The DNA-binding protease,CND41, and the degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase in senescent leaves of tobacco

Plastids bear their own genome, organized into DNA–protein complexes (nucleoids). Recently, we identified a DNA-binding protease (CND41) in the chloroplast nucleoids of cultured tobacco (Nicotiana tabacum L.) cells. In this study, we examine the biochemical function of this novel DNA-binding protease, particularly in senescent leaves, because antisense tobacco with a reduced amount of CND41 showed retarded senescence. Nitrogen-depletion experiments clearly showed that CND41 antisense tobacco maintained green leaves and constant protein levels, especially ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), throughout the whole plant, whereas wild-type tobacco showed marked senescence and the reduction of protein levels in the lower leaves. In vitro analyses confirmed that CND41 showed proteolytic activity at physiological pH when denatured Rubisco was used as the substrate. These results suggest that CND41 is involved in Rubisco degradation and the translocation of nitrogen during senescence. The possible regulation of protease activity of CND41 through DNA-binding is discussed.Abbreviations CABP 2-Carboxyarabinitol-1,5-bisphosphate - CBB Coomassie Brilliant Blue - GS Glutamine synthetase - OEC33 The extrinsic 33-kDa protein in the oxygen-evolving complex - Rubisco Ribulose 1,5-bisphosphate carboxylase/oxygenase     

Post-translational regulation of CND41 protease activity in senescent tobacco leaves

The degradation of chloroplast proteins is an important occurrence in the mobilization of nutrients from senescing leaves to reproductive organs during senescence. Recently, we proved that tobacco CND41 protease is involved in Rubisco degradation and the translocation of nitrogen during senescence. In this study, we show the post-translational regulation of CND41 protease. Using very specific antibodies that were prepared against CND41-specific peptide (anti-Val 186 to Ser 206), immunoblot analysis clearly indicated a change in the accumulation and processing of CND41 during the maturation of leaves in whole plants. The developmental modification of CND41 was also observed in transgenic tobacco with constitutive expression of CND41 under cauliflower mosaic virus 35S promoter. Further studies of seedlings under senescence induced by combined treatment with nitrogen-starvation and high sucrose confirmed that the processing of CND41 was important for protease activity and senescence. A possible mechanism for the regulation of CND41 activity is discussed.     

CND41, a chloroplast nucleoid protein that regulates plastid development, causes reduced gibberellin content and dwarfism in tobacco

CND41, a DNA binding protein of chloroplast nucleoids, may function as a negative regulator of chloroplast gene expression. The reduction of CND41 in an antisense transformant accelerated plastid development in shoot apex cells and early young leaves, and caused a dwarf phenotype and altered leaf morphology. Plant height and leaf shape could be restored almost to those of the wild type by application of gibberellins (GAs), clearly indicating that a reduction in GA content was a prime cause of the dwarf phenotype in CND41 antisense transformants. The transformants had reduced endogenous levels of active gibberellin (GA₁), a biologically active GA, compared to those of wild-type plants. Possible relationships between chloroplast development affected by CND41 function and GA biosynthesis are discussed.     

Repression of isoperoxidase formation in excised tobacco pith by exogenous, auxin-controlled RNA

Previous work has shown that tobacco pith tissue contains two constitutive isoperoxidases migrating toward the anode at pH 9·0. Within 24 hr of aseptic culture on basal medium, such tissue develops five new isoperoxidases, three cathodic and two anodic. The appearance of the new isoperoxidases involves de novo protein formation; it is inhibited by anaerobic conditions, by such inhibitors as Actinomycin D, and by the plant hormone indole-3-acetic acid (IAA). We now find that phenol RNA extracted from parent pith and injected or vacuum infiltrated into cultured pith explants prevents the appearance of the new isozymes; RNA from cultured pith has no such effect. Hydrolysis with 0·3 N KOH, ribonuclease or proteolytic enzymes partially destroys this activity, while treatment with both ribonuclease and proteolytic enzymes completely destroys it. Fractionation of the RNA indicates that part of the repressor activity is associated with an mRNA-like fraction.     

Rapid alkalinization factors in poplar cell cultures. Peptide isolation,cDNA cloning,and differential expression in leaves and methyl jasmonate-treated cells

A family of peptides inducing rapid pH alkalinization in hybrid poplar (Populus trichocarpa x Populus deltoides) cell culture medium was isolated from hybrid poplar leaves. Five related approximately 5-kD peptides were purified by high-performance liquid chromatography and analyzed by matrix-assisted laser desorption ionization-mass spectrometry. The N-terminal sequence of one of the isolated peptides was very similar to a previously characterized peptide from tobacco (Nicotiana tabacum), rapid alkalinization factor (RALF), which causes a rapid increase in culture medium pH when added to tobacco cell cultures (G. Pearce, D.S. Moura, J. Stratmann, C.A. Ryan [2001] Proc Natl Acad Sci USA 98: 12843-12847). Two unique poplar RALF cDNAs (PtdRALF1 and PtdRALF2) were isolated from a poplar cDNA library and used to study RALF expression in poplar saplings and cultured poplar cells. Both genes were found to be expressed constitutively in poplar saplings and cultured cells. However, PtdRALF2 was expressed in leaves at very low levels, and its expression in suspension culture cells was transiently suppressed by methyl jasmonate (MeJa). Although the function of these novel peptides remains enigmatic, our experiments suggest their role may be developmental rather than stress related. Overall, our study confirms the presence of active RALF peptides in other plants, and provides new data on the complexity of the RALF gene family in poplar.     

Chromatographic separation, of extracellular acid phosphatase of tobacco cells cultured under Pi-supplied and omitted conditions

An extracellular acid phosphatase preparation of tobacco XD-6cells cultured in suspension was resolved into three fractionsby sequential chromatography. Two of these were neutral pyrophosphatasewith diesterase activity, having optimum pH at 6.8. The otheris a nonspecific acid phosphatase having optimum pH at 5.8.The latter was concluded to be involved in the increase in extracellularactivity upon Pi-depletion. (Received August 31, 1976; )     

DNA topoisomerase I from rice: Enzyme synthesis in germination, and partial purification from cultured cells

DNA topoisomerase I activity was observed in two-day-old seeds of rice when the seeds started germination at 30°. Partially purified enzyme from cultured rice cells showed maximum activity at pH 7.0 with 75 mM NaCl in the absence of ATP, and showed resistance to camptothecin and DNA-intercalating reagents. The M_r was ca 80 000 using gel permeation on a Sephacryl S-200 column. After fractionation of the homogenate from cultured rice cells by centrifugation, the activity was observed mainly in the crude nuclear fraction.     

Characterization of thiol-, aspartyl-, and thiol-metallo-peptidase activities in Madin-Darby canine kidney cells

We combined fluorogenic substrates or internally quenched fluorescent peptides with specific inhibitors in the pH profile of proteolytic activity experiments in order to detect proteolytic activities in lysates of MDCK cells. Hydrolytic activities related to cathepsin B, L, and D were observed. Serine-proteinase was not detected; however, we clearly demonstrated the presence of a thiol-metallo-endo-oligopeptidase, also called thimet-oligopeptidase (TOP). This peptidase from MDCK cells has substrate and inhibitor specificities as well as an activation profile with mercaptoethanol that are indistinguishable from the recombinant rat testis TOP (EC 3. 4.24.15). In addition, polyclonal purified antibodies to this enzyme depleted the TOP activity of MDCK cells in whole homogenate. Although we present only preliminary data, TOP is secreted by MDCK cells. The presence of TOP in a phenotype polarized MDCK cells can have special significance in the cytoplasmic selection, transport, or clearance of short peptides due to restriction of the enzyme to sequences from 6 to 17 amino acids. Therefore, the MDCK cell could be a very useful cellular model with which to study some of the suggested TOP biological functions as processing of biological active peptides and antigen presentation.     

Intramitochondrial proteolytic activity of rat liver]

Highly purified mitochondria and lysosomes are isolated from rat liver homogenate. pH optimum of proteolytic activity with respect to proteins of own structures and to mitochondrial structural protein is investigated. The purification of mitochondria from lysosomes is found to be accompanied by the change of proteolytic activity pH optimum from 5.0 to 6.0 in coarse and purified mitochondria respectively. Comparative study of structural protein hydrolysis products with enzyme preparations from purified mitochondria and lysosomes has revealed differences in the spectrum of the reaction products. The data obtained suggest a presence of a proteolytic enzyme in rat liver mitochondria.     

Cytoplasmic origin of the so-called nuclear neutral histone protease.

1. We have investigated the origin of proteolytic activity which causes degradation of histones in chromatin isolated from Xenopus liver and the rat liver at neutral pH. Polyacrylamide disc gel electrophoresis was used for detection of proteolytic products of histones. 2. No proteolytic degradation of histones occurs in chromatin isolated from Xenopus erythrocytes and rat liver according to our procedure even after prolonged incubation at pH 8.0 and pH 5.0. However with chromatin isolated from Xenopus liver a high level of histone degradation is observed under similar conditions. 3. Mixing isolated nuclei from Xenopus erythrocytes with a crude cytoplasmic fraction from Xenopus liver causes histone proteolysis in isolated chromatin at pH 8.0. In similar experiments with corresponding fractions from rat liver histone proteolysis can be introduced only after repeated freezing and thawing of the cytoplasmic fraction. 4. A purified lysosomal preparation from rat liver causes a similar type of histone degradation upon incubation with chromatin from Xenopus erythrocytes and rat liver. 5. The neutral proteolytic activity that can be introduced in isolated chromatin by a crude cytoplasmic fraction and by a purified lysosomal erythrocytes and rat liver. 5. The neutral proteolytic activity that can be introduced in isolated chromatin by a crude cytoplasmic fraction and by a purified lysosomal fraction from rat liver is inhibited by sodium bisulphite. 6. We conclude that the neutral proteolytic activity which causes degradation of histones in isolated chromatin is due to a contamination with neutral protease(s) originating from cytoplasmic organelles.     

Protease activity, organisation of vegetative primordia and effect of putrescine in tobacco thin layers

Since in tobacco thin layers exogenous putrescine alters the physiological and mor-phogenic responses induced by IAA (indole-3-acetic acid) and/or BA (benzylade-nine), the effect of this polyamine on protease activity and on the formation of meristemoids and vegetative primordia was studied during morphogenesis. Superficial thin layer explants, excised from the stem of tobacco (Nicotiana tabacum L. cv. Samsun) plants in the vegetative stage, were cultured under various hormonal conditions (IAA, IAA+BA, BA) and in a hormone-free medium, in the presence or absence of 100 μM putrescine. Histological analysis showed that no meristemoids were formed on the control medium or with putrescine alone and only a few were formed on IAA-treated explants with or without putrescine. An increasing number of meristemoids was observed in IAA+BA and BA treatments during culture; in both cases this number was enhanced by the presence of exogenous putrescine. Protease activity was evaluated spectrophotometrically using two synthetic substrates, azocasein and N-benzoyl-DL-arginine-p-nitroanilide (BAPNA). In the former, maximum protease activity was observed in IAA+BA- and BA-treated explants on days 10 and 15, respectively, while with IAA activity was lowest, the maximum occurring on days 5–10. In this case exogenous putrescine enhanced protease activity in the presence of IAA alone or with BA, while it decreased it in the presence of BA. BAPNA-mediated proteolytic activity (serine-proteases) was highest in IAA+BA-treated explants, intermediate in BA- and not different from controls in IAA-treated explants. Putrescine only affected proteolytic activity in IAA+BA treatments. The use of specific inhibitors of protease activities indicated that these enzymes belong to two main classes of proteases, that is serine- and thiol-proteases. The pattern of proteolytic activities during culture appeared to be related to the differentiation of meristemoids into vegetative primordia. The effect of exogenous putrescine on protease activity was different depending on different synthetic substrates, developmental patterns, pH and ionic strength.     

Photosynthetic Characteristics and Carbonic Anhydrase Activity in Cells Cultured Photoautotrophically and Mixotrophically and Cells Isolated from Leaves

Cultured green cells of Nicotiana tabacum var. Samsun, Cytisusscoparius Link and Hyoscyamus niger which were grown photoautotrophicallyunder a stream of air enriched with 1% CO₂ or mixotrophicallyin the presence of 3% sucrose and ordinary air showed very lowcarbonic anhydrase activity, which was only 0–9% of thatin the respective intact leaves. The CO₂ compensation pointfor photosynthesis of autotrophically and mixotrophically culturedgreen cells of tobacco was higher than 0.3 mM NaHCO₃ at pH 7.8,but that of the cells isolated from tobacco leaves was lowerthan 0.1 mM NaHCO₃ at pH 7.8. The fact that the cultured cellscannot grow photoautotrophically under ordinary air is due toa high CO₂ compensation point in photosynthesis. The dark respiratoryactivity in both photoautotrophically and mixotrophically culturedtobacco cells was more than 7-fold that in the cells isolatedfrom tobacco leaves. We therefore could not conclude whetherthe high CO₂ compensation point in the cultured cells is dueto the low carbonic anhydrase activity or simply reflects thehigh respiratory activity. (Received July 10, 1980; Accepted November 25, 1980)     

3-Dehydroshikimate dehydratase in mung bean cultured cells

Summary The activity of 3-dehydroshikimate dehydratase was detected in an extract prepared from cells of mung bean (Vigna mungo) that had been cultured in the presence of shikimate while such activity was not detectable in an extract prepared from cells cultured without shikimate. The enzyme was partially purified and characterized. The maximum activity of the enzyme was observed at pH 7.4. The activity was inhibited to a small extent by EDTA and sulfhydryl inhibitors. The partially purified enzyme was sensitive to thermal denaturation but was stabilized by Mg²⁺ ions. These results suggest that 3-dehydroshikimate dehydratase might be induced in mung bean cultured cells in the presence of shikimic acid.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - DHS 3-dehydroshikimic acid - PCA protocatechuic acid - QA quinic acid - SA shikimic acid - SORase shikimate - NAEP oxidoreductase     

Extracellular acid and alkaline proteases from Candida olea

Candida olea 148 secreted a single acid protease when cultured at acidic pH. In unbuffered medium, the culture pH eventually became alkaline and a single alkaline protease was produced. This was the only proteolytic enzyme produced when the organism was grown in buffered medium at alkaline pH. Both proteolytic enzymes were purified to homogeneity (as assessed by SDS-PAGE). The Mr of the acid protease was 30900, the isoelectric point 4.5; optimum activity against haemoglobin was at 42 degrees C and pH 3.3. This enzyme was inactivated at temperatures above 46 degrees C and was inhibited by either pepstatin and diazoacetyl-norleucine methyl ester but was insensitive to inhibition by either 1,2-epoxy-3-(p-nitrophenoxy)-propane or compounds known to inhibit serine, thiol or metallo proteases. The acid protease contained 11% carbohydrate. The alkaline protease had an Mr of 23400 and isoelectric point of 5.4. The activity of this enzyme using azocoll as substrate above 42 degrees C and was inhibited by phenylmethyl-sulphonyl fluoride and irreversible inactivated by EDTA. The enzyme was also partially inhibited by DTT but was insensitive to either pepstatin or p-chloromercuribenzoic acid.     

A plant caspase-like protease activated during the hypersensitive response

To test the hypothesis that caspase-like proteases exist and are critically involved in the implementation of programmed cell death (PCD) in plants, a search was undertaken for plant caspases activated during the N gene-mediated hypersensitive response (HR; a form of pathogen-induced PCD in plants) in tobacco plants infected with Tobacco mosaic virus (TMV). For detection, characterization, and partial purification of a tobacco caspase, the Agrobacterium tumefaciens VirD2 protein, shown here to be cleaved specifically at two sites (TATD and GEQD) by human caspase-3, was used as a target. In tobacco leaves, specific proteolytic processing of the ectopically produced VirD2 derivatives at these sites was found to occur early in the course of the HR triggered by TMV. A proteolytic activity capable of specifically cleaving the model substrate at TATD was partially purified from these leaves. A tetrapeptide aldehyde designed and synthesized on the basis of the elucidated plant caspase cleavage site prevented fragmentation of the substrate protein by plant and human caspases in vitro and counteracted TMV-triggered HR in vivo. Therefore, our data provide a characterization of caspase-specific protein fragmentation in apoptotic plant cells, with implications for the importance of such activity in the implementation of plant PCD.     

Protease production by the ontjom fungus,Neurospora sitophila

Summary The production of protease and mycelium byNeurospora sitophila cultured on solid and liquid potato dextrose media was studied. Maximal activity of protease extracted from 4-day-old cultures occurred at pH 6.5 when an unfractionated peanut (groundnut) protein substrate was used. The greatest protease activity and mycelium production occurred during the first day of the 4-day test period. Potato dextrose media containing more than 0.2 M NaCl resulted in decreased protease and mycelium production, while tapioca starch was without affect at concentrations up to 1.4%. Addition of up to 0.3 M sucrose to growth media greatly stimulated protease production and mycelial growth. Maximal proteolytic activity was observed in extracts from mycelium cultured in potato dextrose media adjusted from pH 6.0 to 7.5. Activity was greater when soluble peanut protein was used as a substrate, compared to unfractionated or globulin protein substrates.     

Purification and properties of acid phosphatase from cultured tobacco cells

One component of acid phosphatase was purified from cultured tobacco cells. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate. The enzyme possesses high activity toward nucleoside di- and triphosphate, much less activity toward nucleoside monophosphates and sugar esters. The MWs of the phosphatase determined by Sephadex G-100 gel filtration and dodecyl sulfate gel electrophoresis were 74000 and 76000, respectively. The phosphatase showed high affinity for concanavalin A-Sepharose and single superimposed bands of protein and carbohydrate on gel electrophoresis, suggesting that it is a glycoprotein.     

Purification and characterization of furin, a Kex2-like processing endoprotease, produced in Chinese hamster ovary cells.

Furin, a mammalian homolog of the yeast Kex2 protease, is associated with Golgi membranes and is involved in cleavage of precursor proteins at sites marked by the Arg-X-Lys/Arg-Arg (RXK/RR) motif. We have recently shown that a furin mutant lacking the transmembrane domain can be secreted from cDNA-transfected cells with proteolytic activity for the fluorogenic peptide t-butoxycarbonyl-Arg-Val-Arg-Arg-4-methylcoumarin-7- amide. In this study, we purified and characterized the recombinant furin from the conditioned medium of these cells. Furin was purified as a mixture of 83- and 81-kDa forms and a 96-kDa form. The differences in molecular mass were not due to differences in molecular mass were not due to differences in glycosylation. Moreover, all forms had the same NH2-terminal sequence beginning at the residue after the Arg-Ala-Lys-Arg sequence. These data suggest that the three different forms may be produced by differential COOH-terminal processing of a furin molecule and that mature furin may be autocatalytically produced. Both enzyme preparations showed a pH optimum at 7.0, required Ca2+ for the activity, and showed essentially the same inhibitor profile. These properties resembled those of the Kex2 protease. Both preparations efficiently cleaved fluorogenic peptides with an RXK/RR sequence and moderately cleaved a peptide with an RXXR sequence, but did not cleave dibasic peptides. The sequence requirements determined in vitro were compatible with those determined by expression studies in cultured cells. These data unequivocally demonstrate that furin is an endogenous cellular protease responsible for cleavage of precursor proteins mainly at RXK/RR sites.     

球形芽孢杆菌C_3—41菌株胞外蛋白酶特性

球形芽孢杆菌能够合成具杀蚊活性的蛋白晶体,该晶体在蚊中肠碱性条件下降解产生毒性,尽管球形芽孢杆菌蛋白酶与杀蚊毒素的降解无关,但它在球形芽孢杆菌杀蚊制剂的产生中有重要意义。同时球形芽孢杆菌产生的碱性蛋白酶具有潜在的医疗价值。 我们以本实验室分离的高效杀蚊菌C_3—41菌株为材料,研究了球形芽孢杆菌蛋白酶的产生特性及其理化性质,在国内尚属首次报道。     

Preparation and Characterization of a Truncated Caricain Lacking 41 Residues from the N-terminal

We purified an 18.8 kD protease from caricain solution. This protease was derived from caricain. It does not have the first 41 residues of the N-terminal sequence of caricain, and its N-terminal residue is Thr. Also, one of the disulfide bonds of caricain (cys²²–cys⁶³) was opened during the formation of the protease. We named this 18.8 kD protease caricain II. Caricain II has a wide pH range, and it is more sensitive to temperature changes than caricain. The proteolytic activity of caricain II is twice as much as that of caricain using casein as a substrate. However, caricain II has a low hydrolytic activity with N-benzoyl-l-arginine ethyl ester (BAEE) that is one of the special substrates of caricain. Our results indicate that caricain II is remarkably different from caricain and it can provide an improvement over caricain on the proteolytic activity.