Inflorescence bud proteins of Pistacia vera

 The Pistacia vera L. (common name pistachio) is a unique dioecious and deciduous tree species, which is productive under harsh desert climates. We have identified and purified an Inflorescence Bud Protein of 32 kDa (IBP32) from male pistachio trees. There is a close correlation between its accumulation and inflorescence bud development and its disappearance and flowering. Using antibodies raised against this protein, we have identified in female trees the IBP32 and in addition a 27 kDa protein (IBP27), which appears to be specific to female inflorescence buds. The accumulation and disappearance of IBP27 follows the same pattern as that of IBP32. These proteins are glycoproteins rich in glycine and alanine and are highly hydrophilic. Based on the analytical results and immunological cross-reactivity between dehydrin antibodies and the IBPs, it is assumed that the latter are dehydrin-like and may protect inflorescence bud meristems against cold injury during dormancy. The IBPs are the major proteins of the pistachio bud, therefore they may also serve as nitrogen storage during winter for inflorescence bud growth in spring. Received: 17 October 1997 / Accepted: 6 March 1998     

Effect of protease inhibitors on protein degradation in rat hepatoma cells I. Effect on general protein degradation

Tosyllysine chloromethyl ketone and tosylphenylalanine chloromethyl ketone in vitro are active-site specific and irreversible inhibitors of trypsin (EC 3.4.21.4) and chymotrypsin (EC. 3.4.21.1) respectively. Using rat hepatoma cells in suspension culture, both inhibitors were found to partially inhibit breakdown of prelabelled cell proteins ot amino acids, the effect being greastest in the absence of serum. Protein synthesis in rat hepatoma cells, reticulocytes and reticulyte lysates was also irreversibly inhibited by these compounds. Reduction of ATP levels with antimycin a inhibited protein degradation, but neither tosylphenylalanine chloromethyl ketone nor tosyllysine chloromethyl ketone had any effect on ATP concentration in rat hepatoma cells. These results suggest that the degradation of at least some proteins in animal cells may involve the action of serine protease(s).     

Identification of protease IV of E.coli: An outer membrane bound enzyme

The proteolytic activity of $\text{E.coli}$ measured using ¹²⁵I-labelled αS1 casein as substrate, is mainly localised in the outer membrane and is due to an intrinsic outer membrane protein which can be solubilized by deoxycholate. This enzyme exhibits maximum activity at pH 7,5 in Tris-HCl buffer, is resistant to thermal denaturation with a half-life of 28 min. at 90°C in deoxycholate-NaCl buffer and is inhibited by ethylene-diamine tetraacetate, high concentrations of p-aminobenzamidine, tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalaninechloromethyl ketone and by two inhibitors of the processing of the secreted protein precursors, procaine and phenehylalcohol. Whole cells do not exhibit proteolytic activity, nevertheless, some is unmasked when the outer membrane is permeabilized by Tris or ethylenediamine tetraacetate or when vesicles are sonicated. This suggests that the protease is on the inner side of the outer membrane. Because the protease is different from the soluble proteases described in $\text{E.coli}$, and especially from proteases I,II and III, it has been called protease IV.     

Influence of the excision shock on the protein metabolism ofVicia faba L. meristematic root cells

UsingVicia faba root meristems we have shown that protein synthesis was dramatically changed after excision. The amino-acid incorporation dropped to 13% of the level in the unexcised control. This downshift was a direct consequence of the breakdown of polysomes which are converted into monosomes. In order to perform an analysis of the protein pattern by two-dimensional gel electrophoresis, endogenous proteolytic activity, which is high in broad bean root, had to be inhibited. Therefore, several protease inhibitors were tested and a very efficient inhibitor pool was obtained which could be used during the preparation of meristematic cell extracts. Protein-pattern analysis showed important differences between the unexcised control and excised apices. The number of proteins synthesized after excision droped from 250 in the control to 80, as a consequence of polysome breakdown. Futhermore, we present evidence that new and apparently specific proteins are synthesized in response to this excision shock.Abbreviations NEM N-ethylmaleimide - PMSF phenylmethylsulfonyl fluoride - TLCK N-tosyl-L-lysin chloromethyl ketone - TPCK N-tosyl-L-phenylalanine chloromethyl ketone     

Breaking bud dormancy in apple with a plant bioregulator,thidiazuron

The effects of N-phenyl-N′-1,2,3,-thidiazol-5-ylurea (thidiazuron; Dropp; SN49537; TDZ) on metabolic changes in apple buds during dormancy break were determined. The data showed that thidiazuron has the capacity to release lateral buds from dormancy. Decreasing degree of bud break and bud growth with thidiazuron treatment occurred in a basipetal direction, suggesting a gradient of increasingly deep rest from shoot apex to base. The breaking of dormancy by thidiazuron is correlated with increase in DNA, RNA, protein, 1-aminocyclopropane-1-carboxylic acid (ACC), 1-(malonylamino) cyclopropane-1-carboxylic acid (MACC), S-adenosylmethionine (SAM) as well as with greater polyamine formation. Polyamine and ethylene biosynthesis did not seem to be competing for SAM, their common substrate, during bud break and bud development. The release of dormancy in apple bud by thidiazuron was inhibited by cordycepine, 5-fluorouracil, 6-methylpurine and cycloheximide. Inhibition of bud break and bud growth also resulted from treatment with α-difluoromethylarginine (DFMA) and α-difluoromethylornithine (DFMO). DFMO was more inhibitory than DFMA.     

Cleavage of membrane-anchored growth factors involves distinct protease activities regulated through common mechanisms.

The membrane-anchored forms of transforming growth factor-alpha (TGF-alpha) and stem cell growth factors (Kit ligands) KL-1 and KL-2 are converted to soluble growth factor forms by a regulated proteolytic cleavage process. Each of these proteins is cleaved at a distinct site, however their cleavage is activated via a common set of intracellular signaling mechanisms. By using a panel of protease inhibitors, we show here that at least two cell-associated serine protease activities with distinct specificities participate in membrane growth factor cleavage. Two serine protease inhibitors of broad specificity, diisopropylfluorophosphate and 3,4-dichloroisocoumarin, prevent the cleavage of proTGF-alpha and KL-1 but not that of KL-2. Of the agents tested, N-tosyl-L-phenylalanine chloromethyl ketone and various haloenol lactone derivatives are the most potent inhibitors of cleavage of all three membrane growth factors. It is concluded that cleavage of membrane-anchored growth factors involves a proteolytic system with multiple serine protease activities regulated through common mechanisms.     

Characterization of an alkaline protease from Bacillus sp. no. AH-101

Summary The Bacillus sp. no. AH-101 alkaline protease showed higher hydrolysing activity against insoluble fibrous natural proteins such as elastin and keratin in comparison with subtilisins and Proteinase K. The optimum pH of the enzyme toward elastin and keratin was pH 10.5 and pH 11.0–12.0 respectively. The specific activity toward elastin and keratin was 10 600 units/mg protein and 3970 units/mg protein, respectively. The enzymatic activity was not inhibited by p-chloromercuribenzoic acid and iodoacetic acid. Carbobenzoxy-glycyl-glycyl-L-phenylalanyl chloromethyl ketone completely inhibited the caseinolytic activity, but 36% elastolytic activity remained. No inhibitory effect on caseinolytic and elastolytic activity was shown by tosyl-L-phenylalanyl-chloromethyl ketone, tosyl-L-lysine chloromethyl ketone, carbobenzoxy-L-phenylalanyl chloromethyl ketone, and elastatinal. The amino acid composition and amino terminal sequence of the enzyme were determined. The no. AH-101 alkaline protease was compared with subtilisin BPN', subtilisin Carlsberg, no. 221, and Ya-B alkaline proteases. Extensive sequence homology existed among these enzymes. Offprint requests to: H. Takami     

Vegetative axillary bud dormancy induced by shade and defoliation signals in the grasses

Vegetative axillary bud dormancy and outgrowth is regulated by several hormonal and environmental signals. In perennials, the dormancy induced by hormonal and environmental signals has been categorized as eco-, endo- or para-dormancy. Over the past several decades para-dormancy has primarily been investigated in eudicot annuals. Recently, we initiated a study using the monoculm phyB mutant (phyB-1) and the freely branching near isogenic wild type (WT) sorghum (Sorghum bicolor) to identify molecular mechanisms and signaling pathways regulating dormancy and outgrowth of axillary buds in the grasses. In a paper published in the January 2010 issue of Plant Cell and Environment, we reported the role of branching genes in the inhibition of bud outgrowth by phyB, shade and defoliation signals. Here we present a model that depicts the molecular mechanisms and pathways regulating axillary bud dormancy induced by shade and defoliation signals in the grasses.Key words: axillary bud, dormancy, shade, phytochrome, defoliation, shoot branching, teosinte branched1, MAX2, cell cycle, sorghumThe dormancy and outgrowth of axillary buds is regulated by several plant hormones such as auxin, cytokinins, abscisic acid and strigolactones, and by environmental factors such as light quality, quantity and duration as well as water, temperature and nutrient status.^1–3 Since the fate of an axillary bud is regulated by such diverse hormonal and environmental signals and their interactions, the type of dormancy induced varies. In perennials, three types of bud dormancy have been identified.^4,5 Dormancy mediated by factors within the bud is known as endo-dormancy; while dormancy induced by factors within the plant but outside the bud is called paradormancy or correlative inhibition; the best known example being apical dominance. Dormancy induced due to unfavorable environmental conditions is known as eco-dormancy. Although there is an indepth knowledge about para-dormancy in annuals,⁶ few studies have been conducted on eco-dormancy. Similarly, studies of endo-dormancy have largely been restricted to low-temperature mediated growth-cessation of axillary buds of perennial plants.^7,8 To understand the regulation of dormancy and outgrowth of axillary buds in monocots, we initiated a study on the molecular mechanisms inhibiting bud outgrowth by shade and defoliation signals in sorghum. Our results published in the January 2010 issue of Plant, Cell & Environment indicate that different types of dormancy may be induced in axillary buds of annual grasses by various signals and there may be overlapping and independent molecular mechanisms mediating induction of axillary bud dormancy.     

Chromatin-bound protease: degradation of chromosomal proteins under chromatin dissociation conditions.

A chromatin-bound protease, active in 2 M NaCl-5 M urea or 5 M urea alone, was demonstrated in rat liver, kidney, testes, brain, rabbit bone marrow, chicken reticulocyte, and Ehrlich ascites chromatin. Chicken erythrocyte chromatin did not possess any detectable proteolytic activity in salt and urea. The proteolytic activity of rat liver chromatin in salt and urea was found to be independent of the methods of chromatin preparation. The protease can be inhibited by the serine specific reagents phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate and the alkylating reagent, carbobenzoxyphenylalanine chloromethyl ketone, in the presence of organic solvents at 1 mM concentration. The inhibitions of chromatin-bound protease in rat liver by these compounds are irreversible. On the other hand, carbobenzoxyphenylalanine and p-nitrophenyl acetate were shown to be reversible inhibitors of rat liver chromatin-bound protease. The application of these inhibitors during the dissociation of chromatin by salt and urea may be useful to researchers interested in purifying various chromosomal proteins or to those researchers doing reconstitution studies with labile chromatins.     

SERINE PROTEASE FROM MIDGUT OF Bombus terrestris MALES

A serine protease was isolated from midguts of the bumblebee male Bombus terrestris by a combination of precipitation procedures with column chromatography. The purified enzyme exhibited two bands with molecular masses of 25 and 26 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These bands showed a proteolytic activity in zymography assay. Midgut enzymes showed optimum proteolytic activity at pH 9 and 35°C using N‐succinyl‐L‐alanyl‐L‐alanyl‐L‐prolyl‐L‐phenyl‐alanine 4‐nitroanilide as a substrate. The Michaelis constant (Km) and maximum reaction rate (Vmax) were 0.55 ± 0.042 mM and 0.714 ± 0.056 μmol p‐nitroalanine produced min^?1 mg protein^?1, respectively. Inhibition was affected by trypsin inhibitor, but not by phenylmethylsulfonyl fluoride and N‐tosyl‐L‐phenylalanine chloromethyl ketone, which indicated the trypsin‐like but not chymotrypsin‐like specificity. The identity of the serine protease was confirmed by nanoliquid‐tandem mass spectrometry. Eleven unique peptides of the B. terrestris serine protease were found. It shows high homology to a previously reported B. ignitus serine protease covering more than 65% of the protein amino acid sequence.     

Effect of proteases and protease inhibitors on adenylate cyclase activity in rat cerebral cortical membranes

Mild proteolysis of membrane preparations from rat cerebral cortex with low concentrations of endopeptidases such as trypsin or chymotrypsin caused a 50–400% increase in the basal adenylate cyclase activity. Maximal activation of adenylate cyclase was obtained by including the protease in the adenylate cyclase assay, although an activated preparation could be obtained by pretreatment of the membranes with proteolytic enzymes. The proteolytically activated enzyme showed an increased V, with very little change in the K_m for the substrate, ATP. The proteolytically activated enzyme retained responsiveness to activation by sodium fluoride and 5′-guanylylimidodiphosphate (GppNHp), but was no longer activated by gangliosides or calcium-dependent activator protein. Activation by alcohols and detergent was lost or reduced in magnitude. The activity of adenylate cyclase after protease treatment showed a very marked temperature dependence, with maximal activity expressed in the 30–40 °C range and no activation due to the prior protease treatment expressed at either 10 or 50 °C. Basal adenylate cyclase activity was usually slightly inhibited in the presence of various protease inhibitors. Activation by fluoride, gangliosides, or GppNHp was little affected by protease inhibitors although one inhibitor, N-α-tosyl-l-lysine chloromethyl ketone, caused an inhibition of the ganglioside and GppNHp responses, slightly inhibited the fluoride response, and blocked the norepinephrine response normally seen in the presence of gangliosides or GppNHp. This inhibitor caused a loss of β-adrenergic binding sites for dihydroalprenolol in rat cortical membranes which paralleled the loss of the responsiveness of adenylate cyclase to a GppNHp-norepinephrine combination.     

Characterization of a trypsin-like serine protease of activated B cells mediating the cleavage of surface proteins

Activated B cells may cleave their surface receptors due to the proteolytic activity on the cell membrane or in its vicinity. We attempted to isolate and characterize the protease(s) responsible for this cleavage. Zymograms prepared from the supernatant and the plasma membrane fraction of activated human B cells and BL41/95 cell line exhibited a 85-90 kDa doublet band with protease activity, while that of resting B cells did not. Soybean trypsin inhibitor (STI), Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) and EDTA treatment abolished the activity of this protease. The excess of Zn(2+) ions in EDTA did not restore the enzymatic activity, while it was completely recovered in the presence of Ca(2+). We affinity-purified a 85-90 kDa protease from the supernatant of BL41/95 cells using STI coupled to Sepharose 4B beads, and measured its kinetic parameters. For the arginyl substrate K(M) was 358+/-59 microM and for the lysyl substrate 582+/-103 microM. TLCK and benzamidine inhibited the protease at micromolar, while STI at nanomolar concentrations. Both the inhibition profile and the substrate specificity suggest that it is a trypsin-like serine protease. We assume that the 85-90 kDa serine protease expressed on and secreted by activated B cells and BL41/95 cell line is responsible for the cleavage of various membrane proteins, including Fcgamma receptors; thus it may play a crucial role in regulating B cell's function.     

Reversible Inhibition of Poliovirus RNA Synthesis In Vivo and In Vitro by Viral Products

In poliovirus-infected HeLa-S3 cells, the protease inhibitors tolylsulfonyl-phenylalanyl chloromethyl ketone and iodoacetamide cause an accumulation of large precursor proteins, and they block viral RNA synthesis most probably via these products. Viral RNA polymerase activity can, however, be extracted by detergent containing buffer (Tris/Nonidet P-40, deoxycholate) from the inhibited cells. Only cytoplasmic extracts from infected cells treated with tolylsulfonyl-phenylalanyl chloromethyl ketone or iodoacetamide contain a protein which inhibits the in vitro polymerase reaction.     

Changes in the expression of carbohydrate metabolism genes during three phases of bud dormancy in leafy spurge

Underground adventitious buds of leafy spurge (Euphorbia esula) undergo three well-defined phases of dormancy, para-, endo-, and ecodormancy. In this study, relationships among genes involved in carbohydrate metabolism and bud dormancy were examined after paradormancy release (growth induction) by decapitation and in response to seasonal signals. Real-time PCR was used to determine the expression levels of carbohydrate metabolism genes at different phases of bud dormancy. Among differentially-regulated genes, expression of a specific Euphorbia esula β-amylase gene (Ee-BAM1) increased 100-fold after growth induction and 16,000-fold from July (paradormancy) to December (ecodormancy). Sequence data analysis indicated that two genes, Ee-BAM1 and Ee-BAM2, could encode this β-amylase. However, real-time PCR using gene-specific primer pairs only amplified Ee-BAM1, indicating that Ee-BAM2 is either specific to other organs or not abundant. The deduced amino acid sequences of these two genes are very similar at the N-terminal but differ at the C-terminal. Both contain a nearly identical, predicted 48-amino acid plastid transit peptide. Immunoblot analyses identified a 29 kD (mature Ee-BAM1 after cleavage of the transit peptide) and a 35 kD (unprocessed EeBAM1) protein. Both 35 and 29 kD proteins were constitutively expressed in growth-induced and seasonal samples. Immunolocalization indicated that Ee-BAM1 is in the cytosol of cells at the shoot tip of the bud. Ee-BAM1 also surrounds the amyloplasts in mature cells toward the base of the bud. These observations suggests that Ee-BAM1 may have dual functions; serving as reserve protein in the cytosol and as a degrading enzyme at the surface of amyloplasts.     

Freezing exposure releases bud dormancy in Betula pubescens and B. pendula

Bud dormancy in woody plants is released by long-term exposure to non-freezing chilling temperatures, whereas freezing temperatures have been considered to have little or no effect. However, the present results demonstrate that short-term exposure to freezing can release bud dormancy in Betula pubescens (Ehrh.) and B. pendula (Roth). Short-term freezing during the dormancy induction phase improved the release of bud dormancy only if an adequate level of dormancy had been reached. In fully dormant or chilled plants both the percentage and the speed of bud-burst increased, the more so the lower the temperature. Our results rule out the possibility that endogenous abscisic acid could be directly involved in the physiological control of bud dormancy release. The fast, easily applicable method presented here for bud dormancy release could further investigations into the biochemical and biophysical background to the process. The mechanisms of bud dormancy release and its relationship to cold acclimation are discussed in the light of these results, as also are the implications of the findings for modelling of bud dormancy.     

Bacillus subtillis RTSBA6 6.00, a new strain isolated from gut of Helicoverpa armigera (Lepidoptera: Noctuidae) produces chymotrypsin-like proteases

Exploring bacterial communities with proteolytic activity from the gut of the Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) insect pests was the purpose of this study. As initial efforts to achieve this goal here we report the isolation of new Bacillus subtillis RTSBA6 6.00 strain from the gut of H. armigera and demonstrated as proteases producer. Zymographic analysis revealed 12 proteolytic bands with apparent molecular weights ranging from 20 to 185 kDa. Although some activity was detected at acidic pH, the major activity was observed at slight alkaline pH (7.8). The optimum temperature was found to be 35 °C with complete loss of activity at 70 °C. All proteases were completely inactivated by PMSF (phenylmethylsulfonyl fluoride) and TPCK (N-tosyl-l-phenylalanine chloromethyl ketone), suggesting that proteases secreted by B. subtillis RTSBA6 6.00 belong to serine proteases class with chymotrypsin-like activity. The occurrence of protease producing bacterial community in the gut of the H. armigera advocates its probable assistance to insect in proteinaceous food digestion and adaptation to protease inhibitors of host plants.     

Effect of protease inhibitors on protein degradation in rat hepatoma cells II. Effects on ornithine decarboxylase and tyrosine aminotransferase

Tosyllysine chloromethyl ketone and tosylphenylalanine chloromethyl ketone in vitro are active-site specific and irreversible inhibitors of trypsin (EC 3.4.21.4) and chymotrypsin (EC. 3.4.21.1) respectively. Using rat hepatoma cells in suspension culture, both inhibitors were found to partially inhibit breakdown of prelabelled cell proteins ot amino acids, the effect being greastest in the absence of serum. Protein synthesis in rat hepatoma cells, reticulocytes and reticulyte lysates was also irreversibly inhibited by these compounds. Reduction of ATP levels with antimycin a inhibited protein degradation, but neither tosylphenylalanine chloromethyl ketone nor tosyllysine chloromethyl ketone had any effect on ATP concentration in rat hepatoma cells. These results suggest that the degradation of at least some proteins in animal cells may involve the action of serine protease(s).     

Iodination of peptidyl chloromethyl ketones for protease affinity labels

The specificity of peptidyl chloromethyl ketones has been used to label proteases in complex biological systems by incorporating tyrosine into the structure for eventual radioiodination. Contrary to results with iodination of proteins, a mild reagent, that is, one which iodinates at neutrality, was unsuitable, giving complex mixtures with poor reproducibility, apparently because of side reactions at the chloromethyl ketone group. On the other hand, iodine monochloride in acetic acid provided clean products. In the cases examined where a tyrosine residue was not appropriate for the specificity of the target protease, this residue was located well displaced from the primary specificity site. The resultant diiodotyrosine-containing derivatives were generally highly active as protease inhibitors. The p-aminobenzoyl group was used as an alternative to tyrosine as an iodinatable component.