High resolution X-ray structure of yeast hexokinase,an allosteric protein exhibiting a non-symmetric arrangement of subunits

The structure determination of yeast hexokinase has been extended to 3.5 Å resolution for the dimer and to 2.7 Å resolution for the monomer using multiple isomorphous replacement. The electron density maps of both the monomer and dimer crystal forms have been substantially improved by an averaging procedure. From these maps the course of the polypeptide backbone and some aspects of the dimer interaction have been established.The hexokinase subunit arrangement is contrary to a major tenet of the Monod et al. (1965) theory of allosteric proteins which postulated that only symmetric or isologous interactions of subunits would occur in oligomeric proteins. One subunit of the dimer is related to the other by a 156 ° rotation about and a 13.8 Å translation along a molecular screw axis. In the hexokinase dimer the set of residues in one subunit that is interacting with the other subunit is different from the set of residues in the second subunit that is interacting with the first subunit. This heterologous or non-symmetric interaction of subunits is associated with some small differences in the structure of the two subunits, particularly at the subunit interface, and accounts for some of this enzyme's non-symmetric interactions with substrates and activators. Indeed, the non-symmetric subunit association may play an important role in the control of this enzyme's activity.The overall structure of hexokinase is considerably different than the known structures of the other enzymes in the glycolytic pathway. Although there is a striking similarity between the domain of hexokinase that binds AMP and the domain of lactate dehydrogenase that binds NAD, the former structure contains both antiparallel and parallel β-pleated strands, while the latter contains only parallel β-structure. In an attempt to assess the significance of this structural similarity, the structure of the nucleotide binding domains of hexokinase and lactate dehydrogenase are compared to a portion of carboxypeptidase A. The observed similarities among these structures suggests that a central β-pleated sheet flanked by α-helices is a common supersecondary structure that probably arose by convergent as well as divergent evolution. Thus, there appears to be no compelling evidence at this time to support the hypothesis that a part of hexokinase has evolved from the same gene as the dinucleotide binding domain of lactate dehydrogenase.     

Porphyrins in the perienteric fluid of Ascaris lumbricoides.

Cain G. D. and Bassow F. 1976. Porphyrins in the perienteric fluid of Ascaris lumbricoides. International Journal for Parasitology6: 79–82. Porphyrins in the perienteric fluid of adult female A. lumbricoides were esterified in methanolic H₂SO₄, extracted in chloroform, separated by thin-layer chromatography, and identified spectrophotometrically before and after conversion to their zinc and copper chelates. Protoporphyrin IX was the major component, comprising 95·4% of the total; the remaining 4·6 % was coproporphyrin III. Uroporphyrin was not detected; no porphyrins were recovered from other worm tissues. Fluid from worms with light and dark colored guts varied in protoporphyrin content from 0·58 to 4·08 nmoles/ml, respectively, but fluid from both groups contained similar molar ratios of protoporphyrin, coproporphyrin and heme.     

Proceedings: Differential induction of chromosome aberrations in mammalian cell lines.

Mutagenesis was studied in repair- and recombination-deficient strains of Haemophilus influenzae after treatment with N-nitrosocarbaryl (NC). Three different strains of H. influenzae carrying mutations affecting excision-repair of UV-induced pyrimidine dimers exhibited normal repair of premutational lesions (as detected by decreased mutation yield resulting from post-treatment DNA synthesis delay) and normal nonreplicative mutation fixation. This indicates that neither of these phenomena are caused by the same repair mechanism that removes UV-induced pyrimidine dimers from the DNA.The recombination-deficient mutant rec1 is apparently deficient in the replication-dependent mode of NC-induced mutation fixation. This conclusion is based on the following results: (1) NC-induced mutagenesis is lower in the rec1 strain than in rec⁺ cells. (2) Repair of premutational lesions (which depends on the existence of replication-dependent mutation fixation for its detection) was not detected in the rec1 strain. (3) When nonreplicative mutation fixation and final mutation frequency were measured in the same experiment, about $\text{1}\text{4}$ to $\text{1}\text{3}$ of the final mutation yield could be accounted for by nonreplicative mutation fixation in the rec⁺ strain, whereas all of the mutation could be accounted for in the rec1 strain by the nonreplicative mutation fixation. (4) When mutation fixation in strain dna9 rec1 was followed at the permissive (36°) and nonpermissive (41°) temperatures, it became apparent that in the rec1 strain replication-dependent mutation fixation occurs at early times, but these newly fixed mutations are unstable and disappear at later times, leaving only the mutations fixed by the nonreplicative process.The rec1 strain exhibits normal repair of NC-induced single-strand breaks or alkali-labile bonds in the DNA labeled before treatment, but is slow in joining discontinuities present in DNA synthesized after treatment. The results are consistent with the idea that in NC-treated H. influenzae cells the replication-dependent mode of mutation fixation occurs by error-prone joining of interruptions present in the DNA synthesized after treatment. The possibility still exists, however, that during DNA replication mispairing occurs opposite certain alkylation-induced lesions and that mutations arising during replication of strain rec1 later disappear as a result of degradation of newly synthesized DNA, which is excessive in this strain.     

The measurement of chemically-induced DNA repair synthesis in human cells by BND-cellulose chromatography.

Repair synthesis in human cells in tissue culture can be readily separated from semi-conservative DNA synthesis with the aid of a benzoylated naphthoylated DEAE cellulose (BND-cellulose) column. Cells are incubated with a radioactive DNA precursor during treatment with a repair-inducing agent. An inhibitor of semi-conservative DNA synthesis (hydroxyurea) is added to slow the progression of the DNA growing point. The cells are lysed and after treatment with ribonuclease and pronase the lysates are sheared and passed through a BND-cellulose column. Native DNA is eluted with I M NaCl. Any increase in radioactivity in the native DNA is due to repair synthesis and the specific repair activity (nucleotides inserted per mug of DNA) can be determined from radioactivity and absorbancy measurements. Repair can also be measured in the region of the DNA growing point by fractionation of the material eluted from BND-cellulose with 50% formamide. Repair was not detected in N-acetoxy-2-acetylaminofluorene (AAAF)-treated lymphoblasts derived from an individual with xeroderma pigmentosum although methyl methanesulfonate (MMS)-induced repair was observed in these cells.     

Mutagenicity of organophosphorus compounds in bacteria and drosophila

140 Organophosphorus compounds (OP's) have been tested for mutagenic activity in bacteria, principally by using two specially constructed sets of tester strains of the bacteria Salmonella typhimurium and Escherichia coli. It was found that 20% gave positive mutagenic responses and that this group of chemicals produce base substitutions rather than frame-shift mutations. In most cases the DNA repair genes exrA⁺ and recA⁺ were for mutagenic activity.Seven compounds were further tested in Drosophila melanogaster for the ability to induce recessive lethal mutations. In some of these cases the doses administered to the flies had to be very low due to the highly toxic nature of the compounds. To overcome this problem, the accumulation of recessive lethal mutations was measured in populations which were continually exposed to the compounds over a period of some 18 months. During this time the populations developed increased resistance to the compound and so the dose administered could gradually be increased. Six of the compounds were mutagenic.Of the compounds tested in both systems, those showing mutagenic activity in bacteria were also mutaganic in Drosophila, those mutagenic in bacteria were not mutagenic in Drosophila.     

New organic bases from amazonian Banisteriopsis caapi

Three new bases were isolated from Banisteriopsis caapi; they are harmine N-oxide, harmic acid methyl ester (methyl 7-methoxy-β-carboline 1-carboxylate) and harmalinic acid (7-methoxy-3,4-dihydro-β-carboline 1-carboxylic acid).     

糖皮质激素抑制加压素释放的细胞膜机制

利用离休孵育脑薄片和放射免疫测定其释放的精氨酸加压素（ＡＶＰ）方法,探讨糖皮质激素（ＧＣ）在不能进入细胞内的情况下,对去肾上腺大鼠的下丘脑薄片释放ＡＶＰ的快速影响及其可能的细胞膜机制。结果如下：（１）下丘脑薄片能够稳定地释放ＡＶＰ（２ｈ）,其释放量为１５．４２±１．２８ｐｇ／ｍｉｎ;（２）牛血清白蛋白耦联皮质酮（Ｂ－ＢＳＡ）对ＡＶＰ的释放具有快速的（２０ｍｉｎ）抑制性效应,在１０￣（－７）─１０￣（－４）ｍｏｌ／Ｌ范围内呈剂量一效应关系;（３）ＧＣ细胞内受体拮抗剂ＲＵ４８６（１０￣（－４）─１０￣（－３）ｍｏｌ／Ｌ）能部分地阻断Ｂ─ＢＳＡ的快速抑制效应;（４）孵育液中Ｃａ￣（２＋）程度升高,Ｂ─ＢＳＡ的快速抑制效应明显增强;反之,孵育液中无Ｃａ￣（２＋）则Ｂ－ＢＳＡ的快速抑制效应有所减弱。表明ＧＣ在未进入细胞内的情况下也可快速地抑制大鼠下丘脑薄片释放ＡＶＰ,因此没有通过传统的基因组机制,而是由非基因组机制介导的,其作用部位在细胞膜水平上,可能是影响Ｃａ￣（２＋）的跨细胞膜内流通量或／和影响有Ｃａ￣（２＋）参与的ＡＶＰ释放过程的结果。     

Expression and purification of recombinant cynomolgus monkey cholesteryl ester transfer protein from Chinese hamster ovary cells

Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl ester from high- and low-density lipoproteins to triglyceride-rich lipoproteins, and reciprocally mediates triglyceride transfer. The gene for cynomolgus monkey CETP was expressed in serum-free CHO culture with 2g/ml insulin as its only exogenous protein supplement. Cell growth was facilitated by immobilizing the CHO cells in alginate beads. Recombinant CETP (rCETP) was purified 176-fold with a three-step protocol resulting in a 60% final yield as measured by a fluorescent CETP activity assay. Typically, 3.4 mg of rCETP was purified from 1700 ml of media by affinity-gel chromatography involving Reactive Red 120 (RR120) followed by concanavalin A Sepharose 4B and rechromatography on RR120. SDS-PAGE shows a single broad band ofM _r , ranging from 68,000 to 74,000 which immunoreacts in Western blot analysis. Amino acid analysis and protein sequencing of the purified protein agree with the theoretical amino acid composition and sequence of cynomolgus CETP.     

Synthesis,evaluation and CoMFA/CoMSIA study of nitrofuranyl methyl N-heterocycles as novel antitubercular agents

A series of novel nitrofuranyl methyl N-heterocycles based on the structure of IIIM-MCD-211 were designed and synthesized. Compounds 6d, 8b and 12a show excellent activity against MTB H37Rv strain (MIC: 0.031–0.062?μg/mL) roughly comparable to INH and IIIM-MCD-211. In addition, a three-dimensional quantitative structure-activity relationship (3D-QSAR) study was performed on the above mentioned chemical series employing comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) techniques. The developed CoMFA and CoMSIA models display high external predictability (r²_pred of 0.954 and 0.935, respectively) and good statistical robustness. More importantly, the newly designed compounds 16a and 16b (MIC: <0.016?μg/mL) based on the two models, as expected, were found to be more active than 12a and IIIM-MCD-21. Design and synthesis of more potent nitrofuranyl methyl N-heterocycles as anti-TB agents are currently in progress.