Isothermal titration calorimetry and stopped flow circular dichroism investigations of the interaction between lomefloxacin and human serum albumin in the presence of amino acids

The present study was designed to investigate the influence of two indispensable and two dispensable amino acids, including methionine, histidine, cysteine and proline, on the binding interaction between human serum albumin (HSA) and an antibiotic agent lomefloxacin (LMF). The fluorescence quenching experiments showed that the intrinsic emission of HSA was considerably quenched following binding to LMF in all the systems. Furthermore, in all the interactions the maximum wavelength of HSA was slightly decreased. The spectral changes observed in the binding systems we e all attributed to the alteration of the micro-environment around the tryptophan and tyrosine residues of HSA. The K_b values o HSA-LMF complex in the absence and presence of histidine, methionine, cysteine and proline have been obtained 6.02 × 10⁵, 4.83 × 10⁵, 5.05 × 10⁵, 4.94 × 10⁵ and 6.20 × 10⁵ M^?1 respectively. The various kind of Kb values showed the different interaction behavior between HSA and LMF in the absence and presence of amino acids mentioned. The data gathered by isothermal titration calorimetry (ITC) studies revealed that although all the binding interactions were exothermic, the amount of the heat exchanged during the HSA-LMF interaction increased in the presence of the amino acids especially cysteine. In the present study, the binding kinetics and affinity of LMF to HSA in the absence and presence of the amino acids were studies using stopped-flow circular dichroism and ITC techniques respectively. The results of these two techniques revealed that the bindig affinity and binding rate of the LMF-HSA interaction decreased in the presence of histidine, methionine and cysteine. In the presence of proline, the binding process of LMF-HSA was sped up and the affinity of LMF to HSA slightly increased. All the experimental results were then supported by the data collected from molecular modeling studies using density functional theory.

Communicated by Ramaswamy H. Sarma     

L-苯丙氨酸生产的代谢工程研究

L-苯丙氨酸是一种重要的食品和医药中间体。工业上一般采用酶法和发酵法来生产L-苯丙氨酸。代谢工程的兴起,使得更加理性的改造菌株成为可能,这更加促进了发酵法的广泛应用。主要介绍了代谢工程在L-苯丙氨酸生产菌的改造中的应用情况,其中涉及苯丙氨酸生物合成途径中相关基因及其酶的调控、中央代谢途径的改造和芳香族氨基酸生物合成支路的修饰。并探讨了将来的发展前景。     

氨基酸高产菌株创制的关键技术

氨基酸是构成蛋白质的基本单元,被广泛应用于食品、医药、饲料和化工等多个领域。利用微生物细胞工厂生产氨基酸,具备原料可再生、生产过程条件温和、产品纯度高、环境污染小等优点,能够助力实现碳中和。借助代谢工程和合成生物学技术,对大肠杆菌(Escherichia coli)和谷氨酸棒杆菌(Corynebacterium glutamicum)进行定向设计、改造与优化,创制了高产氨基酸的微生物细胞工厂,实现了支链氨基酸、天冬氨酸族氨基酸、谷氨酸族氨基酸和芳香族氨基酸的生物炼制。本文对高产氨基酸的大肠杆菌细胞工厂和谷氨酸棒杆菌细胞工厂创制过程进行分析,以期对高性能微生物细胞工厂的创制提供参考。     

Effect of unstirred layers on binding and reaction kinetics at a membrane surface

The effects of solution unstirred layers on the time course of chemical reactions and transport processes at a membrane surface are determined. A set of equations which describes non-steady-state diffusion through an unstirred layer coupled with chemical reaction at a membrane surface or transport through a membrane is developed. A numerical solution to the equations is obtained by uncoupling diffusive and chemical processes in an iterative manner. The diffusive process is solved by the Crank-Nicolson method; the chemical process is solved by integrating the differential equations describing the kinetics. Diffusive processes in one dimension, in three dimensions, and in the presence of an arbitrary potential near the membrane surface are solved. General characteristics of the calculated reaction time course are discussed using surface binding and membrane transport examples. Small, neglected, unstirred layers are shown to sometimes yield erroneous values of rate parameters for a surface reaction and to simulate competitive reaction kinetics. Experimental approaches for measuring unstirred layer thickness are reviewed.     

Regulation of chorismate mutase activity of various yeast species by aromatic amino acids

The regulatory properties of chorismate mutase, its cellular localization and isoenzyme pattern were investigated in 23 yeast species. All yeasts contained only a single form of the enzyme, which is localized exclusively in the cytosol. The enzyme activity from all sources was activated 3-(Rhodotorula aurantiaca) to 185-fold (Candida maltosa) by tryptophan. The tryphtophan concentration, which was necessary to obtain half maximum velocity was determined to be between 2 (Pichia guilliermondii) and 95 M (Yarrowia lipolytica). Ten yeast species possessed an enzyme that was inhibited by both phenylalanine and tyrosine. The chorismate mutase from four strains was inhibited only by tyrosine and the enzyme from two species was inhibited by phenylalanine alone. The enzyme inhibition by phenylalanine and tyrosine was completely reversed by tryptophan. Six enzyme sources were not inhibited and theY. lipolytica chorismate mutase was slightly activated by both amino acids.     

转氨酶催化不对称合成芳香族L-氨基酸

芳香族L-氨基酸是合成许多药物、农药、精细化学品和食品添加剂的重要手性砌块(Chiral buildingblocks)。利用酶催化具有高活性和高立体选择性的特点合成手性砌块是目前不对称合成领域重要的研究方向。通过对不同来源转氨酶的进化分析,选择分别源自原核生物大肠杆菌Escherichia coli和真核生物酿酒酵母Saccharomyces cerevisia中的两种具有代表性Ⅰ型芳香族转氨酶TyrB和Aro8,比较研究了两种转氨酶通过平衡逆转不对称氨化催化合成芳香族L-氨基酸的反应过程和催化效率。重组转氨酶TyrB和Aro8都能有效地合成天然芳香族氨基酸苯丙氨酸和酪氨酸以及非天然氨基酸苯甘氨酸。手性HPLC分析表明,合成的氨基酸都是L-构型的,e.e值等于100%。L-丙氨酸是适宜的氨基供体,转氨酶TyrB和Aro8都不能利用D-型氨基酸作为氨基供体。反应体系中氨基供体L-丙氨酸和氨基受体芳香族α-酮酸的最适摩尔比为4∶1。底物芳香族α-酮酸分子结构中芳香环上的取代基以及脂肪酸碳链部分的长度都对酶催化的转氨效率有显著的影响。在制备规模试验中,TyrB催化不对称转氨反应合成L-苯甘氨酸、L-苯丙氨酸和L-酪氨酸的比生产速率为0.28 g/(g.h)、0.31 g/(g.h)和0.60 g/(g.h),Aro8催化上述反应的比生产速率分别为0.61 g/(g.h)、0.48 g/(g.h)和0.59 g/(g.h)。研究结果对利用转氨酶通过平衡逆转不对称催化合成芳香族L-氨基酸的工业化应用具有指导意义。     

Ⅱ型抗癌晶体蛋白抗肝癌作用的关键芳香族氨基酸

为探讨Ⅱ型抗癌晶体蛋白(Parasporin-2)上与抗肝癌作用相关的关键氨基酸,利用5-BU对Parasporin-2活性区编码DNA(P2Y)进行PCR诱变,之后在大肠杆菌中表达,产物纯化后经MTT法检测其对肝癌细胞系和正常肝细胞系的作用。获得的9个突变体其抗肝癌活性差异极大,其中P2M1和P2M8对两种肝癌细胞系SMMC7721和Bel7402均有较强的细胞毒杀作用而不影响正常肝细胞系Chang-liver。比较了P2M1、P2M8和P2Y的二级结构与三级结构,发现二级结构上的变化如β折叠变长或α螺旋增加影响着Ⅱ型抗癌晶体蛋白的抗肝癌活性。基于突变体间氨基酸序列比对、突变体与受体间分子对接以及模拟突变等研究的结果表明,位点52、56、58和208上的氨基酸残基特别是芳香族氨基酸在Parasporin-2与受体间的互作中可能起着重要作用。     

人生长激素和转移的人生长激素基因对去垂体鱼的生长代偿效应

通过显微注射技术,将小鼠重金属螯合蛋白(MT-1)基因启动顺序与人生长激素基因顺序的重组体pMThGH注入鲤鱼(Cyprinus carpio)的受精卵内,由此发育的转基因鱼及其后代F1和F2均显示出快速生长效应。去垂体后,转基因鲤鱼F2持续生长,而非转基因鲤鱼和鲫鱼(Carassius auratus)的生长停止。给去垂体的鲫鱼腹腔注射生物合成的人生长激素(hGH),可恢复其生长。实验结果表明,转基因鱼体内表达和体外生物合成的hGH均能代偿鲤鱼和鲫鱼的内源生长激素并刺激去垂体鱼的生长。     

Involvement of lysine residues in the binding of hGH and bGH to somatotropic receptors

The biological activities of human (hGH) and bovine (bGH) growth hormone derivatives obtained by chemical modification of the lysine residues were studied by radioreceptor assays using rabbit liver homogenates for somatotropic activity (SA). Control treatment with BH₄⁻ had a very slight effect on the SA, whereas the methylation and ethylation drastically reduced the acitivty of both hormones. Guanidination of these hormones and even acetimidination at a lower rate are accompanied by a considerable loss of biological activity. These results show the involvement of lysine residues in the interaction of hGH and bGH with somatotropic receptors. The structure-function relationship of these molecules is discussed, suggesting that the lysine or arginine residues in positions 41, 64, 70 and 115 might be particularly implicated.     

The influence of canavanine and related compounds on the water balance system of locusts

In vivo increase in haemolymph volume of canavanine-treated locusts substantiates our previous in vitro findings that canavanine inhibits fluid secretion by locust Malpighian tubules. Furthermore when diuretic hormone is applied in vivo after canavanine treatment haemolymph volume is drastically reduced below levels retained in locusts untreated with canavanine. Again this is in accord with canavanine potentiation of semi-isolated Malpighian tubules and enhanced fluid secretion in vitro. The response is specific to canavanine; compounds similar in structure (arginine, argininic acid, citrulline, canaline, ornithine and homoserine) have no effect on the rate of fluid secreted by Malpighian tubules. Only partial competition is obtained with uridine homoserine.     

The influence of albumin on vitamin D metabolism in fetal chick osteoblast-like cells

Human pancreatic growth hormone releasing factor (1-29)-amide [hpGRF (1-29)-NH2] and the following analogs: [D-Tyr-1]-hpGRF(1-29)-NH2, [D-Ala-2]-hpGRF(1-29)-NH2, [D-Asp-3]-hpGRF(1-29)-NH2, and [N-Ac-Tyr-1]-hpGRF (1-29)-NH2 were synthesized using solid phase methodology and tested for their ability to stimulate growth hormone (GH) secretion in the rat and the pig in vivo. [D-Ala-2]-hpGRF (1-29)-NH2 was approximately 50 times more potent than the parent molecule in eliciting GH secretion in the rat. The other analogs were less active, but all were more potent than the 1-29 amide in the rat. [D-Tyr-1]-hpGRF(1-29)-NH2 was 10 times more potent, [D-Asp-3]-hpGRF(1-29)-NH2 7 times more potent, and the acetylated molecule approximately 12 times more potent than hpGRF(1-29)-NH2.     

Physiological roles of somatocrinin and somatostatin in the regulation of growth hormone secretion

Somatocrinin, a 44 amino acid peptide with potent growth hormone (GH) releasing activity in anesthetized rats, was tested in conscious freely-moving rats. When high doses of 1 to 10 μg were administered (iv) at random times between spontaneous GH pulses, the responses were inconsistent. When similar doses were tested under identical conditions but in rats pretreated with antibodies against somatostatin, all animals demonstrated a marked and immediate increase in plasma GH of 5 to 10 fold. Similarly, a 1 μg dose of somatocrinin was also ineffective in increasing plasma GH when administered to rats subjected to a 72 h fast, a paradigm known to enhance endogenous somatostatin secretion. However, plasma GH increased over 20 fold if rats were pretreated with antibodies against somatostatin. These results demonstrate the dynamic and opposite roles exerted by somatocrinin and somatostatin in regulating GH secretion.     

Somatocrinin,growth hormone releasing factor,stimulates secretion of growth hormone in anesthetized rats

The synthetic replicate of a 44 amino acid peptide isolated from a human pancreatic tumor which had caused acromegaly possesses high specific activity to release growth hormone (GH) in anesthetized male rats. The GH secretion induced by this peptide is dose-dependent from 50 ng to 1 μg, with plasma GH concentrations increasing more than 10-fold within 5 min of iv administration at the higher doses. Two enzymatic degradation products of the 44 residue peptide were also isolated and consist of the first 37 and 40 amino acids. All three peptides appear to possess similar potency, on a molar basis, $\text{in}\text{vivo}$, contrary to $\text{in}\text{vitro}$ results. The specificity of these peptides on GH release was shown by their failure to alter plasma concentrations of prolactin (PRL), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and corticosterone. Based on these $\text{in}\text{vivo}$ results, the three peptides with serve as powerful tools with which to investigate the mechanisms of GH secretion.     

Divalent metal cation chelators enhance chromatographic separation of structurally similar macromolecules: separation of human growth hormone isoforms

Human GH isoforms were separated by anion-exchange chromatography using a linear NaCl gradient in the presence and absence of EDTA and EGTA. SDS–PAGE showed that glycosylated 24-kDa hGH did not appreciably separate from other hGH variants in the absence of metal chelators. However, in the presence of metal chelators, glycosylated 24-kDa hGH separated from the bulk of the hGH isoforms. Human GH isoforms were also separated by size-exclusion chromatography in the presence and absence of metal chelators. Glycosylated 24-kDa hGH eluted with the bulk of the hGH isoforms in both separations. The inclusion of metal chelators in chromatographic buffers to alter the charge and/or size of proteins by stripping their metals may be a generally useful strategy in their fractionation.