固定化生长酵母连续生产酒精的研究

采用固定化生长细胞方法,以柱式生物反应器连续发酵甜菜糖蜜酒精。酒精能力为39.45g/L凝胶/h,停留时间1.8小时。生物反应器具有良好的稳定性,连续工作50天,发酵醪酒精含量在8.5％(v/v)以上。系统研究了最适固定化条件,用L_(16)(4~5)正交试验确定了最佳发酵条件。     

用原子力显微技术研究受体-配体间的相互作用

原子力显微镜(AFM)不仅能对纳米生物结构进行实时动态的形态和结构观察,而且还能以10^-12N(pN)的精度对溶液中生物分子表面的相互作用力进行直接测量,逐渐成为一种研究受体-配体间相互作用的良好工具。本简要综述用AFM研究受体-配体间作用力、受体-配体间相互作用的影响因素及对这些因素的处理方法。     

微水相超声波协同固定化脂肪酶催化酯交换过程优化

超声波协同固定化脂肪酶催化制备生物柴油的最佳工艺条件为：超声波功率70W、叔丁醇为反应介质、叔丁醇用量3％（v／v）、醇油比3：1且甲醇分三批加入、反应温度40℃、水含量为2％（v／v）。副产物甘油对固定化脂肪酶使用寿命影响最大,使用后的固定化脂肪酶用丙酮洗去表面的甘油,进行酯交换反应,酶的稳定性大为提高,可连续使用16批次。     

若干外源凝集素对胰岛素与其受体相互作用的影响

本文研究了若干外源凝集素对胰岛素与其脂肪细胞胰岛素受体相互作用的影响。结果表明与D-甘露糖和D-葡萄糖专一结合的外源凝集素,如ConA(伴刀豆球蛋白A)、豌豆及蚕豆凝集素可抑制~(125)Ⅰ-胰岛素与其受体的结合。与D-半乳糖专一结合的外源凝集素,如蓖麻凝集素Ⅰ和天花粉凝集素基本上不抑制~(125)Ⅰ-胰岛素与其受体的结合,在低浓度时有一定程度的促进胰岛素与受体结合的能力。另外两个和低聚糖专一结合的半夏凝集素和PHA(菜豆凝集素)的效应和蓖麻凝集素Ⅰ等类似。这些结果说明在胰岛素受体分子表面可能存在着与ConA等专一结合的糖,而且它们在胰岛素受体与胰岛素结合部位的附近。而和其他几个外源凝集素专一结合的糖或低聚糖链则和胰岛素受体与胰岛素结合的部位相距较远,并就这些凝集素对胰岛素与受体结合的促进作用进行了讨论。     

若干外源凝集素对胰岛素与其受体相互作用的影响

本文研究了若干外源凝集素对胰岛素与其脂肪细胞胰岛素受体相互作用的影响。结果表明与D-甘露糖和D-葡萄糖专一结合的外源凝集素,如ConA (伴刀豆球蛋白A)、豌豆及蚕豆凝集素可抑制~(125)I-胰岛素与其受体的结合。与D-半乳糖专一结合的外源凝集素,如蓖麻凝集素I 和天花粉凝集素基本上不抑制~(125)I-胰岛素与其受体的结合,在低浓度时有一定程度的促进胰岛素与受体结合的能力。另外两个和低聚糖专一结合的半夏凝集素和PHA (菜豆凝集素)的效应和蓖麻凝集素I 等类似。这些结果说明在胰岛素受体分子表面可能存在着与ConA等专一结合的糖,而且它们在胰岛素受体与胰岛素结合部位的附近。而和其他几个外源凝集素专一结合的糖或低聚糖链则和胰岛素受体与胰岛素结合的部位相距较远,并就这些凝集素对胰岛素与受体结合的促进作用进行了讨论。     

叔丁醇体系中复合酶催化餐饮废油制备生物柴油工艺优化

以叔丁醇为反应体系,研究固定化Novozym 435 和Lipozyme TLIM 脂肪酶协同催化餐饮废油合成生物柴油.采用5 因素5 水平响应面法优化工艺参数,最佳工艺条件为:复合酶用量4%( wt.)、复合酶配比1:1(w/w),油/醇摩尔比1:5,反应温度50℃,叔丁醇用量50%(油体积比v/v).在此条件下反应10 h,生物柴油转化率为83.65 %.复合酶操作稳定性较高,重复使用10 个批次,生物柴油转化率仍保持在80% 以上.     

糖及其衍生物对胰岛素与其受体相互作用的影响

Cuatrecasas报道除去细胞表面的神经氨酸并不影响被处理细胞与胰岛素的结合能力,但继神经氨酸后再除去半乳糖,就使被处理细胞与胰岛素的结合能力降低。前文报道若干外源凝集素对胰岛素与其受体相互作用的影响,推测胰岛素受体分子上与胰岛素结合部位或其附近存在着甘露糖或葡萄糖。Kazten曾研究了一些糖类或其衍生物对胰岛素和其受体结合的影响,但结论似乎不很明确。本文报告若干单糖、低聚糖及糖苷对胰岛素与人胎盘细胞膜胰岛素受体相互作用的影响。     

纳米载体固定化脂肪酶及其在生物柴油转化中的应用进展

随着全球能源需求量的不断上升和日益加剧的环境压力,固定化脂肪酶在可持续生物柴油合成中的应用受到广泛关注。纳米材料,包括纳米粒子(磁性和非磁性)、碳纳米管和纳米静电纺丝,具有比表面积大、结构稳定、易于功能化修饰等优势,是固定化脂肪酶领域的重要载体之一。综述了纳米材料作为载体在脂肪酶固定化中的应用,重点介绍这类生物催化剂在生物柴油合成中的最新进展,并对纳米材料固定化脂肪酶发展前景进行展望,旨在为固定化脂肪酶的研究和工业化应用奠定基础。     

硫酸乙酰肝素与口蹄疫病毒感染

受体是病毒宿主嗜性和致病机制的主要决定因素。硫酸乙酰肝素(HS)是一种多聚阴离子碳水化合物, 广泛存在于真核细胞的细胞膜和细胞基质。HS是许多病毒在细胞膜上的特异受体或辅助受体。目前发现口蹄疫病毒可利用HS和整联蛋白(αvβ3、αvβ6、αvβ1、αvβ8)作为病毒受体。口蹄疫病毒可能在不同的感染阶段利用不同类型的受体与宿主细胞相互作用。研究病毒受体的结构和功能对理解病毒与宿主细胞的关系具有重要意义。本文主要论述了HS的生物学特性及其与口蹄疫病毒感染的关系。     

胰岛素的促生长作用

除了经典的代谢调节作用之外,胰岛素还具有重要的促生长作用：在体外胰岛素能够刺激众多细胞的增殖与分化,一些实验证明胰岛素在体内可能也是一种重要的生长调节因子.胰岛素的促生长作用通过细胞表面的胰岛素受体介导,但在较高的胰岛素浓度下也可以通过类胰岛素生长因子Ⅰ(IGF-Ⅰ)受体进行,在不同细胞体系中可能会有所不同.受体后的信号转导经过了一系列磷酸化和去磷酸化等途径,其中有胰岛素受体底物1(IRS-1)、Shc蛋白、Ras蛋白以及磷酸肌醇3激酶(PI3-K)等的参与.在胰岛素的分子表面很可能存在一些区域或位点,对其促生长作用有着更大的贡献,通过对一些高促生长活性的胰岛素类似物的研究已揭示出一些初步的证据.     

Purification of the catalytically active phosphorylated form of insulin receptor kinase by affinity chromatography with O-phosphotyrosyl-binding antibodies

The catalytically active, tyrosyl-phosphorylated form of insulin receptor kinase was isolated from human placenta by a procedure which exploits the propensity for the intact alpha 2 beta 2 form of insulin receptor to undergo insulin-promoted autophosphorylation at tyrosyl residues and concomitant activation as a tyrosyl kinase. Purification of tyrosyl-phosphorylated insulin receptor was effected by adsorption on and elution (with a hapten) from a column of O-phosphotyrosyl-binding antibody immobilized on protein A-Sepharose (Ab-protein A). The starting material for the purification process was protein which had been solubilized from placental membranes and purified by chromatography on immobilized wheat germ agglutinin. After chromatography on Ab-protein A to remove preexisting O-phosphotyrosyl-containing proteins, the fraction which did not adsorb to the Ab-protein A column was incubated with insulin and briefly treated with ATP so as to maximize selective autophosphorylation of insulin receptor. This material was then subjected to chromatography on Ab-protein A. Although the amount of the intact alpha 2 beta 2 form of insulin receptor present in the starting material was only a small fraction of the protein (approximately 0.2%) and only approximately 20% of the insulin-binding forms of the receptor present, it was eluted (with 10 mM p-nitrophenyl phosphate) from the column in greater than or equal to 80% purity. Chromatography on Ab-protein A appears to have an advantage over the alternative affinity chromatographic procedures which utilize immobilized insulin or antiinsulin receptor antibody to adsorb insulin receptor, since these procedures do not resolve the intact alpha 2 beta 2 form of insulin receptor from the nicked insulin-binding forms of the receptor which do not undergo insulin promoted autophosphorylation.     

Thin-layer chromatography can resolve phosphotyrosine, phosphoserine, and phosphothreonine in a protein hydrolyzate

A solution of propionic acid, 1 M ammonium hydroxide, and isopropyl alcohol (45/17.5/17.5, v/v) was the ascending solvent in the separation of phosphotyrosine, phosphothreonine, and phosphoserine by thin-layer chromatography. The immobile phase was cellulose. The relative migrations were 0.44, 0.38, and 0.2, respectively. A previously described thin-layer system consisting of isobutyric acid and 0.5 M ammonium hydroxide (50/30, v/v) gave very similar relative migrations. To determine the usefulness of thin-layer chromatography in phosphoamino acid analysis, the propionic acid/ammonium hydroxide/isopropyl alcohol solution was used to characterize phosphorylated residues in a plasma membrane protein which is a substrate for the insulin receptor kinase, in insulin receptor phosphorylated histone H2B, and in an in vivo phosphorylated 90000-Da protein from IM9 cells. 32P-labeled proteins were separated by dodecyl sulfate-gel electrophoresis, digested with trypsin, and then hydrolyzed with 6 N HCl, 2 h, 110 degrees C. Following thin-layer chromatography of the hydrolyzates and autoradiography, phosphotyrosine was detected in insulin receptor substrates, and phosphoserine and phosphothreonine were found in the in vivo-phosphorylated protein. This study supports previous reports about the practicality of thin-layer chromatography in phosphoamino acid analysis and it demonstrates that a propionic acid, ammonium hydroxide, isoprophyl alcohol solution may be a useful ascending solvent mixture for this purpose.     

Myricetin, a naturally occurring flavonol, ameliorates insulin resistance induced by a high-fructose diet in rats

The present study was conducted to explore the effects of myricetin on insulin resistance in rats fed for 6 weeks with a diet containing 60% fructose. Repeated intravenous (i.v.) injection of myricetin (1 mg/kg per injection, 3 times daily) for 14 days was found to significantly decrease the high glucose and triglyceride levels in plasma of fructose chow-fed rats. Also, the higher degree of insulin resistance in fructose chow-fed rats as measured by homeostasis model assessment of basal insulin resistance was significantly decreased by myricetin treatment. Myricetin increased the whole-body insulin sensitivity in fructose chow-fed rats, as evidenced by the marked elevation of composite whole-body insulin sensitivity index during the oral glucose tolerance test. Myricetin was found to reverse the defect in expression of insulin receptor substrate-1 (IRS-1) and the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) in soleus muscle of fructose chow-fed rats under the basal state, despite the protein expression of insulin receptor (IR). Increased basal phosphorylation of IR and IRS-1 as well as Akt was observed in parallel. The reduced level of insulin action on phosphorylation of IR, IRS-1 and Akt in soleus muscle of fructose chow-fed rats was reversed by myricetin treatment. Furthermore, myricetin treatment improved the defective insulin action on the translocation of glucose transporter subtype 4 (GLUT 4) in insulin-resistant soleus muscle. These findings indicate that myricetin improves insulin sensitivity through the enhancement of insulin action on IRS-1-associated PI 3-kinase and GLUT 4 activity in soleus muscles of animals exhibiting insulin resistance.     

A BRET assay for monitoring insulin receptor interactions and ligand pharmacology

The insulin receptor (IR) belongs to the receptor tyrosine kinase super family and plays an important role in glucose homeostasis. The receptor interacts with several large docking proteins that mediate signaling from the receptor, including the insulin receptor substrate (IRS) family and Src homology-2-containing proteins (Src). Here, we applied the bioluminescence resonance energy transfer 2 (BRET2) technique to study the IR signaling pathways. The interaction between the IR and the substrates IRS1, IRS4 and Shc was examined in response to ligands with different signaling properties. The association between IR and the interacting partners could successfully be monitored when co-expressing green fluorescent protein 2 (GFP2) tagged substrates with Renilla reniformis luciferase 8 (Rluc8) tagged IR. Through additional optimization steps, we developed a stable and flexible BRET2 assay for monitoring the interactions between the IR and its substrates. Furthermore, the insulin analogue X10 was characterized in the BRET2 assay and was found to be 10 times more potent with respect to IRS1, IRS4 and Shc recruitment compared to human insulin. This study demonstrates that the BRET2 technique can be applied to study IR signaling pathways, and that this assay can be used as a platform for screening and characterization of IR ligands.     

Differential modulation of the tyrosine phosphorylation state of the insulin receptor by IRS (insulin receptor subunit) proteins.

In response to insulin, tyrosine kinase activity of the insulin receptor is stimulated, leading to autophosphorylation and tyrosine phosphorylation of proteins including insulin receptor subunit (IRS)-1, IRS-2, and Shc. Phosphorylation of these proteins leads to activation of downstream events that mediate insulin action. Insulin receptor kinase activity is requisite for the biological effects of insulin, and understanding regulation of insulin receptor phosphorylation and kinase activity is essential to understanding insulin action. Receptor tyrosine kinase activity may be altered by direct changes in tyrosine kinase activity, itself, or by dephosphorylation of the insulin receptor by protein-tyrosine phosphatases. After 1 min of insulin stimulation, the insulin receptor was tyrosine phosphorylated 8-fold more and Shc was phosphorylated 50% less in 32D cells containing both IRS-1 and insulin receptors (32D/IR+IRS-1) than in 32D cells containing only insulin receptors (32D/IR), insulin receptors and IRS-2 (32D/IR+IRS-2), or insulin receptors and a form of IRS-1 that cannot be phosphorylated on tyrosine residues (32D/IR+IRS-1F18). Therefore, IRS-1 and IRS-2 appeared to have different effects on insulin receptor phosphorylation and downstream signaling. Preincubation of cells with pervanadate greatly decreased protein-tyrosine phosphatase activity in all four cell lines. After pervanadate treatment, tyrosine phosphorylation of insulin receptors in insulin-treated 32D/IR, 32D/ IR+IRS-2, and 32D/IR+IRS-1F18 cells was markedly increased, but pervanadate had no effect on insulin receptor phosphorylation in 32D/IR+IRS-1 cells. The presence of tyrosine-phosphorylated IRS-1 appears to increase insulin receptor tyrosine phosphorylation and potentially tyrosine kinase activity via inhibition of protein-tyrosine phosphatase(s). This effect of IRS-1 on insulin receptor phosphorylation is unique to IRS-1, as IRS-2 had no effect on insulin receptor tyrosine phosphorylation. Therefore, IRS-1 and IRS-2 appear to function differently in their effects on signaling downstream of the insulin receptor. IRS-1 may play a major role in regulating insulin receptor phosphorylation and enhancing downstream signaling after insulin stimulation.     

Identification of tyrosines 154 and 307 in the extracellular domain and 653 and 766 in the intracellular domain as phosphorylation sites in the heparin-binding fibroblast growth factor receptor tyrosine kinase (flg).

Four tyrosine residues have been identified as phosphorylation sites in the tyrosine kinase isoform of the heparin-binding fibroblast growth factor receptor flg (FGF-R1). Baculoviral-insect cell-derived recombinant FGF-R1 was phosphorylated and fragmented with trypsin while immobilized on heparin-agarose beads. Phosphotyrosine peptides were purified by chromatography on immobilized anti-phosphotyrosine antibody and analyzed by Edman degradation and electrospray tandem mass spectrometry. Tyrosine residue 653, which is in a homologous spatial position to major autophosphorylation sites in the catalytic domain of the src and insulin receptor kinases, is the major intracellular FGF-R1 phosphorylation site. Residue 766 in the COOH-terminus outside the kinase domain is a secondary site. Tyrosine residues 154 and 307, which are in the extracellular domain of transmembrane receptor isoforms and are in an unusual sequence context for tyrosine phosphorylation, were also phosphorylated.     

Internalization and activation of the rat liver insulin receptor kinase in vivo

The preparation of clearly delineated plasmalemma (PM) and endosomal subcellular fractions from rat liver has allowed us to compare insulin receptor (IR) kinase activity at the cell surface and in hepatic endosomes (ENs) as a function of dose and time after injected insulin. Tyrosine kinase activity in PM and ENs was measured, after solubilization and partial purification by wheat germ agglutinin chromatography (lectin-purified), using poly(Glu:Tyr) as substrate. Following the injection of a subsaturating dose of insulin (1.5 micrograms/100 g body weight), lectin-purified receptor showed peak activation at 30 s in PM and at 2 min in ENs. As observed previously (Khan, M. N., Savoie, S., Bergeron, J. J. M., and Posner, B. I. (1986) J. Biol. Chem. 261, 8462-8472) autophosphorylation activity was also augmented following insulin injection. In a pattern virtually identical to that of exogenous kinase activity, autophosphorylation attained peak activity at 30 s in PM and at 2 min in ENs. The time course of IR autophosphorylation in intact membranes was very similar to that observed for lectin purified receptors and was seen with an injected insulin dose as low as 150 ng/100 g body weight. Phosphatase treatment of the solubilized endosomal receptor abolished its enhanced activity. Hence, insulin treatment led to in vivo receptor phosphorylation which was reflected in the enhancement of both tyrosine kinase and autophosphorylation activities. Significant differences in the phosphorylation activities of PM and ENs were observed. Phosphoamino acid analyses revealed that the activated IR of intact PM was autophosphorylated in vitro, at both serine (55%) and tyrosine (45%) residues; whereas the activated IR of intact ENs was phosphorylated in vitro exclusively on tyrosine autophosphorylation specific activity for the activated IR of ENs was 3- to 4-fold that of the IR of PM. This was observed for the lectin purified IRs as well as for IRs of intact cell fractions. The reduced level of IR autophosphorylation in PM was not due to occlusion of tyrosine acceptor sites by prior in vivo phosphorylation. The rapidity with which activated IR accumulates in ENs as well as the sensitivity of endosomal IR kinase to activation by injected insulin are consistent with the endosomal apparatus serving a physiologically significant site for the regulation of transmembrane signaling.     

Insulin receptors internalize by a rapid, saturable pathway requiring receptor autophosphorylation and an intact juxtamembrane region

The effect of receptor occupancy on insulin receptor endocytosis was examined in CHO cells expressing normal human insulin receptors (CHO/IR), autophosphorylation- and internalization-deficient receptors (CHO/IRA1018), and receptors which undergo autophosphorylation but lack a sequence required for internalization (CHO/IR delta 960). The rate of [125I]insulin internalization in CHO/IR cells at 37 degrees C was rapid at physiological concentrations, but decreased markedly in the presence of increasing unlabeled insulin (ED50 = 1-3 nM insulin, or 75,000 occupied receptors/cell). In contrast, [125I]insulin internalization by CHO/IRA1018 and CHO/IR delta 960 cells was slow and was not inhibited by unlabeled insulin. At saturating insulin concentrations, the rate of internalization by wild-type and mutant receptors was similar. Moreover, depletion of intracellular potassium, which has been shown to disrupt coated pit formation, inhibited the rapid internalization of [125I]insulin at physiological insulin concentrations by CHO/IR cells, but had little or no effect on [125I]insulin uptake by CHO/IR delta 960 and CHO/IRA1018 cells or wild-type cells at high insulin concentrations. These data suggest that the insulin-stimulated entry of the insulin receptor into a rapid, coated pit-mediated internalization pathway is saturable and requires receptor autophosphorylation and an intact juxtamembrane region. Furthermore, CHO cells also contain a constitutive nonsaturable pathway which does not require receptor autophosphorylation or an intact juxtamembrane region; this second pathway is unaffected by depletion of intracellular potassium, and therefore may be independent of coated pits. Our data suggest that the ligand-stimulated internalization of the insulin receptor may require specific saturable interactions between the receptor and components of the endocytic system.     

Specificity of insulin and insulin-like growth factor I receptors investigated using chimeric mini-receptors. Role of C-terminal of receptor alpha subunit

We have investigated the role of the C-terminal of the alpha-subunit in the insulin receptor family by characterizing chimeric mini-receptor constructs comprising the first three domains (468 amino acids) of insulin receptor (IR) or insulin-like growth factor I receptor (IGFIR) combined with C-terminal domain from either insulin receptor (IR) (residues 704-719), IGFIR, or insulin receptor-related receptor (IRRR). The constructs were stably expressed in baby hamster kidney cells and purified, and binding affinities were determined for insulin, IGFI, and a single chain insulin/IGFI hybrid. The C-terminal domain of IRRR was found to abolish binding in IR and IGFIR context, whereas other constructs bound ligands. The two constructs with first three domains of the IR demonstrated low specificity for ligands, all affinities ranging from 3.0 to 15 nM. In contrast, the constructs with the first three domains of the IGFIR had high specificity, the affinity of the novel minimized IGFIR for IGFI was 1.5 nM, whereas the affinity for insulin was more than 3000 nM. When swapping the C-terminal domains in either receptor context only minor changes were observed in affinities (<3-fold), demonstrating that the carboxyl-terminal of IR and IGFIR alpha-subunits are interchangeable and suggesting that this domain is part of the common binding site.     

Similar control mechanisms regulate the insulin and type I insulin-like growth factor receptor kinases. Affinity-purified insulin-like growth factor I receptor kinase is activated by tyrosine phosphorylation of its beta subunit

Insulin-like growth factor I (IGF-I) receptors are partially purified from human placenta by sequential affinity chromatography with wheat germ agglutinin-agarose and agarose derivatized with an IGF-I analog. Adsorption specificity to this affinity matrix demonstrates that low coupling ratios of IGF-I analog to agarose yield preparations that are highly selective in purifying IGF-I receptor with minimal cross-contamination by the insulin receptor present in the same placental extracts. Incubation of the immobilized IGF-I receptor preparation with [gamma-32P]ATP results in a marked phosphorylation of the receptor beta subunits, which appear as a doublet of Mr = 93,000 and 95,000 upon electrophoresis on dodecyl sulfate-polyacrylamide gels. The 32P-labeled receptor beta subunit doublet contains predominantly phosphotyrosine and to a much lesser extent phosphoserine and phosphothreonine residues. The immobilized IGF-I receptor preparation exhibits tyrosine kinase activity toward exogenous histone. The characteristics of the IGF-I receptor-associated tyrosine kinase are remarkably similar to those of the insulin receptor kinase. Thus, prior phosphorylation of the immobilized IGF-I receptor preparation with increasing concentrations of unlabeled ATP followed by washing to remove the unreacted ATP results in a progressive activation of the receptor-associated histone kinase activity. A maximal (10-fold) activation is achieved between 0.25 and 1 mM ATP. The concentration of ATP required for half-maximal (30 microM) activation of the IGF-I receptor kinase is similar to that of the insulin receptor kinase. Like the insulin receptor kinase, the elevated kinase activity of the phosphorylated IGF-I receptor is reversed following dephosphorylation of the receptor beta subunit with alkaline phosphatase. Furthermore, the phosphorylation of the IGF-I receptor beta subunit doublet is enhanced by 7-8-fold when reductant is included in the reaction medium, as is observed for the insulin receptor kinase. Significantly, the dose responses of both receptor types to reductant are identical. Both of the 32P-labeled IGF-I receptor beta subunit bands are resolved into six matching phosphopeptide fractions when the corresponding tryptic hydrolysates are resolved by reverse phase high pressure liquid chromatography. Significantly, four out of the six phosphopeptide fractions derived from the trypsinized IGF-I receptor beta subunits are chromatographically identical to those from the tryptic hydrolysates of 32P-labeled insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)