Amino acid sequence and some properties of phytolacain G, a cysteine protease from growing fruit of pokeweed, Phytolacca americana

A protease, phytolacain G, has been found to appear on CM-Sepharose ion-exchange chromatography of greenish small-size fruits of pokeweed, Phytolacca americana L, from ca. 2 weeks after flowering, and increases during fruit enlargement. Reddish ripe fruit of the pokeweed contained both phytolacain G and R. The molecular mass of phytolacain G was estimated to be 25.5 kDa by SDS-PAGE. Its amino acid sequence was reconstructed by automated sequence analysis of the peptides obtained after cleavage with Achromobacter protease I, chymotrypsin, and cyanogen bromide. The enzyme is composed of 216 amino acid residues, of which it shares 152 identical amino acid residues (70%) with phytolacain R, 126 (58%) with melain G, 108 (50%) with papain, 106 (49%) with actinidain, and 96 (44%) with stem bromelain. The amino acid residues forming the substrate binding S(2) pocket of papain, Tyr67, Pro68, Trp69, Val133, and Phe207, were predicted to be replaced by Trp, Met, His, Ala, and Ser in phytolacain G, respectively. As a consequence of these substitutions, the S(2) pocket is expected to be less hydrophobic in phytolacain G than in papain.     

Amino acid sequence and some properties of phytolacain R, a cysteine protease from full-growth fruits of pokeweed, Phytolacca americana.

A cysteine protease, phytolacain R from full-growth greenish fruits of pokeweed, Phytolacca americana L, was purified to electrophoretic homogeneity by a simple purification procedure employing CM-Sepharose ion-exchange chromatography. The enzyme was present in low content in the young fruits about 50 d after flowering but gradually accumulated in growing fruits. Its molecular mass was estimated to be ca. 23 kDa by SDS-PAGE, and its sugar content was zero. Its amino acid sequence was established by automated sequence analysis of the peptides obtained by cleavage with Achromobacter protease I, chymotrypsin, trypsin, and cyanogen bromide. The enzyme is composed of 218 amino acid residues, of which it shares 110 residues (50%) with papain, 104 (47%) with actinidain, and 87 (40%) with stem bromelain. The amino acid residues forming the substrate-binding the S2 pocket of papain, Tyr61, Tyr67, Pro68, Trp69, Val133, and Phe207, were predicted to be replaced by Gly, Trp, Met, His, Ala, and Met in phytolacain R, respectively. As a consequence of these substitutions, the S2 pocket is expected to be less hydrophobic in phytolacain R than in papain.     

Betacyanin-Decolorizing Enzymes from Phytolacca americana

Betacyanin-decolorizing enzymes (BDEs) in Phytolacca americanawere partially purified and characterized. Acidic enzymes, BDE₁and BDE₂, and basic enzymes, BDE₃ and BDE₄, were partially purifiedby column chromatography on DEAE-Sepharose CL-6B and CM-SepharoseCL-6B. These enzymes degraded betanin to its aglycon (betanidin)and then to a product with a peak of absorbance at 485 nm. Theoptimum pH values were 5.0 for BDE₁, 3.5 for BDE₂, and 5.5 forBDE₃ and BDE₄. BDE activity was detected in extracts of theleaf, stem, seed, and root. The highest specific activity ofBDE₂ was found in the red epidermis of the stem. The activityof BDE₂ was inhibited by NH₄NO₃, KNO₃, KC1, and NaCl, but theactivities of the other BDEs were not inhibited. These resultssuggest that BDEs other than BDE₂ control the degradation ofbetacyanin in the intracellular space. (Received March 27, 1989; Accepted December 8, 1989)     

Protoplasts from Phytolacca dodecandra L'Herit (endod) and P. americana L. (pokeweed)

Summary Pokeweed (Phytolacca americana L.) and endod (P. dodecandra L'Herit) produce ribosome-inactivating proteins which are sequestered in leaf cell walls. These proteins display strong antiviral activity. To aid in studying the antiviral mechanism, we developed protocols to isolate protoplasts from suspension culture cells and leaves. Ninety-five percent of pokeweed or endod culture cells were converted to protoplasts using 2% cellulase, 0.25% pectinase, 0.2 M mannitol, 2% sucrose, 15 mM CaCl₂ Murashige and Skoog salts, pH 5.7. Viability was >85% after 24 h. Culture-derived protoplasts were purified by centrifugation through a 15% sucrose pad. Protoplasts collected from the supernatant were then pelleted in 0.3 M mannitol. Pokeweed leaves provided respectable yields (4×10⁶ protoplasts/g f w) of partially-purified viable protoplasts when digested in solution containing 1% cellulase, 0.2% Pectolyase, 0.4 M mannitol, CPW salts, 0.5 mM MES, pH 5.6. We were unable to completely separate cell debris from mesophyll protoplasts, which were small and easily damaged by centrifugation. Endod leaves were found to be resilient to several digestion enzymes tested.     

Amino acid sequences of heterotrophic and photosynthetic ferredoxins from the tomato plant (Lycopersicon esculentum Mill.)

Several forms (isoproteins) of ferredoxin in roots, leaves, and green and red pericarps in tomato plants (Lycopersicon esculentum Mill.) were earlier identified on the basis of N-terminal amino acid sequence and chromatographic behavior (Green et al. 1991). In the present study, a large scale preparation made possible determination of the full length amino acid sequence of the two ferredoxins from leaves. The ferredoxins characteristic of fruit and root were sequenced from the amino terminus to the 30th residue or beyond. The leaf ferredoxins were confirmed to be expressed in pericarp of both green and red fruit. The ferredoxins characteristic of fruit and root appeared to be restricted to those tissue. The results extend earlier findings in demonstrating that ferredoxin occurs in the major organs of the tomato plant where it appears to function irrespective of photosynthetic competence.Abbreviations CBB Coomassie brilliant blue R-250 - Cm Carboxymethylated - Fd Ferredoxin - FNR ferredoxin-NADP⁺ oxidoreductase - FPLC Fast protein liquid chromatography - HPLC High performance liquid chromatography - rt root     

Amino acid sequences of two nonspecific lipid-transfer proteins from germinated castor bean

The amino acid sequence of two nonspecific lipid-transfer proteins (nsLTP) B and C from germinated castor bean seeds have been determined. Both the proteins consist of 92 residues, as for nsLTP previously reported, and their calculated Mr values are 9847 and 9593 for nsLTP-B and nsLTP-C, respectively. The sequences of nsLTP-B and nsLTP-C, compared to the known sequence of nsLTP-A from the same source, are 68% and 35% similar, respectively. No variation was found at the positions of the cysteine residues, indicating that they might be involved in disulfide bridges.     

Amino acid sequences of the two major isoforms of troponin C from crayfish

The primary structure of the two major isoforms (alpha and gamma) of troponin C (TnC) from crayfish tail muscle has been determined by the application of manual and automated Edman degradation procedures to fragments generated by suitable chemical and proteolytic cleavages. Both amino acid sequences commence with an acetylated methionyl residue and contain 150 amino acid residues, including a single proline residue at position 29 and 2 residues of tyrosine at positions 95 and 102. No cysteine or tryptophan are present. The molecular weights calculated for alpha- and gamma-TnC are 17,157 and 16,974, respectively. The two crayfish proteins are invariable at 129 positions and conserved at 11 others. Pairwise comparisons show that the two sequences are 33-39% identical with those of seven TnCs reported so far and 39% identical with that of bovine brain calmodulin. The N-terminal end of about 10 residues, found in vertebrate TnCs, is absent in crayfish TnCs. In the latter proteins, domains I and III appear as abortive Ca2+-binding sites due to nonconservative amino acid replacements at the key Ca2+-coordinating positions in their loops. The remaining two Ca2+-binding loops (II and IV) show a remarkable similarity with the Ca2+-specific loops (I and II) found in vertebrate TnCs. These findings are consistent with the Ca2+-binding data (Wnuk, W. (1989) J. Biol. Chem. 264, 18240-18246) which indicate the presence of two Ca2+-specific sites in crayfish TnCs. These two sites display the same affinity for Ca2+ (log KCa = 4.3) on gamma-TnC but differ in their affinity (log KCa = 6.0 and 4.1) on alpha-TnC. The only structural difference between the dodecapeptide loops II and IV in both alpha- and gamma-TnC, which correlates with the existence of the high affinity (log KCa = 6.0) Ca2+-specific site on alpha-TnC, is position 11 occupied by a methionyl residue in the loop IV of alpha-TnC as opposed to negatively charged residues found in the other three loops. This suggests that the high affinity Ca2+-specific site on alpha-TnC is located in domain IV. Since the Ca2+-binding studies show that the formation of the complex of crayfish troponin I (TnI) with alpha- and gamma-TnC increases significantly the affinity of only one of their two Ca2+-specific sites and this TnI-sensitive site is not the high affinity Ca2+-specific site on alpha-TnC, we conclude that the binding of Ca2+ to site II controls the Ca2+-dependent interaction between crayfish TnCs and TnI.     

Purification and partial characterization of another form of the antiviral protein from the seeds of Phytolacca americana L. (pokeweed).

1. The pokeweed antiviral protein, previously identified in two forms (PAP and PAP II) in the leaves of Phytolacca americana (pokeweed) [Obrig. Irvin & Hardesty (1973) Arch. Biochem. Biophys. 155, 278-289; Irvin, Kelly & Robertus (1980) Arch. Biochem. Biophys. 200, 418-425] is a protein that prevents replication of several viruses and inactivates ribosomes, thus inhibiting protein synthesis. 2. PAP is present in several forms in the seeds of pokeweed. One of them, which we propose to call 'pokeweed antiviral protein from seeds' (PAP-S) was purified in high yield (180 mg per 100 g of seeds) by chromatography on CM-cellulose, has mol.wt. 30 000, and is similar to, but not identical with. PAP and PAP II. 3. PAP-S inhibits protein synthesis in a rabbit reticulocyte lysate with an ID50 (concentration giving 50% inhibition) of 1.1 ng/ml (3.6 x 10(-11) M), but has much less effect on protein synthesis by whole cells, with an ID50 of 1 mg/ml (3.3 x 10(-5) M), and inhibits replication of herpes simplex virus type 1.     

A new antifungal peptide from the seeds of Phytolacca americana: characterization, amino acid sequence and cDNA cloning

An antifungal peptide from seeds of Phytolacca americana, designated PAFP-s, has been isolated. The peptide is highly basic and consists of 38 residues with three disulfide bridges. Its molecular mass of 3929.0 was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation, and cDNA cloning was successfully performed by 3'-RACE. The deduced amino acid sequence of a partial cDNA corresponded to the amino acid sequence from chemical sequencing. PAFP-s exhibited a broad spectrum of antifungal activity, and its activities differed among various fungi. PAFP-s displayed no inhibitory activity towards Escherichia coli. PAFP-s shows significant sequence similarities and the same cysteine motif with Mj-AMPs, antimicrobial peptides from seeds of Mirabilis jalapa belonging to the knottin-type antimicrobial peptide.     

Large differences in amino acid sequences among ferredoxins from several species of genus Solanum

The complete amino acid sequences of [2Fe-2S] ferredoxins from four species of genus Solanum (S. nigrum, S. lyratum, S. indicum, and S. abutiloides) were determined by automated Edman degradation of the entire S-carboxymethylcysteinyl proteins and of the peptides obtained by enzymatic digestion. The amino acid sequences of these four ferredoxins differed from each other by 12-19, whereas 0-4 differences have been observed among ferredoxins from plants in the same genus and 14-40 differences were seen between different families. This suggests that these Solanum plants are distantly related to each other taxonomically.     

Amino acid sequences of proteins from Leptospira serovar pomona

This report describes a partial amino acid sequences from three putative outer envelope proteins from Leptospira serovar pomona. In order to obtain internal fragments for protein sequencing, enzymatic and chemical digestion was performed. The enzyme clostripain was used to digest the proteins 32 and 45 kDa. In situ digestion of 40 kDa molecular weight protein was accomplished using cyanogen bromide. The 32 kDa protein generated two fragments, one of 21 kDa and another of 10 kDa that yielded five residues. A fragment of 24 kDa that yielded nineteen residues of amino acids was obtained from 45 kDa protein. A fragment with a molecular weight of 20 kDa, yielding a twenty amino acids sequence from the 40 kDa protein.     

Amino acid sequences of ferredoxin isoproteins from radish roots

Three ferredoxin isoproteins (R-Fd A, R-Fd B-1, and R-Fd B-2) were purified from white roots of radish (Raphanus sativus L. var. acantiformis cultivar Miyashige) and two isoproteins (L-Fd A and L-Fd B) from leaves. The amino acid sequences of three of them (L-Fd A, R-Fd B-1, and R-Fd B-2) were determined and compared with one another and with those of other higher plant ferredoxins so far studied. L-Fd A and R-Fd B-1 had heterogeneities at four and two amino acid sites, respectively. Two isoprotein (R-Fd B-1 and R-Fd B-2) were deduced to be expressed only in root tissue on the basis of sequence studies and amino acid compositions of all isoferredoxins isolated from the radish plant. The root ferredoxins sequenced in this study were similar to each other, but quite different from other higher plant ferredoxins, all of which were isolated from leaf tissue. The coupling activities of these ferredoxin isoproteins were measured in the NADP+-photoreduction system of radish chloroplasts and glutamate synthase [EC 1.4.7.1] systems isolated from radish leaf and root tissues. No distinctive physiological characteristics were observed among these isoferredoxins.     

Two distinct ferredoxins from Rhodobacter capsulatus: complete amino acid sequences and molecular evolution

Two distinct ferredoxins were purified from Rhodobacter capsulatus SB1003. Their complete amino acid sequences were determined by a combination of protease digestion, BrCN cleavage and Edman degradation. Ferredoxins I and II were composed of 64 and 111 amino acids, respectively, with molecular weights of 6,728 and 12,549 excluding iron and sulfur atoms. Both contained two Cys clusters in their amino acid sequences. The first cluster of ferredoxin I and the second cluster of ferredoxin II had a sequence, CxxCxxCxxxCP, in common with the ferredoxins found in Clostridia. The second cluster of ferredoxin I had a sequence, CxxCxxxxxxxxCxxxCM, with extra amino acids between the second and third Cys, which has been reported for other photosynthetic bacterial ferredoxins and putative ferredoxins (nif-gene products) from nitrogen-fixing bacteria, and with a unique occurrence of Met. The first cluster of ferredoxin II had a CxxCxxxxCxxxCP sequence, with two additional amino acids between the second and third Cys, a characteristics feature of Azotobacter-[3Fe-4S] [4Fe-4S]-ferredoxin. Ferredoxin II was also similar to Azotobacter-type ferredoxins with an extended carboxyl (C-) terminal sequence compared to the common Clostridium-type. The evolutionary relationship of the two together with a putative one recently found to be encoded in nifENXQ region in this bacterium [Moreno-Vivian et al. (1989) J. Bacteriol. 171, 2591-2598] is discussed.     

Stimulation by 2,4-dichlorophenoxyacetic acid of betacyanin accumulation in suspension cultures of Phytolacca americana

2,4-Dichlorophenoxyacetic acid (2,4-D) strongly promoted betacyanin accumulation in suspension cultures of Phytolacca americana L. The betacyanin accumulation attained a maximum at 5 μ M 2,4-D, when betacyanin content per cell reached 252% as compared to the control (2,4-D free). 2,4-D elevated the level of free tyrosine, which is the precursor of betacyanin. The addition of 1 m M tyrosine to the medium partially reversed the reduction of betacyanin accumulation caused by the removal of 2,4-D. Tracer experiments using labelled tyrosine showed that 2,4-D activated the biosynthetic pathway from tyrosine to betacyanin. These results indicate that a sufficient supply of tyrosine and the activation of biosynthesis of betacyanin from tyrosine by 2,4-D elevate the level of betacyanin.     

Amino acid sequences of the two heme c-binding sites of Pseudomonas cytochrome-c peroxidase

The amino acid sequences of the two heme c-containing tryptic peptides of Pseudomonas cytochrome-c peroxidase have been determined. The tryptic peptides were isolated from two cyanogen bromide fragments of the protein. Both heme-binding sites have the Cys-X-Y-Cys-His structure characteristic of c-type cytochromes. The sequences of the two peptides show distinct homology with each other, suggesting the occurrence of gene doubling during evolution of the protein molecule. The function of the heme c moieties in the catalytic cycle of the enzyme is discussed on the basis of their homology with the proximal histidine region of peroxidase (horseradish peroxidase and yeast cytochrome-c peroxidase) and cytochromes (horse cytochrome c and Pseudomonas cytochrome c-551).     

Comparison of Phytolacain G,a Cysteine Protease from Fruit of Phytolacca americana,with Phytolacain R

The enzymatic properties of phytolacain G, a protease isolated from green fruit of pokeweed, were compared with those of phytolacain R, a protease obtained from ripe fruit. The optimum pH of phytolacain G was 7.5-8.0 at 37°C using casein as the substrate. The enzyme was strongly inhibited by iodoacetic acid and p-chloromercuribenzoic acid, but not by diisopropyl fluorophosphate or EDTA. These results indicated that phytolacain G was a cysteine protease, like phytolacain R. Nine sites of oxidized insulin B-chain were cleaved by phytolacain G during 20 h of hydrolysis. The six sites cleaved by phytolacain G were also cleaved by phytolacain R. The substrate specificity of phytolacain G was broad, but the preference for hydrophobic residues at the P₂ position was similar to the substrate specificity of papain. The amino-terminal sequence of phytolacain G was not identical with that of phytolacain R; however, the amino acid residues conserved in the papain family were also conserved in this enzyme.