Cis proline mutants of ribonuclease A. I. Thermal stability.

A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60-89). The expressed protein, which contains an additional N-terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification. Site-directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made. In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing. Thermal unfolding experiments on four single mutants give delta Tm approximately equal to 10 degrees C and delta delta G0 (apparent) = 2-3 kcal/mol. The reason is that either the substituted amino acid goes in cis, and cis<==>trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild-type conformation, that allows the substituted amino acid to form a trans peptide bond.     

水分胁迫对植物线粒体结构和脯氨酸氧化酶活性的影响

水分胁迫期间,小麦幼苗芽鞘和棉花幼苗胚轴细胞内游离脯氨酸浓度增加;但复水后又恢复正常。电镜观察发现线粒体肿大,嵴消失。胞质中出现脂肪滴。气相层析技术分析,发现水分胁迫使线粒体脂肪酸组成及含量有明显变化;不饱和脂肪酸含量增加。随着水分胁迫时间的延长,脯氨酸氧化酶活性也明显下降。设想水分胁迫使线粒体结构和组分发生了不利于脯氨酸氧化酶活性表达的变化,因而抑制了酶活性。     

Free proline accumulation in drought-stressed plants

Summary Free-proline accumulation was measured in leaves of intact wheat (Triticum vulgare L. cv. Kalyan Sona), plantago (Plantago ovata Forsk-Isabgool), papavar (Papaver somnifera L. Opium poppy) and mustard (Brassica juncea L. var. Varuna) grown in the field with low to high field water content and thus they were subjected to water stress. Leaf water deficit in percentage was used to determine the degree of stress at the time of proline anlysis.Free proline content was higher in mustard leaves as compared to wheat, plantago and papavar leaves. Water stress enhances the proline content but at same water deficit level the content differ in the leaves of the plants studied.     

Metabolism of polyamines in NaCl-resistant cell lines from Nicotiana sylvestris

The polyamine titers in three cell lines of Nicotiana sylvestris were compared: Type 1, rapidly adapting to NaCl; Type 2, constantly resistant to NaCl; Type 3, a saltsensitive wild strain. During short-term cultivation in MS medium in the presence of 170 mM NaCl (1 passage, 14 d) the changes in polyamine titer in cell suspensions of type 1 (in a slightly adapted state) and non-adapted wild strain (type 3) showed a considerable increase in spermidine and spermine and a decrease in putrescine. After prolonged adaptation to NaCl (20 passages) the putrescine content in the cells of type 1 and of type 2 was increased at the expense of the polyamines. This suggests that the pattern of polyamine titer varies under short- and long-term adaptation to NaCl. The inverse ratio between growth processes and changes in polyamine and proline level indicates that polyamines fulfil primarily a protective and osmorepulatory function in plant cells under NaCl stress.     

Effects of weak acids on proline accumulation in barley leaves: a comparison between abscisic acid and isobutyric acid

Abstract. When isobutyric acid (IBA) or abscisic acid (ABA) are supplied to leaf sections a similar rapid and marked decrease in the intracellular pH is observed. This acidification is accompanied by an increase in proline level which is about the same for both 3 mol m⁻³ IBA and 1 mol m⁻³ ABA treatments.
Fusicoccin (FC), known to act at the proton pump level, almost completely suppresses the ABA-induced acidification of the cell sap, whereas it only partially counteracts the acidifying effect of IBA, in particular during short periods of treatment. This effect of FC is paralleled by a similar inhibition of the induced proline accumulation: in fact, FC completely suppresses the ABA-induced increase in proline during short treatment periods, whereas it is only effective in inhibiting the IBA-induced proline accumulation after long treatment periods.
These data seem to suggest that the ABA- and IBA-induced changes in proline level might be mediated by changes in the intracellular pH.     

脯氨酸对缺钾植株的保护作用

在缺钾培养条件下,高粱苗地上部单位鲜重含钾量由于地上部鲜重的增加而下降。从高梁苗钾缺乏症出现的时间、频率和程度来看,Pro培养似乎有利于缓和或减轻缺钾对植物造成的伤害。在同样含钾量情况下,以Ca(NO_3)_2为氮源的苗受到缺钾伤害要重于以Pro为氮源的苗受到的伤害,其钾缺乏症植株的百分数增加值与含钾量降低值之比为0.26,以Pro为氮源的缺钾营养液培养的苗为0.09,即下降同样量的钾前者苗的钾缺乏症的增加要大大地超过后者。Pro并不减轻在缺钾情况下叶绿素含量的下降。     

Total nitrogen and free amino acids in Morus alba stems from autumn through spring

During leaf senescence and abscission, total nitrogen in leaves of mulberry ( Morus alba L. ev. Shin-ichinose) declined substantially whereas total nitrogen in buds, bark and stem wood increased markedly, suggesting translocation of nitrogen from senescent leaves in the autumn. After leaf abscission the winter buds and stems remained almost unchanged with respect to fresh and dry weight and total nitrogen until bud break in spring. In burst buds these parameters then increased drastically during the new growth while they decreased markedly in stems. Free arginine in the stem bark accumulated in parallel with the accumulation of total nitrogen in buds and stems in the autumn. Accumulation of proline in the wood, bark and buds also started in October but continued even after leaf-fall, increasing until mid-January (wood), mid-February (bark) and the new growth (buds). Prior to and in the early stage of bud break, proline in bark and wood decreased significantly and arginine in stem bark decreased slightly. Simultaneously, proline and arginine in the dormancy-releasing buds and asparagine, aspartic acid and glutamic acid in the buds and stems increased appreciably, suggesting that this increase in free amino acids was mainly derived from free amino acids (proline and arginine) stored in stems. The resulting marked decrease in total nitrogen and the drastic increase in asparagine in the stems and sprouting buds/new shoots were primarily due to a breakdown of protein stored in stems.     

The Utilization of Glucose and Proline by Culture Forms of Trypanosoma brucei*

Exponentially dividing culture forms of Trypanosoma brucei did not utilize glucose provided in the culture medium. The inclusion of 2-deoxyglucose in the medium had no effect on the growth of the trypanosomes. Glucose could be replaced by proline in the liquid phase of biphasic medium without affecting the doubling time of the organisms. Proline added to the culture medium in this way disappeared during the log phase of growth. Glucose in the culture medium was used by the trypanosomes only when the stationary growth phase had been reached. Lipid accumulated in stationary phase trypanosomes grown in glucose-containing medium, but there was no lipid accumulation in log phase organisms or in those which had been grown in proline-containing medium. Bloodstream trypanosomes transferred to liquid medium rapidly utilized glucose over the first 12 hr of culture, and this was accompanied by an accumulation of free pyruvate in the medium. The rate of glucose utilization fell off over the next 36 hr; this was accompanied by a lowering of free pyruvate in the medium and a rise in the proline oxidase activity of the trypanosomes. The possible biologic significance of proline to trypanosomes developing in the midgut of the tsetse vector is discussed.     

Selection and characterization of an L-thiazolidine-4-carboxylic acid resistant callus culture of Vigna radiata (L.) Wilczek var. radiata

Callus cultures were established from seedling root tips of mungbean (Vigna radiata (L.) Wilczek var. radiata) cv. K 851. The growing calli were exposed to increasing concentrations of thioproline — an analog of proline, in the medium. A concentration of 3.0 mM thioproline completely inhibited the growth of the cells. However, after 25 days incubation 5 cell clones were obtained which could grow on this concentration of thioproline. Out of them one vigorously growing cell clone was further characterized. This selected clone contained higher endogenous levels of free proline (5 fold) and K⁺ (1.5 fold) and exhibited elevated tolerance, not only to thioproline but also to exogenously applied NaCl in the growth medium, as compared to the normal sensitive callus cells. Higher endogenous levels of free proline and K⁺ appear to impart dual resistance to thioproline and NaCl to the selected cell strain.     

Uptake of lysine and prolinevia separate α-neutral amino acid transport pathways inMytilus gill brush border membranes

Summary Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel,Mytilus edulis. These membranes contained two distinct pathways for cotransport of Na⁺ and -neutral amino acids. The major pathway in mussel gill BBMV was the alanine-lysine (AK) pathway, which had a high affinity for alanine and for the cationic amino acid, lysine. The AK pathway was inhibited by nonpolar -neutral amino acids and cationic amino acids, but was not affected by -neutral amino acids or imino acids. The kinetics of lysine transport were consistent with a single saturable process, with aJ _max of 550 pmol/mg-min and aK _t of 5 m. The AK pathway did not have a strict requirement for Na⁺, and concentrative transport of lysine was seen in the presence of inwardly directed gradients of Li⁺ and K⁺, as well as Na⁺. Harmaline inhibited the transport of lysine in solutions containing either Na⁺ or K⁺. The alanine-proline (AP) pathway transported both alanine and proline in mussel gill BBMV. The AP pathway was strongly inhibited by nonpolar -neutral amino acids, proline, and -(methylamino)isobutyric acid (Me-AIB). The kinetics of proline transport were described by a single saturable process, with aJ _max of 180 pmol/mg-min andK _t of 4 m. In contrast to the AK pathway, the AP pathway appeared to have a strict requirement for Na⁺. Na⁺-activation experiments with lysine and proline revealed sigmoid kinetics, indicating that multiple Na⁺ ions are involved in the transport of these substrates. The transport of both lysine and proline was affected by membrane potential in a manner consistent with electrogenic transport.