The effect of serine and thiol protease inhibitors on the chemiluminescence of human neutrophils in investigations In Vitro

We have studied an indirect role of serine and thiol proteases in the activation of human neutrophils in vitro. Stimulation was evaluated using a chemiluminescence (CL) generation system. Receptor-dependent and receptor-independent stimuli were studied, e.g. opsonized zymosan, formyl-methionyl-leucyl-phenylalanine, platelet activating factor, phorbol myristate acetate, and calcium ionophore A23187. The serine protease inhibitors TPCK and TLCK, and thiol protease inhibitor PHMB, diminished the CL with different potencies and in a dose-dependent manner after treatment of cells with the various stimuli. Non-specific serine protease inhibitor, PMSF, and trypsin substrate TAME, showed a low inhibitory potency with respect to CL generation. Synthetic substrates for chymotrypsin (BTEE, ATEE) significantly inhibited CL with the various stimuli used with some differences in susceptibility to their inhibition. Specific chymotrypsin inhibitors diminished both the resting and activator-induced CL. We suggest that cell-bound chymotrypsin-like protease(s) is involved in the activation of signal transduction in human neutrophils after both receptor-dependent and receptor-independent stimulation.     

Evidence that elastase is the TNF-R75 shedding enzyme in resting human polymorphonuclear leukocytes

We previously showed that a metalloprotease and a serine protease mediate shedding of the TNF-R75 (75-kDa tumor necrosis factor receptor) in neutrophils. Here we show that elastase is the TNF-R75 solubilizing serine protease. Release of the TNF-R75 by resting cells was almost totally inhibited by the serine protease inhibitor diisopropylfluorophosphate (DFP), by two synthetic, chemically unrelated, elastase-specific inhibitors and by alpha1-protease inhibitor. Release after TNF or FMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) stimulation was blocked by DFP and a metalloprotease inhibitor used in combination. Supernatants from resting neutrophils contained a 28-kDa fragment of the receptor, compatible with that generated by elastase, whose appearance was inhibited by DFP. Upon FMLP stimulation, the release of 28-kDa and 40-kDa fragments was observed, which was inhibited by DFP and a metalloprotease inhibitor, respectively. We conclude that elastase is the TNF-R75 sheddase of resting neutrophils and that it contributes to shedding of this receptor in stimulated cells.     

Studies on the effects of protease substrate analogues on some of the actions of insulin

Added TAME (N alpha-p-tosyl-1-anginine methyl ester) or BAME (benzoyl-anginine methyl ester) inhibited insulin induced activation of glucose oxidation and fat cell PDH activation without affecting spermine action on PDH activation and glucose oxidation in fat cells. BAME inhibited insulin-induced generation of both PDH stimulator and PDH inhibitor from liver particulate fraction. In contrast, insulin-induced internalization of insulin receptors and negative cooperativity of insulin receptors were not affected by protease substrate inhibitors. These results suggest that certain actions of insulin (glucose oxidation, generation of PDH regulators) are mediated by proteolytic events, while insulin-induced down regulation and negative cooperativity of insulin receptors are not mediated by activation of endogenous proteases.     

The requirement for esterase activation in the anti-immunoglobulin-triggered movement of B lymphocytes.

In this paper we bring evidence suggesting that there is activation of an esterase upon reaction of anti-immunoglobulin antibodies (anti-Ig) with murine B lymphocytes. B lymphocytes upon exposure to anti-Ig cap the ligand-receptor complexes and immediately afterward become briefly motile. It is this latter step which is inhibitable by exposure to di-isopropyl phosphofluoridate (DFP). Various experimental manipulations indicated that treatment with anti-Ig activates the cell for motility which, however, is not manifested until the temperature is raised to 37 degrees C. The cell incubated with anti-Ig at cold temperatures becomes susceptible to the effect of DFP, suggesting that the antibody-treated cells are activated up to but not beyond the DFP inhibitable step. Exposure of cells to DFP and removal of it before their treatment with anti-Ig does not affect the anti-Ig-induced response. Four lines of evidence indicate that the reduction of lymphocyte movement of DFP is due to the inhibition of an esterase activated by the combination of antibody and cell: 1) The inhibition by DFP is irreversible; once DFP has reacted it can be washed away and the antibody-treated cell is still inhibited. 2) The inhibition increases with time of contact of lymphocytes and DFP and with the concentration of DFP. 3) A very poorly phosphorylating phosphonate, phenyl ethyl pentylphosphonate is completely inactive under conditions where an excellent phosphorylating phosphonate, p nitrophenyl ethyl pentylphosphonate maximally inactivated the cells' movement. 4) The amino acid esters, tosyl L arginine methyl ester and benzoyl arginine methyl ester specifically prevent the inactivation by DFP. The last finding suggests that tosyl L arginine methyl ester and benzoyl L arginine methyl ester might be substrates for the putative antibody-induced lymphocyte esterase. Lymphocytes incubated with antibody in the cold for more than 30 min lose their ability to move when the temperature is raised, suggesting that there is a time-dependent deactivation of the cell.     

Inhibition by phorbol esters of antiimmunoglobulin-induced calcium signalling and B-cell activation

The inhibitory effect of phorbol dibutyrate (PDB) on B-cell stimulation was evaluated using a model in which activation is induced by modest doses of antiimmunoglobulin antibody (anti-Ig) and progression to DNA synthesis is induced by cytochalasin. PDB preferentially inhibited anti-Ig-induced activation and did so during brief (2 hr) preincubation with anti-Ig. Activation was inhibited whether PDB was added before or shortly after anti-Ig. Since activation for cytochalasin responsiveness appears to be mediated by Ca2+, the effect of PDB on the anti-Ig-induced rise in intracellular Ca2+ was evaluated. PDB (and other phorbol esters that activate protein kinase C) inhibited the rise in Ca2+ normally associated with anti-Ig treatment; moreover, PDB reversed an established anti-Ig-induced Ca2+ response. These data suggest that phorbol esters inhibit B-cell activation by interfering with the elevated levels of intracellular Ca2+ produced by cross-linking of surface immunoglobulin by anti-Ig. This could represent a "feedback inhibition" type of response, but it remains to be seen if this occurs under physiological conditions of protein kinase C activation.     

Rapid killing of actinomycin D-treated tumor cells by human monocytes. II. Cytotoxicity is independent of secretion of reactive oxygen intermediates and is suppressed by protease inhibitors

Pretreatment with Actinomycin D (ActD, 1 microgram/ml for 3 hr) rendered WEHI 164 tumor cells susceptible to killing by human monocytes in a 6-hr 51Cr release assay. The present study was designed to elucidate the role of reactive oxygen intermediates (ROI) and of proteolytic enzymes in this reactivity. ActD-treated WEHI 164 cells did not trigger any measurable release of O-2 or H2O2 from monocytes. Monocytes exposed to phorbol-12-myristate-13-acetate, which enhanced release of ROI, did not show augmented killing of ActD-treated tumor cells. Scavengers of oxygen metabolites (catalase, superoxide dismutase, gluthatione, and mannitol), which inhibited ROI-mediated PMA-induced monocyte cytotoxicity against erythrocytes, did not affect monocyte killing of ActD-treated WEHI 164 cells. Enzymatically generated ROI with xanthine/xanthine-oxidase glucose/glucose-oxidase did not show preferential killing of ActD-treated WEHI 164 cells. Two patients with chronic granulomatous disease had normal levels of monocyte cytotoxicity against ActD-treated tumor cells. To determine the possible role of proteolytic enzymes in mediating this reactivity, we studied various antiproteases. Organophosphorous agents (DFP and PMSF), chloromethyl-ketone derivatives of tosylamino acids (TLCK and TPCK), Actinomyces products (pepstatin and chymostatin), and the synthetic protease substrate TAME inhibited monocyte-mediated cytotoxicity against ActD-treated WEHI 164 cells. The macromolecular protease inhibitors alpha-1 antitrypsin, bovine pancreatic trypsin inhibitor (BPTI), soybean trypsin inhibitor, and the synthetic protease substrate ATEE had little effect on monocyte cytotoxicity. When monocytes were preincubated with drugs for 1 hr and washed, TLCK, TPCK, and PMSF inhibited cytolysis, whereas the less effective chymostatin and TAME and the inactive BPTI had no effect under these conditions. Inhibition by preincubation with TLCK, PMSF, and TPCK was completely reversed after 6 hr of culture. Supernatants of monocyte cultures had lytic activity against ActD-treated WEHI 164 but not against untreated cells. Antiproteases inhibited the lytic activity of monocyte supernatants. These results strongly suggest that ROI do not play a critical role in monocyte-mediated rapid killing of drug-treated tumor cells, and that proteolytic enzymes are involved in this reactivity.     

The effects of various serine protease inhibitors on estrogen receptor steroid binding

Two serine protease inhibitors, phenylmethanesulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP), were utilized to investigate the possible involvement of serine hydroxyl groups on 17 beta-estradiol binding to the rat estrogen receptor (ER). Single point saturation analysis and Scatchard analysis demonstrated that both 5 mM PMSF and 5 mM DFP were able to inhibit steroid binding to the ER after incubation at 37 degrees C, but neither were able to inhibit steroid binding of the nonactivated ER (0-4 degrees C). The reducing agent dithiothreitol (DTT) was used to differentiate between the interaction of PMSF with serine groups or with sulfhydryl groups of the receptor. When incubated in the presence of 5 mM PMSF, various concentrations of DTT up to 25 mM were not able to overcome the inhibition of this agent, indicating that there was no interaction of PMSF with sulfhydryl groups. Thus, these findings indicate that serine hydroxyl groups are involved in steroid binding of the rat ER.     

The role of nuclear serine proteases in the degradation of newly synthesized histones and ribosomal proteins

To examine whether serine proteases of rat liver chromatin are also involved in the degradation of newly synthesized and unbound ribosomal proteins and histones, like the nuclear thiol protease which we reported previously (Tsurugi, K. & Ogata, K. (1979) Eur. J. Biochem. 101, 205-213), in vivo experiments were carried out with serine protease inhibitor, PMSF. The following results were obtained. When normal rats received an intraperitoneal injection of PMSF (10 mg per 100 g body weight), nuclear serine proteases were inhibited almost completely for at least 90 min. PMSF did not affect the synthesis of proteins and RNAs of ribosomes and other subcellular fractions. The effects of PMSF treatment in vivo on the degradation of newly synthesized ribosomal proteins and histones in regenerating rat liver pretreated with a low dose of actinomycin D, which preferentially inhibited rRNA synthesis, were examined by using the double-isotope method. It was found that PMSF treatment did not affect their degradation. On the other hand, administration of E-64, a thiol protease inhibitor, to partially hepatectomized rats inhibited the degradation of those proteins markedly. From these results, it is concluded that the nuclear thiol protease, but not serine proteases, is preferentially involved in the degradation of newly synthesized ribosomal proteins and histones which are not associated with rRNA and DNA, respectively.     

Suppression of human leukocyte tumor necrosis factor secretion by the serine protease inhibitor p-toluenesulfonyl-L-arginine methyl ester (TAME)

The addition of the serine protease inhibitor p-toluenesulfonyl-L-arginine methyl ester (TAME) to human peripheral blood mononuclear cells suppressed TNF secretion in a concentration dependent manner. At a concentration 10 mM TAME leukocyte TNF release was completely inhibited without decreasing the secretion of IL-1 alpha. Simultaneously exposing leukocytes to 10 mM TAME and either 1000 U/ml IFN-gamma or 10 micrograms/ml LPS reduced the quantity of TNF secreted by 75% and 47%, respectively, when compared with the effect of either IFN-gamma or LPS alone. TAME was most effective when added to leukocytes at the initiation of culture and the suppressive effects of this protease inhibitor were reversible by washing the cells. TAME suppressed TNF secretion without affecting either the level of TNF mRNA or the expression of cell surface cytokine. These findings suggest that leukocyte TNF secretion is dependent upon the action of one or more serine proteases.     

Detection and partial characterization of lymphoid cell surface proteases

Cultures of viable thymocytes and lymph node cells (LNC) were found to exhibit neutral protease activity toward radiolabeled protein substrates. Proteases were not actively secreted in serum-free culture. Thymocyte surface proteases were not affected by incubation of the cells in 1 mM ethylenediaminetetraacetic acid (EDTA) or 1 mM ethylene glycol bis(aminoethyl ether) N, N'-tetraacetic acid (EGTA); however, approximately 25% of lymph node cell surface protease activity was released from the cells by EDTA. It was concluded that the majority of protease activity displayed by both cell types was tightly associated with the cell surface. The inhibitor sensitivity of the cell surface proteases detected on hamster thymocytes and LNC and rat thymocytes was very similar. Cell surface protease activity was inhibited (85%) by the serine protease inhibitors diisopropylfluorophosphate (DFP) and phenylmethylsulfonylfluoride (PMSF) and was partially inhibited by l-1-tosylamide-2-phenylethylchloromethyl ketone(TPCK) and soybean trypsin inhibitor (SBTI), but not by N-α-p-tosyl-l-lysine-chloromethyl ketone (TLCK) or ?-aminocaproic acid (EACA). The bacterial protease inhibitor antipain was strongly inhibitory whereas leupeptin was less effective and elastinal did not inhibit cell surface protease activity. Thymocyte surface proteases were also inhibited (65%) by ZnCl₂, but not be several other divalent cations. In LNC, both ZnCl₂ and NiCl₂ were inhibitory to a lesser extent (32% inhibition). At least one surface protease in both thymocytes and LNC could function as a plasminogen activator.     

Proteolysis of cardiac gap junctions during their isolation from rat hearts

Summary Gap junctions (GJ) isolated from rat hearts in presence of the protease inhibitor phenylmethylsulfonylfuoride (PMSF) contain a M_r 44,000 to 47.000 major polypeptide and have a urea-resistant layer of fuzz on their cytoplasmic surfaces, whereas junctions isolated without PMSF are proteolyzed to a M_r 29.500 polypeptide by a serine protease and have smooth cytoplasmic surfaces (C.K. Manjunath, G.E. Goings & E. PageAm. J. Physiol. 246:H865–H875, 1984). Rat liver GJ isolated with or without PMSF contain a M_r 28,000 polypeptide and have smooth cytoplasmic surfaces. Here we examine the origin, type and inhibitor sensitivity of the heart protease; why similar proteolysis is absent during isolation of rat liver gap junctions; and whether the M_r 44.000 to 47,000 cardiac GJ polypeptide is the precursor of the M_r 29,500 subunit. We show that the M_r 44,000 to 47,000 polypeptide corresponds to the unproteolyzed connexon subunit; that proteolysis of this polypeptide occurs predominantly during exposure to high ionic strength solution (0.6m KI) which releases serine protease from mast cell granules; that this protease is inhibitable with PMSF and (less completely) soybean trypsin inhibitor and chymostatin; and thatin vivo degranulation of mast cells by injecting rats with compound 48/80 fails to prevent breakdown of cardiac GJ during isolation. The results support the concept that GJ from rat heart and liver differ in protein composition.     

Presence of Organelle-Associated Serine Enzymes in Spinach Leaves

Particulate fractions prepared from spinach leaves by differentialcentrifugation were analyzed for proteins capable of bindingdi-isopropyl phosphorofluoridate (DFP); [3H]-labeled DFP andSDS-polyacrylamide gel electrophoresis were used. The chloroplast-richfraction contained one kind of DFP-binding protein, whose bindingwas inhibited by phenylmethylsulfonyl fluoride (PMSF). The mitochondrion-richfraction contained another DFP-binding protein, whose bindingwas sensitive to PMSF and L-1-tosylamide-2-phenylethyl chloromethylketone (TPCK). The microsome-rich fraction contained three PMSF-sensitiveDFP-binding proteins; one was sensitive to both Np-tosyl-L-lysinechloromethyl ketone (TLCK) and TPCK, one was sensitive to TLCKand one to TPCK. These DFP-binding proteins are believed tobe serine proteases. (Received April 30, 1983; Accepted October 17, 1983)     

Time-dependent inhibition of tuberculin-induced lymphocyte DNA synthesis by a serine protease inhibitor

Diisopropylfluorophosphate (DFP), a group-specific irreversible inhibitor of serine proteases, has been shown to exert time-dependent inhibition of DNA synthesis of lymphocytes stimulated by three different B lymphocyte mitogens: purified protein derivative of tuberculin (PPD), endotoxin protein (EP), and lipopolysaccharide (LPS). The time-dependent inhibition profile found in B lymphocytes is absent in concanavalin A (Con A)-stimulated T lymphocytes. Structural analogs of DFP, which have lost the phosphorylating ability, are not inhibitory. Inhibition of DNA synthesis by DFP is reversible in the first 8 hr of mitogenic stimulation. Maximal and irreversible inhibition by DFP occurs around the 16th hour of stimulation. These data support the postulate that a mitogenesis-linked protease, or proteases, in B lymphocytes is absent in the resting cells but is made available several hours before the initiation of DNA synthesis in the late G1 phase of the cell cycle.     

Proteases and Their Involvement in the Infection and Immobilization of Nematodes by the Nematophagous Fungus Arthrobotrys oligospora

The nematophagous fungus Arthrobotrys oligospora produced extracellular proteases when grown in a liquid culture, as revealed by measuring the hydrolysis of the chromogenic substrate Azocoll. The extracellular protease activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and other serine protease inhibitors and partly inhibited by the aspartate protease inhibitor pepstatin and by a cysteine protease inhibitor [l-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane, or E-64]. Substrate gel electrophoresis showed that the fungus produced several different proteases, including multiple serine proteases. The function of proteases in the infection of nematodes was examined by treating the fungus with various protease inhibitors. None of the inhibitors tested affected the adhesion of nematodes to the traps, but incubating trap-bearing mycelium with a serine protease inhibitor, PMSF, antipain, or chymostatin, or the metalloprotease inhibitor phenanthroline significantly decreased the immobilization of nematodes captured by the fungus. Inhibitors of cysteine or aspartic proteases did not affect the immobilization of captured nematodes. The effects of PMSF on the immobilization of nematodes were probably due to serine proteases produced by the fungus, since the effects were observed when unbound inhibitor was washed away from the fungus before the nematodes were added to the system. No effects were observed when the nematodes only were pretreated with PMSF.     

Characterization of cerastobin, a thrombin-like enzyme from the venom of Cerastes vipera (Sahara sand viper)

Cerastobin, a thrombin-like enzyme, was isolated from the venom of Cerastes vipera (Sahara sand viper) in homogeneous form. Cerastobin had a molecular weight of 38,000 with 348 amino acid residues. It had an isoelectric point of 7.7 (a pH optimum of 7.9 and a temperature optimum of 45 degrees C). Cerastobin hydrolyzed arginine-containing synthetic substrates such as TAME, BAME, and BAEE, but BAPNA was not hydrolyzed. Cerastobin had thrombin-like activity, producing fibrin from fibrinogen and also hydrolyzing chromogenic substrates for thrombin such as 2AcOH.H-D-CHG-But-Arg-pNA (CBS 34.47) and H-D-Phe-Pip-Arg-pNA (S-2238). It showed kallikrein-like activity and hydrolyzed kallikrein substrates 2AcOH.H-D-Phe-Gly-Arg-pNA (CBS 33.27) and H-D-Pro-Phe-Arg-pNA (S-2302). It produced bradykinin from bradykininogen, as uterus contraction was observed. A serine inhibitor, DFP, exerted a pronounced inhibitory effect, suggesting that cerastobin is a serine-type protease. The sequence of 37 residues from the amino-terminal end was investigated. The amino-terminal amino acid was valine as it is in most other thrombin-like enzymes. The amino acid sequence of cerastobin was similar to that of thrombin in some residues and had some homology with that of kallikrein. However, cerastobin showed a high degree of homology to thrombin-like enzymes isolated from various snake venoms. Factor X was partially degraded by cerastobin. It was also found that antithrombin III was degraded by the enzyme. The alpha and beta chains of fibrin monomer were preferentially hydrolyzed by cerastobin, but the gamma chain was quite resistant.     

Purification and characterization of an extracellular alkaline serine protease with dehairing function from Bacillus pumilus

An extracellular alkaline serine protease (called DHAP), produced by a Bacillus pumilus strain, demonstrates significant dehairing function. This protease is purified by hydrophobic interaction chromatography, ion exchange, and gel filtration. DHAP had a pI of 9.0 and a molecular weight of approximately 32,000 Dalton. It shows maximal activity at pH 10 and with a temperature of 55 degrees C; the enzyme activity can be completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFP). The first 20 amino acid residues of the purified DHAP have been determined with a sequence of AQTVPYGIPQIKAPAVHAQG. Alignment of this sequence with other alkaline protease demonstrates its high homology with protease from another B. pumilus strain.     

Lethal attribute of serine protease secreted by Vibrio alginolyticus strains in kuruma prawn Penaeus japonicus

Toxicity of the extracellular products (ECP) and the lethal attribute of serine protease secreted by five pathogenic Vibrio alginolyticus strains from various sources in kuruma prawn Penaeus japonicus were studied. The ECPs of organisms originally isolated from diseased kuruma prawn or small abalone Haliotis diversicolor supertexta were more lethal (LD50 value of 0.48 or 0.41 microg protein/g prawn) than those from diseased tiger prawn P. monodon, yellowfin porgy Acanthopagrus latus or horse mackerel (LD50 value of 0.98-1.17 microg protein/g prawn). All the ECPs manifested strong, weak and no activities against gelatin, sheep erythrocytes and chitin, respectively. In immunodiffusion tests using rabbit antiserum to a purified 33 kDa serine protease of strain Swy against ECP of each tested strain produced one single precipitation band in each treatment. Furthermore, the serine protease was suggested to be the dominant protease secreted by V. alginolyticus strains tested since the majority of enzymatic activity of the respective ECP was inhibited by phenylmethanesulfonyl fluoride (PMSF). A higher inhibition of serine protease activity by PMSF resulted in lower mortality rate of the ECPs injected into the prawns suggesting that the protease is one of the major lethal factor(s) secreted by V. alginolyticus.     

Autocatalytic cleavage of myelin basic protein: an alternative to molecular mimicry

Although multiple sclerosis (MS) is thought to be an autoimmune disease, the mechanisms by which immunodominant epitopes are generated and lymphocytes are activated are not known. Here, myelin basic protein-component 1 (MBP-C1) from MS tissue was shown to undergo autocatalytic cleavage at slightly alkaline pH. Importantly, one of the major peptides released contained the immunodominant epitope 84-89. Interestingly, MBP isolated from MS patients showed a faster time course of cleavage and a more robust release of epitope 84-89 than MBP isolated from normal individuals. The cleavage reaction was not inhibited by protease inhibitors, except for phenylmethanesulfonyl fluoride (PMSF), a serine protease inhibitor. Since PMSF inhibition suggested a role for a serine residue in the cleavage, we labeled myelin basic protein with diisopropyl fluorophosphate (DFP), known to bind active site serine residues. Mass spectrometry was used to identify the labeled peptide, which consisted of residues 140-152. Since this peptide contained a single serine residue, we concluded it to be the active serine. The importance of this cleavage mechanism is that it provides for a ready source of the immunodominant peptide for sensitization of T-cells. It is not necessary to invoke other mechanisms such as molecular mimicry.     

Molecular events in B cell activation. I. Signals required to stimulate G0 to G1 transition of resting B lymphocytes

Anti-immunoglobulin antibodies (anti-Ig) can stimulate a majority of resting B cells via their receptor Ig. Evidence suggests that the signals generated after this ligand-receptor interaction may be transduced via hydrolysis of inositol phospholipids. In other systems, the ability of inositol phospholipid hydrolysis to link receptor-ligand interactions to subsequent activational events has been suggested to relate to the ability of metabolic intermediates of this hydrolytic process to facilitate activation of protein kinase C and mobilization of Ca+2. In this study, we investigated the importance of protein kinase C and Ca+2 mobilization in the signaling mechanism by which anti-Ig drives B cells to undergo G0 to G1 transition. Our results show that pharmacologic inhibition of either protein kinase C activity or channel-mediated Ca+2 influx completely abrogates the increase in RNA synthesis associated with B cell activation after stimulation by anti-Ig. This suggests that pathways leading to both protein kinase C activation and elevation of intracellular Ca+2 are critical for receptor Ig-mediated G0 to G1 transition. Furthermore, studies in which anti-Ig-induced signaling could be bypassed by directly facilitating Ca+2 mobilization and protein kinase C activation using Ca+2 ionophore and phorbol diester show that these events are sufficient to drive the majority of resting B cells into G1 in the absence of additional signaling from accessory cells or extra-cellular factors. However, like anti-Ig-induced stimulation, Ca+2 ionophore and phorbol diester are relatively inefficient in driving B cells that have entered G1 into S phase. We discuss the relevance of these results towards the transduction mechanism linking B cell membrane-associated Ig-generated signals with subsequent activation events.     

Identification, gene cloning and expression of serine proteases in the extracellular medium of Nicotiana tabacum cells

Recombinant proteins secreted from plant suspension cells into the medium are susceptible to degradation by host proteases secreted during growth. Some degradation phenomena are inhibited in the presence of various protease inhibitors, such as EDTA or AEBSF/PMSF, suggesting the presence of different classes of proteases in the medium. Here, we report the results of a proteomic analysis of the extracellular medium of a Nicotiana tabacum bright yellow 2 culture. Several serine proteases belonging to a Solanaceae-specific subtilase subfamily were identified and the genes for four cloned. Their expression at the RNA level during culture growth varied depending on the gene. An in-gel protease assay (zymography) demonstrated serine protease activity in the extracellular medium from cultures. This was confirmed by testing the degradation of an antibody added to the culture medium. This particular subtilase subfamily, therefore, represents an interesting target for gene silencing to improve recombinant protein production. Key message The extracellular medium of Nicotiana tabacum suspension cells contains serine proteases that degrade antibodies.