Changes in activity of proteolytic enzymes (BAPAases) in germinating buckwheat and rye seeds

The interrelationship between the activity of proteolytic enzymes (BAPAases) from buckwheat and rye seeds hydrolyzing Nalpha-benzoyl-DL-arginine-p-nitroanilide (BAPA) and the amount of the antiserum to these enzymes necessary to obtain a certain inhibition level has been studied at different stages of seed germination. The data obtained show that the increase of the BAPAase activity in germinating rye seeds is due to de novo synthesis of this enzyme. During this process antigenically identical enzyme molecules are synthesized in roots and shoots of the developing plant.     

Purification and characterization of a 24 kDa protein from tartary buckwheat seeds

A 24 kDa protein was isolated from tartary buckwheat seeds by using chromatography of Superdex 75 gel filtration and Resource Q ion-exchange column. SDS-PAGE and Sephacryl S-200 gel filtration were used to provide information about the molecular mass of the protein purified from tartary buckwheat. The protein was composed of 215 amino acid residues and showed strong IgE binding activity in an ELISA test to the sera colleted from two patients allergic to buckwheat. These results suggested that the purified 24 kDa protein from tartary buckwheat seeds was an important functional protein and was relatively specific for buckwheat-allergic patients. It should be a very useful tool in the diagnosis of buckwheat allergy in the future.     

Isolation and properties of a metalloproteinase from buckwheat (Fagopyrum esculentum) seeds.

A homogeneous preparation of metalloproteinase, purified 1000-fold, was obtained from buckwheat (Fagopyrum esculentum) seeds. The Mr of the enzyme, determined by SDS/PAGE, was 34,000 (it was 39,000 by gel chromatography). Its pH optimum was 8.0-8.2 with 13 S globulin, from buckwheat seeds, as substrate. Atomic-absorption spectroscopy revealed the presence of one Zn2+ ion per enzyme molecule. The enzyme was completely inhibited by EDTA (1 mM), zincone (1 mM) and 1, 10-phenanthroline (1 mM). The metalloproteinase performed limited proteolysis of the following seed storage proteins: 13 S globulin from buckwheat seeds and 11 S globulin from soybean (Glycine max) seeds. It hydrolysed three peptide bonds formed by the amino groups of Leu15, Tyr16 and Phe25 in the oxidized B-chain of insulin. In its main properties the enzyme is similar to metalloproteinases of animal and bacterial origin.     

Isolation and characteristics of benzoyl-DL-arginl-p-nitroanilide-hydrolysing enzyme (BAPAase) from vetch seedlings]

The enzyme hydrolysing N-benzoyl-D,L-arginine-p-nitroanilide (BAPA). is isolated from vetch seedlings and 1600-fold purified by means of chromatography on DEAE-cellulose, hdroxyapatite and gel filtration through Sephadex G-100. The preparation is chromatographically homogenous, but disc electrophoresis in polyacrylamide gel revealed an insignificant contamination by inactive proteins. The data of disc electrophoresis in polyacrylamide gel in the presence of sodium dodecylsulphate have shown that BAPAase has a quaternary structure containing, probably, four subunits identical in their molecular weight. BAPAase has a narrow substrate specificity: it hydrolyses BAPA, benzoyl-D,L,-argininenaphtylamide, benzoyl-L-arginyglycine CBZ-L-arginylglycine histones and protamine, but does not attack L-arginyl-p-nitroanilide benzoyl-L-arginineamide, tosyl-L-arginine methyl ester and casein.     

Properties and substrate specificity of proteinase from buckwheat seeds]

A thiol proteinase was isolated from buckwheat seeds and purified 300-fold, using ammonium sulfate, acetone fractionation ion-exchange chromatography on Sephadex CM-50 and electrofocussing. The proteinase preparation obtained was found homogenous after polyacrylamide gel electrophoresis at pH 4.5. The molecular weight of the enzyme (75.000) was determined by gel-filtration through Sephadex G-100. The activation of proteinase by cysteine, 2-mercaptoethanol and dithiothreitol, its inhibition by p-chloromercurybenzoate and the absence of inhibition by diisopropyl fluorophosphate and EDTA suggest that the enzyme isolated is a thiol proteinase. The enzyme hydrolyzed many peptide bonds in the B-chain of insulin, showing high substrate specificity. The glutelin and globulin fractions of buckwheat seed proteins were hydrolyzed by the enzyme. It is assumed that the hydrolysis of reserve proteins of buckwheat seeds is the main function of the proteinase isolated.     

Identification and characterization of granule bound starch synthase (GBSS-I) from common buckwheat (Fagopyrum esculentum Moench)

Starch grains present in the endosperm of grains of common buckwheat (Fagopyrum esculentum Moench) show a monomodal distribution with size ranging from 4 to 10 μm. SDS-PAGE analysis of starch granule bound proteins revealed the presence of a single band corresponding to molecular mass of 59.7 kDa. The protein is localized within the central core of the starch grains. Antisera raised against the 59.7 kDa protein cross reacted with the 61 kDa GBSS-I from endosperm starches of maize and the 60 kDa GBSS-I from endosperm starches of rice and wheat, thereby indicating serological homology between the 59.7 kDa buckwheat starch granule bound protein and GBSS-I of wheat, maize and rice. 2D-PAGE of starch granule bound proteins of common buckwheat resolved the fraction into 7 spots with pI ranging from 5.2 to 5.6. N-terminal amino acid sequence for 25 residues of two immunoreactive proteins separated by 2D PAGE showed 94 % homology with N-terminal amino acid sequence of GBSS-I from Hordeum vulgare, Triticum spp. and Phaseolus vulgaris. Even though analysis of the sequence alignment revealed a clear diversification into monocotyledonous and dicotyledonous groups, the protein from buckwheat showed similarities with GBSS-I from both dicots as well as monocots. As is the case with dicots, the sequence of GBSS-I from buckwheat has valine as the 11th residue. GBSS-I from majority of monocots has methionine at this position. The sequence also showed similarities with monocots with valine at P’5 from the N-terminus. GBSS-I from majority of dicots has isoleucine at this position. The significance of these substitution remains to be ascertained.     

Purification and partial characterization of a proteolytic enzyme from Cephalosporium acremonium (Corda)

A proteolytic enzyme was isolated fromCephalosporium acremonium (Corda). The enzyme had a molecular weight of 28,200 and a pH optimum of 8.4–9.2. Proteolytic activity was inhibited by diisopropylfluorophosphate and mercuric chloride. Of the substrates tested, casein was most rapidly hydrolyzed.     

Isolation and partial characterization of wheat-germ-agglutinin-like lectins from rye (Secale cereale) and barley (Hordeum vulgare) embryos

Lectins have been isolated from embryos of Secale cereale (rye) and Hordeum vulgare (barley) by affinity chromatography on immobilized N-acetylglucosamine. Both lectins are dimeric proteins of two identical subunits of mol.wt. 18000. They resemble strongly wheat-germ agglutinin with respect to their chemical, physical, biological and immunological properties.     

Strongyloides ransomi: proteolytic enzymes from larvae

The filariform larvae of Strongyloides ransomi can infect their hosts by penetration through skin. In this report, homogenates of these organisms were prepared and their proteolytic enzymes examined. Homogenates prepared in 0.2 M citrate, pH 4.0, contain two thiol-dependent proteinases with molecular weights of approximately 32,000 and 28,000. These proteinases have an acidic pH optimum and show substrate preferences and inhibitor susceptibilities similar to the vertebrate acidic cysteinyl proteinases. Homogenates prepared in 0.1 M Tris, pH 7.5, contain multiple proteolytic enzymes, active against both Azocoll and synthetic substrates. These enzymes do not require thiols for activity and they have an alkaline pH optimum. The enzymes are inhibited by both chelating agents and heavy metals, but not by serine-proteinase inhibitors. Extracts prepared in 0.1 M Tris-HCl, pH 7.5, contain endogenous proteinase inhibitors.     

Purification and characterization of proteolytic enzymes from normal and opaque-2Zea mays L. developing endosperms

Purification and characterization of proteases from developing normal maize endosperm and high lysine opaque-2 maize endosperm have been carried out with a view to understand their role in storage protein modification. At day 15, normal maize endosperm had two types of proteolytic enzymes, namely, protease I and protease II, while at day 25 protease n disappeared and in place protease III appeared. However, in opaque-2 maize endosperm at both the stages only one type of enzyme (protease I) was present. These proteases had many properties in common-optimum pH and temperature were respectively, 5.7and 40°C; their activity was inhibited to the extent of 75 –93 % by p-chloromercuribenzoate; trypsin inhibitor inhibited the activity more at early stages of endosperm development; all proteases cleaved synthetic substrates p-tosyl-L-arginine methylesler and N-benzoyl-L-tyrosine ethyl ester and poly-L-glutamic acid. TheKm values of day 15 and 25 normal maize endosperm proteases ranged from 2.73–3.30, while for opaque-2 maize endosperm protease I it was 3.33 mg azocasein per ml assay medium. These enzymes, however, differed with respect to proteolytic activity towards poly-L-lysine. Only normal maize endosperm protease III at day 25 followed by protease II at day 15 showed high activity towards this homopolypeptide suggesting thereby their role in determining the quality of normal maize endosperm protein. Part of Ph.D. thesis submitted by the first author     

Purification and characterization of proteinase inhibitors from wild soja (Glycine soja) seeds

Nine proteinase inhibitors, I-VIIa, VIIb, and VIII, were isolated from wild soja seeds by ammonium sulfate fractionation and successive chromatographies on SP-Toyopearl 650M, Sephacryl S-200SF, and DEAE-Toyopearl 650S columns. Reverse-phase HPLC finally gave pure inhibitors. All of the inhibitors inhibited trypsin with dissociation constants of 3.2-6.2 x 10(-9) M. Some of the inhibitors inhibited chymotrypsin and elastase as well. Two inhibitors (VIIb and VIII) with a molecular weight of 20,000 were classified as a soybean Kunitz inhibitor family. Others (I-VIla) had a molecular weight of about 8,000, and were stable to heat and extreme pH, suggesting that these belonged to the Bowman-Birk inhibitor family. Partial amino acid sequences of four inhibitors were also analyzed. The complete sequence of inhibitor IV was ascertained from the nucleotide sequences of cDNA clones encoding isoinhibitors homologous to soybean C-II.     

Purification and characterization of proteolytic enzymes of Leishmania mexicana mexicana amastigotes and promastigotes

Leishmania mexicana mexicana amastigote and promastigote soluble proteinases were purified using gel filtration and ion exchange chromatography. For the amastigotes, two main proteinase activity peaks were separated with both methods. These accounted for approximately 10% and 90% of the total activity. Characterization of the two activities for substrate specificity and sensitivity to inhibitors indicated that the major peak from both column methods contained enzymes with the characteristics of cysteine proteinases. SDS-polyacrylamide gel electrophoresis of the enzyme from the major peak purified by gel filtration revealed one polypeptide with a molecular weight in the region of 31 000. In contrast, the activity of the minor peak eluted from the columns was of higher molecular weight (67 000) and was similar to metalloproteinases. Purification of the soluble proteinases in the promastigote of L. m. mexicana produced only one activity peak from both column techniques. This activity (mol. wt 67 000) corresponded to the high molecular weight proteinase of the amastigote. The purified proteinases were active on 4-nitroanilide and 7-amino-4-methylcoumarin derivatives of various small peptides. The high molecular weight proteinases of both amastigotes and promastigotes were similarly active against most of the peptides, suggesting a low specificity of the enzymes. In contrast, the low molecular weight amastigote proteinases were particularly active against two of the substrates, namely BZ-Pro-Phe-Arg-Nan and Z-Phe-Arg-MCA. These results indicate that a highly active, substrate-specific, soluble proteinase, with characteristics of a cysteine proteinase, is produced upon transformation of the L. m. mexicana promastigote to amastigote. The discovery and characterization of this enzyme offers opportunities for the development of new antileishmanial agents.     

Some properties of proteolytic enzymes from Tribolium confusum

The digestive tract of Tribolium confusum Duv. larvae was studied for proteolytic enzymes properties. The pH optima are determined for the enzymes effect on various substrates. Proteases were partially purified by gel chromatography on Sephadex G = 100 and investigated for thyol compound influence on their activity. The activity of the enzymes is shown to increase considerably with addition of cystein, glutathione, 2-mercaptoethanol. dithiotreitol and EDTA. Dithiotreitol produces the strongest restoring effect and in concentration of 10(-6) M it activates the enzyme almost twice. Storage for 48 h at 4 degrees C induced a 2.5-fold decrease in the proteolytic enzyme activity; SH-groups in the catalytic action of enzymic solutions is shown. The maximum proteolytic activity is found in extracts from 14-day insects.