Regio- and stereo-selective metabolism of 4-methylbenz[a]anthracene by the fungus Cunninghamella elegans.

Metabolism of 4-methylbenz[a]anthracene by the fungus Cunninghamella elegans was studied. C. elegans metabolized 4-methylbenz[a]anthracene primarily at the methyl group, this being followed by further metabolism at the 8,9- and 10,11-positions to form trans-8,9-dihydro-8,9-dihydroxy-4-hydroxymethylbenz[a]anthracene and trans-10,11-dihydro-10,11-dihydroxy-4-hydroxymethylbenz[a]anthracene. There was no detectable trans-dihydrodiol formed at the methyl-substituted double bond (3,4-positions) or at the 'K' region (5,6-positions). The metabolites were isolated by reversed-phase high-pressure liquid chromatography and characterized by the application of u.v.-visible-absorption-, 1H-n.m.r.- and mass-spectral techniques. The 4-hydroxymethylbenz[a]anthracene trans-8,9- and -10,11-dihydrodiols were optically active. Comparison of the c.d. spectra of the trans-dihydrodiols formed from 4-methylbenz[a]anthracene by C. elegans with those of the corresponding benz[a]anthracene trans-dihydrodiols formed by rat liver microsomal fraction indicated that the major enantiomers of the 4-hydroxymethylbenz[a]anthracene trans-8,9-dihydrodiol and trans- 10,11-dihydrodiol formed by C. elegans have S,S absolute stereochemistries, which are opposite to those of the predominantly 8R,9R- and 10R,11R-dihydrodiols formed by the microsomal fraction. Incubation of C. elegans with 4-methylbenz[a]anthracene under 18O2 and subsequent mass-spectral analysis of the metabolites indicated that hydroxylation of the methyl group and the formation of trans-dihydrodiols are catalysed by cytochrome P-450 mono-oxygenase and epoxide hydrolase enzyme systems. The results indicate that the fungal mono-oxygenase-epoxide hydrolase enzyme systems are highly stereo- and regio-selective in the metabolism of 4-methylbenz[a]anthracene.     

Conformational aspect of polypeptide structure. XLIV. Conformational transitions of poly(N-methyl-alanines) induced by trifluoroacetic acid

The synthesis of poly(N-methyl-L -alanine) and poly (N-methyl-DL -alanine) are described. The polymers were examined by 220 MHz high-resolution nuclear magnetic resonance (nmr) and circular dichroism (CD). The results demonstrate that poly(N-methyl-L -alanine) exists as an ordered helical structure with all the amide bonds in the trans configuration in appropriate solvents. As trifluoroacetic acid (TFA) is added to the solutions of the polymer in helix-supporting solvents, resonances corresponding to both trans and cis amide conformations of N-methyl, C-methyl, and α-CH are observed. The presence of both the trans and the cis peptide bonds in a polymer chain disrupts the ordered structures. Our conclusions from CD data are in agreement with the nmr results. Ultracentrifugation shows that degradation of the polymer chain does not occur during the TFA treatment.     

Enzymatic synthesis of 5-bromo-2’-deoxyuridine-2-14C and of 5-iodo-2’-deoxyuridine-2-14C and their incorporation into deoxyribonucleic acid (Allium cepa)

5-Bromo-2’-deoxyuridine-2-¹⁴C was prepared from 5-bromouracil-2-¹⁴C and 2’-de-oxyguanosine using trans-N-deoxyribosylase fromLactobacillus helveticus and incorporated into DNA ofAllium cepa roots. After isolating the DNA and hydrolyzing it enzymatically to deoxynucleoside-5’-phosphates a radioactive nucleotide was detected which yielded 5-bromo-2’-deoxyuridine-2-¹⁴C on enzymatic dephosphorylation. The incorporation of 5-iodo-2’-deoxy-uridine-2-¹⁴C was followed only by microautoradiography.     

L-乳酸脱氢酶热变性的热力学研究

用差示扫描量热法对L-乳酸脱氢酶的热变性进行了研究(温度扫描范围为290—390K,酶蛋白溶液浓度为0.28—0.72mg蛋白/mg溶液)。实验观察到当酶溶液浓度在0.62—0.72mg蛋白/mg溶液范围内有一个吸热转变,酶溶液浓度小于0.62mg蛋白/mg溶液时有两个未完全分开的吸热转变。 这个酶的量热焓与范德霍夫焓的比远大于1,而接近于2,这表明乳酸脱氢酶的变性过程不是一个简单的两态转变,从热力学和吸热峰的形状、大小分析,可以推断乳酸脱氢酶分子是由两个以弱相互作用相连结的合作结构区组成,而每一个结构区是由两个相互作用很强的亚基组成。也就是说乳酸脱氨酶的变性过程包括两个半独立的合作结构区的转变,每一个结构区的转变都近似一个两态转变,ΔHeal与ΔHvh的比值是随着两个半独立部分相互作用的增强,即蛋白浓度的增加而减小。随着蛋白浓度的减小,蛋白质周围水分子增多,酶分子中两个半独立部分的相对独立性增强,这可由热谱图上一个吸热转变变成两个半独立的转变得到证实。     

Integration and expression of theEscherichia coli xylose isomerase gene inSchizosaccharomyces pombe

Summary The xyclose isomerase gene inEscherichia coli was cloned complementarily into a Leu2-negativeSchizosaccharomyces pombe mutant (ATCC 38399). The subsequent integration of the plasmid into the chromosomal DNA of the host yeast was verified by using the dot blot and southern blot techniques. The expressed xylose isomerase showed activity on a nondenaturing polyacrylamide gel. The expression of xylose isomerase gene was influenced by the concentration of nutrients in the fermentation broth. The yeast possessed a xylose isomerase activity of 20 nmol/min/mg by growing in an enriched medium containing yeast extract-malt extract-peptone (YMP) andd-xylose. The conversion ofd-xylose tod-xylulose catalyzed by xylose isomerase in the transformed yeast cells makes it possible to fermentd-xylose with ethanol as a major product. When the fermentation broth contained YMP and 5% (w/v)d-xylose, the maximal ethanol yield and productivity reached 0.42 g/g and 0.19 g/l/h, respectively.     

Functional expression of a Rhodospirillum rubrum gene encoding dinitrogenase reductase ADP-ribosyltransferase in enteric bacteria

The function of the cloned draT gene of Rhodospirillum rubrum was studied by placing it under the control of the tac promoter in the vector, pKK223-3. After induction with isopropyl-beta-D-thiogalactopyranoside, dinitrogenase reductase ADP-ribosyltransferase (DRAT) activity was detected in crude extracts of the heterologous hosts Escherichia coli and Klebsiella pneumoniae. In addition, the expression of draT produced a Nif- phenotype in the otherwise wild-type K. pneumoniae strains, the result of the ADP-ribosylation of accumulated dinitrogenase reductase (DR). DR from a nifF- background was also susceptible to ADP-ribosylation, indicating that the oxidized form of DR will serve as a substrate for DRAT in vivo. A mutation that changes the Arg-101 residue of DR, the ADP-ribose attaching site, eliminates the ADP-ribosylation of DR in vivo, confirming the necessity of this residue for modification.     

Oncogenic activation of the human trk proto-oncogene by recombination with the ribosomal large subunit protein L7a.

The trk-2h oncogene, isolated from the human breast carcinoma cell line MDA-MB 231 by genomic DNA-transfection into NIH3T3 cells, consists of the trk proto-oncogene receptor kinase domain fused to a N-terminal 41 amino acid activating sequence (Kozma, S.C., Redmond, S.M.S., Xiao-Chang, F., Saurer, S.M., Groner, B. and Hynes, N.E. (1988) EMBO J., 7, 147-154). Antibodies raised against a bacterially produced beta gal-trk receptor kinase fusion protein recognized a 44 kd phosphoprotein phosphorylated on serine, threonine and tyrosine in extracts of trk-2h transformed NIH3T3 cells. In vitro, in the presence of Mn2+/gamma ATP, this protein became phosphorylated extensively on tyrosine. Cells transformed by trk-2h did not, however, show an elevation in total phosphotyrosine. We have cloned and sequenced the cDNA encoding the amino terminal activating sequences of trk-2h (Kozma et al., 1988). The encoded protein has a high basic amino acid content and the gene is expressed as an abundant 1.2 kb mRNA in human, rat and mouse cells. Antipeptide antibodies raised against a C-terminal peptide recognized specifically a 30 kd protein on Western blots of human, rat and mouse cell extracts. Immunofluorescence revealed, in addition to granular cytoplasmic fluorescence, intense nucleolar staining. The high basic amino acid content and nucleolar staining prompted us to investigate whether the 30 kd protein could be a ribosomal protein. Western immunoblotting analysis of 2D-electrophoretically resolved ribosomal proteins indicated that the 30 kd protein is the ribosomal large subunit protein L7a.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ⅱ型限制性核酸内切酶NcrⅠ的研究

 在肉色诺卡氏菌C-212株Nocardia carnea C-212中筛选到一种Ⅱ型限制性核酸内切酶NcrⅠ,经与BglⅡ的λDNA降解物的酶谱比较,以及酶识别特异性和切割位点的检测,证明了NcrⅠ是已知的限制酶BglⅡ的同切限制酶,而且其切割位点也与BglⅡ相同,其为: