Microbial transformation of rifamycin B: A new extracellular oxidase fromCurvularia lunata

Summary A highly active extracellular rifamycin oxidase was isolated fromCurvularia lunata var.aeri. The enzyme has a pH optimum of 6.5 and temperature optimum of 50°C.     

Detection of antimycin-binding subunits of Complex III by photoaffinity-labeling with an azido derivative of antimycin

Deformamidoazidoantimycin A (DAA), a photoactive derivative of antimycin A containing an azido group substituting for the formamido group attached to the phenyl ring, was synthesized. The ultraviolet spectrum of DAA was almost identical to that of antimycin A, indicating little alteration of the electronic structure of the substituted phenyl ring by the azido substitution. However, the inhibitory effectiveness of DAA toward ubiquinol-cytochromec reductase (Complex III) purified from bovine heart (K _i =ca. 0.5 µM) was considerably less than that of antimycin (K _i 3 pM), indicating a direct rather than a supporting role of the formamido group in the inhibitory activity of antimycin. Exposure of purified Complex III to [³H]DAA plus ultraviolet light caused a major labeling by tritium of SDS-PAGE band 7 (m=13 kDa by SDS-PAGE) and lesser but significant labeling of bands 3, 6, 8, and 9. Pretreatment of Complex III with antimycin greatly suppressed the labeling of bands 5, 6, and 7 but caused an apparent increased labeling of bands 8 and 9 by [³H]DAA, respectively. The labeling of band 7 by [³H]DAA also was strongly suppressed by reduction of Complex III by either sodium borohybride or ascorbate. Based on magnitude of labeling by [³H]DAA and the degree of suppression of labeling by antimycin, the protein of band 7 qualified as the principal component for specific binding of antimycin with the protein of band 6 (m=16 kDa) showing a lesser but significant amount of specific binding.     

Purification and characterization of catalase from goat (Capra capra) lung

Catalase plays a major role in the protection of tissues from toxic effects of H₂O₂ and partially reduced oxygen species. In the present study catalase was extracted and purified 330-fold from goat lung by acetone fractionation and successive chromatographies on DEAE-cellulose, Sephadex G-200, Blue Sepharose CL-6B and Ultrogel AcA-34. The purified enzyme was almost homogeneous as judged by polyacrylamide gel electrophoresis and FPLC. The molecular weight and Stokes' radius of the purified enzyme were 339 kDa and 127±2 Å. The enzyme had 11 sulfhydryl groups and 15 tryptophan groups per mol of the enzyme. A broad pH optimum in the range 5.2 to 7.8 was obtained. Sulfhydryl group binding agents, thiol reagents and N-Bromosuccinimide inhibited the enzyme activity. The kinetic data show no cooperativity between the substrate binding sites. Tryptophan, indole acetic acid, cysteine, formaldehyde and sodium azide inhibited the enzyme non-competitively with K_i values of 1.5, 1.6, 6.7, 0.55 and 0.0017 mM, respectively.     

Primary structure and properties of the Na+/glucose symporter (Sg1S) of Vibrio parahaemolyticus.

Previously, we cloned and sequenced a DNA fragment from Vibrio parahaemolyticus and found four open reading frames (ORFs). Here, we clearly demonstrate that one of the ORFs, ORF1, is the gene (sglS) encoding a Na+/glucose symporter (SglS). We characterize the Na+/glucose symporter produced in Escherichia coli mutant (JM1100) cells which lack original glucose transport activity and galactose transport activity. We also show that phlorizin, a potent inhibitor of the SGLT1 Na+/glucose symporter of animal cells, inhibited glucose transport, but not galactose transport, via the SglS system.     

Initiation of Protein Synthesis Without Formylation in a Mutant of Escherichia coli That Grows in the Absence of Tetrahydrofolate

Starting from a p-aminobenzoate-requiring strain of Escherichia coli (E. coli K-12 AB3292), we have isolated mutants that can grow in the absence of p-aminobenzoate (and thus tetrahydrofolate). The following lines of evidence suggest that at least one of these mutants is capable of initiating protein synthesis without formylation of methionyl-transfer ribonucleic acid (methionyl-tRNA(fMet)). (i) tRNA isolated (and charged in vivo with [(35)S]methionine) from this mutant grown in a p-aminobenzoate-free medium contained less than 0.4% of the total methionine charged to the tRNA as formylmethionine. However, when the mutant was grown in the presence of p-aminobenzoate, 40 to 50% of the total [(35)S]methionine was detected as formylmethionine. (ii) Extracts of the mutant grown in the absence of p-aminobenzoate contained no formyl-tetrahydrofolate, but such extracts did contain formylatable methionyl-tRNA and a functional transformylase. (iii) Tetrahydrofolate-free extracts of the mutant were capable of supporting protein synthesis with viral RNA (from f2) as messenger, but the resulting synthesized proteins contained no formylmethionine, and methionine residues were detected where formylmethionine residues are normally found. In the presence of formyl-tetrahydrofolate, use of a similar extract resulted in the detection of 30 to 40% of the total polypeptide methionine as formylmethionine. (iv) Initiation of protein synthesis in vitro occurred more readily with formyl-tetrahydrofolate-free extracts of the mutant than with similar extracts prepared from the parent strain. However, in the presence of formyl-tetrahydrofolate, initiation of protein synthesis proceeded equally well with both kinds of extracts. tRNA from this mutant and another spontaneously derived mutant was found to be partially deficient in the modified nucleoside ribothymidine (rT). Analysis of extracts showed that the mutants contained decreased levels of the methylase that results in the formation of ribothymidine. In vivo studies with an independently isolated rT(-) strain suggest that the lack of rT in tRNA facilitates the growth of E. coli under conditions where protein synthesis is forced to take place without formylation.     

Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor tRNA genes

We describe the generation of mammalian cell lines carrying amber suppressor genes. Nonsense mutants in the herpes simplex virus thymidine kinase (HSV tk) gene, the Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco-gpt) gene and the aminoglycoside 3′ phosphotransferase gene of the Tn5 transposon (NPT-II) were isolated and characterized. Each gene was engineered with the appropriate control signals to allow expression in both E. coli and mammalian cells. Expression in E. coli made possible the use of well developed bacterial and phage genetic manipulations to isolate and characterize the nonsense mutants. Once characterized, the nonsense mutants were transferred into mammalian cells by microinjection and used, in turn, to select for amber suppressor genes. Xenopus laevis amber suppressor genes, prepared by site-specific mutagenesis of a normal X. laevis tRNA gene, were microinjected into the above cell lines and selected for the expression of one or more of the amber mutant gene products. The resulting cell lines, containing functional amber suppressor genes, are stable and exhibit normal growth rates.     

Secondary structure of mouse and rabbit α- and β-globin mRNAs: Differential accessibility of α and β initiator AUG codons towards nucleases

The nucleotide sequence from the 5′ terminus inward of one third of mouse α- and β^maj-globin messenger RNAs has been established. In addition, using 5′ ³²P end-labeled mRNAs as substrates and S1 and T1 nucleases as probes for single-stranded regions, the secondary structures of mouse and rabbit α- and β-globin mRNAs have been analyzed. Our results indicate that the AUG initiator codon in both mouse and rabbit β-globin mRNA is quite susceptible to cleavage with S1 and T1 nucleases, suggesting that it resides in a single-stranded exposed region. In contrast, the initiator AUG in the α-globin mRNA of both species is inaccessible to cleavage, indicating that it is either buried by tertiary structure or is base-paired. Since the rate of initiation of protein synthesis with β-globin mRNA in rabbit reticulocyte is 30–40% faster than for α-globin mRNA, these results imply a possible correlation between the differential rates of initiation with these two mRNAs and the accessibility of the respective AUG initiator codons.     

Detection of conformational changes in Complex III of the respiratory chain by a maleimido spin label

Changes in the conformation of Complex III (CoQH₂-cytochromec reductase) of the mitochondrial respiratory chain were detected upon oxidoreduction using the nitroxide spin label, 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl. EPR spectra of the spin label show a transition from a greater to a lesser degree of immobilization when the labeled enzyme, reduced either with ascorbate or sodium dithionite, is oxidized with potassium ferricyanide or ferricytochromec. These observations are interpreted to indicate that Complex III is more compact in the reduced state at least in the locality of the spin label. An apparent increase in the concentration of total spins during oxidation of the complex suggests change in the interaction between the spin label and other paramagnetic centers and not an oxidation of spin label, itself, since reduced free spin label could not be reoxidized. Addition of antimycin A had no effect on the EPR spectrum of the spin-labeled enzyme, indicating that this inhibitor does not initiate a conformational change in the region of the spin label. Experiments in which N-ethyl-[2-³H] maleimide was bound to Complex III show that binding occurs primarily to a subunit with a molecular weight of 45,000. Although no qualitative differences were observed, it was found that less radioactivity appears in samples reduced with dithionite than in those reduced with ascorbate. This difference appears to be caused by decomposition products of dithionite.     

Virtual Screening to Identify Novel Inhibitors of Pan ERBB Family of Proteins from Natural Products with Known Anti-tumorigenic Properties

International Journal of Peptide Research and Therapeutics - Overexpression of ERBBB family of receptors (ERBB1, ERBB2, ERBB3 and ERBB4) has been found to be hyper-activated in a number of...