A chitinase with antifungal activity from the mung bean

A chitinase with antifungal activity was isolated from mung bean (Phaseolus mungo) seeds. The procedure entailed aqueous extraction, (NH₄)₂SO₄ precipitation, ion-exchange chromatography on CM-Sepharose, high-performance liquid chromatography (HPLC) on Poros HS-20, and gel filtration on Sephadex G-75. The protein exhibited a molecular mass of 30.8 kDa in SDS–polyacrylamide gel electrophoresis. Its pI was 6.3 as determined by isoelectric focusing. The specific activity of the chitinase was estimated to be 3.81 U/mg. The enzyme expressed its optimum activity at pH 5.4 and was stable from 40 to 50 °C. It exerted antifungal action toward Fusarium solani, Fusarium oxysporum, Mycosphaerella arachidicola, Pythium aphanidermatum, and Sclerotium rolfsii.     

Superprecipitation of actomyosin with p-chloromercuribenzoate-modified myosin reconstituted from rabbit skeletal muscle

Myosin head modified with p-chloromercuribenzoate (CMB) forms rigor-like complex with actin in the presence of ATP. Actomyosins with CMB-modified myosin were reconstituted to study the effect of rigor-like complexes on superprecipitation. As native myosin was increasingly replaced by CMB-modified myosin, superprecipitation of the actomyosin was strongly suppressed. Further, the suppression of superprecipitation occurred in a different fashion depending on how CMB-modified myosin was incorporated in myosin filaments of the reconstituted actomyosin. The present results indicate that superprecipitation requires the dissociation of actin and myosin head to take place (i.e., the presence of molecular rearrangements of actomyosin network), and further suggest that superprecipitation is associated with dynamic rearrangements of actomyosin network along myosin filaments.     

Isolation and characterization of two 70 kDa modulator-complexes from rabbit skeletal muscle

The activation as well as the inactivation of the ATP,Mg-dependent protein phosphatase has been shown to be totally dependent upon the presence of the modulator subunit. This modulator (inhibitor-2) is a heat stable protein and its isolation in pure form (32 kDa) always includes a boiling step. The boiled modulator fractions are known to be inhibitory to the phosphatase activity. Unboiled rabbit skeletal muscle preparations do not contain "free modulator", but two higher molecular weight complexes (70 kDa) can be isolated which have the 32 kDa modulator together with a 38 kDa protein. One complex is the already characterized inactive ATP,Mg-dependent phosphatase [FCM] while the second one, [MX], although seemingly of identical composition, does not exhibit phosphatase activity when measured under the usual conditions. The MX-complex does not inhibit the phosphatase activity unless subjected to a boiling step which dissociates the modulator subunit. The unboiled [MX] exhibits the activation as well as the inactivation characteristics of the free modulator.     

Dissociation and reassociation of rabbit skeletal muscle myosin

Whereas dissociation of rabbit skeletal muscle myosin light chains occurs at an increased temperature (25°) and in the obsence of divalent cations, reassociation of the myosin oligomer requires a low temperature (4°C) and the presence of divalent cations, thus resulting in the original light to heavy chain stoichiometry. With a 5–10 per cent release of alkali light chains, LC₁ and LC₃, and a 50 per cent dissociation of the Ca²⁺ binding light chain, LC₂, there is no significant decrease in myosin ATPase activity irrespective of the cation activator, however, there is an approximate 15–20 per cent decrease in actomyosin ATPase activity. With reassociation of the myosin oligomer, actomyosin ATPase activity is partially restored as well as the original number of Ca²⁺ binding sites.     

Tropomyosin shares immunologic epitopes with group A streptococcal M proteins

Tropomyosin is an alpha-helical coiled-coil protein with structural similarities to the streptococcal M protein. In order to show serologic cross-reactivity between streptococcal M proteins and tropomyosin, we selected from a panel of murine mAb those which reacted with M proteins and tropomyosins in the ELISA. Western blots were used to study the reactions of each mAb with human and rabbit cardiac and rabbit skeletal tropomyosins. The antibodies were further characterized for their reactions with the additional autoantigens myosin, actin, keratin, and DNA. Five mAb were found which reacted with either PepM5 or ColiM6 protein and tropomyosin in Western blots or ELISA. Two of the tropomyosin positive mAb were also antinuclear antibodies and were inhibited with DNA. In Western blots of cardiac tropomyosins, the mAb reacted with either the 70-kDa dimer of tropomyosin, the 35-kDa monomer, or both. Some differences were observed in the reactions of the mAb with the different tropomyosins in Western blots. The heart cross-reactive epitopes shared between M proteins and tropomyosin were in most instances shared with cardiac myosin. Differences were observed among the reactions of the mAb with the different tropomyosins. This report constitutes the first evidence of serologic cross-reactivity between streptococcal M proteins and tropomyosins.     

绿豆皮中黄酮类化合物提取工艺

研究了绿豆总黄酮的提取工艺。通过对固液比、乙醇体积分数、提取温度与提取时间的单因素实验确定水平点,设计4因素3水平实验,选用L9（3^4）正交表,优选绿豆总黄酮的最佳提取工艺为固液比1：50,φ（乙醇）为40％,提取温度70℃,提取时间120min。在此基础上得到绿豆皮中总黄酮的提取量为27．57mg／g,且5次平行的相对标准偏差为0．75％,总黄酮的平均回收率达97．5％。     

A non-specific lipid transfer protein with antifungal and antibacterial activities from the mung bean

A non-specific lipid transfer peptide (nsLTP) with antimicrobial activity was isolated from the mung bean (Phaseolus mungo) seeds. The procedure entailed aqueous extraction, ion exchange chromatography on CM-Sephadex and high performance liquid chromatography (HPLC) on POROS-HS-20. The peptide exhibited a molecular mass of 9.03 kDa in mass spectrometry. It exerted antifungal action toward Fusarium solani, Fusarium oxysporum, Pythium aphanidermatum and Sclerotium rolfsii, and antibacterial action against Staphylococcus aureus but not against Salmonella typhimurium. The lipid binding of this peptide was very similar to that of a previously described lipid transfer protein extracted from wheat seeds and maize seeds, indicating that it possessed lipid transfer activity. The present findings add to the scarcity of the literature on leguminous nsLTPs.     

Trinitrophenylation of rabbit skeletal myosin by 2,4,6-trinitrobenzene sulfonate and treatment of trinitrophenyl myosin with dithiothreitol

Rabbit skeletal myosin was trinitrophenylated with 2,4,6-trinitrobenzene sulfonate (TNBS) in the presence or absence of inorganic pyrophosphate (PP1). When myosin trinitrophenylated either in the presence or absence of PP1 was treated with dithiothreitol (DTT), the absorbance at 345 nm of both trinitrophenylated myosins was decreased, as though the trinitrophenyl groups bound to myosin were removed. The DTT treatment also essentially reversed the inhibition of the EDTA-ATPase and Ca-ATPase activities that was caused by trinitrophenylation of myosin. These effects of trinitrophenylation and of DTT treatment were independent of the presence or absence of PP1 during the trinitrophenylation. In contrast, the PP1-induced formation of a difference spectrum of trinitrophenylated myosin was not affected by the DTT treatment. On the basis of these observations, it is suggested that the "reactive lysine residues," trinitrophenylation of which resulted in inhibition of the ATPase activities, are different from those whose trinitrophenyl groups show an altered spectrum on addition of PP1.     

Phosphorylation of myosin light chain by a protease-activated kinase from rabbit skeletal muscle

A protease-activated protein kinase that phosphorylates the P light chain of myosin in the absence of Ca2+ and calmodulin has been isolated from rabbit skeletal muscle. The enzyme has properties similar to protease-activated kinase I from rabbit reticulocytes [S. M. Tahara and J. A. Traugh (1981) J. Biol. Chem. 256, 11588-11564], which has been shown to phosphorylate the P light chain of myosin [P. T. Tuazon, J. T. Stull, and J. A. Traugh (1982) Biochem. Biophys. Res. Commun. 108, 910-917]. The protease-activated kinase from skeletal muscle has been partially purified by chromatography on DEAE-cellulose, phosphocellulose and hydroxyapatite. The enzyme phosphorylates histone as well as the P light chain of myosin following activation by proteolysis. Stoichiometric phosphorylation of myosin light chain was observed with the protease-activated kinase and myosin light chain kinase. The sites phosphorylated by the protease-activated kinase and myosin light chain kinase were examined by two-dimensional peptide mapping following chymotryptic digestion. The phosphopeptides observed with the protease-activated kinase were different from those obtained with the Ca2+-dependent myosin light chain kinase, indicating that the two enzymes phosphorylated different sites on the P light chain of skeletal muscle myosin. When actomyosin from skeletal muscle was examined as substrate, the P light chain was phosphorylated following activation of the protease-activated kinase by limited proteolysis.     

Purification and properties of glucosidase I from mung bean seedlings

The microsomal enzyme fraction from mung bean seedlings was found to contain glucosidase activity capable of releasing [3H]glucose from the glucose-labeled Glc3Man9GlcNAc. The enzymatic activity could be released in a soluble form by treating the microsomal particles with 1.5% Triton X-100. When the solubilized enzyme fraction was chromatographed on DE-52, it was possible to resolve glucosidase I activity (measured by the release of [3H]glucose from Glc3Man9GlcNAc) from glucosidase II (measured by release of [3H]glucose from Glc2Man9GlcNAc). The glucosidase I was purified about 200-fold by chromatography on hydroxylapatite, Sephadex G-200, dextran-Sepharose, and concanavalin A-Sepharose. The purified enzyme was free of glucosidase II and aryl-glucosidase activities. Only a single glucose residue could be released from the Glc3Man9GlcNAc by this purified enzyme and the other product was the Glc2Man9GlcNAc. Furthermore, this enzyme was inhibited in a dose-dependent manner by kojibiose, an alpha-1,2-linked glucose disaccharide, but not by other alpha-linked glucose disaccharides. These data indicate that this glucosidase is a specific alpha-1,2-glucosidase. The pH optimum for the glucosidase I was about 6.3 to 6.5, and no requirements for divalent cations were observed. The enzyme was inhibited strongly by the glucosidase processing inhibitors, castanospermine and deoxynojirimycin, and less strongly by the plant pyrrolidine alkaloid, 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine. However, the enzyme was not inhibited by the mannosidase processing inhibitors, swainsonine, deoxymannojirimycin or 1,4-dideoxy-1,4-imino-D-mannitol. The stability of the enzyme under various conditions and other properties of the enzyme were determined.     

Mg++ -activated and -inhibited ATPases from mung bean hypocotyls

Mg⁺⁺-activated and inhibited ATPases were isolated from dark-grownmung bean hypocotyls. The enzymes hydrolyzed nucleoside tri-,di- and monophosphates and ß-glycerophosphate. Theeffect of Mg⁺⁺ was most marked when ATP and other nucleosidetriphosphates were used as substrates. Mg⁺⁺-activated ATPases: The activity of enzyme-I was localizedin the membranes and was not released by treatment with 0.1%deoxycholate. Enzyme-II was released and separated by CM-cellulosecolumn chromatography. Enzyme-V was separated from the solublefraction of the cell homogenate by DEAE-cellulose column chromatography.The rates of activivation by Mg⁺⁺ of enzyme-II and enzyme-Vwere very small compared to that of enzyme-I. Mg⁺⁺-inhibited ATPases: Enzyme-II and -IV were precipitatedwith 50–80% ammonium sulfate from the soluble fractionof the cell homogenate and were separated by successive columnchromatographies on Sepharose 6B and DEAE-cellulose. The activitiesof enzyme-III and -IV were inhibited by Mg⁺⁺, when ATP, UTPand GTP were used as substrates. Enzyme-III was purified approximately38-fold, and was more remarkably inhibited by Mg⁺⁺ than wasenzyme-IV. ¹Present address: Institute for Plant Virus Research, 959 Aobacho,Chiba 280, Japan. (Received January 7, 1974; )     

Calmodulin and myosin light-chain kinase of rabbit fast skeletal muscle.

1. It is confirmed that myosin light-chain kinase is a protein of mol.wt. about 80,000 that is inactive in the absence of calmodulin. 2. In the presence of 1 mol of calmodulin/mol of kinase 80-90% of the maximal activity is obtained. 3. Crude preparations of the whole light-chain fraction of rabbit fast-skeletal-muscle myosin contain enough calmodulin to activate the enzyme. A method for the preparation of calmodulin-free P light chain is described. 4. A procedure is described for the isolation of calmodulin from rabbit fast skeletal muscle. 5. Rabbit fast-skeletal-muscle calmodulin is indistinguishable from bovine brain calmodulin in its ability to activate myosin light-chain kinase. The other properties of these two proteins are also very similar. 6. Rabbit fast-skeletal-muscle troponin C was about 10% as effective as calmodulin as activator for myosin light-chain kinase. 7. By chromatography on a Sepharose-calmodulin affinity column evidence was obtained for the formation of a Ca2+-dependent complex between calmodulin and myosin light-chain kinase. 8. Troponin I from rabbit fast skeletal muscle and histone IIAS were phosphorylated by fully activated myosin light-chain kinase at about 1% of the rate of the P light chain.     

Intrinsic multiple antibiotic resistance markers for competitive and effectiveness studies with various strains of mung bean rhizobia

Ten strains ofRhizobium sp. with multiple antibiotic resistance markers were used for competitive and ef ficiency studies with mung bean var. ML 5. All the strains showed significant increase in grain yield and so also for nitrogenase activity except MO 5. Nitrogenase activity correlated very well with grain yeild. The compatibility of strains varied from 17 to 50%. The intrinsic multiple antibiotic markers for strain identification were found to be stable after passing through soil and host conditions and could be used for ecological studies. It was further revealed that the overall efficiency of a strain is the combined effect of characters like compatability, competitiveness and inherent capacity to fix nitrogen.     

Reovirus protein sigmaNS binds in multiple copies to single-stranded RNA and shares properties with single-stranded DNA binding proteins

Reovirus nonstructural protein sigmaNS interacts with reovirus plus-strand RNAs in infected cells, but little is known about the nature of those interactions or their roles in viral replication. In this study, a recombinant form of sigmaNS was analyzed for in vitro binding to nucleic acids using gel mobility shift assays. Multiple units of sigmaNS bound to single-stranded RNA molecules with positive cooperativity and with each unit covering about 25 nucleotides at saturation. The sigmaNS protein did not bind preferentially to reovirus RNA over nonreovirus RNA in competition experiments but did bind preferentially to single-stranded over double-stranded nucleic acids and with a slight preference for RNA over DNA. In addition, sigmaNS bound to single-stranded RNA to which a 19-base DNA oligonucleotide was hybridized at either end or near the middle. When present in saturative amounts, sigmaNS displaced this oligonucleotide from the partial duplex. The strand displacement activity did not require ATP hydrolysis and was inhibited by MgCl(2), distinguishing it from a classical ATP-dependent helicase. These properties of sigmaNS are similar to those of single-stranded DNA binding proteins that are known to participate in genomic DNA replication, suggesting a related role for sigmaNS in replication of the reovirus RNA genome.     

Structural studies of rabbit skeletal muscle myosin light chain kinase with monoclonal antibodies

Monoclonal antibodies directed against rabbit skeletal muscle myosin light chain kinase have been used to study the domains of this kinase. Specificity of nine monoclonal antibodies against rabbit skeletal muscle myosin light chain kinase was demonstrated by immunoblot analysis and immunoadsorption of kinase activity. None of the antibodies reacted by immunoblot analysis with either chicken skeletal or rabbit smooth muscle myosin light chain kinases. Epitope mapping of trypsin-digested rabbit skeletal muscle myosin light chain kinase showed that antibodies 2a, 9a, 9b, 12a, 12b, 16a, and 16b are directed against the 40-kDa catalytic domain. In addition, these seven antibodies reacted with sites that are clustered within a 14-kDa fragment of the kinase generated by Staphylococcus aureus V8 protease digestion. Two monoclonal antibodies, 14a and 19a, reacted with two distinct epitopes located within the inactive, asymmetric trypsin fragment. Six of nine monoclonal antibodies (2a, 9a, 9b, 12a, 12b, and 14a) inhibited kinase activity. Kinetic analyses demonstrated that antibodies 2a, 12a, and 14a inhibited kinase activity competitively with respect to myosin phosphorylatable light chain; 2a, 12a, and 14a exhibit noncompetitive inhibition with respect to calmodulin. These data suggest that monoclonal antibodies 2a, 12a, and 14a bind at or adjacent to the active site of the kinase.