Expression of Psychrophilic Genes in Mesophilic Hosts: Assessment of the Folding State of a Recombinant α-Amylase

α-Amylase from the antarctic psychrophile Alteromonas haloplanktis is synthesized at 0 ± 2°C by the wild strain. This heat-labile α-amylase folds correctly when overexpressed in Escherichia coli, providing the culture temperature is sufficiently low to avoid irreversible denaturation. In the described expression system, a compromise between enzyme stability and E. coli growth rate is reached at 18°C.     

Redistribution of cobalt and nickel in detached wheat shoots: effects of steam-girdling and of cobalt and nickel supply

Detached wheat shoots (ear with peduncle and flag leaf) were incubated for 4 d in a solution containing 1 mM RbCl and 1 mM SrCl2 as well as 10, 40 or 160 μM NiCl2 and CoCl2. The phloem of some plants was interrupted by steam-girdling the stem below the ear to distinguish between xylem and phloem transport. The phloem-immobile Sr flowed mainly to the leaf lamina and to the glumes via the xylem. The Sr transport was not sensitive to steam-girdling. In contrast, the phloem-mobile Rb accumulated during the incubation time mainly in the stem and the leaf sheath. The Rb transport to the grains was impaired by steam-girdling as well as by elevated Ni and Co concentrations in the incubation solution indicating that Rb was transported via the phloem to the maturing grains and that this transport was affected by the heavy metals. Ni was removed more efficiently from the xylem in the peduncle than Co (but far less efficiently than Rb). It became evident that the two heavy metals can also be transferred from the xylem to the phloem in the stem of wheat and reach the maturing grains via the phloem. This revised version was published online in July 2006 with corrections to the Cover Date.     

Dithiothreitol triggers photooxidative stress and fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase in intact pea chloroplasts

In intact chloroplasts isolated from mature pea leaves (Pisum sativum L.), the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) was rapidly fragmented into several products upon illumination in the presence of 1 mM dithiothreitol (DTT). Very similar effects on LSU stability could be observed when illuminated chloroplasts were poisoned with cyanide which, like DTT, inhibits important plastid antioxidant enzymes, or when a light-dependent hydroxyl radical-producing system was added to the incubation medium. Moreover, DTT-stimulated light degradation of LSU was markedly delayed in the presence of scavengers of active oxygen species (AOS). It is therefore suggested that light degradation of LSU in the presence of DTT is mainly due to inhibition of the chloroplast antioxidant defense system and the subsequent accumulation of AOS in intact organelles. When chloroplasts were isolated from nonsenescent or senescent leaves, LSU remained very stable upon incubation without DTT, indicating that the antioxidant system was still functional in the isolated chloroplasts during leaf ageing. Our data support the notion that AOS might be important for the degradation of Rubisco in vivo under oxidative stress.     

Thermodynamic stability of a cold-active alpha-amylase from the Antarctic bacterium Alteromonas haloplanctis

The thermal stability of the cold-active alpha-amylase (AHA) secreted by the Antarctic bacterium Alteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and differential scanning calorimetry. It was found that this heat-labile enzyme is the largest known multidomain protein exhibiting a reversible two-state unfolding, as demonstrated by the recovery of DeltaHcal values after consecutive calorimetric transitions, a DeltaHcal/DeltaHeff ratio close to unity, and the independence of unfolding thermodynamic parameters of scan rates. By contrast, the mesophilic alpha-amylases investigated here (from porcine pancreas, human salivary glands, yellow meal beetle, Bacillus amyloliquefaciens, and Bacillus licheniformis) unfold irreversibly according to a non-two-state mechanism. Unlike mesophilic alpha-amylases, the melting point of AHA is independent of calcium and chloride binding while the allosteric and structural functions of these ions are conserved. The thermostability of AHA at optimal conditions is characterized by a Tm of 43.7 degrees C, a DeltaHcal of 238 kcal mol-1, and a DeltaCp of 8.47 kcal mol-1 K-1. These values were used to calculate the Gibbs free energy of unfolding over a wide range of temperatures. This stability curve shows that (a) the specific DeltaGmax of AHA [22 cal (mol of residue)-1] is 4 times lower than that of mesophilic alpha-amylases, (b) group hydration plays a crucial role in the enzyme flexibility at low temperatures, (c) the temperature of cold unfolding closely corresponds to the lower limit of bacterial growth, and (d) the recombinant heat-labile enzyme can be expressed in mesophilic hosts at moderate temperatures. It is also argued that the cold-active alpha-amylase has evolved toward the lowest possible conformational stability of its native state.     

Nitrogen Addition Increases Freeze Resistance in Black Mangrove (Avicennia germinans) Shrubs in a Temperate-Tropical Ecotone

Ecosystems - Low temperature stress is the primary factor determining the latitudinal limits of tropical plants. As the climate warms, tropical species are migrating poleward, displacing native...     

Structural hierarchy of mechanical extensibility in human von Willebrand factor multimers

The von Willebrand factor (VWF) is a multimeric glycoprotein composed of 80- to 120-nm-long protomeric units and plays a fundamental role in mediating platelet function at high shear. The exact nature of the shear-induced structural transitions have remained elusive; uncovering them requires the high-resolution quantitative analysis of gradually extended VWF. Here, we stretched human blood-plasma-derived VWF with molecular combing and analyzed the axial structure of the elongated multimers with atomic force microscopy. Protomers extended through structural intermediates that could be grouped into seven distinct topographical classes. Protomer extension thus progresses through the uncoiling of the C_1–6 domain segment, rearrangements among the N-terminal VWF domains, and unfolding and elastic extension of the A₂ domain. The least and most extended protomer conformations were localized at the ends and the middle of the multimer, respectively, revealing an apparent necking phenomenon characteristic of plastic-material behavior. The structural hierarchy uncovered here is likely to provide a spatial control mechanism to the complex functions of VWF.     

Isomeric-Activity ratios of trimetoquinol enantiomers on β-adrenergic receptor subtypes: Functional and biochemical studies

Trimetoquinol [1-(3′,4′,5′-trimethoxybenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, TMQ] exists as two enantiomers, and the (?)-(S)-isomer is a potent β-adrenergic receptor (β-AR) agonist. Experiments were conducted to examine the functional and biochemical potencies of the (S)- and (R)-enantiomers of TMQ for interaction with β-AR subtypes in tissues, membrane fractions, and cell systems. The isomeric-activity ratios (IARs) of the TMQ isomers [(S)-isomer ? (R)-isomer] for stimulation of β₁- and β₂-AR of guinea pig right atria and trachea were 224 and 1585, respectively; these IARs were similar to those observed on atypical β-AR systems of rat distal colon (575), rat brown adipocytes (398), but differed from that of rat esophageal smooth muscle (2884) in the presence of pindolol. In the absence of pindolol, the potencies for the TMQ enantiomers were slightly increased; however, the IARs remained unchanged in rat distal colon, rat brown adipocytes, and rat esophageal smooth muscle. Similarly, radioligand binding studies demonstrated that the TMQ isomer β-AR affinities were stereoselective for the (?)-(S)-isomer in membranes of guinea pig left ventricle (β₁) and lung (β₂) giving IARs of 115 and 389, respectively; and in E. coli expressing human β₁- and β₂-AR giving IARs of 661 and 724, respectively. Corresponding IARs of receptor affinities and stimulation of cAMP accumulation in Chinese hamster ovary cells expressing human β₂-AR and rat β₃-AR were 331 and 282, and 118 and 4678, respectively. These results indicate that the (?)-(S)-isomer of TMQ exhibits high affinity, and is a potent and highly stereoselective agonist for each β-AR subtype, that the isomers generally fail to differentiate between the β-AR subtypes, and that, based upon differences in IAR within β₃-AR containing systems, subtypes of atypical β (or β₃)-AR may exist in adipose tissue and smooth muscle. © 1994 Wiley-Liss, Inc.     

High and low affinity binding sites for Concanavalin A on normal human fibroblasts in vitro

The binding of high specific activity, radioactive Concanavalin A to cultured normal human fibroblasts was investigated. We report the presence of two classes of Concanavalin A binding sites on the plasma membranes of these cells. These classes of binding sites are distinguished by their affinities for the lectin. Scatchard analysis of the binding data indicates the presence of a class of high affinity sites which are saturated at about 0.25 μg/ml of Concanavalin A. The other, lower affinity binding sites are not saturated until 50–100 μg/ml Concanavalin A levels are achieved. At 4°C the K_a for the high affinity sites varies between 1.5 – 5 × 10⁹ M^?1 depending on the method used to label the Concanavalin A. For the lower affinity sites K_a varies between 1 – 4 × 10⁶ M^?1. The average number of high affinity sites per cell is 8 × 10⁵ representing less than 1% of the total receptor sites for the lectin.     

Patterns of proteolytic enzyme activities in different tissues of germinating corn (Zea mays L.)

Profiles of pH dependence and activities of live proteolytic enzymes, amino- and carboxypeptidase and endopeptidases active at pH 3.8, 5.4 and 7.5, with casein as substrate, were determined in crude extracts from the various organs of corn seedlings during germination and early development (30°C, dark, 8 d). With respect to the endopeptidases, caseolytic activities at pH 3.8, 5.4 and 7.5 in extracts from endosperm increased concurrently with loss of endosperm N during germination; however, the relative amounts of the pH 7.5 activity were very small. In scutellum extracts, caseolytic activities at both pH 5.4 and 7.5 increased during the initial stages of development but only the increase at pH 5.4 was concurrent with loss of scutellar N. In shoot extracts, caseolytic activities at pH 5.4 and 7.5 were very low and remained relatively constant. There was a progressive increase in shoot N with time. In root extracts, caseolytic activities at pH 5.4 and 7.5 were higher (3-fold) than in shoot extracts. The activity at pH 5.4 remained constant while the activity at pH 7.5 increased during germination. The rate of accumulation of N by the root was low after day 5. The pattern and ratio but not the amounts of the pH 5.4 and 7.5 caseolytic activities of the root were similar to those observed in senescing leaves of field-grown corn. Addition of mercaptoethanol increased (several-fold) the caseolytic activities at pH 3.8 and 5.4, especially the latter, but not the pH 7.5 activity in endosperm extracts and increased the pH 5.4 activity in extracts from scutellum (30%) and roots (30%) while the effect in shoot extracts was negligible. Carboxypeptidase activity was relatively low in young tissue (root tip, 3-d-old shoots) and increased with development of the various organs except the roots (whole) where the activity remained relatively constant. The increases in carboxypeptidase activities were concurrent with decreases in N from endosperm and scutellum; this result indicates that this enzyme in these tissues may be involved (cooperatively with endopeptidases) in the mobilization of reserve protein.Of all the enzymes tested, only carboxypeptidase activity was markedly (in excess of 50%) inhibited by phenylmethylsulfonylfluoride. Only aminopeptidase activity was found in appreciable amounts in endosperm and scutellum of dry kernels. Aminopeptidase activity was highest in organs with high metabolic activity (scutella, shoot, root tips) and decreased in plant parts undergoing rapid loss of nitrogen (endosperm, senescing leaves).Abbreviations AP aminopeptidase - CA caseolytic activity - CP carboxypeptidase - ME mercaptoethanol     

Dual regulation of the synthesis of the arginine pathway carbamoylphosphate synthase of Saccharomyces cerevisiae by specific and general controls of amino acid biosynthesis.

Summary The synthesis of the arginine pathway carbamoylphosphate synthase (CPSase A) of Saccharomyces cerevisiae is subject to two control mechanisms. One mechanism is specific for CPSase A and is exerted by arginine; it probably involves a repressoroperator type of interaction. This specific mechanism regulates the expression of gene cpaI coding for the small glutaminase subunit of CPSase A but has little influence on the production of the large subunit of the enzyme, a product of gene cpaII. This large component, which alone has no biological significance, accumulates freely under conditions of arginine repression. The second mechanism is general: it controls enzyme synthesis in a number of amino acid biosynthetic pathways in addition to the arginine sequence. Two types of evidence that this general mechanism participates in the control of CPSase A synthesis are presented: (1) Derepression upon starvation for any amino acid of which the synthesis is subject to this general control; and (2) repression during growth in amino acid-rich medium. In contrast to the specific mechanism, the general mechanism regulates the expression of both the cpaI and cpaII genes.