An experiment in enzyme evolution studies withPseudomonas aeruginosa amidase

The regulation of amidase synthesis inP. aeruginosa is under positive control. This review describes the experimental evolution of amidase and its regulator protein for the hydrolysis of novel substrates and experiments to elucidate the mechanism of the control system.     

Synthesis of active Olisthodiscus luteus ribulose-1,5-bisphosphate carboxylase in Escherichia coli

The ribulose-1,5-bisphosphate carboxylase (Rubisco) large- and small-subunit genes are encoded on the chloroplast genome of the eukaryotic chromophytic alga Olisthodiscus luteus. Northern blot experiments indicate that both genes are co-transcribed into a single (>6 kb) mRNA molecule. Clones from the O. luteus rbc gene region were constructed with deleted 5 non-coding regions and placed under control of the lac promoter, resulting in the expression of high levels of O. luteus Rubisco large and small subunits in Escherichia coli. Sucrose gradient centrifugation of soluble extracts fractionated a minute amount of carboxylase activity that cosedimented with native hexadecameric O. luteus Rubisco. Most of the large subunit synthesized in E. coli appeared insoluble or formed an aggregate with the small subunit possessing an altered charge: mass ratio compared to the native holoenzyme. The presence in O. luteus of a polypeptide that has an identical molecular mass and cross reacts with antiserum generated against pea large-subunit binding protein may indicate that a protein of similar function is required for Rubisco assembly in O. luteus.     

Growth and activities of enzymes of primary metabolism in batch cultures of Catharanthus roseus cell suspension under different pCO2 conditions

In vitro enzyme activities of glycolysis, pentose-phosphate pathway and dark CO₂ fixation were assayed in batch cultures of heterotrophic Catharanthus roseus cells under various gassing rates and partial pressures of carbon dioxide. Detrimental effects of low pCO₂ culture conditions on the growth characteristics could be linked to marked changes in levels of enzymes of primary metabolism during growth. The enzyme levels observed during the early stages of growth were found to be more stable when a constant pCO₂ (20 mbar) was maintained and enabled exponential growth to be reached more rapidly.The importance of carbon dioxide as a conditioning factor of the culture medium is discussed.     

Response of aromatic-pathway enzymes to changing physiological states of growth in suspension cultures of Nicotiana silvestris

The activity levels of enzymes of aromatic amino acid biosynthesis respond to changing physiological states of growth, as illustrated by results obtained from suspension-cultured cells of Nicotiana silvestris Speg. et Comes line ANS 1 (2N=24). The experimental system provides a foundation for interpretations about overall regulation of enzyme levels in relationship to growth physiology. Levels of activity for shikimate dehydrogenase (EC 1.1.1.25), prephenate aminotransferase and arogenate dehydrogenase were followed throughout a growth cycle obtained by a conventional subculture protocol. Enzyme date were also obtained from cell cultures maintained in continuous exponential growth for greater than 10 generations (EE cells). Both shikimate dehydrogenase and prephenate aminotransferase exhibited elevated stationary-phase levels of enzyme, much of which was carried over into a subsequent subculture. At least 4 generations of exponential growth were required before diminution of the latter two enzymes to the levels characteristic of truly exponential-phase growth (EE cells) occurred. This is reminiscent of the overall behavior of 3-deoxy-D- arabino -heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15), specifically attributed to the properties of the cytosolic isozyme species (DAHP synthase-Co). Elevation of arogenate dehydrogenase also occurred in stationary-phase cells, but diminished rapidly during lag phase to reach the level characteristic of EE cells.     

Leaf ultrastructure of C4 species possessing different Kranz anatomical types in the cyperaceae

The leaf ultrastructure of NADP-malic enzyme type C₄ species possessing different anatomical features in the Cyperaceae was examined: types were the Rhynchosporoid type, a normal Kranz type in which mesophyll cells are adjacent to Kranz cells, and Fimbristyloid and Chlorocyperoid types, unusual Kranz types in which nonchlorophyllous mestome sheath intervenes between the two types of green cells. They show structural characteristics basically similar to the NADP-malic enzyme group of C₄ grasses, that is, centrifugally located chloroplasts with reduced grana and no increase of mitochondrial frequency in the Kranz cells. However, the Kranz cell chloroplasts of the Fimbristyloid and Chlorocyperoid types exhibit convoluted thylakoid systems and a trend of extensive development of peripheral reticulum, although those of the Rhynchosporoid type do not possess such particular membrane systems. The suberized lamella, probably a barrier for CO₂ diffusion, is present in the Kranz cell walls of the Rhynchosporoid type and in the mestome sheath cell walls of the other two types, and tightly surrounds the Kranz cells (sheaths) that are the sites of the decarboxylation of C₄ acids. These ultrastructural features are discussed in relation to C₄ photosynthetic function.     

猪肺血管紧张素转换酶的提纯

本文报道了猪肺血管紧张素转换酶(ACE)的提纯方法及其鉴定,并讨论了方法的改进。肺匀浆经1.6—2.6mol/L硫酸铵沉淀,Sephadex G-200凝胶过滤,DEAE-Sephaeel及羟基磷灰石柱层析步骤,从168克肺中获得4.5毫克酶蛋白纯品。活力回收45.2％,比活力15.6单位/毫克蛋白;和匀浆上清比较,提纯390倍。经聚丙烯酰胺凝胶电泳(pH8.3)鉴定为一条带。按SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)测得其分子量为132,000道尔顿。酶蛋白在-30℃貯存10月,比活力丢失30％。     

Purification of the Lewis blood-group gene associated α-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to Type 1 and lactose-based oligosaccharide chains

A soluble Lewis blood-group gene associated -3/4-L-fucosyltransferase has been purified from human milk by a series of steps involving hydrophobic chromatography on Phenyl Sepharose 4B, ion exchange chromatography on CM-Sephadex C-50, affinity chromatography on GDP-hexanolamine Sepharose 4B and gel filtration on Sephacryl S-200. The first step separated -3-L-fucosyltransferase activity directed towardsN-acetylglucosamine in Type 2 (Gal1-4GlcNAc-R) acceptors from an -3/4-fucosyltransferase fraction acting on both Type 1 (Gal1-3GlcNAc-R) and Type 2 acceptors. Further purification of this latter fraction on CM-Sephadex and GDP-hexanolamine Sepharose gave a single peak of fucosyltransferase activity that catalysed the addition of fucose toN-acetylglucosamine in both Type 1 and Type 2 acceptors and to theO-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described -3/4-fucosyltransferase preparations purified from human milk. However, gel filtration of this preparation on Sephacryl S-200 or Sephadex G-150 separated further amounts of -3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual -3/4-fucosyltransferase that retained strong -4 activity with the Type 1 acceptor, lacto-N-biose 1, and -3 activity with 2-fucosyllactose, but had relatively little -3 activity withN-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins withN-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.     

Regulation of fat body glycogen phosphorylase in larval tobacco hornworm,Manduca sexta

Activation and inactivation of fat body glycogen phosphorylase was investigated in ligated abdomens of larval Manduca sexta and in vitro. After maximal activation through Manduca adipokinetic hormone (AKH) or chilling, inactivation of glycogen phosphorylase commenced as soon as the stimulus for the activation was removed indicating that the enzyme system in the fat body is fine-tuned to low phosphorylase activities which is necessary to allow glycogen synthesis. In intact ligated abdomens phosphorylase can be activated repeatedly by either stimulus showing that the fat body system does not lose its responsiveness. It was impossible to achieve complete conversion of the inactive form of phosphorylase into the active form even after administration of AKH and simultaneous chilling. © 1992 Wiley-Liss, Inc.