一种具有立体选择性新酯酶的菌种筛选和基因克隆

从土样分离到一株产生具有立体选择性酯水解酶的恶臭假单孢菌(Pseudomonas putida NH33)。构建P.putida NH33的基因组文库,并在E.coli中进行酯酶活性筛选,得到一个含有4.7kb插入片段的阳性克隆。对这个克隆的DNA片段进行序列分析,表明它含有一个1142个碱基的开放阅读框,为编码381个氨基酸的酯酶。推测的酯酶氨基酸序列与其它丝氨酸酯水解酶具有共同的保守基序GXSXG。把该蛋白在E.coli BL21(DE3)中进行表达,并用金属亲和层析纯化至单一条带。利用纯化的酶水解2-芳基丙酸乙酯制备2-芳基丙酸的S型异构体,产物的光学纯度ee_p>99%,说明此酶可8用于手性药物的合成。该酯酶是一个新酶,其基因序列已递交GenBank,登记号为AY896293。     

Polymorphism and antigenic specificity of monocytic acid esterase (EC 3.1.1.6)

With the aim to provide a biochemical or immunological marker for human blood monocytes, their differentiational derivatives, and neoplastic variants the polymorphism of acid α-naphthyl acetate esterase was investigated. Using an isoelectric focusing technique it could be shown that the five enzyme variants of normal human blood monocytes, which do not occur in other human blood cell types, were regularly detectable in human peritoneal and alveolar macrophages. Antisera, raised against isoenzymes of monocytic acid esterase in rabbits, reacted in all cases with every five isoenzymes of blood monocytes; no cross-reactivity was observed with the isoenzymes of acid esterase obtained from purified human granulocytes, peripheral lymphocytes, B lymphocytes, and thymocytes as shown by immunoprecipitation. The results indicate that monocytic acid esterase and its variants could be regarded as a biochemical as well as immunological marker for this cell line.     

A tumor cell line producing granulocyte colony-stimulating factor and an immune suppressive factor

From a patient, both a cell line incapable of secreting granulocyte colony-stimulating factor (G-CSF) (TC873) and a cell line capable of secreting G-CSF (TCM902) were established. The effector cells induced, with TC873 cells showed a high lytic capacity against two types of tumor cells. The effector cells induced by TCM902 cells did not show such capacity. Furthermore, the TCM902 cells excreted a factor suppressing the proliferation of lymphokine activated killer (LAK) cells and the autologous tumor cell lysis of tumor associated lymphocytes. This factor probably is TFG- ₁.Abbreviations CSF colony stimulating factor - ELISA enzyme-linked immunosorbent assay - G granulocyte - GM granulocyte-monocyte - IFN interferon - IL interleukin - LAK lymphokine activated killer - M monocyte - MLTC mixed lymphocyte tumor cell culture - TGF transforming growth factor - TILs tumor infiltrating lymphocytes - TNF tumor necrosis factor     

Population balance between producing and nonproducing hybridoma clones is very sensitive to serum level, state of inoculum, and medium composition

Secreting and nonsecreting hybridoma populations derived from the murine hybridoma cell line 167.4G5.3 were each grown in batch culture in low serum and serum-free media. Under serum-free conditions, a secreting population gained on a predominantly nonsecreting population and competed with the existing antibody-deficient cells effectively. It was found that this competition was sensitive to state of inoculum and medium composition. We conclude that the competition between a secreting and nonsecreting, or more generally, a producing and nonproducing, population is important; the appearance of the latter may not be a significant setback in terms of expected product titer.     

The mucus producing glands and the distribution of the cilia of the pulmonate slug Limax pseudoflavus

The gross anatomy and histochemistry of the mucus-producing glands of Limax pseudoflavus Evans were investigated. The body mucus can be divided into three areas. The dorsal body surface is covered with a sulphated acid mucopolysaccharide/protein mixture secreted largely by five cell types. The pedal mucus is a mixture of neutral mucopolysaccharide from the suprapedal gland. The dorsal and pedal mucus sheets are separated by the peripodal groove whose cells secrete a weakly acid mucus. The duct of the suprapedal gland, the epidermis around the pneumostome, the ventral surface of the peripodal groove and the centre of the underside of the foot are ciliated. The dorsal and pedal mucus remain stationary relative to the body and the substrate respectively and the only rejection currents seen in the mucus are around the pneumostome.
It is suggested that the pedal mucus is formed by the mixture of the products of the suprapedal gland and the mucoprotein secreting gland in the leading edge of the foot, thus producing a mucus suitable for locomotion. Many areas of the animal (e.g. the head, pneumostome, sole and the leading edge of the foot) are capable of producing both a fluid (neutral or weakly acid) and a viscous (acid) mucus. It is postulated that such an arrangement allows for both adhesion and lubrication at different times.     

A cinnamoyl esterase from Aspergillus niger can break plant cell wall cross-links without release of free diferulic acids.

A cinnamoyl esterase, ferulic acid esterase A, from Aspergillus niger releases ferulic acid and 5-5- and 8-O-4-dehydrodiferulic acids from plant cell walls. The breakage of one or both ester bonds from dehydrodimer cross-links between plant cell wall polymers is essential for optimal action of carbohydrases on these substrates, but it is not known if cinnamoyl esterases can break these cross-links by cleaving one of the ester linkages which would not release the free dimer. It is difficult to determine the mechanism of the reaction on complex substrates, and so we have examined the catalytic properties of ferulic acid esterase A from Aspergillus niger using a range of synthetic ethyl esterified dehydrodimers (5-5-, 8-5-benzofuran and 8-O-4-) and two 5-5-diferulate oligosaccharides. Our results show that the esterase is able to cleave the three major dehydrodiferulate cross-links present in plant cell walls. The enzyme is highly specific at hydrolysing the 5-5- and the 8-5-benzofuran diferulates but the 8-O-4-is a poorer substrate. The hydrolysis of dehydrodiferulates to free acids occurs in two discrete steps, one involving dissociation of a monoesterified intermediate which is negatively charged at the pH of the reaction. Although ferulic acid esterase A was able to release monoesters as products of reactions with all three forms of diesters, only the 5-5- and the 8-O-4-monoesters were substrates for the enzyme, forming the corresponding free diferulic acids. The esterase cannot hydrolyse the second ester bond from the 8-5-benzofuran monoester and therefore, ferulic acid esterase A does not form 8-5-benzofuran diferulic acid. Therefore, ferulic acid esterase A from Aspergillus niger contributes to total plant cell wall degradation by cleaving at least one ester bond from the diferulate cross-links that exist between wall polymers but does not always release the free acid product.     

Role of membrane associated serine esterase in the activation of phospholipase A2 by calcium ionophore (A23187) in pulmonary arterial smooth muscle cells

Exposure of rabbit pulmonary arterial smooth muscle cells to 10 M of the calcium ionophore A23187 dramatically stimulates cell membrane-associated phospholipase A₂ activity and arachidonic acid release. In addition, A23187 also enhances cell membrane-associated serine esterase activity. Serine esterase inhibitors phenylmethylsulfonylfuoride and diisopropyl fluorophosphate prevent the increase in serine esterase and phospholipase A₂ activities and arachidonic acid release caused by A23187. A23187 still stimulated serine esterase and phospholipase A₂ activities and arachidonic acid release in cells pretreated with nominal Ca²⁺ free buffer. Treatment of the cell membrane with A23187 does not cause any appreciable change in serine esterase and phospholipase A₂ activities. Pretreatment of the cells with actinomycin D or cycloheximide did not prevent the increase in the cell membrane associated serine esterase and phospholipase A₂ activities, and arachidonic acid release caused by A23187. These results suggest that (i) a membrane-associated serine esterase plays an important role in stimulating the smooth muscle cell membrane associated phospholipase A₂ activity (ii) in addition to the presence of extracellular Ca²⁺, release of Ca²⁺ from intracellular storage site(s) by A23187 also appears to play a role in stimulating the cell membrane-associated serine esterase and phospholipase A₂ activities, and (iii) the increase in the cell membrane-associated serine esterase and phospholipase A₂ activities does not appear to require new RNA or protein synthesis.Abbreviations A23187 calcium ionophore - AA arachidonic acid - PMSF phenylmethyl sulfonylfuoride - DFP diisopropyl-fluorophosphate - DMEM Dulbecco's modified Eagles medium - FCS fetal calf serum - PBS phosphate buffered saline - HBPS Hank's buffered physiological saline - PLA₂ phospholipase A₂     

Mammalian-like differentiation of gastric cells in the shark Hexanchus griseus

Summary Gastric glands of submammalian vertebrates are formed by one single cell type known as the oxyntopeptic cell. This cell secretes both hydrochloric acid and pepsinogen. In mammals, this cell differentiates into an acid secreting cell and a pepsinogen secreting one. In the elasmobranch fish Hexanchus griseus we observed, by means of histological studies at the light-and electron-microscopic levels, two different cell types for the secretion of acid and zymogen. This organization represents an evolutionary divergence in a primitive animal, i.e., the appearance of a feature that is acquired much later in evolution, in mammals.     

A novel strategy for producing chimeric bispecific antibodies by gene transfection

We have used genetic manipulation to produce chimeric bispecific antibodies. Plasmids containing variable regions of immunoglobulin from a murine hybridoma secreting anti-hepatitis B surface antigen were joined to human constant regions. These chimeric plasmids were introduced into transfectomas, secreting chimeric antibodies to iodo-hydroxy-nitrophenyl, by electroporation. Transfectomas secreting bispecific antibodies were identified. This approach has advantages over the fusion of hybridomas or chemical linking of two antibody molecules and will enable the use of bispecific antibodies in vivo.     

The nature of the inhibition of cholesterol esterase by delta 1-tetrahydrocannabinol

An improved assay for cholesterol esterase based on the use of fatty acid radiolabelled cholesterol esters has been developed. The method was used to demonstrate the effects of delta 1-tetrahydrocannabinol on a crude Leydig cell esterase preparation and on crystalline pancreatic esterase. Both enzymes were inhibited and the Km values determined (6.6 mumol/1 for the Leydig cell esterase and 6.25 mumol/1 for the pancreatic enzyme). While the former exhibited a mixed type of inhibition, the latter clearly was competitive.     

Comparison of mesophilic and thermophilic feruloyl esterases: characterization of their substrate specificity for methyl phenylalkanoates

The active sites of feruloyl esterases from mesophilic and thermophilic sources were probed using methyl esters of phenylalkanoic acids. Only 13 out of 26 substrates tested were significant substrates for all the enzymes. Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished activity for both enzymes, which demonstrates the importance of the correct distance between the aromatic group and the ester bond. Maintaining the phenylpropanoate structure but altering the substitutions of the aromatic ring demonstrated that the type-A esterase from the mesophilic fungus Fusarium oxysporum (FoFaeA) showed a preference for methoxylated substrates, in contrast to the type-B esterase from the same source (FoFaeB) and the thermophilic type-B (StFaeB) and type-C (StFaeC) from Sporotrichum thermophile, which preferred hydroxylated substrates. All four esterases hydrolyzed short chain aliphatic acid (C2-C4) esters of p-nitrophenol, but not the C12 ester of laurate. All the feruloyl esterases were able to release ferulic acid from the plant cell wall material in conjunction with a xylanase, but only the type-A esterase FoFaeA was effective in releasing the 5,5' form of diferulic acid. The thermophilic type-B esterase had a lower catalytic efficiency than its mesophilic counterpart, but released more ferulic acid from plant cell walls.     

Effects of cell density and cell proliferation on acid cholesterol esterase and cathepsin activity of cultured human skin fibroblasts

We tested the effects of fibroblast cell density and proliferation on the activities of acid cholesterol esterase and cathepsins, the lysosomal enzymes which degrade low-density lipoprotein. Rates of cell proliferation were increased by: (1) fibroblast conditioned medium, (2) increasing the time since subculture from 3 to 7 days, and (3) decreasing the plating density of cells. Cathepsin activity was consistently decreased as cellular proliferation was increased by these various methods. Changes in acid cholesterol esterase activity were more variable. For example, acid cholesterol esterase activity was consistently a positive function of cell density only at densities under 3 micrograms protein/cm2, while cathepsin activity increased up to densities of 16 micrograms protein/cm2. However, the activities of both enzymes were lower at cell densities of under 3 micrograms cell protein/cm2 compared to confluent cultures. Sparse fibroblast cultures may provide a unique model system to study low-density lipoprotein metabolism since, at low cell density, LDL receptor activity is high while lysosomal activity is low, making it possible that lysosomal degradation could become the rate-limiting step in the process of LDL degradation rather than receptor-mediated internalization of the lipoprotein. This might then allow an accumulation of lipoprotein-derived cholesteryl esters in the cell. Such a model could be relevant to the propensity of arterial cells to become foam cells during atherogenesis.     

Differences in distribution of esterase between cell fractions of rat liver homogenates prepared in various media. Relevance to the lysosomal location of the enzyme in the intact cell

The distribution of esterase in subcellular fractions of rat liver homogenates was compared with that of the lysosomal enzyme acid phosphatase and the microsomal enzyme glucose 6-phosphatase. Most of the esterase from sucrose homogenate sediments with glucose 6-phosphatase and about 8% is recovered in the supernatant. However, up to 53% of the esterase can be washed from microtome sections of unfixed liver, in which less cellular damage would be expected than that caused by homogenization. About 40% of both esterase and acid phosphatase are recovered in the soluble fraction after homogenization in aqueous glycerol or in a two-phase system (Arcton 113-0.25m-sucrose), although glucose 6-phosphatase is still recovered in the microsomal fraction of such homogenates. The esterase of the microsomal fraction prepared from a sucrose homogenate is much more readily released by treatment with 0.26% deoxycholate than are other constituents of this fraction. The release of esterase from the microsomal fraction by the detergent and its concomitant release with acid phosphatase after homogenization in glycerol or the two-phase system suggests that a greater proportion of esterase may be present in lysosomes of the intact cell than is indicated by the results of standard fractionation procedures.     

Relationship between the culturability of stressed Listeria monocytogenes cells in non-selective and selective culture media and the cellular esterase activity measured by solid phase cytometry

Solid phase cytometry in conjunction with fluorescent probe was applied to rapidly quantify cellular esterase activity of Listeria monocytogenes cells. Viability of cells stressed by several treatments (starvation, NaCl, lactic acid and peracetic acid) was assessed simultaneously by their esterase activity estimated by fluorescence intensity and by their ability to multiply in liquid and solid, non-selective and selective, culture media. It was determined that cell physiological state has a significant impact on the cellular fluorescence intensity which was very dependent on the stress suffered by cells. No general relationship was observed between the bacterial populations observed by cytometry and the populations able to grow on culture media. The link between the cell culturability in non-selective and selective media and the esterase activity was always dependent on the stress suffered. Nevertheless, it was also established that solid phase cytometry is an efficient, sensitive and accurate tool to characterize the ability of non-selective and selective enrichment broths to allow the repair of stressed L. monocytogenes cells by examining the increase in the fraction of the most esterase active cells during the course of resuscitation.     

Hydrolysis of diethyl diferulates by a tannase from Aspergillus oryzae

Diferulic acid forms cross-links in naturally occurring plant cell wall polymers such as arabinoxylans and pectins. We have used model ethyl esterified substrates to find enzymes able to break these cross-links. A tannase from Aspergillus oryzae exhibited esterase activity on several synthetic ethyl esterified diferulates. The efficiency of this esterase activity on most diferulates is low compared to that of a cinnamoyl esterase, FAEA, from Aspergillus niger. Of the diferulate substrates assayed, tannase was most efficient at hydrolysing the first ester bond of the 5–5- type of dimer. Importantly and unlike the cinnamoyl esterase, tannase from A. oryzae is able to hydrolyse both ester bonds from the 8–5-benzofuran dimer, thus forming the corresponding free acid product. These results suggest that tannases may contribute to plant cell wall degradation by cleaving some of the cross-links existing between cell wall polymers.     

Chemical and biochemical studies for the differentiation of coagulase-positive staphylococci.

The cell wall composition, the configuration of lactic acid produced from glucose under anaerobic conditions, the occurrence of fructose-1,6-diphosphate (FDP) activated L-lactate dehydrogenase (L-LDH), and the esterase pattern were determined from more than 80 strains of coagulase-positive staphylococci isolated from man and animal. Strains isolated from man, swine, bovines and hares form a rather homogeneous group. They exhibit a similar cell wall composition, produce predominantly D,L-lactate and have a characteristic and simple esterase pattern. Coagulase-positive staphylococci isolated from dogs, horses, minks and pigeons are quite distinct from typical Staphylococcus aureus strains. They exhibit a different cell wall composition, produce only L-lactate, possess an L-LDH which is specifically activated by FDP, and have a quite complex esterase pattern.     

Spectrophotometric assay and electrophoretic detection of trans-feruloyl esterase activity.

Wheat bran cell walls were subjected to mild acid hydrolysis and the major phenolic product was purified and identified as 5-O-(trans-feruloyl)-arabinofuranose. Sensitive continuous and stopped, microtiter plate-based spectrophotometric assays for trans-feruloyl esterase activity were developed using this compound as substrate. Procedures were also developed for the detection of trans-feruloyl esterase activities on gels following electrophoresis using this compound. These procedures are applicable to other natural feruloyl esters derived from plant cell walls by enzymatic hydrolysis. The extracellular trans-feruloyl esterases of Aspergillus niger 814 grown on 1% wheat bran were fractionated by anion-exchange chromatography and isoelectric focusing. These studies indicate that there are multiple forms of trans-feruloyl esterase but that most activity is associated with a major isozyme with a pI of 3.2.     

Role of serine esterase in hydrogen peroxide-mediated activation of phospholipase A2 in rabbit pulmonary arterial smooth muscle cells.

Exposure of rabbit pulmonary arterial smooth muscle cells to hydrogen peroxide cause dose-dependent stimulation of [14C] arachidonic acid (AA) release and enhancement of the cell membrane-associated phospholipase A2 activity as well as of the cell membrane-bound serine esterase activity tested against synthetic substrate p-tosyl-L-arginine methyl ester. While pretreatment of cells with serine protease inhibitors, viz. phenyl methyl sulphonyl fluoride, diisopropyl fluorophosphate and alpha-1-proteinase inhibitor, and antioxidant vitamin E prevents H2O2 stimulation of AA release and the cell membrane-bound serine esterase and PLA2 activities, that with actinomycin D and cycloheximide is devoid of any effect on H2O2 caused stimulation of AA release and the smooth muscle cell membrane associated serine esterase and PLA2 activities. Treatment of the smooth muscle cell membrane suspension with the serine protease trypsin markedly stimulates PLA2 activity. These results suggest that on exposure to H2O2 the smooth muscle cell membrane-bound serine esterase plays an important role in stimulating the cell membrane associated PLA2 activity thereby resulting in an increase in AA release.     

Surface binding, uptake and fate of cationic ferritin in a steroid producing ovarian cell

Ovarian granulosa cells (GC) were exposed to cationic ferritin (CF) in an effort to determine the binding, intracellular fate of endocytosed negatively charged plasma membrane. Following labeling at zero degrees or after pre-fixation, CF accumulated in patches over the cell surface. Exposure to methylamine (MA) resulted in an even distribution of CF over the GC surface. Endocytosis occurred in non-clathrin coated regions of the GC surface and CF was subsequently observed in a variety of smooth surfaced vesicles. Following a 60 min exposure to CF many of the CF containing vesicles appeared to fuse with each other forming larger vesicles. Numerous examples of small CF containing vesicles surrounding large CF containing vesicles were observed. Also observed at 60 min were CF containing multivcsicular and vesicular bodies. Tubular evaginations of the large vesicular structures were often observed; some containing CF. Acid phosphatase activity was observed in multivesicular bodies and the large CF filled vesicles. CF-containing vesicles were also observed in the Golgi region, but CF was never observed in the saccules of this organelle. Our study suggests that endocytosed CF does not pass through the Golgi complex. Many of the internalized vesicles become associated with the lysosomal system. Since GC's secrete progesterone in culture, these observations may indicate that membrane recycling in steroid secreting cells differs from protein secreting cells.