Oxidizing side of the cyanobacterial Photosystem I: Mutational analysis of the luminal H loop of the PsaB subunit

PS I core proteins are expected to interact with the electron donor proteins plastocyanin or cytochrome c ₆. To investigate the role of the luminal H loop of PsaB in the assembly and function of the PS I complex, we generated 15 deletion and repetition mutations in the H loop of the PsaB protein from Synechocystis sp. PCC 6803. The mutant strains differed in their photoautotrophic growth. The PS I proteins could not be detected in the membranes of mutants in which the N438–E448, I453–T464, or S500–G512 region was deleted from the PsaB protein, indicating the essential role of these segments in proper folding of the PsaB protein. Mutants with partial or complete deletion of the L469–D496 segment contained the PS I proteins. These results indicate that the regions near the transmembrane helices are more important for the assembly of PsaB than the middle region of the H loop. The L469-D496 segment in the H loop of PsaB is dispensable in the interaction between the PS I complex and the soluble donor proteins. These results suggested that sections of the H loop of PsaB are crucial for the structural integrity of the PsaB protein.     

The crystal structure of the N-terminal region of the alpha subunit of translation initiation factor 2 (eIF2alpha) from Saccharomyces cerevisiae provides a view of the loop containing serine 51, the target of the eIF2alpha-specific kinases

The alpha subunit of translation initiation factor 2 (eIF2alpha) is the target of specific kinases that can phosphorylate a conserved serine residue as part of a mechanism for regulating protein expression at the translational level in eukaryotes. The structure of the 20 kDa N-terminal region of eIF2alpha from Saccharomyces cerevisiae was determined by X-ray crystallography at 2.5A resolution. In most respects, the structure is similar to that of the recently solved human eIF2alpha; the rather elongated protein contains a five-stranded antiparallel beta-barrel in its N-terminal region, followed by an almost entirely helical domain. The S.cerevisiae eIF2alpha lacks a disulfide bridge that is present in the homologous protein in humans and some of the other higher eukaryotes. Interestingly, a conserved loop consisting of residues 51-65 and containing serine 51, the putative phosphorylation site, is visible in the electron density maps of the S.cerevisiae eIF2alpha; most of this functionally important loop was not observed in the crystal structure of the human protein. This loop is relatively exposed to solvent, and contains two short 3(10) helices in addition to some extended structure. Serine 51 is located at the C-terminal end of one of the 3(10) helices and near several conserved positively charged residues. The side-chain of serine 51 is sufficiently exposed so that its phosphorylation would not necessitate a substantial change in the protein structure. The structures and relative positions of residues that have been implicated in kinase binding and in the interaction with guanine nucleotide exchange factor (eIF2B) are described.     

Aspartyl protease inhibitor pepstatin binds to the presenilins of Alzheimer's disease.

Mutations in the presenilin genes PS1 and PS2 cause early-onset Alzheimer's disease by altering gamma-secretase cleavage of the amyloid precursor protein, the last step in the generation of Abeta peptide. Ablation of presenilin (PS) genes, or mutation of two critical aspartates, abolishes gamma-secretase cleavage, suggesting that PS may be the gamma-secretases. Independently, inhibition experiments indicate that gamma-secretase is an aspartyl protease. To characterize the putative gamma-secretase activity associated with presenilins, lysates from human neuroblastoma SH-SY5Y and human brain homogenates were incubated with biotin derivatives of pepstatin, followed by immunoprecipitation of PS and associated proteins, and biotin detection by Western blotting. Precipitation with PS1 antibodies, directed to either N-terminal or loop regions, yielded the same 43 kDa band, of apparent molecular mass consistent with that of full-length PS1, although it may represent an aspartyl protease complexed with PS1. Incubation of cell lysates with pepstatin-biotin, followed by streptavidin precipitation and PS1 Western blotting, revealed PS1 fragments and full-length protein, indicating that pepstatin-biotin bound to both cleaved and uncleaved PS1. Binding could be competed by gamma-secretase inhibitor L-685,458 and could not be achieved with a PS1 mutant lacking the two transmembrane aspartates. Pepstatin-biotin was also shown to bind to PS2. PS1 was specifically absorbed to pepstatin-agarose, with an optimal pH of 6. Binding of pepstatin-biotin to PS1 from lymphocytes of a heterozygous carrier of pathologic exon 9 deletion was markedly decreased as compared to control lymphocytes, suggesting that this PS1 mutation altered the pepstatin binding site.     

Isolation and characterization of Photosystem I from two strains of the marine oxychlorobacterium Prochlorococcus

Photosystem I (PS I) complexes from two strains of the marine photosynthetic prokaryote Prochlorococcus, MED4 (= clone CCMP1378) and SS120 (= clone CCMP1375), were isolated by centrifugation on sucrose gradients after detergent treatment. The PS I-enriched fractions of both strains contained about 100 chlorophyll molecules per P700. Electron microscopy showed that the PS I complexes were in a trimeric form. The characteristic long wavelength fluorescence emission of PS I at 77 K, currently observed in chloroplasts and most cyanobacteria was absent both in intact cells and in PS I preparations of both strains. The major proteins of the PS I-enriched fractions were identified immunologically as PsaA and PsaB. Two proteins with apparent molecular masses of about 21 and 25 kDa were present in PS I preparations of Prochlorococcus, whereas the small PS I subunits in cyanobacteria all have molecular masses below 18 kDa. The 25 kDa protein showed a strong cross-reaction with a heterologous antibody against PsaL. Relatedness of the 21 kDa protein to PsaF was demonstrated by internal protein sequencing. Although only trace amounts of the major divinyl-Chl a/b-binding antenna complexes were present in the PS I preparations, significant amounts of divinyl-Chl b were observed in this fraction. The putative organization of this Chl b in PS I is discussed.     

Isolation, characterization, and recombinant expression of multiple serpins from the cat flea, Ctenocephalides felis

Several clones encoding serine protease inhibitors were isolated from larval and adult flea cDNA expression libraries by immunoscreening and PCR amplification. Each cDNA contained an open reading frame encoding a protein of approximately 45 kDa, which had significant sequence similarity with the serpin family of serine protease inhibitors. The thirteen cDNA clones isolated to date encode serpin proteins, which share a primary structure that includes a nearly identical constant region of about 360 amino acids, followed by a C-terminal variable region of about 40-60 amino acids. The variable C-terminal sequences encode most of the reactive site loop (RSL) and are generated by mutually exclusive alternative exon splicing, which may confer unique protease selectivity to each serpin. Utilization of an alternative exon splicing mechanism has been verified by sequence analysis of a flea serpin genomic clone and adjacent genomic sequences. RNA expression patterns of the cloned genes have been examined by Northern blot analysis using variable region-specific probes. Several putative serpins have been overexpressed using the cDNA clones in Escherichia coli and baculovirus expression systems. Two purified baculovirus-expressed recombinant proteins have N-terminal amino acid sequences identical to the respective purified native mature flea serpins indicating that appropriate N-terminal processing occurred in the virus-infected insect cells.     

Specific Degradation of D1 Protein during Exposure of Thylakoid Membranes to High Temperature in the Dark

Exposure of thylakoid membranes to high temperature in dark leads to the degradation of D1 protein. Maximum degradation of D1 protein occurred at 45 °C. Using N-terminal specific D1 antibody, a 23 kDa fragment of D1 protein was detected. The degradation of D1 protein could be prevented both by radical scavengers and inhibitors of serine protease and metallo-protease. These results suggest that degradation of D1 protein during exposure of thylakoid membranes to high temperature in dark is catalyzed by protease. This revised version was published online in June 2006 with corrections to the Cover Date.     

Negatively charged residues in the H loop of PsaB subunit in Photosystem I from Synechocystis sp. PCC 6803 appear to be responsible for electrostatic repulsions with plastocyanin*

Wild-type plastocyanin from the cyanobacterium Synechocystis sp. PCC 6803 does not form any kinetically detectable transient complex with Photosystem I (PS I) during electron transfer, but the D44R/D47R double mutant of copper protein does [De la Cerda et al. (1997) Biochemistry 36: 10125–10130]. To identify the PS I component that is involved in the complex formation with the D44R/D47R plastocyanin, the kinetic efficiency of several PS I mutants, including a PsaF–PsaJ-less PS I and deletion mutants in the lumenal H and J loops of PsaB, were analyzed by laser flash absorption spectroscopy. The experimental data herein suggest that some of the negative charges at the H loop of PsaB are involved in electrostatic repulsions with mutant plastocyanin. Mutations in the J loop demonstrate that this region of PsaB is also critical. The interaction site of PS I is thus not as defined as first expected but much broader, thereby revealing how complex the evolution of intermolecular electron transfer mechanisms in photosynthesis has been. This revised version was published online in June 2006 with corrections to the Cover Date.     

Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle

Bacillus thuringiensis (Bt) Cry8D insecticidal proteins are unique among Cry8 family proteins in terms of its insecticidal activity against adult Scarab beetles, such as Japanese beetle (Popillia japonica Newman). From the sequence homology with other Bt Cry proteins especially those active against beetles, such as Cry3Aa whose 3D structure is available, the structure of the Cry8D protein has been predicted to be a typical three-domain Cry protein type. In addition, the activation process of Cry8D in gut juice of susceptible insects is presumed to be similar to that of Cry3A (Yamaguchi et al., 2008). In this study, the activation process of Cry8Da in insect gut juice was closely examined. Japanese beetle gut juice proteases digested the 130 kDa Cry8Da protein to produce a 64 kDa protein. This 64 kDa protein was active against both adult and larval Japanese beetle and considered to be an activated toxin. N-terminal sequencing of this 64 kDa protein revealed that the Cry8Da leader sequence consisting of 63 amino acid residues from M¹ to F⁶³ was removed. As in the case of Cry3Aa, the proteases further digested the 64 kDa protein to two 8 kDa and 54 kDa fragments. N-terminal amino acid analysis of these smaller fragments indicated that the proteases digested the loop between Alpha Helix (Alpha for short) 3 and Alpha 4. This means that the 8 kDa fragment consists of Alpha 1-3 of Domain I and that the 54 kDa fragment contains the remaining Domain I and full Domain II and Domain III. Size exclusion chromatography and anion exchange chromatography could not separate these 64, 54 and 8 kDa proteins suggesting that the 54 kDa and 8 kDa fragments are still forming the toxin complex equivalent to the 64 kDa protein by size and ionic charge. The sequencing and chromatography results suggest that the gut juice proteases merely nicked the loop between Alpha 3 and Alpha 4. This nicking process appeared to be essential for receptor binding of the Cry8Da toxin. BBMV binding assay revealed that the Cry8Da toxin bound to BBMV preparations from both adult and larval Japanese beetle only after the loop was nicked. Only the 54 kDa fragment bound to the BBMV preparations but not the 64 kDa protein. Ligand blot showed that the protease activated Cry8Da toxin, presumably the 54 kDa fragment, bound to specific BBMV proteins, one or more of those would be receptor(s). The sizes and binding affinities of these Cry8Da-bound proteins of Japanese beetle BBMV differed between larvae and adults.     

Regulation of Lens Connexin 45.6 by Apoptotic Protease,Caspase-3

Gap junctions are important in maintaining lens homeostasis. Here we report that connexin 45.6 (Cx45.6) was partially truncated to a 46 kDa fragment during chicken lens development. This specific truncation initiated during embryonic days and the truncated fragment accumulated towards the later developmental stages. When membranes of the embryonic lens were subjected to caspase-3 treatment, the 46 kDa fragment of Cx45.6 was reproduced, suggesting apoptotic protease caspase-3 is a potential protease involved. The COOH-terminus of Cx45.6 in GST-fusion protein was also cleaved by caspase-3, confirming that Cx45.6 is a direct substrate of caspase-3. Induction of apoptosis in lens primary cultures regenerated the 46 kDa fragment and this cleavage was blocked by a caspase-3 inhibitor. Alteration of amino acid residue Asp³⁶⁴or Glu³⁶⁷ to Ala prevented Cx45.6 from cleavage by caspase-3, suggesting that the cleavage site of Cx45.6 is likely to be between Glu³⁶⁷ and Gly³⁶⁸. Phosphorylation of Ser³⁶³, a known substrate for casein kinase II (CKII) in vivo, inhibited the cleavage of Cx45.6 by caspase-3. Thus, this study demonstrates that a lens connexin can be a direct target of caspase-3 and the cleavage by caspase-3 leads to the development-associated truncation of Cx45.6. Finally, caspase-3 mediated truncation can be modulated by the specific connexin phosphorylation.     

The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer’s disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of γ-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as β-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.     

Massive breakdown of the Photosystem II polypeptides in a mutant of the cyanobacterium Synechocystis sp. PCC 6803

Degradation of the D1 protein of the Photosystem II (PS II) complex was studied in the Fad6/desA::Km^r mutant of a cyanobacterium Synechocystis sp. PCC 6803. The D1 protein of the mutant was degraded during solubilization of thylakoid membranes with SDS at 0°C in darkness, giving rise to the 23 kDa amino-terminal and 10 kDa carboxy-terminal fragments. Moreover, the D2 and CP43 proteins were also degraded under such conditions of solubilization. Degradation of the D2 protein generated 24, 17 and 15.5 kDa fragments, and degradation of the CP43 protein gave rise to 28, 27.5, 26 and 16 kDa fragments. The presence of Ca²⁺ and urea protected the D1, D2 and CP43 proteins against degradation. Degradation of the D1 protein was also inhibited by the presence of a serine protease inhibitor suggesting that the putative protease involved belonged to the serine class of proteases. The protease had the optimum activity at pH 7.5; it was active at low temperature (0°C) but a brief heating (65°C) during solubilization destroyed the activity. Interestingly, the protease was active in isolated thylakoid membranes in complete darkness, suggesting that proteolysis may be a non-ATP-dependent process. Proteolytic activity present in thylakoid membranes seemed to reside outside of the PS II complex, as demonstrated by the 2-dimensional gel electrophoresis. These results represent the first (in vitro) demonstration of strong activity of a putative ATP-independent serine-type protease that causes degradation of the D1 protein in cyanobacterial thylakoid membranes without any induction by visible or UV light, by active oxygen species or by any chemical treatments.     

Activity of Lymphostatin,A Lymphocyte Inhibitory Virulence Factor of Pathogenic Escherichia coli,is Dependent on a Cysteine Protease Motif

Lymphostatin (LifA) is a 366 kDa protein expressed by attaching & effacing Escherichia coli. It plays an important role in intestinal colonisation and inhibits the mitogen- and antigen-stimulated proliferation of lymphocytes and the synthesis of proinflammatory cytokines. LifA exhibits N-terminal homology with the glycosyltransferase domain of large clostridial toxins (LCTs). A DTD motif within this region is required for lymphostatin activity and binding of the sugar donor uridine diphosphate N-acetylglucosamine. As with LCTs, LifA also contains a cysteine protease motif (C1480, H1581, D1596) that is widely conserved within the YopT-like superfamily of cysteine proteases. By analogy with LCTs, we hypothesised that the CHD motif may be required for intracellular processing of the protein to release the catalytic N-terminal domain after uptake and low pH-stimulated membrane insertion of LifA within endosomes. Here, we created and validated a C1480A substitution mutant in LifA from enteropathogenic E. coli strain E2348/69. The purified protein was structurally near-identical to the wild-type protein. In bovine T lymphocytes treated with wild-type LifA, a putative cleavage product of approximately 140 kDa was detected. Appearance of the putative cleavage product was inhibited in a concentration-dependent manner by bafilomycin A1 and chloroquine, which inhibit endosome acidification. The cleavage product was not observed in cells treated with the C1480A mutant of LifA. Lymphocyte inhibitory activity of the purified C1480A protein was significantly impaired. The data indicate that an intact cysteine protease motif is required for cleavage of lymphostatin and its activity against T cells.     

YfiD of Escherichia coli and Y06I of bacteriophage T4 as autonomous glycyl radical cofactors reconstituting the catalytic center of oxygen-fragmented pyruvate formate-lyase.

Reaction of oxygen with the glycyl radical in pyruvate formate-lyase (PFL) leads to cleavage of the polypeptide backbone between N-Calpha of Gly734. A recombinant protein comprising the core of PFL (Ser1-Ser733) is shown here to associate with the YfiD protein (14 kDa) of Escherichia coli and likewise with the homologous T4 encoded Y06I protein, yielding upon reaction with PFL activase a heterooligomeric PFL enzyme that has full catalytic activity (35 U/nmol). Treatment of the activated complexes with oxygen led to cleavage of the 14 kDa proteins into 11 and 3 kDa polypeptides as expected for the localization of the putative glycyl radical at Gly102 (YfiD) or Gly95 (Y06I). For the isolated fragments from Y06I, mass spectrometric analysis (nanoESI-MS) determined a C-terminal serine carboxamide in the 11 kDa fragment, and a N-terminal oxalyl modification in the 3 kDa fragment. We speculate that YfiD in E. coli and other facultative anaerobic bacteria has evolved as a "spare part" for PFL's glycyl radical domain, utilized for rapid recovery of PFL activity (and thus ATP generation) in cells that have experienced oxidative stress.     

The fate of newly synthesized V-ATPase accessory subunit Ac45 in the secretory pathway.

The vacuolar H+-ATPase (V-ATPase) is a multimeric enzyme complex that acidifies organelles of the vacuolar system in eukaryotic cells. Proteins that interact with the V-ATPase may play an important role in controlling the intracellular localization and activity of the proton pump. The neuroendocrine-enriched V-ATPase accessory subunit Ac45 may represent such a protein as it has been shown to interact with the membrane sector of the V-ATPase in only a subset of organelles. Here, we examined the fate of newly synthesized Ac45 in the secretory pathway of a neuroendocrine cell. A major portion of intact approximately 46-kDa Ac45 was found to be N-linked glycosylated to approximately 62 kDa and a minor fraction to approximately 64 kDa. Trimming of the N-linked glycans gave rise to glycosylated Ac45-forms of approximately 61 and approximately 63 kDa that are cleaved to a C-terminal fragment of 42-44 kDa (the deglycosylated form is approximately 23 kDa), and a previously not detected approximately 22-kDa N-terminal cleavage fragment (the deglycosylated form is approximately 20 kDa). Degradation of the N-terminal fragment is rapid, does not occur in lysosomes and is inhibited by brefeldin A. Both the N- and C-terminal fragment pass the medial Golgi, as they become partially endoglycosidase H resistant. The Ac45 cleavage event is a relatively slow process (half-life of intact Ac45 is 4-6 h) and takes place in the early secretory pathway, as it is not affected by brefeldin A and monensin. Tunicamycin inhibited N-linked glycosylation of Ac45 and interfered with the cleavage process, suggesting that Ac45 needs proper folding for the cleavage to occur. Together, our results indicate that Ac45 folding and cleavage occur slowly and early in the secretory pathway, and that the cleavage event may be linked to V-ATPase activation.     

Detection of point mutations in chloroplast genes of Antirrhinum majus L. I. Identification of a point mutation in the psaB gene of a photosystem I plastome mutant

A point mutation in the plastome-encoded psaB gene of the mutant en:alba-1 of Antirrhinum majus L. was identified by an analysis of chloroplast DNA with a modified PCR-SSCP technique. Application of this technique is indicated when a gene or a group of genes is known in which the point mutation is located. Analysis of primary photosynthetic reactions in the yellowish white plastome mutant indicated a dysfunction of photosystem (PS) 1. The peak wavelength of PS I-dependent chlorophyll (Chl) fluorescence emission at 77 K was shifted by 4 nm to 730 nm, as compared to fluorescence from wild-type. There were no redox transients of the reaction center Chl P₇₀₀ upon illumination of leaves with continuous far-red light or with rate-saturating flashes of white light. The PS I reaction center proteins PsaA and PsaB are not detectable by SDS-PAGE in mutant plastids. Hence, plastome encoded PS I genes were regarded as putative sites of mutation. In order to identify plastome mutations we developed a modified SSCP (single-strand conformation polymorphism) procedure using a large PCR fragment which can be cleaved with various restriction enzymes. When DNA from wild-type and en:alba-1 was submitted to SSCP analysis, a single stranded Hinf I fragment of a PCR product of the psaB gene showed differences in electrophoretic mobility. Sequence analysis revealed that the observed SSCP was caused by a single base substitution at codon 136 (TAT → TAG) of the psaB gene. The point mutation produces a new stop codon that leads to a truncated PsaB protein. The results presented indicate that the mutation prevents the assembly of a functional PS I complex. The applicability to other plastome mutants of the new method for detection of point mutations is discussed.     

Hydrogen peroxide cleavage of the prion protein generates a fragment able to initiate polymerisation of full length prion protein

The prion protein is central to the disease pathogenesis of a variety of neurodegenerative diseases such as CJD. The protein is only able to initiate the disease process following post-translational modification. The main characteristic of this change is the ability of this altered isoform to polymerise. We wish to determine if altered cleavage of the protein could generate a protein fragment able to initiate polymerisation. During normal metabolic breakdown the protein is initially cleaved at a single site at around amino acid residue 111/112 in the mouse sequence. A second site before amino acid residue 90 has been postulated as an alternative cleavage point. We have provided evidence that hydrogen peroxide as low as 50 microM in the presence of copper, iron or manganese (but not nickel, magnesium or zinc) can cleave the recombinant protein near this site and requires a GXXH motif in the protein sequence. This reaction results in the production of 6 and 19 kDa fragments of the protein. This cleavage pattern occurs in prion proteins from different species (mouse, chicken and turtle) and is enhanced by modification of the octameric repeat region. The 19 kDa fragment produced by this reaction is protease sensitive. This fragment in a pure form caused the polymerisation of wild-type prion protein by a seeding mechanism. Therefore our results provide a possible mechanism by which altered cleavage of the prion protein could result in the kind of protein polymerisation associated with prion diseases.     

Processing of the AIDA-I precursor: removal of AIDAC and evidence for the outer membrane anchoring as a β-barrel structure

The AIDA-I adhesin known to be responsible for the diffuse adherence (DA) phenotype of the diarrhoea-genie Escherichia coli (DAEC) strain 2787 has been shown previously to be synthesized as a precursor protein and to undergo additional C-terminal processing. Here, the C-terminal processing of the AIDA-I precursor and the outer membrane topology of the cleaved C-terminal fragment, AIDA^C, were investigated. By isolation of the cleaved AIDA^C fragment and N-terminal sequencing, the C-terminal cleavage site was identified between Ser-846 and Ala-847 thereby indicating a molecular mass of 47.5 kDa for AIDA^C. The correct processing to AIDA-I and AIDA^C in OmpT, OmpP and DegP protease-deficient E. coli strains as well as in avirulent salmonellae and shigellae points to an autocatalytic cleavage mechanism. The cleaved AIDA^C was localized in the outer membrane. A leader sequence-AIDA^C fusion was efficiently routed to the outer membrane. Analysis by protease digestion, secondary-structure prediction and modelling, by comparison with structurally related bacterial proteins like the lgA1 protease from neisseria, the vacuolating toxin from Helicobacter pylori, and the VirG protein of Shigella flexneri, strongly indicates that AIDA^C is present in the outer membrane as a β-barrel structure.     

Protease from a strain of bacteria E. coli A2, specifically cleaving actin

A novel bacterial protease specifically hydrolyzing actin with the formation of a stable fragment with Mr of 36 kDa was obtained. This protease was shown to be synthesized at the stationary phase of bacterial culture growth. The actin hydrolysis by bacterial protease was inhibited by o-phenanthroline, EDTA and p-chloromercuribenzoate but not by N-ethyl-maleimide, phenylmethylsulfonylfluoride, Leu-peptin, pepstatin and other serine proteinase inhibitors. The protease was stable within the pH range of 4.5-8.5 and had an activity optimum at pH 7.0-8.0. The protease activity was maintained for 40 min at 45 degrees C and for 30 min at 50 degrees C; at 65 degrees C the enzyme was fully inactivated by 5 min heating. The protease preparations causing quantitative conversion of actin into a 36 kDa fragment did not hydrolyze casein, albumin, ovalbumin, lysozyme, DNAase I, RNAase, myosin, alpha-actinin, tropomyosin and troponin. It was assumed that the protease under consideration is a neutral metalloprotease specifically hydrolyzing actin.     

A 38 kDa precursor protein of aqualysin I (a thermophilic subtilisin-type protease) with a C-terminal extended sequence: its purification and in vitro processing

The precursor of aqualysin I, an extracellular subtilisin-type protease produced by Thermus aquaticus, consists of four domains: an N-terminal signal peptide, an N-terminal pro-sequence, a protease domain, and a C-terminal extended sequence. In an Escherichia coli expression system for the aqualysin I gene, a 38 kDa precursor protein consisting of the protease domain and the C-terminal extended sequence is accumulated in the membrane fraction and processed to a 28 kDa mature enzyme upon heat treatment at 65°C. The 38 kDa precursor protein is separated as a soluble form from denatured E. coli proteins after heat treatment. Accordingly, purification of the 38 kDa proaqualysin I was performed using chromatography. The purified precursor protein gave a single band on SDS-polyacrylamide gels. The precursor protein exhibited proteolytic activity comparable to that of the mature enzyme. The purified precursor protein was processed to the mature enzyme upon heat treatment. The processing was inhibited by diisopropyl fluorophosphate. The processing rate increased upon either the addition of mature aqualysin I or upon an increase in the concentration of the precursor, suggesting that the cleavage of the C-terminal extended sequence occurs through an intermolecular self-processing mechanism.     

Detection of point mutations in chloroplast genes of Antirrhinum majus L. I. Identification of a point mutation in the psaB gene of a photosystem I plastome mutant

A point mutation in the plastome-encoded psaB gene of the mutant en:alba-1 of Antirrhinum majus L. was identified by an analysis of chloroplast DNA with a modified PCR-SSCP technique. Application of this technique is indicated when a gene or a group of genes is known in which the point mutation is located. Analysis of primary photosynthetic reactions in the yellowish white plastome mutant indicated a dysfunction of photosystem (PS) 1. The peak wavelength of PS I-dependent chlorophyll (Chl) fluorescence emission at 77 K was shifted by 4 nm to 730 nm, as compared to fluorescence from wild-type. There were no redox transients of the reaction center Chl P₇₀₀ upon illumination of leaves with continuous far-red light or with rate-saturating flashes of white light. The PS I reaction center proteins PsaA and PsaB are not detectable by SDS-PAGE in mutant plastids. Hence, plastome encoded PS I genes were regarded as putative sites of mutation. In order to identify plastome mutations we developed a modified SSCP (single-strand conformation polymorphism) procedure using a large PCR fragment which can be cleaved with various restriction enzymes. When DNA from wild-type and en:alba-1 was submitted to SSCP analysis, a single stranded Hinf I fragment of a PCR product of the psaB gene showed differences in electrophoretic mobility. Sequence analysis revealed that the observed SSCP was caused by a single base substitution at codon 136 (TAT TAG) of the psaB gene. The point mutation produces a new stop codon that leads to a truncated PsaB protein. The results presented indicate that the mutation prevents the assembly of a functional PS I complex. The applicability to other plastome mutants of the new method for detection of point mutations is discussed.