Site-directed mutagenesis of glycine-14 and two "critical" cysteinyl residues in Drosophila alcohol dehydrogenase

Three amino acid residues (glycine-14, cysteine-135, and cysteine-218) previously speculated to be important for the structure and function of Drosophila melanogaster alcohol dehydrogenase have been investigated by using site-directed mutagenesis followed by kinetic analysis and chemical modification. Mutating glycine-14 to valine (G14V) virtually inactivates Drosophila ADH, and substitution of alanine at this position (G14A) causes a 31% decrease in activity. Thermal denaturation and kinetic and inhibition studies further demonstrate that replacing glycine-14 with either alanine or valine leads to structural changes in the NAD binding domain. These results provide direct evidence for the role played by glycine-14 in maintaining the correct conformation in the NAD binding domain. On the other hand, changing of cysteine-135, -218, or both to alanine (C135A, C218A, and C135A/C218A) causes no decrease in the catalytic activity of the enzyme, indicating that neither of the cysteinyl residues is essential for catalysis. C135A and wild-type enzyme are both inactivated by DTNB. In contrast, C218A and C135A/C218A are unaffected by DTNB treatment. DTNB modification of cysteine-218 can be prevented by the substrates NAD and 2-propanol, suggesting that cysteine-218 may be in the vicinity of the active site. Cysteine-135 which is normally insensitive to DTNB becomes accessible in the presence of 2-propanol and/or NAD, suggesting a conformational change induced by binding of these substrates.     

Surface-enhanced resonance raman scattering spectroscopy of bacterial photosynthetic membranes: orientation of the carotenoids of Rhodobacter sphaeroides 2.4.1

Surface-enhanced resonance Raman scattering (SERRS) spectra were obtained from carotenoids, in the all-trans configuration, located on the antenna complexes of Rhodobacter sphaeroides 2.4.1 membranes. Since resonance Raman (RR) spectra are barely detectable at the concentration that SERRS signals saturate, SERRS represents a very sensitive means of detecting pigments in biological systems. Prominent SERRS spectra of sphaeroidenone were detected in chromatophores (cytoplasmic side out) but not in spheroplast-derived vesicles (periplasmic side out), demonstrating that the carotenoid is asymmetrically located on the cytoplasmic side of the cell membrane. Comparison of peak frequencies from SERRS and RR spectral data suggests that the carotenoids are oriented into the membrane with the methoxy end of the isoprenoid chains located closest to the cytoplasmic side of the intracytoplasmic membrane. This work not only shows that SERRS spectroscopy can provide information on the location of a chromophore in a biological membrane but also for the first time demonstrates that SERRS data can be used to ascertain the orientation of a chromophore within the membrane. This observation greatly increases the potential of this technique for structural analysis of intact membranes at the molecular level.     

Controlled potential enzymology of methyl transfer reactions involved in acetyl-CoA synthesis by CO dehydrogenase and the corrinoid/iron-sulfur protein from Clostridium thermoaceticum

Many anaerobic bacteria fix CO2 via the Wood pathway of acetyl-CoA synthesis. Carbon monoxide dehydrogenase (CODH), also called acetyl-CoA synthase, accepts the methyl group from the methylated corrinoid/iron-sulfur protein (C/Fe-SP), binds a carbonyl group from CO, CO2, or the carboxyl of pyruvate, and binds coenzyme A. Then CODH catalyzes the synthesis of acetyl-CoA from these enzyme-bound groups. Here, we have characterized the methyl transfer steps involved in acetyl-CoA synthesis. We have studied the reactions leading to methylation of CODH by methyl iodide and shown an absolute requirement of the C/Fe-SP in this reaction. In addition, we have discovered and partly characterized two previously unknown exchange reactions catalyzed by CODH: between the methylated C/Fe-SP and methylated CODH and between methylated CODH and the methyl moiety of acetyl-CoA. We have performed these two exchange reactions, methylation of the C/Fe-SP, and methylation of CODH at controlled potentials. The rates of all these reactions except the exchange between methylated C/Fe-SP and methylated CODH are accelerated (from 1 to 2 orders of magnitude) when run at low potentials. Our results provide strong evidence for a nucleophilic redox-active metal center on CODH as the initial acceptor of the methyl group from the methylated C/Fe-SP. This metal center also is proposed to be involved in the cleavage of acetyl-CoA in the reverse reaction.     

Ca2(+)-activated K+ channels from rabbit kidney medullary thick ascending limb cells expressed in Xenopus oocytes.

Ca2(+)-activated K+ channels are present in muscle, nerve, pancreas, macrophages, and renal cells. They are important in such diverse functions as neurotransmitter release, muscle excitability, pancreatic secretion, and cell volume regulation. Although much is known about the biophysics of Ca2(+)-activated K+ channels, the molecular structure, cDNA and amino acid sequences are unknown. We injected size-fractionated mRNA isolated from cultured rabbit kidney medullary thick ascending limb cells in Xenopus oocytes and observed newly expressed K+ currents using two-microelectrode voltage-clamp technique. The expressed K+ currents are Ca2+ dependent and inhibited by charybdotoxin, a specific blocker of Ca2(+)-activated K+ channels. Amplitudes of the current ranged from 30 nA to more than 1 microA at a membrane potential of +30 mV. Reversal potential of the current suggested a K(+)-selective channel. The peak activity of Ca2(+)-activated K+ channels were observed in fractions corresponding to a message RNA with size of approximately 4.5 kilobases.     

The effect of sugars on inverse relation between the growth and chlorophy11 synthesis in tobacco tissue

Cytokinin-dependent and cytokinin-autonomous strains of tobacco callus tissue (Nicotiana tabacum L. cv. ‘Wisconsin 38’) were grown on media containing sucrose, glucose and fructose, respectively. The tissues were kept 14 days in darkness and then transferred for 9 days to continuous light after which time the fresh weight and chlorophyll content were estimated. The highest chlorophyll concentration was recorded at sugar levels which were either suboptimal (sucrose in the case of cytokinin-dependent strain) or supraoptimal (all other sugars for both strains and sucrose for the cytokinin-autonomous strain) for tissue growth. The chlorophyll concentration was increased when the tissue was cultured on media containing glucose or fructose,i.e. sugars whioh did not support the growth as well as sucrose. Chlorophyll synthesis in the cytokinin-autonomous strain is significantly lower than in the cytokinin-dependent strain. This difference was independent of either sugar source or concentration. These results support the observed inverse relationship between tissue growth and plastid development and the limited metabolic activity of plastids in cytokinin-autonomous tissues.     

Effects of chloramine on Bacillus subtilis deoxyribonucleic acid.

The lesions induced in Bacillus subtilis deoxyribonucleic acid (DNA) after treating bacterial cells (in vivo) and bacterial DNA (in vitro) with chloramine were studied biologically and physically. Single-strand breaks and a few double-strand scissions (at higher chloramine doses) accompanied loss of DNA-transforming activity in both kinds of treatments. Chloramine was about three times more efficient in vitro than in vivo in inducing DNA single-strand breaks. DNA was slowly chlorinated; the subsequent efficiency of producing DNA breaks was high. Chlorination of cells also reduced activity of endonucleases in cells; however, chlorinated DNA of both treatments was sensitized to cleavage by endonucleases. The procedure of extracting DNA from cells treated with chloramine induced further DNA degradation. Both treatments introduced a small fraction of alkali-sensitive lesions in DNA. DNA chlorinated in vitro showed further reduction in transforming activity as well as further degradation after incubation at 50 C for 5 h whereas DNA extracted from chloramine-treated cells did not show such a heat sensitivity.     

l-Ornithine:2-Oxoacid Aminotransferase from Squash (Cucurbita pepo, L.) Cotyledons: Purification and Properties

ORNITHINE: 2-oxoacid aminotransferase (EC 2.6.1.13) has been purified over 400-fold with a total recovery of 14% from acetone powders of cotyledons of germinating squash (Cucurbita pepo, L.) seedlings. The pH optimum of the transamination between l-ornithine and alpha-ketoglutarate is 8 and the Michaelis constants are 4.7 mm and 6.3 mm, respectively. The enzyme has a molecular weight of 48,000 as determined by gel filtration. The reaction is essentially specific for alpha-ketoglutarate as the amino group acceptor. The enzyme is inhibited very strongly by hydroxylamine, and less severely by NaCN and isonicotinylhydrazide. No inhibition is observed in the presence of 10 mml-cysteine. The energy of activation is 7.6 kcal/mole. The stability of the enzyme preparation is enhanced by the presence of dithioerythritol and glycerol. The enzyme activity of the most purified fraction is stimulated 30% by the addition of pyridoxal phosphate; however, the evidence for the unequivocal involvement of pyridoxal phosphate was inconclusive.     

ALKBH5/MAP3K8 axis regulates PD-L1+ macrophage infiltration and promotes hepatocellular carcinoma progression

Hepatocellular carcinoma is one of the most common malignant tumors.M6A is a novel epigenetic modification that have been emerged as vital regulators for the progression of HCC. However, the regulatory role, clinical significance and the details of the modification, such as the impact on the local tumor environment, remain largely unclear. Our study showed that ALKBH5 was highly expressed in HCC and high ALKBH5 expression predicted a worse prognosis of HCC patients. Prediction of ALKBH5 function by tissue samples and single cell sequencing Gene Set Variation Analysis. Primary CD3 + T lymphocytes and bone marrow-derived macrophages were used to evaluate the effect of ALKBH5 on immune microenvironment. The results indicated that ALKBH5 promote HCC cell proliferation, metastasis and PD-L1+macrophage recruitment. Mechanistically the results showed that ALKBH5 regulates MAP3K8 expression in a m6A dependent manner which mediates the proliferation and metastasis of HCC cells. ALKBH5 also promotes the activation of JNK and ERK pathways through upregulating MAP3K8, thus regulating the expression of IL-8 and promoting macrophage recruitment. Taken together, these data show that ALKBH5 promotes HCC growth, metastasis and macrophage recruitment through ALKBH5/MAP3K8 axis and it may serve as a potential diagnostic marker and target for treatment of HCC patients.