-SGP-IV were located in the same cluster of the medulla terminalis ganglionic X-organ (MTGX), and that three kinds of neurosecretory cells, which were stained with anti-PejSGP-III antiserum and/or anti-Pej-SGP-IV antiserum were present. The number of neurosecretory cells which stained with both antisera was much fewer than that of neurosecretory cells which stained with one of the antisera only. The axon and axon terminals in the sinus gland were also stained and the staining density of the sinus gland was always deeper than that of the neurosecretory cells.

Structural analysis of apolipoprotein A-I: limited proteolysis of methionine-reduced and -oxidized lipid-free and lipid-bound human apo A-I.

The domain structures of lipid-free and lipid-bound apolipoprotein A-I (apo A-I) containing reduced and oxidized methionines were analyzed by limited proteolysis. Lipid-free apo A-I is cleaved primarily in the extreme carboxy-terminus and, to a much lesser extent, in the central region of the protein between residues 115 and 136. Oxidation of methionines 112 and 148 to the corresponding sulfoxides in putative amphipathic helices 4 (P99-E120) and 6 (P143-A164), respectively, causes helices 1 (L44-G65), 2 (P66-S87), and 7 (P165-G186) to become susceptible to protease digestion. These results are consistent with a discrete, globular tertiary structure for the lipid-free protein minimally formed from amphipathic helices 1, 2, 4, 6, and 7. In distinct contrast to lipid-free apo A-I, lipid-bound apo A-I is most susceptible to cleavage in the extreme amino-terminus and, to a lesser extent, in both the central and carboxy-terminal regions. The observed cleavage pattern for the reduced lipid-bound protein supports the existence of many of the turns between helices predicted by sequence analysis of the lipid-bound protein. Methionine oxidation of lipid-bound protein results in a decreased protease susceptibility in the extreme amino-terminus and a concomitant increase in protease susceptibility in the central and carboxy-terminal regions. The results from methionine oxidation indicate the oxidation state of the protein is an important determinant in defining the conformation of both lipid-free and lipid-bound apo A-I.

4-Demethylpenclomedine, an antitumor-active, potentially nonneurotoxic metabolite of penclomedine.

Penclomedine [3,5-dichloro-4,6-dimethoxy-2-(trichloromethyl)pyridine], an antitumor agent, is currently in Phase I clinical trials and is believed to be a prodrug. In these studies, cerebellar effects have been dose limiting. Previous studies identified 4-demethylpenclomedine (4-DM-PEN) as the major plasma metabolite in rodents and humans. 4-DM-PEN was demonstrated to be an antitumor-active metabolite of penclomedine in vivo when evaluated against the penclomedine-sensitive MX-1 human breast tumor xenograft implanted either s.c. or intracerebrally and is believed to be on the metabolic activation pathway of penclomedine. Because earlier studies revealed an absence of neurotoxic cerebellar effects for 4-DM-PEN in contrast to penclomedine in a rat model, this metabolite may be a candidate for an alternative to penclomedine in the clinic for treatment of breast cancer or brain tumors, if the cerebellar effects of penclomedine preclude its further clinical development. Because neither penclomedine nor 4-DM-PEN were very active in vitro, the metabolism of penclomedine was also investigated using rat liver microsomes in an attempt to identify the ultimate active form of the drug. Metabolites and putative metabolites were prepared by chemical synthesis for antitumor evaluation in vitro and in vivo. A reductive metabolite, alpha,alpha-didechloro-PEN, was observed to be much more cytotoxic than penclomedine or 4-DM-PEN in vitro, but evaluation of this and the other metabolites and putative metabolites in vivo against the MX-1 tumor failed to identify any active metabolite among the structures evaluated other than 4-DM-PEN. The limited activity of 4-DM-PEN in vitro indicates that it, like penclomedine, is also a prodrug, demonstrating a need for additional studies on the metabolic activation of penclomedine to identify the ultimate active form of the drug.

Nonenzymatic isomerization of 9-cis-retinoic acid catalyzed by sulfhydryl compounds.

Certain thiol-containing compounds catalyze, in a chemical reaction, the isomerization of 9-cis-retinoic acid to a mixture of all-trans-retinoic acid, 9-cis-retinoic acid, 13-cis-retinoic acid, and 9,13-dicis-retinoic acid. In the presence of such catalysts, all-trans-retinoic acid gives rise to the same mixture. Reactions approaching equilibrium contain more all-trans-retinoic acid than either of the other isomers. Small molecules effective as catalysts are mercaptoethanol, L-cysteine methyl ester, glutathione, and N-acetyl-L-cysteine. Apoferritin (a thiol-containing protein), native microsomes, and, to a lesser extent, boiled microsomes catalyze the reaction. In intact cells, these interconversions also occur in a process inhibited by a sulfhydryl-specific reagent. The thiol-catalyzed isomerization of 9-cis-retinoic acid may be relevant in the biological activity of this compound.

Isolation and characterization of the female-specific protein (vitellogenin) in mature female hemolymph of the freshwater prawn, Macrobrachium rosenbergii: comparison with ovarian vitellin.

Purification and characterization of the female-specific protein (vitellogenin) from the hemolymph of mature female prawn, Macrobrachium rosenbergii, were the objectives of this study. The comparison of biochemical characteristics between vitellogenin and ovarian vitellin was also conducted. Hemolymph vitellogenin was purified with DEAE, hydroxylapatite, and another DEAE chromatographic column. The specific protein (vitellogenin) was shown in the fractions of chromatographic columns on the basis of ELISA, Western blotting, and immunoprecipitation. A purified vitellogenin was obtained with an apparent molecular weight of 700 kDa as determined by PAGE. The purified vitellogenin was considered as a lipoglycoprotein on the basis of staining data. Three subunits (170, 100, and 89 kDa) in purified vitellogenin and two subunits (100 and 89 kDa) in vitellin were detected with SDS-PAGE. Nondisulfide bonds were found in the binding of polypeptide subunits. Only the 89-kDa subunit was a glycopolypeptide in both vitellogenin and vitellin. The amino acid composition of vitellogenin differed from that of vitellin in a few amino acids. Eight amino acid sequences from the N-terminal end of 89- and 100-kDa subunits were determined and they were identical between vitellogenin and vitellin. Seven amino acid sequence from the N-terminal end of the 170-kDa subunit were also identical to the 100-kDa subunit. Purified vitellogenin was more susceptible to precipitation in a solution with low ionic strength than vitellin. This study suggests a close relationship between vitellogenin and vitellin in M. rosenbergii in their biochemical characteristics.

Retinoyl beta-glucuronide: lack of binding to receptor proteins of retinoic acid as related to biological activity.

Retinoid beta-glucuronides have emerged as biologically active, water-soluble, natural retinoids with relatively few toxic and teratogenic effects. The mechanism of action of these glucuronides in the control of epithelial differentiation, growth, and tumorigenesis is unknown. Since retinoyl beta-glucuronide (RAG) contains a free carboxyl group, we studied the interactions of RAG with cellular retinoic acid-binding protein (CRABP) and nuclear receptors of retinoic acid (RARs), the possible mediators of the biological action of retinoic acid (RA). RAG did not exhibit any significant affinity to bind either CRABP or RARs. During 24- and 48-hr incubations of RAG in chick cytosol, detectable amounts of RA were generated which interacted with the RA receptors. In chick skin, the biological activity of RAG may be due to this slowly released RA. Other possible modes of action of RAG are suggested.

Conversion of retinoid ethers to alcohols by enzymatic activity present in rat liver microsomes.

An enzyme present in rat liver microsomes catalyzes the conversion of retinyl methyl ether (RME) to retinol; NADPH is required for activity. The optimum pH for the reaction is 7.4; the KM and Vmax values are 120 microM RME and 14.3 nmol of retinol/mg protein/hr, respectively. As a substrate, the 2,3,6-trimethyl-4-methoxyphenyl analog of RME is as effective as RME. There is, however, no measurable activity for dealkylation of retinyl ethyl ether or retinyl butyl ether. Hepatic enzyme activity for the metabolism of RME is induced by 3-methylcholanthrene but not by phenobarbital or RME itself. The induced activity also requires NADPH as a cofactor. The optimum pH for the induced enzyme is 8.4; the KM and Vmax values are 50 microM RME and 111 nmol of retinol/mg protein/hr, respectively. For this enzyme, RME is a better substrate than the 2,3,6-trimethyl-4-methoxyphenyl analog of RME; retinyl ethyl ether is less effective; and again, there is no measurable activity with retinyl butyl ether as a substrate. Neither constitutive nor induced activity is detectable in microsomes from lung, spleen, stomach, kidney, small intestine, or large intestine. The enzyme activity that cleaves retinoid ethers appears to be similar to other microsomal NADPH-requiring O-dealkylases and different from a reported tetrahydropteridine-requiring dealkylase.

Conversion of retinol to retinal by rat liver microsomes.

An NADPH-requiring enzyme present in rat liver microsomes catalyzes the conversion of retinol to retinal; the enzyme activity is induced by 3-methylcholanthrene (MC). The optimum activities for both the constitutive and induced reactions occur within a pH range of 8.2-8.7. For the constitutive enzyme, the KM and Vmax values are 285 microM retinol and 18 nmol of retinal/mg protein/hr, respectively; for the MC-induced activity, the corresponding values are 133 microM retinol and 33 nmol of retinal/mg protein/hr. Neither the constitutive nor the induced activities are detectable in microsomes from seven other tissues. Both hepatic activities are inhibited by citral, ketoconazole, SKF 525-A, and alpha-naphthoflavone; both are stimulated by divalent cations. Neither is inhibited by 3-amino-1,2,4-triazole, pyrazole, or sodium azide or stimulated by monovalent cations. The enzyme appears to be a previously unreported retinol oxidase, distinct from the known cytosolic enzymes, retinal reductase and retinol dehydrogenase.

Enzymatic hydrolysis of retinamides.

Enzymatic activity present in liver microsomes from rats slowly hydrolyzed N-(4-hydroxyphenyl)retinamide (4HPR). A product of the reaction was all-trans-retinoic acid. The reaction, which had a pH optimum greater than 8.6, was stimulated by divalent cations, particularly Mn2+. Enzyme activity was highest in liver microsomes but was also present in kidney microsomes, liver cytoplasm, and spleen cytoplasm. Of 10 possible substrates tested, the 13-cis- and all-trans-forms of N-ethylretinamide were most active. The all-trans-form of 4HPR was much more active than the 13-cis-form. Neither 13-cis- nor all-trans-retinoyl leucine was a substrate. Because no detectable [14C]all-trans-retinoic acid could be found in the livers of rats after doses of [14C]4HPR, we conclude that this enzyme is not extensively active in intact animals.

Nonenzymatic isomerization of all-trans- and 13-cis-retinoids catalyzed by sulfhydryl groups.

Certain thiol-containing compounds catalyze, in a chemical reaction, the isomerization of all-trans-retinoic acid (RA) to 13-cis-RA and of 13-cis-RA to RA. Reactions approaching equilibrium contain more RA than 13-cis-RA. Small molecules effective as catalysts are glutathione, mercaptoethanol, and L-cysteine methyl ester. L-Cysteine is not a catalyst and inhibits the reaction catalyzed by glutathione or mercaptoethanol. Apoferritin (a thiol-containing protein), native microsomes, and, to a lesser extent, boiled microsomes catalyze the reaction, but their activity is reduced or eliminated by prior incubation with iodoacetate. Other cis and trans isomeric retinoids are also substrates for this reaction; the reactions proceed more readily for the cis isomers. The thiol-catalyzed isomerization of RA and 13-cis-RA may account for the observations of both cis and trans forms of retinoids in tissues of animals after administration of either.

Disposition and metabolism of the carcinogen reduced Michler's ketone in rats.

Studies on the in vivo and in vitro disposition of 4,4'-[14C]-methylenebis(N,N-dimethyl)benzamine (reduced Michler's ketone, RMK) were performed. Osborne-Mendel rats retained, after 24 hr, 78% of a p.o. dose of [14C]RMK. At 24 hr after an i.p. dose, fat, liver, and intestine represented major sites for deposition of radioactivity. The major urinary metabolite of RMK, representing 36% of the total radioactivity recovered in the urine, was N,N'-diacetyl-4,4'-(hydroxymethylene)dianiline. In vitro microsomal metabolism of RMK involved demethylation. Products included N,N-dimethyl-4,4'-methylenedianiline, N,N'-dimethyl-4,4'-methylenedianiline, N-methyl-4,4'-methylenedianiline, and 4,4'-methylenedianiline, representing 44.7, 5.3, 11.8, and 6.9%, respectively, of the total radioactivity recovered from the reaction mixture. Although none of the microsomal metabolites was a direct-acting mutagen in the standard Salmonella typhimurium assay, all could be activated to mutagens when incubated with 9000 X g liver supernatants and reduced nicotinamide adenine dinucleotide phosphate. The activation of 4,4'-methylenedianiline to a mutagen suggests that the methyl groups of RMK are not required for the conversion of RMK to a reactive electrophile.

Metabolic activation of 1,2-dibromoethane by glutathione transferase and by microsomal mixed function oxidase: further evidence for formation of two reactive metabolites.

In liver cytosolic preparations containing added glutathione, radioactivity from [14C]1,2-dibromoethane becomes bound preferentially to added polycytidylic acid. When microsomes and NADPH are present, however, the radioactivity becomes bound selectively to microsomal proteins.

Metabolism and macromolecular binding of the carcinogen Michler's ketone in rats.

1. Studies on the metabolism of 14C-Michler's ketone (4,4'-bis-(dimethylamino)[carbonyl- 14C]benzophenone) in rats have revealed that this carcinogen is subject to demethylation, ring-hydroxylation and N-acetylation after adjacent methyl groups have been removed. 2 As identified by mass spectral analysis, microsomal metabolites are the mono-, di-, tri- and tetra-demethylated derivatives. 3. The major metabolites appearing in the bile are the di- and tri-demethylated derivatives and the N-acetylated tetra-demethylated compound; minor metabolites, tentatively identified, are the ring-hydroxylated derivatives of di- and tri-demethylated Michler's ketone and of N-acetylated tri- and tetra-demethylated Michler's ketone. 4. The major urinary metabolite is tentatively identified as a ring-hydroxylated derivative of N-acetylated, di-demethylated Michler's ketone; a minor urinary metabolite lacks the hydroxyl group. 5. Injection of 14C-Michler's ketone into rats resulted in the irreversible binding of radioactivity to liver proteins. 6. When the rats were pretreated with phenobarbital, this binding was increased and extended to proteins of some other tissues and to DNA and RNA of liver and DNA of kidney.

Macromolecular binding and metabolism of the carcinogen 4-chloro-2-methylaniline.

Biochemical investigations relating to the mechanism of action and mechanism of activation have been made for the carcinogen, 4-chloro-2-methylaniline. Radioactivity from 4-chloro-2-[methyl-14C]methylaniline became extensively bound to protein, DNA, and RNA of rat liver, but macromolecules of some of the other tissues examined contained little radioactivity. Enzymatic activity dependent upon reduced nicotinamide adenine dinucleotide and leading to irreversible binding to radioactivity from labeled 4-chloro-2-methylaniline to macromolecules in the reaction system was present in microsomes from rat liver. The activity was inducible by phenobarbital. Two soluble products of microsomal enzymes were identified by mass spectral analysis and chemical synthesis as 5-chloro-2-hydroxylaminotoluene and 4,4'-dichloro-2,2'-dimethylazobenzene. The hydroxylamino compound appears to be a more activated form of 4-chloro-2-methylaniline.

Selective inhibition and kinetics of enzymatic reactions leading to irreversible binding of benzo[a]pyrene to microsomal macromolecules of mouse lung.

Two enzymatic reactions, catalyzed by mouse lung microsomes and distinguishable by selective inhibition and kinetic studies, lead to irreversible binding of benzo[a]pyrene to macromolecules present in vitro reaction systems. One type (low Km) is inducible in the lungs of mice by treatment with benz[a]anthracene and is subject to inhibition by 7,8-benzoflavone. The other type (high Km) is predominant in lungs of untreated mice, but a small amount of low-Km activity is also present. The high-Km activity may be involved in carcinogenesis by benzo[a]pyrene, for it is inhibited by butylated hydroxytoluene, retinol or disulfiram, each of which is reported to have anticarcinogenic activity in intact animals.

Inhibition of benzo(alpha)pyrene metabolism catalyzed by mouse and hamster lung microsomes.

Induced and constitutive microsomal enzymes of mouse and hamster lungs catalyze both the hydroxylation of benzo(alpha)pyrene and reactions that lead to its irreversible binding to macromolecules. For mouse and hamster, the induced lung hydroxylases have Km values of 1.10 and 0.52 muM, respectively. The induced hydroxylases are strongly inhibited by 7,8-benzoflavone and are stimulated by cyclohexene oxide, an inhibitor of epoxide hydrase. Formation of the macromolecular product by the induced "binding" enzyme follows. Michaelis-Menten kinetics, except for substrate inhibition, and has Km values of 0.52 and 0.25 muM for lung microsomes from mouse and hamster, respectively. These reactions are also inhibited by 7,8-benzoflavone. The reaction catalyzed by the constitutive hydroxylase of mouse lungs is characterized by a brief lag period but proceeds in a linear fashion after the lag. The enzyme requires 60 muM benzo(alpha)pyrene to achieve maximum reaction velocity. Above this concentration, strong substrate inhibition is observed; accurate values for Vmax and Km cannot be derived. The constitutive hydroxylases are moderately inhibited by butylated hydroxytoluene, retinol, cyclohexene oxide, and 7,8-benzoflavone. The product of the constitutive "binding" enzyme is formed in a reaction that follows Michaelis-Menten kinetics. The Km value for enzymes from mouse and hamster lungs are 11.8 and 4.9 muM, respectively. Formation of this product is strongly inhibited by butylated hydroxytoluene and by retinol but not strongly by 7,8-benzoflavone or cyclohexene oxide. Since other evidence indicates that a constitutive enzyme may be involved in carcinogenesis by benzo(alpha)pyrene and since this reaction is inhibited by two known anticarcinogens, we suggest that it may be involved in this process.