Use of 6-fluoroderivatives of pyridoxal and pyridoxal phosphate in the study of the coenzyme function in glycogen phosphorylase

6-Fluoropyridoxal phosphate (6-FPLP) has been synthesized. Its properties were studied, and it was used, along with 6-fluoropyridoxal (6-FPAL), to reconstitute apophosphorylase b. Kinetic studies of the resulting enzymes showed that phosphorylases reconstituted with 6-FPLP and 6-FPAL have characteristics similar to those of native and pyridoxal enzymes, respectively, except that the former two enzymes have lower Vmax values. 19F NMR and UV spectra of 6-FPLP phosphorylase showed that the coenzyme forms a neutral enolimine Schiff base. Because the UV and fluorescence spectra of 6-FPLP phosphorylase are comparable to those obtained with native phosphorylase, it further confirms the postulate that pyridoxal phosphate forms a neutral enolimine Schiff base in phosphorylase. The results suggest that the 3-OH group is protonated and the pyridine nitrogen unprotonated in both 6-FPLP phosphorylase and native enzyme. 19F NMR study of 6-FPLP- and 6-FPAL-reconstituted phosphorylases in the inactive and active states indicates that the protein structure near the coenzyme binding site undergoes certain changes when these enzymes are activated by the substrates and AMP. The comparison of the properties of 6-FPLP-reconstituted and native phosphorylases implies that the ring nitrogen of the coenzyme PLP in phosphorylase may interact with the protein during catalysis, and this interaction is important for efficient catalysis by phosphorylase.     

Modification and processing of internalized signal sequences of prolipoprotein in Escherichia coli and in Bacillus subtilis

We have cloned the Escherichia coli lipoprotein structural gene (lpp) into a shuttle vector and studied its expression in both E. coli and in Bacillus subtilis. Using in vitro gene fusion techniques, the lpp gene was placed under the control of the promoter for the erythromycin-resistance (ery) gene. This fusion gene directed the synthesis of Braun's prolipoprotein which can be subsequently processed into the mature lipoprotein. In addition to the prolipoprotein, two ery-lpp hybrid proteins containing a 45- and a 22-amino acid extension preceding the NH2 terminus of prolipoprotein, respectively, are also synthesized in E. coli. The synthesis of these three proteins appears to involve the utilization of three distinct translation initiation sites. In B. subtilis, only two proteins are synthesized, the hybrid protein with a 45-amino acid extension and the prolipoprotein. In both E. coli and B. subtilis, the precursor forms of the hybrid proteins are lipid-modified, and they are processed to mature lipoprotein in vivo. These results indicate that internalized signal sequence containing the prolipoprotein modification and processing site (Leu-Ala-Glys-Cys) can function normally and permit the modification of hybrid proteins to lipid-modified precursors which can be subsequently processed by the globomycin-sensitive prolipoprotein signal peptidase.     

Segmental pulmonary vascular resistance in progressive hydrostatic and permeability edema

We used the in situ blood-perfused left lower lobe preparation of the dog to examine the effect of hydrostatic and permeability edema on the slope and intercept of the vascular pressure-flow (P/Q) relationship and on the longitudinal distribution of vascular resistance with the arterial and venous occlusion technique. Hydrostatic edema (HE) was induced by raising the venous pressure, and permeability edema (PE) was induced with alpha-naphthylthiourea. When the hematocrit (Hct) of the perfusate was kept normal (approximately 40%), HE had no significant effect on either the slope or the intercept of the P/Q relationship or on the distribution of vascular resistance. PE caused a small increase in the intercept of the P/Q relationship and a small rise in the resistance of the vessels in the middle segment. In another series of HE experiments in which Hct was allowed to increase during edema formation, there was a marked increase in vascular resistance. We conclude that edema per se does not increase vascular resistance significantly and that the increases in vascular resistance which were observed previously by other investigators in the isolated lungs may be due to increases in blood hematocrit.     

中国人ε珠蛋白基因5′侧序列中多态性Hinc Ⅱ位点的进一步研究

本文进一步研究了我国不同民族的正常个体以及β地中海贫血患者θ珠蛋白基因5′侧序列中的多态性HincⅡ位点及其遗传性质。在广西壮族正常个体和β地中海贫血纯合子中,该多态性位点的发生频率均为75％,与正常汉族人测得值相近。家系分析资料表明,该多态性位点完全按照孟德尔规律进行遗传。     

光呼吸代谢物乙醇酸、乙醛酸和草酸对烟草叶片硝酸还原的影响

乙醇酸、乙醛酸和草酸能明显促进烟草(Nicotiana rustica)叶片在黑暗中的硝酸还原,光呼吸抑制剂a-羟基吡啶甲烷磺酸能消除前二者的促进作用而不能完全消除草酸的作用。草酸+NAD~+能显著促进离体的硝酸还原。烟叶提取液加入草酸和NAD~+后生成NADH和CO_2认为活体内由乙醛酸氧化生成的草酸是经脱氢生成NADH供硝酸还原之用。未能证明在烟叶内存在乙醇酸脱氨酶,因此排除由乙醇酸直接脱氢以还原硝酸的可能。     

玉米黄早4雄性不育系、保持系过氧化物酶同工酶的比较研究

在玉米黄早4雄性不育系、保持系的10个组织(叶、根、茎、芽鞘、胚轴、苞叶、穗轴、花丝、雌蕊、花药)中共检测出18种正、负极向过氧化物酶,其中有7个组织不育系与保持系间过氧化物酶没有差异,只有在3个组织中(叶、茎、花药)不育系与保持系间的过氧化物酶存在差异,说明玉米黄早4雄性不育系及保持系的过氧化物酶可能由细胞核基因编码。不育系与保持系个别组织内过氧化物酶存在差异,可能是由于核内编码过氧化物酶的基因表达异常所引起,而这种表达异常,可能是与不育系中不育细胞质基因调控核基因的表达有关。     

Computer control for continuous cultivation ofSaccharomyces cerevisiae

Summary An on-line respiratory quotient control system has been developed for the continuous cultivation of baker's yeast. This system is based on moving identification of the microbial dynamics. The optimal dilution rate that was selected as the control variable was determined by minimizing a performance index. Without resorting to complicated microbial analysis, a simple and practical moving model is obtained by continually updating the input and output data. The experimental results indicate the satisfactory controllability of the present system and the possible extention of the proposed method to other bioprocesses.     

A calcium ionophore-inducible cellular promoter is highly active and has enhancerlike properties.

We examined the regulatory/promoter sequence of a calcium ionophore-inducible gene isolated from the rat genome. Whereas the promoter of this ubiquitously expressed gene is active under noninduced conditions, after induction by calcium ionophore A23187 this promoter is 10- to 25-fold more active than the simian virus 40 early promoter, as measured by chloramphenicol acetyltransferase activities. Within this regulatory/promoter region, we have identified a DNA fragment with enhancer-like properties immediately 5' to the TATA sequence. This 291-nucleotide fragment acts in cis to enhance expression of the neomycin phosphotransferase (neo) gene driven by the herpes simplex virus thymidine kinase promoter in an orientation-independent manner. In addition, this fragment can confer A23187 inducibility to the neo gene and effectively compete for positive regulatory factors involved in A23187 induction. Sequence analysis of this promoter reveals homology with viral core enhancer sequences, and the apparent organization of direct repeat domains is similar to those observed in viral enhancers.     

Purification and characterization of yeast topoisomerase I

Yeast topoisomerase I (Mr = 76,000) has been purified to 80% homogeneity using a combination of ion exchange, gel filtration, and DNA-cellulose chromatography. The enzyme was characterized with respect to its ability to relax supercoiled DNA and to catenate nicked circular DNA. Yeast topoisomerase I will remove both positive and negative turns in DNA supercoils in the absence of ATP and magnesium ion. The products of the catenating activity of the enzyme were examined on agarose gels and in the electron microscope. These analyses indicate that yeast topoisomerase I will generate large catenated DNA networks which appear to rearrange to multimeric linear structures upon long incubation time.