Heart phosphofructokinase: allosteric kinetics with fructose 6-sulfate.

The allosteric regulation of heart phosphofructokinase was studied at pH 6.9 with an alternative substrate, fructose 6-sulfate. The alternative substrate allowed kinetic studies to be carried out at high enzyme concentrations (0.1 mg/ml) where the effect of allosteric ligands on enzyme physical structure has been studied. A Km for ATP binding (8-10 muM) in the presence of saturating AMP concentrations was found which agreed well with the value obtained at pH 8.2, ATP inhibitory effects closely followed saturation of its substrate site. Hill plots for ATP inhibition gave an interaction coefficient of 3.5 indicating cooperatively between at least four enzyme subunits. Neither AMP nor fructose 6-sulfate affected the cooperativity between the ATP inhibitory sites but only increased the inhibitory threshold. As the ATP concentration was increased from suboptimal to inhibitory levels, interaction coefficients for AMP and fructose 6-sulfate changed from 1 to 2. Increasing citrate concentration resulted in an increase in the interaction coefficient for fructose 6-sulfate to a value of 1.9. Citrate inhibition was synergistic with ATP inhibition with an interaction coefficient of 2. The data indicate that allosteric kinetics of the enzyme can be shown at high enzyme concentrations with the alternative substrate. ATP inhibition appears to involve interaction between at least four subunits, while citrate, AMP, and fructose 6-sulfate interact minimally with two subunits.     

A modular treatment of molecular traffic through the active site of cholinesterase

We present a model for the molecular traffic of ligands, substrates, and products through the active site of cholinesterases (ChEs). First, we describe a common treatment of the diffusion to a buried active site of cationic and neutral species. We then explain the specificity of ChEs for cationic ligands and substrates by introducing two additional components to this common treatment. The first module is a surface trap for cationic species at the entrance to the active-site gorge that operates through local, short-range electrostatic interactions and is independent of ionic strength. The second module is an ionic-strength-dependent steering mechanism generated by long-range electrostatic interactions arising from the overall distribution of charges in ChEs. Our calculations show that diffusion of charged ligands relative to neutral isosteric analogs is enhanced approximately 10-fold by the surface trap, while electrostatic steering contributes only a 1.5- to 2-fold rate enhancement at physiological salt concentration. We model clearance of cationic products from the active-site gorge as analogous to the escape of a particle from a one-dimensional well in the presence of a linear electrostatic potential. We evaluate the potential inside the gorge and provide evidence that while contributing to the steering of cationic species toward the active site, it does not appreciably retard their clearance. This optimal fine-tuning of global and local electrostatic interactions endows ChEs with maximum catalytic efficiency and specificity for a positively charged substrate, while at the same time not hindering clearance of the positively charged products.     

Proline-specific dipeptidyl peptidase from the blue blowfly Calliphora vicina hydrolyzes in vitro the ecdysiostatic peptide trypsin-modulating oostatic factor (Neb-TMOF)

To elucidate the mechanisms of inactivation of the ecdysiostatic peptide trypsin-modulating oostatic factor (Neb-TMOF) in the blue blowfly Calliphora vicina, we investigated its proteolytic degradation. In homogenates and membrane and soluble fractions, this hexapeptide (sequence: NPTNLH) was hydrolyzed into two fragments, NP and TNLH, suggesting the involvement of a proline-specific dipeptidyl peptidase. The dipeptidyl peptidase activity was highest in the late larval stage. It was purified 240-fold from soluble fractions of pupae of mixed age and classified on the basis of several catalytic properties as an invertebrate homologue of mammalian dipeptidyl peptidase IV (EC 3.4.14.5). Fly dipeptidyl peptidase IV has a molecular mass of 200 kDa, showed a pH optimum of 7.5–8.0 with the chromogenic substrate Gly-Pro-4-nitroanilide, and cleaved other chromogenic substrates with penultimate Pro or, with lower activity, Ala. It liberated Xaa-Pro dipeptides from the N-terminus of several bioactive peptides including substance P, neuropeptide Y, and peptide YY but not from bradykinin, indicating that the peptide bond between the two proline residues was resistant to cleavage. Fly dipeptidyl peptidase belongs to the serine class of proteases as the mammalian enzyme does; the fly enzyme, however, is not inhibited by several selective or nonselective inhibitors of its mammalian counterpart. It is suggested that dipeptidyl peptidases exert a regulatory role for the clearance not only of TMOF in flies but for other bioactive peptides in various invertebrates. Arch. Insect Biochem. Physiol. 37:146–157, 1998. © 1998 Wiley-Liss, Inc.     

Variation in antiviral 2',5'-oligoadenylate synthetase (2'5'AS) enzyme activity is controlled by a single-nucleotide polymorphism at a splice-acceptor site in the OAS1 gene

It is likely that human genetic differences mediate susceptibility to viral infection and virus-triggered disorders. OAS genes encoding the antiviral enzyme 2',5'-oligoadenylate synthetase (2'5'AS) are critical components of the innate immune response to viruses. This enzyme uses adenosine triphosphate in 2'-specific nucleotidyl transfer reactions to synthesize 2',5'-oligoadenylates, which activate latent ribonuclease, resulting in degradation of viral RNA and inhibition of virus replication. We showed elsewhere that constitutive (basal) activity of 2'5'AS is correlated with virus-stimulated activity. In the present study, we asked whether constitutive activity is genetically determined and, if so, by which variants. Analysis of 83 families containing two parents and two children demonstrated significant correlations between basal activity in parent-child pairs (P<.0001) and sibling pairs (P=.0044), but not spousal pairs, suggesting strong genetic control of basal activity. We next analyzed association between basal activity and 15 markers across the OAS gene cluster. Significant association was detected at multiple markers, the strongest being at an A/G single-nucleotide polymorphism at the exon 7 splice-acceptor site (AG or AA) of the OAS1 gene. At this unusual polymorphism, allele G had a higher gene frequency in persons with high enzyme activity than in those with low enzyme activity (0.44 vs. 0.20; P=3 x 10(-11)). Enzyme activity varied in a dose-dependent manner across the GG, GA, and AA genotypes (tested by analysis of variance; P=1 x 10(-14)). Allele G generates the previously described p46 enzyme isoform, whereas allele A ablates the splice site and generates a dual-function antiviral/proapoptotic p48 isoform and a novel p52 isoform. This genetic polymorphism makes OAS1 an excellent candidate for a human gene that influences host susceptibility to viral infection.     

Specific inhibitors prevent proteolytic degradation of recombinant proteins expressed in High Five cells

The insect cell line BTI-TN-5B1-4 (High Five) is frequently used to express recombinant proteins in large amounts using the baculovirus expression system. However, extensive proteolytic degradation of recombinant proteins is often encountered. Furthermore, we have observed that recombinant proteins migrate in SDS-PAGE in agreement with poly-ubiquitinated forms of the protein, suggesting a ubiquitin/proteasome degradation pathway. Here, we describe a systematic study unraveling the effect of adding proteasome inhibitors or specific protease inhibitors to the growth medium of High Five insect cells infected with recombinant baculovirus. Furthermore, protease inhibitors were added to the lysis buffer to establish the most efficient way to inhibit proteolytic activity after lysis of baculovirus-infected cells expressing recombinant proteins. We conclude that a combination of adding protease inhibitors to the growth medium and to the lysis buffer minimizes the proteolytic activity in High Five cells. The most efficient protease inhibitors were E-64 in the growth medium together with Leupeptin in the lysis buffer at concentrations higher than with available cocktails of inhibitors. The optimal treatment of High Five cells is different from the optimal treatment of Sf9 cells. For proteins susceptible to ubiquitinylation, a treatment of insect cell cultures with the proteasome inhibitor MG132 (LLL) leads to a considerable reduction of the yield of production of recombinant protein.     

豆科黄华属植物种子表面特征的研究

在扫描电镜下观察了豆科黄华属Thermopsis 18种植物种子的表面纹饰,发现 T．alpina,T.bar- bata,T．inflata,T．lupinoides,T. licentiana,T.smithiana和T．turkestanica的种子表面为粗网状,T californica,T.divaricarpa,T. macrophylla,T．mollis的种子表面为细网状,T.gracilis,T.montana,T. fabacea的种子表面为相对平滑型纹饰,T．alterniflora的种子表面为不规则条形,T．chinensis的种子表 面为粘膜状,T.rhombifolia的种子表面为条形及 T．viuosa的种子表面为碎屑状纹饰。结果表明黄华属的种子表面特征对属下类群的划分有一定意义,对澄清某些混乱的种有一定价值。     

榆科榉属模式的考证

长期以来,Zelkova crenata Spach被认为是榉属(Zelkova)的模式。作者基于国际植物命名法规中的模式指定原则,并通过有关文献的考证,确认Zelkova carpinifolia(Pall.) K. Koch为榉属的合法模式,而并非Z. crenata Spach或Z. carpinifolia(Pall.) Dipp.。     

Studies on heart phosphofructokinase. Use of fructose 6-sulfate as an alternative substrate to study the mechanism of action and active site specificity.

Fructose 6-sulfate was synthesized by direct sulfurylation of fructose and was isolated by two selective steps: (a) conversion of the 6-sulfuryl ester to fructose 1-phosphate-6-sulfate with phosphofructokinase; (b) conversion of fructose 1-phosphate-6-sulfate to fructose 6-sulfate by fructose-1,6-diphosphatase. Utilizing crystalline sheep heart phosphofructokinase, kinetic studies with the alternative substrate were carried out at pH 8.2 which is optimal for nonallosteric kinetics. The data are consistent with an ordered addition of the two substrates with the first, MgATP, being at thermodynamic equilibrium. The Vmax and Km obtained with fructose 6-sulfate were 0.03- and 100-fold, respectively, that obtained with the natural substrate. The study suggests that the divalent phosphoryl moiety is intimately involved in the active site conformation. Identification of the product of the reaction, fructose 1-phosphate-6-sulfate, was confirmed through studies with aldolase, fructose-1,6-diphosphatase, and by 31P NMR. The utilization of fructose 6-sulfate as a substrate by yeast glucose-6-phosphate isomerase could not be demonstrated.     

Diet-induced obesity in rats leads to a decrease in sperm motility

Background  

Obesity is rapidly becoming a worldwide epidemic that affects children and adults. Some studies have shown a relationship between obesity and infertility, but until now it remains controversial. Thus, the aim of the present study was to investigate the effect of high-fat diet-induced obesity on male reproductive parameters.     

长爪沙鼠ATPase8,ATPase6,COX3基因的克隆及序列分析

目的对长爪沙鼠线粒体ATPase8,ATPase6,COX3基因全序列进行测定,并对其进行鉴定及进化分析。方法根据长爪沙鼠已知基因序列设计引物,采用PCR产物测序法,对目的片段进行测序鉴定。结合已公布啮齿类动物ATPase8,ATPase6,COX3基因序列,分析其碱基组成、遗传距离、并基于最小进化法和UPGMA法构建系统进化树。结果获得长爪沙鼠线粒体ATPase8,ATPase6,COX3基因全序列,其与家鼠、小家鼠和仓鼠均具有较高的同源性（76～98%）;进化分析结果显示,长爪沙鼠与家鼠、黑家鼠和仓鼠遗传距离较近;碱基G的含量分别为6.9%,10.7%,15.2%,符合mtDNA的特点;A＋T含量分别为68.2%,64.1%,59.2%,明显低于G＋C含量,符合哺乳动物的特点。结论本研究为首次获得长爪沙鼠ATPase8,ATPase6,COX3基因全序列,长爪沙鼠与家鼠、黑家鼠和仓鼠具有较近遗传距离,本研究为长爪沙鼠进化研究、线粒体的结构和功能研究奠定基础。