Oxidative stress, protein carbonylation, proteolysis and antioxidative defense system as a model for depicting water deficit tolerance in Indica rice seedlings

Water deficit is an important constraint to rice (Oryza sativa L.) productivity. The present study was undertaken to investigate whether the level of oxidative stress, carbonylation of proteins, proteolysis and status of antioxidative defense could serve as a model to distinguish water deficit tolerant and sensitive rice cultivars. When 10-day-grown seedlings of two rice cultivars, Malviya-36 (drought-sensitive) and Brown Gora (drought-tolerant) were subjected to ?1.0 and ?2.1 MPa water deficit treatments for 24–72 h with polyethylene glycol 6000 in the medium, a greater decline in the growth of the seedlings and levels of leaf water potential, relative water content, Chl a, Chl b, carotenoids and greater increase in leaf water loss were observed in the sensitive cultivar than the tolerant. Under similar level of water deficit seedlings of sensitive cultivar showed higher level of superoxide anion generation, H₂O₂, lipid peroxidation and proteolysis in roots as well as shoots compared to the tolerant. Drought-tolerant cultivar had higher constitutive level of antioxidative enzymes superoxide dismutase and catalase and the activities of these two enzymes alongwith of guaiacol peroxidase showed greater increase in this cultivar under water deficit compared to the sensitive. A significant decline in the level of protein thiol and a higher increase in protein carbonyls content, also confirmed by protein gel blot analysis with an antibody against 2,4-dinitrophenylhydrazine was observed in the seedlings of drought sensitive cv. Malviya-36 compared to the tolerant cv. Brown Gora when subjected to similar level of water deficit. Seedlings of drought sensitive cultivar, under water deficit, showed higher proteolytic activity, higher number of in-gel activity stained proteolytic bands and higher expression of oxidized proteins in roots compared to the tolerant cultivar. Results suggest that poor capacity of antioxidative enzymes could be, at least partly, correlated with water deficit sensitivity of sensitive cultivar and that higher activity of antioxidative enzymes superoxide dismutase, catalase, guaiacol peroxidase, low proteolytic activity, lower level of protein carbonyls and protein thiolation could serve as a model to depict water deficit tolerance in Indica rice seedlings.     

Biochemical and Enzymatic Changes in Apple Leaf Tissue during Autumnal Senescence

The biochemical changes occurring during the natural senescence of apple leaf tissue (Pyrus malus L., Golden Delicious) coincided with specific changes in the environment. Protein, sugars, and total nitrogen began declining in leaf tissue when the daylength first became less than 14 hours in the second week of August. The activity of triose phosphate dehydrogenase declined shortly afterwards, while the activities of malate dehydrogenase, glutamic dehydrogenase, and aspartate aminotransaminase increased. Chlorophyll, DNA, RNA, and fresh weight began declining when the daylength first became less than 12 hours at the end of September. At the same time sugars and the activities of RNase, polyphenol oxidase, and proteolytic enzymes began increasing. Protein synthesis, total nitrogen, and the activities of malate dehydrogenase, glutamic dehydrogenase, and aspartate aminotransaminase began declining rapidly and amino acids began to accumulate after the first frost of the year. RNase, polyphenol oxidase, and proteolytic activity reached their highest specific activities after the first frost.     

Proteinases in molting fluid of the tobacco hornworm,Manduca sexta

Manduca sexta pharate pupal molting fluid contains more than 10 proteolytic enzymes that differ in relative mobility during electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and gelatin. The major gelatin digesting enzyme was an endoprotease with an apparent molecular weight of 100 kDa. Gel filtration on a Sephacryl S-300 column resolved another endoprotease of similar size that digests azocoll and [³H]casein. In addition we found an aminopeptidase-like enzyme (MW_app 500 kDa) and at least three carboxypeptidase-like enzymes (MW_app 10–60 kDa). Use of pseudosubstrates and inhibitors suggested the presence of both trypsin-like and chymotrypsin-like enzymes with the former activity approx. 10-fold greater than the latter. However, none of the proteolytic enzymes were substantially inhibited by diisopropylphosphorofluoridate or phenylmethylsulfonyl fluoride which are poteint inhibitors of trypsin and chymotrypsin. No carboxyl or sulfhydryl proteases were detected. The enzymes were most active in the neutral to alkaline pH range, but they were relatively unstable during storage which precluded their purification to homogeneity. Proteolysis of Manduca cuticular protein appears to involve a rather complex and unique mixture of endo- and exo-cleaving proteolytic enzymes.     

A quantitative technique for determining proteases and their substrate specificities and pH optima in crude enzyme extracts

A zymography technique based on native polyacrylamide gel electrophoresis (PAGE) has been devised, which enables the substrate specificities, content and pH profiles of proteolytic enzymes to be determined in an unfractionated tissue extract. Enzymes were visualized by exogenous application of small molecule substrates that fluoresce when hydrolyzed. The linearity of response, treatment of background fluorescence, and effects of diffusion of substrate and enzyme were taken into account. Based on these studies, successive application of different substrates on the same gel has enabled the presence and specificity of individual enzymes to be determined. Differences in the concentrations and profiles of enzymes, resulting from environmental factors or ontogeny of the organism, can be assessed from crude extracts on a single gel. The technique was applied to aminopeptidases and peptidases in crude Phaseolus vulgaris leaf extracts. One enzyme active against Ala-AMC (7-amino-4-methylcoumarin), one enzyme active against Z-Arg-AMC, several enzymes active against Leu-AMC, and (for the first time in plants) several enzymes active against Phe-AMC were identified. The technique is very sensitive, and microgram quantities of total protein led to picomoles of liberated AMC, with a linear response over a 32-fold range of concentration. The experimental procedure, including electrophoresis, is rapid, taking approximately 1 h.     

Sugarcane glycoproteins may act as signals for the production of xanthan in the plant-associated bacterium Xanthomonas albilineans

Visual symptoms of leaf scald necrosis in sugarcane (Saccharum officinarum) leaves develop in parallel to the accumulation of a fibrous material invading exocellular spaces and both xylem and phloem. These fibers are produced and secreted by the plant-associated bacterium Xanthomonas albilineans. Electron microscopy and specific staining methods for polysaccharides reveal the polysaccharidic nature of this material. These polysaccharides are not present in healthy leaves or in those from diseased plants without visual symptoms of leaf scald. Bacteria in several leaf tissues have been detected by immunogold labeling. The bacterial polysaccharide is not produced in axenic culture but it is actively synthesized when the microbes invade the host plant. This finding may be due to the production of plant glycoproteins, after bacteria infection which inhibit microbial proteases. In summary, our data are consistent with the existence of a positive feedback loop in which plant-produced glycoproteins act as a cell-to-bacteria signal that promotes xanthan production, by protecting some enzymes of xanthan biosynthesis against from bacterial proteolytic degradation.Key words: leaf scald, infectivity, Saccharum officinarum (L.) cv. mayarí 55-14, sugarcane glycoproteins, xanthan-like polysaccharide, Xanthomonas albilineans     

The protease problem in Neurospora. Variable stability of enzymes in aromatic amino acid metabolism.

A proteolytic activity isolated from Neurospora crassa is shown to be responsible for the variable stability observed in vitro for enzymes involved in aromatic amino acid metabolism. For example, the activity of kynurenine formamidase was insensitive to the action of this protease preparation over a 24-h period of incubation at 25 °C, whereas chorismate synthase, anthranilate synthase, kynureninase, and the five activities of the arom multienzyme system were inactivated during this time. Anthranilate synthase and two of the arom system activities (dehydroquinate synthase and shikimate kinase) were inactivated by the protease preparation within 2 h. Phenylmethanesulfonylfluoride and a specific proteolytic inhibitor from N. crassa prevented inactivation of these enzymes. Spontaneous loss of activity at 25 °C of purified samples of anthranilate synthase, dehydroquinate synthase and shikimate kinase was also prevented by the inhibitors. A method for purifying the inhibitor from N. crassa is described, and its use as a reagent in the analysis of proteolytic action is demonstrated.     

Reaction of the main proteolytic fraction of Schistosoma mansoni cercarial enzymes with synthetic substrates and inhibitors of proteolytic enzymes

The purified proteolytic fraction of Schistosoma mansoni cercarial enzymes (PCF) was inhibited by Diisopropylphosphofluoridate (DFP). Its esterolytic activity was not affected by either of two specific active center reagents of proteolytic enzymes: (1) 1-chloro-3-tosylamido-7-ammo-2-heptanone (trypsin) and (2) l-1-tosylamido-2-phenylethyl chloromethyl ketone (chymotrypsin). Furthermore, PCF did not destroy the biological activity of bradykinin on the isolated guinea pig ileum, nor did it release bradykinin from purified dog plasma bradykininogen.     

Absorption Microscopy of Enzymatically Treated Cell-Free Chloroplasts

Monochromatic light microscopy at 435 mµ shows in Euglena gracilis, the distribution of chlorophyll and the general orientation and geometry of chloroplasts in vivo. In addition it discloses, in swelling chloroplasts, a lamellar pigmented structure. Changes in this structure are observed in extruded swollen chloroplasts treated with lipolytic or proteolytic enzymes. Lipolytic enzymes produce an increase in the number of visible lamellae while proteolytic enzymes disrupt the lamellar array. Correlation of chloroplast swelling behavior and the effects of enzymatic degradation with current electron microscope observations support the following: (1) the pigment lamellae observed in vivo consist of component laminae; (2) the lamellae are separated by sites of swelling; and (3) the integrity of the lamellar structure is primarily dependent upon the intact state of the protein.     

Diversity of Proteolytic Clostridium botulinum Strains, Determined by a Pulsed-Field Gel Electrophoresis Approach

Pulsed-field gel electrophoresis (PFGE) was applied to the study of the similarity of 55 strains of proteolytic Clostridium botulinum (C. botulinum group I) types A, AB, B, and F. Rare-cutting restriction enzymes ApaI, AscI, MluI, NruI, PmeI, RsrII, SacII, SmaI, and XhoI were tested for their suitability for the cleavage of DNA of five proteolytic C. botulinum strains. Of these enzymes, SacII, followed by SmaI and XhoI, produced the most convenient number of fragments for genetic typing and were selected for analysis of the 55 strains. The proteolytic C. botulinum species was found to be heterogeneous. In the majority of cases, PFGE enabled discrimination between individual strains of proteolytic C. botulinum types A and B. The different toxin types were discriminated at an 86% similarity level with both SacII and SmaI and at an 83% similarity level with XhoI. Despite the high heterogeneity, three clusters at a 95% similarity level consisting of more than three strains of different origin were noted. The strains of types A and B showed higher diversity than the type F organisms which formed a single cluster. According to this survey, PFGE is to be considered a useful tool for molecular epidemiological analysis of proteolytic C. botulinum types A and B. However, epidemiological conclusions based on PFGE data only should be made with discretion, since highly similar PFGE patterns were noticed, especially within the type B strains.     

Studies on the Function of Intracellular Ribonucleases : II. The Interaction of Ribonucleoprotein and Enzymes

To determine the possible significance of in vivo or in vitro enzyme action in ribonucleoprotein systems, rat liver microsomes and ribonucleoprotein particles (RNP) prepared from them by deoxycholate treatment were incubated for 1 hour at 37°C. with crystalline pancreatic ribonuclease (RNase) or various RNase-free crystalline proteolytic enzymes. The extent of the degradation of the RNA of the microsomes and RNP was determined and the protein degradation estimated in both cases. With either microsomes or RNP, RNase (0.5 to 1.0 mg. per ml.) degraded from 75 to 95 per cent of the RNA, with little protein breakdown being apparent when microsomes were used but with significant protein degradation in the RNP. When microsomes were treated with proteolytic enzymes approximately 40 to 50 per cent of the original microsomal protein became nonsedimentable while at the same time 60 to 80 per cent of the RNA was also found to be non-sedimentable. Of the non-sedimentable RNA, approximately one-third was in the form of acid-precipitable RNA while the remainder was in the form of acid-soluble nucleotides. When RNP was treated with proteolytic enzymes, about 95 per cent of the RNA could no longer be sedimented. About half of this appeared as acid-precipitable RNA and half as acid-soluble nucleotides. Both microsomes and RNP contained significant RNase activity with RNP exhibiting about 10 times the specific activity of microsomes. Some of the characteristics of this RNase activity were determined and the results with proteolytic enzymes interpreted in light of this activity.     

The contribution of fungal enzymes to the digestion of leaves by Gammarus fossarum Koch (Amphipoda)

Summary Gut extracts of Gammarus fossarum liberated reducing substances (at pH values 7) and amino acids (pH7) from freshly shed oak leaves only after removal of soluble leaf phenols. When carboxymethylcellulose was used at a concentration equal to that of leaf cellulose, release of reducing substances was considerably higher. Fungal enzymes extracted from decomposing leaves were active against structural carbohydrates but showed no proteolytic activity. At low pH values, they retained their full activity in the presence of gut enzymes of G. fossarum, at higher pH values they were inhibited. Conditioned leaves released larger amounts of reducing substances and amino acids when exposed to gut enzymes. The improvement varies with the fungal species used for conditioning and with the length of the conditioning period. The digestibility of leaf carbohydrates and proteins reached separate peaks and then declined. Fungal carbohydrases ingested by G. fossarum retained some activity for up to 4h.     

Amino Acid transport and metabolism in relation to the nitrogen economy of a legume leaf

Net balances of amino acids were constructed for stages of development of a leaf of white lupin (Lupinus albus L.) using data on the N economy of the leaf, its exchanges of amino acids through xylem and phloem, and net changes in its soluble and protein-bound amino acids. Asparagine, aspartate, and γ-aminobutyrate were delivered to the leaf in excess of amounts consumed in growth and/or phloem export. Glutamine was supplied in excess until full leaf expansion (20 days) but was later synthesized in large amounts in association with mobilization of N from the leaf. Net requirements for glutamate, threonine, serine, proline, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine were met mainly or entirely by synthesis within the leaf. Amides furnished the bulk of the N for amino acid synthesis, asparagine providing from 24 to 68%. In vitro activity of asparaginase (EC 3.5.1.1) exceeded that of asparagine:pyruvate aminotransferase (EC 2.6.1.14) during early leaf expansion, when in vivo estimates of asparagine metabolism were highest. Thereafter, aminotransferase activity greatly exceeded that of asparaginase. Rates of activity of one or both asparagine-utilizing enzymes exceeded estimated rates of asparagine catabolism throughout leaf development. In vitro activities of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1) were consistently much higher than that of glutamate dehydrogenase (EC 1.4.1.3), and activities of the former two enzymes more than accounted for estimated rates of ammonia release in photorespiration and deamidation of asparagine.     

Photosynthetic Carbon Fixation Characteristics of Fruiting Structures of Brassica campestris L

Activities of key enzymes of the Calvin cycle and C₄ metabolism, rates of CO₂ fixation, and the initial products of photosynthetic ¹⁴CO₂ fixation were determined in the podwall, seed coat (fruiting structures), and the subtending leaf (leaf below a receme) of Brassica campestris L. cv `Toria.' Compared to activities of ribulose-1,5-bisphosphate carboxylase and other Calvin cycle enzymes, e.g. NADP-glyceraldehyde-3-phosphate-dehydrogenase and ribulose-5-phosphate kinase, the activities of phosphoenol pyruvate carboxylase and other enzymes of C₄ metabolism, viz. NADP-malate dehydrogenase, NADP-malic enzyme, glutamate pyruvate transaminase, and glutamate oxaloacetate transaminase, were generally much higher in seed than in podwall and leaf. Podwall and leaf were comparable to each other. Pulse-chase experiments showed that in seed the major product of ¹⁴CO₂ assimilation was malate (in short time), whereas in podwall and leaf, the label initially appeared in 3-PGA. With time, the label moved to sucrose. In contrast to legumes, Brassica pods were able to fix net CO₂ during light. However, respiratory losses were very high during the dark period.     

东北虎幼体消化系统蛋白水解酶的初步研究

蛋白水解酶在许多生命活动中是必需的物质(Vassalli and Pepper,1994)。蛋白质的酶解修饰(Xuet al.,1999)、细胞迁移、组织再生与修复、消化系统对蛋白质的消化等均与蛋白水解酶有关(Baimbridgeet al.,1992),且蛋白水解酶功能失调会导致许多疾病(Teichertet al.,1989)。东北虎(     

Influence of leaf age on photosynthesis, enzyme activity, and metabolite levels in wheat

The rate of photosynthesis under high light (1000 micromole quanta per square meter per second) and at 25°C was measured during development of the third leaf on wheat plants and compared with the activity of several photosynthetic enzymes and the level of metabolites. The rate of photosynthesis reached a maximum the 7th day after leaf emergence and declined thereafter. There was a high and significant correlation between the rate of photosynthesis per leaf area and the activities of the enzymes ribulose 5-phosphate kinase (r = 0.91), ribulose 1,5-bisphosphate (RuBP) carboxylase (r = 0.94), 3-phosphoglycerate (PGA) kinase (r = 0.82), and fructose 1,6-bisphosphatase (r = 0.80) per leaf area. There was not a significant correlation of photosynthesis rate with chlorophyll content. The rate of photosynthesis was strongly correlated with the level of PGA (r = 0.85) and inversely correlated with the level of triose phosphate (dihydroxyacetone phosphate and glyceraldehyde 3-phosphate) (r = 0.92). RuBP levels did not change much during leaf development; therefore photosynthesis rate was not correlated with the level of RuBP. The rate of photosynthesis was at a maximum when the ratio of PGA/triose phosphate was high, and when the ratio of RuBP/PGA was low. Although several enzymes change in parallel with leaf development, the metabolite changes suggest the greatest degree of control may be through RuBP carboxylase. The sucrose content of the leaf was highest under high rates of photosynthesis. There was no evidence that later in leaf development, photosynthesis (measured under high light and at 25°C) was limited by utilization of photosynthate.     

Two proteases from the latex of Elaeophorbia drupifera

Two serine-centred proteolytic enzymes containing catalytically essential histidine residues have been purified to homogeneity from the latex of Elaeophorbia drupifera. The high (117 K) M_r, form, euphorbain d₁, and the low M_r, (65 K) form, euphorbain d₂, are each composed of subunits of weight 30 000. The subunits differ slightly, as is seen by tryptic mapping and in the amino acid compositions reported for the proteases. Both enzymes have five isoelectric forms, and both display two pH maxima for proteolytic activity. Large molar excesses of sulphydryl-blocking reagents produce some activation of euphorbains d₁ and d₂.     

Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate

The mutualism between leaf-cutting ants and their fungal symbionts revolves around processing and inoculation of fresh leaf pulp in underground fungus gardens, mediated by ant fecal fluid deposited on the newly added plant substrate. As herbivorous feeding often implies that growth is nitrogen limited, we cloned and sequenced six fungal proteases found in the fecal fluid of the leaf-cutting ant Acromyrmex echinatior and identified them as two metalloendoproteases, two serine proteases and two aspartic proteases. The metalloendoproteases and serine proteases showed significant activity in fecal fluid at pH values of 5–7, but the aspartic proteases were inactive across a pH range of 3–10. Protease activity disappeared when the ants were kept on a sugar water diet without fungus. Relative to normal mycelium, both metalloendoproteases, both serine proteases and one aspartic protease were upregulated in the gongylidia, specialized hyphal tips whose only known function is to provide food to the ants. These combined results indicate that the enzymes are derived from the ingested fungal tissues. We infer that the five proteases are likely to accelerate protein extraction from plant cells in the leaf pulp that the ants add to the fungus garden, but regulatory functions such as activation of proenzymes are also possible, particularly for the aspartic proteases that were present but without showing activity. The proteases had high sequence similarities to proteolytic enzymes of phytopathogenic fungi, consistent with previous indications of convergent evolution of decomposition enzymes in attine ant fungal symbionts and phytopathogenic fungi.     

Maize leaf proteases and their effect on endogenous inorganic pyrophosphatase

Several different proteolytic enzymes are present in leaf and root tissue of maize seedlings. The activity of these enzymes diminishes to a basal level by the time seedling height reaches 20–30 cm. We have partially characterized an endopeptidase with trypsin-like specificity and two aminopeptidases, all from leaf tissue, and compared them to previously reported proteases from maize. Both the endopeptidase and the aminopeptidases degrade the maize leaf enzyme, inorganic pyrophosphatase. Modification of the pyrophosphatase by the peptidases results in the formation of catalytically active, electrophoretically distinct products. The aminopeptidases have little effect on several other maize leaf enzymes, but also modify yeast inorganic pyrophosphatase.     

The production of toxic protease by the entomopathogenous fungus Metarhizium anisopliae in submerged culture

The production of a toxic complex of proteolytic enzymes by Metarhizium anisopliae was evaluated with 29 nitrogen sources in modified Czapek-Dox medium in submerged cultures. The proteolytic complex is more constitutive than that of Beauveria bassiana and its production is influenced by the quality of complex natural media. The highest activity was attained with Galleria mellonella proteins. The proteolytic complex manifests proteolytic activity of two pH optima, 5.5 and 8.0. The ratio of these two activities differs markedly with the nitrogen source used, but the major proteolytic activity occurs at pH 5.5.