Identification of a 22-kDa protein required for the degradation of Selenomonas ruminantium lysine decarboxylase by ATP-dependent protease

In Selenomonas ruminantium, a strictly anaerobic, Gram-negative bacterium isolated from sheep rumen, a rapid degradation of lysine decarboxylase (LDC) occurred on entry into the stationary phase of cell growth. Here, we identified a 22-kDa protein as a stimulating factor for the degradation of LDC, which was catalyzed by ATP-dependent protease(s) in S. ruminantium. The purified 22-kDa protein preparation itself had no degradation activity towards LDC but it was required for the degradation of LDC by ATP-dependent proteases in a cell-free system. The 22-kDa protein had similar biochemical and biophysical characteristics to those of antizyme, the regulator for the degradation of mammalian ODC, which had been reported only in mammalian cells. From the sequencing data of the N-terminal 30 amino acid residues of the 22-kDa protein preparation, 22-kDa protein was found to be a new protein which was distinguished from antizyme. This is the first report of the presence of an antizyme-like regulator protein in a prokaryote.     

Proteolytic activities during growth and aging in the fungus Podospora anserina: Effect of specific mutations

In Podospora anserina five proteolytic enzymes were characterized by chromatographic procedures. Three of these (proteases A, B and C) were found in the cell extracts of growing cultures and the other two (proteases III and IV) were revealed by studies on protoplasmic incompatibility. During growth, only protease C, an acidic enzyme, was active in crude extracts. From the stationary and the poststationary stages this activity decreased and finally disappeared, whereas a neutral serine protease (activity B) became active in crude extracts. A close relationship was observed between the proteolytic activity of the culture filtrates and the intracellular protease(s) concomitantly active in the crude extracts. None of the proteases associated with protoplasmic incompatibility was detected, both in the extra- and intracellular spaces. Qualitative variations in the proteolytic activities during stationary and post-stationary stages depended on the presence of specific genes and mutations: the mod C mutation suppressing protoplasmic incompatibility, inhibits the progressive decrease of protease C and, furthermore, the presence of non allelic incompatibility genes have for consequence the substitution of serine protease B by serine protease A during the poststationary stage.     

The geminivirus nuclear shuttle protein NSP inhibits the activity of AtNSI, a vascular-expressed Arabidopsis acetyltransferase regulated with the sink-to-source transition

DNA viruses can suppress or enhance the activity of cellular acetyltransferases to regulate virus gene expression and to affect cell cycle progression in support of virus replication. A role for protein acetylation in regulating the nuclear export of the bipartite geminivirus (Begomovirus) DNA genome was recently suggested by the findings that the viral movement protein NSP, a nuclear shuttle protein, interacts with the Arabidopsis (Arabidopsis thaliana) nuclear acetyltransferase AtNSI (nuclear shuttle protein interactor), and that this interaction and NSI expression are necessary for cabbage leaf curl virus infection and pathogenicity. To further investigate the consequences of NSI-NSP interactions, and the potential role of NSI in Arabidopsis growth and development, we used a reverse yeast two-hybrid selection and deletion analysis to identify NSI mutants that failed to interact with NSP, and promoter fusions to a uidA reporter gene to analyze the pattern of NSI expression during plant development. We found that NSI self assembles into highly active enzyme complexes and that high concentrations of NSP, in the absence of viral DNA, can inhibit NSI activity in vitro. Based on our detailed analysis of three NSI missense mutants, we identified an 88-amino acid putative domain, which spans NSI residues 107 to 194, as being required for both NSI oligomerization and its interaction with NSP. Finally, we found that NSI is predominantly transcribed in vascular cells, and that its expression is developmentally regulated in a manner that resembles the sink-to-source transition. Our data indicate that NSP can inhibit NSI activity by interfering with its assembly into highly active complexes, and suggest a mechanism by which NSP can both recruit NSI to regulate nuclear export of the viral genome and down-regulate NSI activity on cellular targets, perhaps to affect cellular differentiation and favor virus replication.     

Intracellular Proteases of Bacillus thuringiensis subsp. kurstaki and a Protease-Deficient Mutant Btk-q

The commencement of intracellular protease synthesis was studied by gelatin zymography in Bacillus thuringiensis (Btk) HD1, Btk HD73, and a protease-deficient mutant Btk-q derived from the former strain. By gelatin zymography, a 92-kDa protease was detected first at 3 h of sporulation, which continued until 48 h, whereas two other proteases of mol wt 78 and 69 kDa were detectable from 6 h onwards and continued until 48 h of growth in Btk HD1. Similar studies revealed the presence of two major intracellular proteases in Btk HD73 by gelatin zymography, which first appeared at 6 h of sporulation and continued until 48 h of growth. The quantitative azocasein assay confirmed that the total protease activity increases from 3 to 21 h, thereafter reaching a plateau up to 48 h of growth examined, in HD1 and HD73 strains. Btk-q, a protease-deficient mutant, showed traces of protease activity by azocasein analysis that could not be detected by gelatin zymography. The free amino acid pool content was also increased parallel to the way that the protease activity increased in all three strains. However, this increase was found to be low (16-fold) in Btk-q when compared with Btk HD1 and HD73 strains. The following amino acids were detected by paper chromatography in Btk HD1: DL-alanine, L-glutamic acid, L-aspartic acid, tyrosine, tryptophan/methionine/valine, arginine, leucine/norleucine/isoleucine, and glycine, whereas only DL-alanine, L-glutamic acid, and L-aspartic acid were in Btk-q at 24 and 48 h, when the protease activity was maximum. Received: 4 January 2002 / Accepted: 6 March 2002     

Secretion of haemolysins and proteases by Aeromonas hydrophila EO63: separation and characterization of the serine protease (caseinase) and the metalloprotease (elastase)

AIMS: To determine the haemolysins and proteases excreted by the virulent strain EO63 of Aeromonas hydrophila grown in complex media and to then fractionate and characterize them, in particular those with elastolytic activity. METHODS AND RESULTS: The amount of haemolytic and proteolytic activity in EO63 culture supernatants was dependent on the culture media used. In all media, haemolysins appeared during the phase of active growth and haemolytic activity decreased quickly thereafter, as previously described for aerolysin. In contrast, proteases were mainly released during the stationary phase. Serine protease activity in EO63 culture supernatants was four times greater than that caused by metalloproteases. Two main proteases were partially purified from EO63 culture supernatants by isoelectrophoresis: a serine protease (68 kDa) active against casein; a mixture of different protein bands (60, 44 and 31 kDa) representing a thermostable metalloprotease active against elastin and casein. This metallo-elastase was also inhibited by dithiothreitol and showed a pH optimum of 8.0. Both exoenzymes were toxic for eels at LD50 doses of 1.1 and 3.5 microg (g fish)(-1), respectively. CONCLUSIONS: A serine caseinase and a metallo-elastase may play a role in the pathogenicity of EO63 for eels. These toxins are excreted in vitro by EO63 in the ratio of 4:1 during the stationary phase of growth. Strain EO63 also produced beta-haemolysins in vitro which could correspond to aerolysin. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the purification of a metallo-elastase excreted by a wild-type A. hydrophila strain.     

Physiological regulation of protease activity in Streptomyces peucetius

Streptomyces peucetius ATCC 29050, a producer of anthracycline antineoplastic agents, was investigated for the expression of intracellular and extracellular azocaseinase activities as a function of growth and medium conditions. When cultures were grown in either nitrate-containing defined medium or glucose-yeast extract complex medium, the intracellular proteolytic activity was greatest during early to mid stationary phase, whereas the extracellular proteolytic activity was produced in late stationary phase. All of the proteolytic activity detected against azocasein was of a serine-type protease activity. These late-occurring proteases may have some function in cellular turnover associated with secondary metabolism and (or) morphogenesis.     

Ontogenetic switchover in Bacillus subtilis. II. The dynamics of the stationary phase processes during growth under conditions of catabolite repression]

To reveal the pecularities of the growth under the conditions of catabolite repression (medium 2) of Bacillus subtilis and the mutants obtained, the investigations of dynamics of the following processes were carried out: alteration of the pH of the culture exhaustion of glucose in the medium, appearance of the activity of both aconitase in the cells and extracellular metal- and serine proteases in the supernatant, and the appearance of the thermoresistant spores. The following features were observed during the growth under the conditions of catabolite repression: 1. Bacillus subtilis WB 746 and cgs mutants: the death of the main part of the culture after the Iogarithmic phase of growth (LPG), the presence of the secondary LPG of the survived cells which have the increasing activity of aconitase, the appearance and sharp increase in the extracellular serine protease activity 6 hours before thermoresistant spore formation. In the case of cgs mutants the activity of metal proteases appears and increases during the secondary LPG; 2. In the culture of cgl mutants the pH is lowered to 5.1 at the end of the LPG and after the glucose exhaustion the death of almost all the culture follows; 3. cgr mutants: a comparatively high activity of aconitase in the cells is found by the time of the early LPG, and at the end of the LPG the activity of both metal- and serine proteases appear in the supernatant of the culture and the secondary induction of the serine protease activity 6 hours before thermoresistant spore formation is observed. The serine protease activity found in the supernatant before and after the secondary induction of the enzyme belongs to the identical protein. During the stationary phase of the growth of cgr mutants, the high rate of 3H-uridine incorporation into the RNA molecules which have the electrophoretic mobility of mRNA was observed. The sporulation of Bac. subtilis strains under investigation, except cgl mutants, occurs when the culture has reached the definite state: the alkaline pH, the presence of the aconitase activity in the cells and the induced activity of serine protease.     

Effects of silicate resupply to silicate‐deprived Thalassiosira weissflogii (Bacillariophyceae) in stationary or senescent phase: short‐term patterns of growth and cell death

The ability of nutrient‐deprived phytoplankton to recover in the short term when nutrients are resupplied has been studied for nitrogen and phosphorus, but the case for silicate (Si) is poorly understood. Si‐limited Thalassiosira weissflogii (Grunow) Fryxell et Hasle (grown in batch culture) was harvested in stationary phase (when cell numbers stopped increasing ~2 d after Si depletion) and senescence (when cell numbers declined ~4 d after Si depletion) and Si was resupplied at different concentrations (from 0 to 100 μM). Cell numbers, proportion of dead cells, variable fluorescence emissions (F_v/F_m), and activities of proteases were measured during Si depletion and for 24 h after Si resupply. As Si was depleted, the specific growth rate declined, dead cells increased from ~2% in log phase, to ~25% in stationary phase to over 35% in senescence, and activities of proteases associated with cell death increased several‐fold. Concentration‐dependent recovery of growth rate was seen after 24 h for cultures resupplied with Si in stationary phase but not in senescence. However, resupply of Si at 100 μM to stationary phase cultures alone increased protease activity to nearly the levels seen in senescence. Differences in the responses to Si resupply suggest that the ability and time to recover from Si depletion depend not only on the growth phase but also on the concentration resupplied.     

Comparison of proteolytic activities produced by entomopathogenic Photorhabdus bacteria: strain- and phase-dependent heterogeneity in composition and activity of four enzymes

Twenty strains (including eight phase variant pairs) of nematode-symbiotic and insect-pathogenic Photorhabdus bacteria were examined for the production of proteolytic enzymes by using a combination of several methods, including gelatin liquefaction, zymography coupled to native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and activity measurement with two chromogen substrate types. Four protease activities (approximately 74, approximately 55, approximately 54, and approximately 37 kDa) could be separated. The N-terminal sequences of three of the proteases were determined, and a comparison with sequences in databases allowed identification of these proteases as HEXXH metallopeptidases. Thus, the 74-kDa protease (described formerly as Php-B [J. Marokhazi, G. Koczan, F. Hudecz, L. Graf, A. Fodor, and I. Venekei, Biochem. J. 379:633-640, 2004) is an ortholog of OpdA, a member the thimet oligopeptidase family, and the 55-kDa protease is an ortholog of PrtA, a HEXXH+H peptidase in clan MB (metzincins), while the 37-kDa protease (Php-C) belongs to the HEXXH+E peptidases in clan MA. The 54-kDa protease (Php-D) is a nonmetalloenzyme. PrtA and Php-C were zymographically detected, and they occurred in several smaller forms as well. OpdA could not be detected by zymography. PrtA, Php-C, and Php-D were secreted proteases; OpdA, in contrast, was an intracellular enzyme. OpdA activity was found in every strain tested, while Php-D was detected only in the Brecon/1 strain. There was significant strain variation in the secretion of PrtA and Php-C activities, but reduced activity or a lack of activity was not specific to secondary-phase variants. The presence of PrtA, OpdA, and Php-C activities could be detected in the hemolymph of Galleria melonella larvae 20 to 40 h postinfection. These proteases appear not to be directly involved in the pathogenicity of Photorhabdus, since strains or phase variants lacking any of these proteases do not show reduced virulence when they are injected into G. melonella larvae.     

Synthesis and release of proteases byBacteroides fragilis

Protease production byBacteroides fragilis ATCC 25285 was determined in batch and continuous cultures. During exponential growth in batch culture, the majority of proteolysis was cell associated. However, as the bacteria reached stationary phase, most of the intracellular proteases were released into the culture medium. Measurements of alkaline phosphatase and -galactosidase, which are respectively periplasmic and cytoplasmic marker enzymes inB. fragilis, showed that secretion of proteases in the stationary phase was a discrete event and was not associated with a general release of cytoplasmic contents. When the bacterium was grown in continuous culture, cell-associated protease activity increased concomitantly with dilution rate (D=0.03–0.23/h). The ratio of intracellular to whole cell protease activity also increased with growth rate (11 at D=0.03/h; 11.7 at D=0.23/h). Extracellular protease activity was detected only in trace amounts in continuous cultures at the lowest dilution rate. Determinations of the distribution of extracellular protease activity in batch culture after 48 h incubation showed that the majority of proteolysis (ca. 90%) was soluble. Nevertheless, a proportion was associated with particulate fractions, which had high specific activities.     

Purification and partial characterization of an elastinolytic proteinase from Aspergillus flavus culture filtrates

 A 23-kDa protein with elastinolytic activity was purified from Aspergillus flavus (NRRL 18543) culture filtrates by gel-filtration chromatography. Severe inhibition of the elastinolytic activity by 1,10-phenanthrolene (5 mM) and EDTA (0.8 mM) indicated that the protein belongs to the metallo class of proteases. The isoelectric point was 9.0. Natural substrates susceptible to cleavage by this protease, in addition to elastin, included cottonseed storage protein, collagen, ovalbumin and bovine serum albumin. The 23-kDa protein was thermostable to 70°C and retained its elastinolytic activity in concentrated form at 4°C for 6 months. Elastinolytic activity was initially secreted into the culture medium as a 35-kDa protein, which was subsequently converted to a 23-kDa protein, presumably through autolysis. This putative proteolytic degradation product appears to be identical to the 23-kDa protein recovered from the gel-filtration column. The 23-kDa protease may confer selective advantage to the fungus in the extracellular environment because of its temperature and pH stability and wide range of potential natural protein substrates. Received: 24 October 1995/Received last revision: 27 March 1996/Accepted: 30 March 1996     

Regulation of Extracellular Chitinases and Proteases in the Entomopathogen and Acaricide <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

Metarhizium anisopliae infects insects and ticks via a combination of specialized structures and cuticle degradation. Hydrolytic enzymes are accepted as key factors for the penetration step. The search for pathogenicity determinants has demonstrated that the process is multifactorial. Host specificity is an important factor to be addressed. The study of the enzymes produced during infection is important to discover those with a role in the process. To address some of the enzymes that take part during the infection of the tick, Boophilus microplus, we have analyzed the secretion of proteases and chitinases in single and combined carbon/nitrogen sources as compared with such complex substrates as chitin and B. microplus cuticles. Two chitinases, endo- and N-acetylglucosaminidases, and two proteases, subtilisin and trypsin-like proteases, were analyzed. Enzyme activities were detected in all carbon sources tested, but higher levels were found when combinations of carbon sources were used. A major 30-kDa protein apparently secreted during M. anisopliae growth on all carbon/nitrogen sources tested was demonstrated by SDS–PAGE. Received: 8 May 2002 / Accepted: 8 June 2002     

Characterization of protease production by a type-III group-BStreptococcus

A type-III group-B streptococcus (Streptococcus agalactiae) isolated from a case of late-onset sepsis was examined for protease production. In broth culture, extracellular proteolytic enzymes were not detected until the late exponential phase of growth with maximal protease production occurring during the stationary phase. Three distinct protease pools were isolated from the supernatant fluids of stationary-phase cultures, employing a combination of ion-exchange chromatography and gel-filtration chromatography. One population of proteases (containing two protease pools separable by gel filtration chromatography) eluted from a diethylaminoethyl cellulose column at a sodium chloride gradient concentration of 0.15M while a second population eluted from the same material at a sodium chloride concentration of 0.35M. These protease pools varied in molecular weights from approximately 25,000 daltons to 160,000 daltons as determined by gel filtration on Sephadex G-200. All three protease preparations had pH optima of 8.0–9.0, and all were active against gelatin, human serum albumin, and casein, but were not active against elastin or collagen. In addition, all three protease preparations completely inactivated purified type-III group-B streptococal neuraminidase. The role of these proteases in the disease process caused by the type-III group-B streptococci must remain speculative at this time.     

Characteristics of protease synthesis in Bacteroides splanchnicus NCTC 10825

Studies to determine the physiological and nutritional characteristics of protease synthesis by Bacteroides splanchnicus NCTC 10825 showed that the proteases were constitutive and cell-associated during exponential growth in batch culture. As growth slowed and the bacteria entered the stationary phase, proteases that had accumulated intracellularly were released into the culture media. In continuous cultures, [dilution rate (D)=0.03 h^–1 to D=0.29 h^–1], protease activity was completely cell-bound and maximal during nitrogen-limited growth at high dilution rates. The proteases hydrolysed a relatively restricted range of protein substrates including casein, azocasein and gelatine (comparative maximum rates of hydrolysis were 1.0, 4.1 and 2.7 units mg^–1 protein respectively). B. splanchnicus proteases exhibited arylamidase activities against leucine p-nitroanilide, valylalanine p-nitroanilide and glycylproline p-nitroanilide. Inhibition experiments indicated that the bacterium produced a mixture of serine, thiol and, possibly, metalloproteases. Protease activities were affected by reducing agents and divalent metal ions. Mercaptoethanol at 1 mm was slightly stimulatory; however, dithiortheitol and dithioerythritol (each 10 mm) respectively inhibited protease activities by 91% and 100%. Calcium ions (5 mm) stimulated protease activity by 30%, whereas Mn²⁺ and Mg²⁺ had little or no effect. Protease and arylamidase activities had neutral to alkaline pH optima. Together, these results show that with respect to the types of protease formed and the physiology of the process, B. splanchnicus proteolysis is similar in many respects to that occurring in species belonging to the B. fragilis group. Correspondence to: G. T. Macfarlane     

Initial characterization of two extracellular autolysins from Pseudomonas aeruginosa PAO1.

Two extracellular autolysins have been detected in the spent culture supernatants of Pseudomonas aeruginosa PAO1 by using renaturing polyacrylamide gel electrophoresis. The two autolysins were isolated from the culture supernatant by trichloroacetic acid precipitation and were shown to have apparent molecular masses of 26 and 29 kDa. The 26-kDa autolysin first appears during the early exponential phase of growth and then declines sharply, while the 29-kDa autolysin first appears in the late exponential phase of growth and continues well into the stationary phase. Fractionation of whole cells indicated that the 26-kDa enzyme was also localized within the periplasm, with a lesser amount of activity associated with the cytoplasmic membrane. The 29-kDa autolytic activity was distributed within the cell equally between the periplasm and the cytoplasmic membrane. The pH optima of the isolated 26- and 29-kDa autolysins are 6.0 and 5.0, respectively. Further evidence from both protease susceptibility and inhibition studies confirms that these two extracellular autolysins isolated from P. aeruginosa PAO1 are separate and distinct.     

Calcium-activated neutral protease purified from beef lung: properties and use in defining structure of epidermal growth factor receptors

Ca2+-Requiring proteases degrade cytosolic and integral membrane proteins as well as alter, by limited proteolysis, the activity of certain protein kinases. When cells are lysed, a Ca2+-requiring protease degrades the epidermal growth factor (EGF) receptor, an integral membrane protein with an intrinsic kinase activity, from its 170-kDa form to a 150-kDa form. This Ca2+-requiring protease has all of the characteristics of calcium-activated neutral protease (CANP). To show that CANP is the protease uniquely responsible for the degradation of the native EGF receptor in vitro, CANP was highly purified from beef lung. This affinity purified CANP had properties previously described for other CANPs: heterodimer of 80 and 30 kDa; neutral pH optimum; activation by millimolar Ca2+; and inhibition by an endogenous, heat-stable proteinaceous inhibitor, by leupeptin, and by sulfhydryl alkylating agents. Using the EGF receptor labeled by covalent attachment to 125I-EGF, this purified CANP quantitatively generated the 150-kDa form from the native receptor in A-431 cell membranes. As with the native receptor, the 150-kDa receptor forms produced by the endogenous Ca2+-requiring protease, by CANP, by chymotrypsin, and by elastase were all capable of EGF-stimulated autophosphorylation. When the 150-kDa receptor forms were generated by the three exogenously added proteases, autophosphorylation with [gamma-32P]ATP followed by trypsinization produced 32P-labeled peptides that were not the same. However, the tryptic 32P-labeled peptides from the autophosphorylated 150-kDa receptor form produced by CANP or by the endogenous Ca2+-requiring protease were identical. These data indicate that CANP is identical to the endogenous Ca2+-requiring protease responsible for producing the autophosphorylating 150-kDa receptor form from the native EGF receptor when cells are lysed.     

Comparison of Proteolytic Activities Produced by Entomopathogenic Photorhabdus Bacteria: Strain- and Phase-Dependent Heterogeneity in Composition and Activity of Four Enzymes

Twenty strains (including eight phase variant pairs) of nematode-symbiotic and insect-pathogenic Photorhabdus bacteria were examined for the production of proteolytic enzymes by using a combination of several methods, including gelatin liquefaction, zymography coupled to native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and activity measurement with two chromogen substrate types. Four protease activities (~74, ~55, ~54, and ~37 kDa) could be separated. The N-terminal sequences of three of the proteases were determined, and a comparison with sequences in databases allowed identification of these proteases as HEXXH metallopeptidases. Thus, the 74-kDa protease (described formerly as Php-B [J. Marokházi, G. Kóczán, F. Hudecz, L. Gráf, A. Fodor, and I. Venekei, Biochem. J. 379:633-640, 2004) is an ortholog of OpdA, a member the thimet oligopeptidase family, and the 55-kDa protease is an ortholog of PrtA, a HEXXH+H peptidase in clan MB (metzincins), while the 37-kDa protease (Php-C) belongs to the HEXXH+E peptidases in clan MA. The 54-kDa protease (Php-D) is a nonmetalloenzyme. PrtA and Php-C were zymographically detected, and they occurred in several smaller forms as well. OpdA could not be detected by zymography. PrtA, Php-C, and Php-D were secreted proteases; OpdA, in contrast, was an intracellular enzyme. OpdA activity was found in every strain tested, while Php-D was detected only in the Brecon/1 strain. There was significant strain variation in the secretion of PrtA and Php-C activities, but reduced activity or a lack of activity was not specific to secondary-phase variants. The presence of PrtA, OpdA, and Php-C activities could be detected in the hemolymph of Galleria melonella larvae 20 to 40 h postinfection. These proteases appear not to be directly involved in the pathogenicity of Photorhabdus, since strains or phase variants lacking any of these proteases do not show reduced virulence when they are injected into G. melonella larvae.     

Purification of alkaline proteases from a Bacillus strain and their possible interrelationship

 Alkalophilic Bacillus sp. KSM-K16 produced three alkaline proteases, as detected by polyacrylamide gel electrophoresis (PAGE). The major protease, designated M protease, was recently purified to homogeneity and its properties were characterized. In the present study, two minor proteases, designated H protease and N protease, were purified to homogeneity from cultures of this organism. H protease had a molecular mass of 28 kDa, as estimated by sodium dodecyl sulfate/PAGE (SDS-PAGE) and its maximum activity against casein was observed at pH 11.0 and at 55^°C. N protease consisted of two polypeptide chains with molecular masses of 12.5 kDa and 14.5 kDa, as estimated by SDS-PAGE, although it migrated as a single protein band during non-denaturing PAGE. Its maximum activity was observed at pH 11.0 and at 60^°C. The amino-terminal sequences of H protease and of the 14.5-kDa polypeptide of N protease were identical to that of M protease. The electrophoretic relationship between the three enzymes was examined after they had been stored at different pH values and at 5^°C. M protease was converted to H protease more rapidly at pH 11 than at pH 8 or below, and H protease was converted to M protease at pH 8 or below but not at pH 11. N protease appeared to be the autolytic product of the M and H proteases. Received: 12 December 1994/Received last revision: 9 June 1995/Accepted: 31 July 1995     

Mutational Analysis of the Amino Acid Residues Essential for the cis and trans Cleavage Activity of the Potato Virus Y 50-kDa Protease

To examine, the proteolytic activities of various truncated derivatives of the potato virus Y (PVY) 50-kDa protease, the derivatives were expressed in Escherichia coli in polyprotein forms fused with coat protein (CP). For the intermolecular cleavage reaction, the truncated proteases were expressed together with the substrate protein containing the polymerase-CP junction. The activity was evaluated by the amount of the mature CP released from the precursor by the intra- and intermolecular cleavage occurring in E. coli. By this experiment, we identified the moiety responsible for the proteolytic activity of the 50-kDa protease to be a 26-kDa polypeptide mapped to the C-terminal half of the protease. Introduction of His234→Tyr, Asp269→Asn, or Cys339→Gly substitution in the putative catalytic triad of the protease abolished its activity. However, the mutated protease with Cys339→Ser replacement retained a reduced proteolytic activity.