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Serine peptidases (SPs) of the chymotrypsin S1A subfamily are an extensive group of enzymes found in all animal organisms, including insects. Here, we provide analysis of SPs in the yellow mealworm Tenebrio molitor transcriptomes and genomes datasets and profile their expression patterns at various stages of ontogeny. A total of 269 SPs were identified, including 137 with conserved catalytic triad residues, while 125 others lacking conservation were proposed as non-active serine peptidase homologs (SPHs). Seven deduced sequences exhibit a complex domain organization with two or three peptidase units (domains), predicted both as active or non-active. The largest group of 84 SPs and 102 SPHs had no regulatory domains in the propeptide, and the majority of them were expressed only in the feeding life stages, larvae and adults, presumably playing an important role in digestion. The remaining 53 SPs and 23 SPHs had different regulatory domains, showed constitutive or upregulated expression at eggs or/and pupae stages, participating in regulation of various physiological processes. The majority of polypeptidases were mainly expressed at the pupal and adult stages. The data obtained expand our knowledge on SPs/SPHs and provide the basis for further studies of the functions of proteins from the S1A subfamily in T. molitor .

Roughly 1% of the global population is susceptible to celiac disease (CD)-inheritable autoimmune inflammation of the small intestine caused by intolerance to gliadin proteins present in wheat, rye, and barley grains, and called gluten in wheat. Classical treatment is a life-long gluten-free diet, which is constraining and costly. An alternative approach is based upon the development and oral reception of effective peptidases that degrade in the stomach immunogenic proline- and glutamine-rich gliadin peptides, which are the cause of the severe reaction in the intestine. In previous research, we have established that the major digestive peptidase of an insect Tribolium castaneum -cathepsin L-hydrolyzes immunogenic prolamins after Gln residues but is unstable in the extremely acidic environment (pH 2-4) of the human stomach and cannot be used as a digestive aid. In this work, using molecular dynamics simulations, we discover the probable cause of the pH instability of cathepsin L-loss of the catalytically competent rotameric state of one of the active site residues, His 275. To "fix" the correct orientation of this residue, we designed a V277A mutant variant, which extends the range of stability of the peptidase in the acidic environment while retaining most of its activity. We suggest this protein as a lead glutenase for the development of oral medical preparation that fights CD and gluten intolerance in susceptible people.

A detailed analysis of the complexes of proline-specific peptidases (PSPs) in the midgut transcriptomes of the larvae of agricultural pests Tenebrio molitor and Tribolium castaneum and in the genome of T. castaneum is presented. Analysis of the T. castaneum genome revealed 13 PSP sequences from the clans of serine and metal-dependent peptidases, of which 11 sequences were also found in the gut transcriptomes of both tenebrionid species' larvae. Studies of the localization of PSPs, evaluation of the expression level of their genes in gut transcriptomes, and prediction of the presence of signal peptides determining secretory pathways made it possible to propose a set of peptidases that can directly participate in the hydrolysis of food proteins in the larvae guts. The discovered digestive PSPs of tenebrionids in combination with the post-glutamine cleaving cysteine cathepsins of these insects effectively hydrolyzed gliadins, which are the natural food substrates of the studied pests. Based on the data obtained, a hypothetical scheme for the complete hydrolysis of immunogenic gliadin peptides by T. molitor and T. castaneum digestive peptidases was proposed. These results show promise regarding the development of a drug based on tenebrionid digestive enzymes for the enzymatic therapy of celiac disease and gluten intolerance.

Wheat gliadins contain a large amount of glutamine- and proline-rich peptides which are not hydrolyzed by human digestive peptidases and can cause autoimmune celiac disease and other forms of gluten intolerance in predisposed people. Peptidases that efficiently cleave such immunogenic peptides can be used in enzyme therapy. The stored product insect pest Tribolium castaneum efficiently hydrolyzes gliadins. The main digestive peptidase of T. castaneum is cathepsin L, which is from the papain C1 family with post-glutamine cleavage activity. We describe the isolation and characterization of T. castaneum recombinant procathepsin L (rpTcCathL1, NP_001164001), which was expressed in Pichia pastoris cells. The activation of the proenzyme was conducted by autocatalytic processing. The effects of pH and proenzyme concentration in the reaction mixture on the processing were studied. The mature enzyme retained high activity in the pH range from 5.0 to 9.0 and displayed high pH-stability from 4.0 to 8.0 at 20 °C. The enzyme was characterized according to electrophoretic mobility under native conditions, activity and stability at various pH values, a sensitivity to various inhibitors, and substrate specificity, and its hydrolytic effect on 8-, 10-, 26-, and 33-mer immunogenic gliadins peptides was demonstrated. Our results show that rTcCathL1 is an effective peptidase that can be used to develop a drug for the enzyme therapy of various types of gluten intolerance.

The lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), is a major global pest of cereal grains. Infestations are difficult to control as larvae feed inside grain kernels, and many populations are resistant to both contact insecticides and fumigants. We sequenced the genome of R. dominica to identify genes responsible for important biological functions and develop more targeted and efficacious management strategies. The genome was assembled from long read sequencing and long-range scaffolding technologies. The genome assembly is 479.1 Mb, close to the predicted genome size of 480.4 Mb by flow cytometry. This assembly is among the most contiguous beetle assemblies published to date, with 139 scaffolds, an N 50 of 53.6 Mb, and L 50 of 4, indicating chromosome-scale scaffolds. Predicted genes from biologically relevant groups were manually annotated using transcriptome data from adults and different larval tissues to guide annotation. The expansion of carbohydrase and serine peptidase genes suggest that they combine to enable efficient digestion of cereal proteins. A reduction in the copy number of several detoxification gene families relative to other coleopterans may reflect the low selective pressure on these genes in an insect that spends most of its life feeding internally. Chemoreceptor genes contain elevated numbers of pseudogenes for odorant receptors that also may be related to the recent ontogenetic shift of R. dominica to a diet consisting primarily of stored grains. Analysis of repetitive sequences will further define the evolution of bostrichid beetles compared to other species. The data overall contribute significantly to coleopteran genetic research.

We previously described the most highly expressed enzymes from the gut of the red flour beetle, Tribolium castaneum, as cathepsins L. In the present study, two C1 family-specific cysteine cathepsin L enzymes from the larval midgut were isolated and identified using MALDI-TOF MS analysis. The isolated T. castaneum cathepsins were characterized according to their specificity against chromogenic and fluorogenic peptide substrates, and the most efficiently hydrolyzed substrate was Z-FR-pNA with Arg in the P1 subsite. The specificity of insect digestive cathepsins was compared with human lysosomal cathepsin L, the well-studied peptidase of the C1 family cathepsins. T. castaneum digestive cathepsins efficiently hydrolyzed substrates with small and uncharged amino acid residues at P1 (Ala, Gln) more than human cathepsin L. In particular, these insect digestive cathepsins cleaved with higher efficiency the analogs of immunogenic peptides of gliadins, which contribute to autoimmune celiac disease in susceptible people, and thus insect enzymes may be useful in enzymatic treatments for this disease. A bioinformatic study supported by the proteomic analysis of the primary structures of the isolated cathepsins was used to compare tertiary models. The phylogenetic analysis of coleopteran and human cathepsins from the L subfamily indicated that insect digestive cathepsins grouped separately from lysosomal cathepsins., (Copyright © 2021. Published by Elsevier Ltd.)

To date, there is no effective treatment for celiac disease (CD, gluten enteropathy), an autoimmune disease caused by gluten-containing food. Celiac patients are supported by a strict gluten-free diet (GFD). However, in some cases GFD does not negate gluten-induced symptoms. Many patients with CD, despite following such a diet, retain symptoms of active disease due to high sensitivity even to traces of gluten. In addition, strict adherence to GFD reduces the quality of life of patients, as often it is difficult to maintain in a professional or social environment. Various pharmacological treatments are being developed to complement GFD. One promising treatment is enzyme therapy, involving the intake of peptidases with food to digest immunogenic gluten peptides that are resistant to hydrolysis due to a high prevalence of proline and glutamine amino acids. This narrative review considers the features of the main proline/glutamine-rich proteins of cereals and the conditions that cause the symptoms of CD. In addition, we evaluate information about peptidases from various sources that can effectively break down these proteins and their immunogenic peptides, and analyze data on their activity and preliminary clinical trials. Thus far, the data suggest that enzyme therapy alone is not sufficient for the treatment of CD but can be used as a pharmacological supplement to GFD.

Tenebrio molitor is an important coleopteran model insect and agricultural pest from the Tenebrionidae family. We used RNA-Seq transcriptome data from T. molitor to annotate trypsin-like sequences from the chymotrypsin S1 family of serine peptidases, including sequences of active serine peptidases (SerP) and their inactive homologs (SerPH) in T. molitor transcriptomes. A total of 63 S1 family tryspin-like serine peptidase sequences were de novo assembled. Among the sequences, 58 were predicted to be active trypsins and five inactive SerPH. The length of preproenzyme and mature form of the predicted enzyme, position of signal peptide and proenzyme cleavage sites, molecular mass, active site and S1 substrate binding subsite residues, and transmembrane and regulatory domains were analyzed using bioinformatic tools. The data can be used for further physiological, biochemical, and phylogenetic study of tenebrionid pests and other animal systems., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article., (© 2021 The Authors. Published by Elsevier Inc.)

New substrates with glutamine in the P1-position are introduced for the assay of peptidases from the C1 papain family, with a general formula of Glp-Phe-Gln-X, where Glp is pyroglutamyl and X is pNA ( p -nitroanilide) or AMC (4-amino-7-methylcoumaride). The substrates have a simple structure, and C1 cysteine peptidases of various origins cleave them with high efficiency. The main advantage of the substrates is their selectivity for cysteine peptidases of the C1 family. Peptidases of other clans, including serine trypsin-like peptidases, do not cleave glutamine-containing substrates. We demonstrate that using Glp-Phe-Gln-pNA in combination with a commercially available substrate, Z-Arg-Arg-pNA, provided differential determination of cathepsins L and B. In terms of specific activity and kinetic parameters, the proposed substrates offer improvement over the previously described alanine-containing prototypes. The efficiency and selectivity of the substrates was demonstrated by the example of chromatographic and electrophoretic analysis of a multi-enzyme digestive complex of stored product pests from the Tenebrionidae family., (Copyright © 2020 Filippova, Dvoryakova, Sokolenko, Simonyan, Tereshchenkova, Zhiganov, Dunaevsky, Belozersky, Oppert and Elpidina.)

Background: Proline specific peptidases (PSPs) are a unique group of enzymes that specifically cleave bonds formed by a proline residue. The study of PSPs is important due to their role in the maturation and degradation of peptide hormones and neuropeptides. In addition, changes in the activity of PSPs can result in pathological conditions, including various types of cancer., Scope of Review: PSPs annotated from the Homo sapiens genome were compared and classified by their physicochemical and biochemical features and roles in vital processes. In addition to catalytic activity, we discuss non-enzymatic functions that may regulate cellular activity., Major Conclusions: PSPs apparently have multiple functions in animals. Two functions rely on the catalytic activity of the enzyme: one involved in a regulatory pathway associated with the ability of many PSPs to hydrolyze peptide hormones and neuropeptides, and the other involved in the trophic pathway associated with the proteolysis of total cellular protein or Pro-containing dietary proteins in the digestive tract. PSPs also participate in signal transduction without proteolytic activity by forming protein-protein interactions that trigger or facilitate the performance of certain functions., General Significance: PSPs are underestimated as a unique component of the normal human peptidase degradome, providing the body with free proline. A comparative analysis of PSPs can guide research to develop inhibitors that counteract the abnormalities associated with changes in PSP activity, and identify natural substrates of PSPs that will enable better understanding of the mechanisms of the action of PSPs in biological processes and disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

The tremendous diversity of Hymenoptera is commonly attributed to the evolution of parasitoidism in the last common ancestor of parasitoid sawflies (Orussidae) and wasp-waisted Hymenoptera (Apocrita). However, Apocrita and Orussidae differ dramatically in their species richness, indicating that the diversification of Apocrita was promoted by additional traits. These traits have remained elusive due to a paucity of sawfly genome sequences, in particular those of parasitoid sawflies. Here, we present comparative analyses of draft genomes of the primarily phytophagous sawfly Athalia rosae and the parasitoid sawfly Orussus abietinus. Our analyses revealed that the ancestral hymenopteran genome exhibited traits that were previously considered unique to eusocial Apocrita (e.g., low transposable element content and activity) and a wider gene repertoire than previously thought (e.g., genes for CO2 detection). Moreover, we discovered that Apocrita evolved a significantly larger array of odorant receptors than sawflies, which could be relevant to the remarkable diversification of Apocrita by enabling efficient detection and reliable identification of hosts., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species due in part to its exceptionally high levels of polyphagy. This species is also a nuisance due to overwintering in human-made structures. It has caused significant agricultural losses in recent years along the Atlantic seaboard of North America and in continental Europe. Genomic resources will assist with determining the molecular basis for this species' feeding and habitat traits, defining potential targets for pest management strategies., Results: Analysis of the 1.15-Gb draft genome assembly has identified a wide variety of genetic elements underpinning the biological characteristics of this formidable pest species, encompassing the roles of sensory functions, digestion, immunity, detoxification and development, all of which likely support H. halys' capacity for invasiveness. Many of the genes identified herein have potential for biomolecular pesticide applications., Conclusions: Availability of the H. halys genome sequence will be useful for the development of environmentally friendly biomolecular pesticides to be applied in concert with more traditional, synthetic chemical-based controls.

A method is described for the direct detection of unstable cysteine peptidase activity in polyacrylamide gels after native electrophoresis using new selective fluorogenic peptide substrates, pyroglutamyl-phenylalanyl-alanyl-4-amino-7-methylcoumaride (Glp-Phe-Ala-AMC) and pyroglutamyl-phenylalanyl-alanyl-4-amino-7-trifluoromethyl-coumaride (Glp-Phe-Ala-AFC). The detection limit of the model enzyme papain was 17 pmol (0.29 μg) for Glp-Phe-Ala-AMC and 43 pmol (0.74 μg) for Glp-Phe-Ala-AFC, with increased sensitivity and selectivity compared to the traditional method of protein determination with Coomassie G-250 staining or detection of activity using chromogenic substrates. Using this method, we easily identified the target digestive peptidases of Tenebrio molitor larvae by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. The method offers simplicity, high sensitivity, and selectivity compared to traditional methods for improved identification of unstable cysteine peptidases in multi-component biological samples., (Copyright © 2018. Published by Elsevier Inc.)

Background: Having conquered water surfaces worldwide, the semi-aquatic bugs occupy ponds, streams, lakes, mangroves, and even open oceans. The diversity of this group has inspired a range of scientific studies from ecology and evolution to developmental genetics and hydrodynamics of fluid locomotion. However, the lack of a representative water strider genome hinders our ability to more thoroughly investigate the molecular mechanisms underlying the processes of adaptation and diversification within this group., Results: Here we report the sequencing and manual annotation of the Gerris buenoi (G. buenoi) genome; the first water strider genome to be sequenced thus far. The size of the G. buenoi genome is approximately 1,000 Mb, and this sequencing effort has recovered 20,949 predicted protein-coding genes. Manual annotation uncovered a number of local (tandem and proximal) gene duplications and expansions of gene families known for their importance in a variety of processes associated with morphological and physiological adaptations to a water surface lifestyle. These expansions may affect key processes associated with growth, vision, desiccation resistance, detoxification, olfaction and epigenetic regulation. Strikingly, the G. buenoi genome contains three insulin receptors, suggesting key changes in the rewiring and function of the insulin pathway. Other genomic changes affecting with opsin genes may be associated with wavelength sensitivity shifts in opsins, which is likely to be key in facilitating specific adaptations in vision for diverse water habitats., Conclusions: Our findings suggest that local gene duplications might have played an important role during the evolution of water striders. Along with these findings, the sequencing of the G. buenoi genome now provides us the opportunity to pursue exciting research opportunities to further understand the genomic underpinnings of traits associated with the extreme body plan and life history of water striders.

The gut is one of the primary interfaces between an insect and its environment. Understanding gene expression profiles in the insect gut can provide insight into interactions with the environment as well as identify potential control methods for pests. We compared the expression profiles of transcripts from the gut of larval stages of two coleopteran insects, Tenebrio molitor and Tribolium castaneum. These tenebrionids have different life cycles, varying in the duration and number of larval instars. T. castaneum has a sequenced genome and has been a model for coleopterans, and we recently obtained a draft genome for T. molitor. We assembled gut transcriptome reads from each insect to their respective genomes and filtered mapped reads to RPKM>1, yielding 11,521 and 17,871 genes in the T. castaneum and T. molitor datasets, respectively. There were identical GO terms in each dataset, and enrichment analyses also identified shared GO terms. From these datasets, we compiled an ortholog list of 6907 genes; 45% of the total assembled reads from T. castaneum were found in the top 25 orthologs, but only 27% of assembled reads were found in the top 25 T. molitor orthologs. There were 2281 genes unique to T. castaneum, and 2088 predicted genes unique to T. molitor, although improvements to the T. molitor genome will likely reduce these numbers as more orthologs are identified. We highlight a few unique genes in T. castaneum or T. molitor that may relate to distinct biological functions. A large number of putative genes expressed in the larval gut with uncharacterized functions (36 and 68% from T. castaneum and T. molitor, respectively) support the need for further research. These data are the first step in building a comprehensive understanding of the physiology of the gut in tenebrionid insects, illustrating commonalities and differences that may be related to speciation and environmental adaptation., (Published by Elsevier Ltd.)

The Colorado potato beetle is one of the most challenging agricultural pests to manage. It has shown a spectacular ability to adapt to a variety of solanaceaeous plants and variable climates during its global invasion, and, notably, to rapidly evolve insecticide resistance. To examine evidence of rapid evolutionary change, and to understand the genetic basis of herbivory and insecticide resistance, we tested for structural and functional genomic changes relative to other arthropod species using genome sequencing, transcriptomics, and community annotation. Two factors that might facilitate rapid evolutionary change include transposable elements, which comprise at least 17% of the genome and are rapidly evolving compared to other Coleoptera, and high levels of nucleotide diversity in rapidly growing pest populations. Adaptations to plant feeding are evident in gene expansions and differential expression of digestive enzymes in gut tissues, as well as expansions of gustatory receptors for bitter tasting. Surprisingly, the suite of genes involved in insecticide resistance is similar to other beetles. Finally, duplications in the RNAi pathway might explain why Leptinotarsa decemlineata has high sensitivity to dsRNA. The L. decemlineata genome provides opportunities to investigate a broad range of phenotypes and to develop sustainable methods to control this widely successful pest.

Prolidase is a proline-specific metallopeptidase that cleaves imidodipeptides with C-terminal Pro residue. Prolidase was purified and characterized from the Tenebrio molitor larval midgut. The enzyme was localized in the soluble fraction of posterior midgut tissues, corresponding to a predicted cytoplasmic localization of prolidase according to the structure of the mRNA transcript. Expression of genes encoding prolidase and the major digestive proline-specific peptidase (PSP)-dipeptidyl peptidase 4-were similar. The pH optimum of T. molitor prolidase was 7.5, and the enzyme was inhibited by Z-Pro, indicating that it belongs to type I prolidases. In mammals, prolidase is particularly important in the catabolism of a proline-rich protein-collagen. We propose that T. molitor larval prolidase is a critical enzyme for the final stages of digestion of dietary proline-rich gliadins, providing hydrolysis of imidodipeptides in the cytoplasm of midgut epithelial cells. We propose that the products of hydrolysis are absorbed from the luminal contents by peptide transporters, which we have annotated in the T. molitor larval gut transcriptome. The origin of prolidase substrates in the insect midgut is discussed in the context of overall success of grain feeding insects., (© 2017 Wiley Periodicals, Inc.)

Tribolium castaneum is a major agriculture pest damaging stored grains and cereal products. The T. castaneum genome contains 26 cysteine peptidase genes, mostly cathepsins L and B, and seven have a major role in digestion. We targeted the expression of the most highly expressed cathepsin L gene on chromosome 10, TC011001, by RNA interference (RNAi), using double-stranded RNA (dsRNA) constructs of different regions of the gene (3', middle, 5' and entire coding regions). RNA sequencing and quantitation (RNA-seq) was used to evaluate knockdown and specificity amongst the treatments. Overall, target gene expression decreased in all treatment groups, but was more severe and specific in dsRNA targeting the 3' and entire coding regions, encoding the proteolytic active site in the enzyme. Additional cysteine cathepsin genes also were down-regulated (off-target effects), but some were up-regulated in response to RNAi treatment. Notably, some serine peptidase genes were increased in expression, especially in dsRNA targeting 5' and middle regions, and the response was similar to the effects of dietary cysteine protease inhibitors. We manually annotated these serine peptidase genes to gain insight into function and relevance to the RNAi study. The data indicate that T. castaneum larvae compensate for the loss of digestive peptidase activity in the larval gut, regardless of the mechanism of disruption., (Published 2016. This article is a U.S. Government work and is in the public domain in the USA.)

Dipeptidyl peptidase 4 (DPP 4) is a proline specific serine peptidase that plays an important role in different regulatory processes in mammals. In this report, we isolated and characterized a unique secreted digestive DPP 4 from the anterior midgut of a stored product pest, Tenebrio molitor larvae (TmDPP 4), with a biological function different than that of the well-studied mammalian DPP 4. The sequence of the purified enzyme was confirmed by mass-spectrometry, and was identical to the translated RNA sequence found in a gut EST database. The purified peptidase was characterized according to its localization in the midgut, and substrate specificity and inhibitor sensitivity were compared with those of human recombinant DPP 4 (rhDPP 4). The T. molitor enzyme was localized mainly in the anterior midgut of the larvae, and 81% of the activity was found in the fraction of soluble gut contents, while human DPP 4 is a membrane enzyme. TmDPP 4 was stable in the pH range 5.0-9.0, with an optimum activity at pH 7.9, similar to human DPP 4. Only specific inhibitors of serine peptidases, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, suppressed TmDPP 4 activity, and the specific dipeptidyl peptidase inhibitor vildagliptin was most potent. The highest rate of TmDPP 4 hydrolysis was found for the synthetic substrate Arg-Pro-pNA, while Ala-Pro-pNA was a better substrate for rhDPP 4. Related to its function in the insect midgut, TmDPP 4 efficiently hydrolyzed the wheat storage proteins gliadins, which are major dietary proteins of T. molitor., (Published by Elsevier Ltd.)

The red flour beetle, Tribolium castaneum, is a major agricultural pest responsible for considerable loss of stored grain and cereal products worldwide. T. castaneum larvae have a highly compartmentalized gut, with cysteine peptidases mostly in the acidic anterior part of the midgut that are critical to the early stages of food digestion. In previous studies, we described 26 putative cysteine peptidase genes in T. castaneum (types B, L, O, F, and K) located mostly on chromosomes 3, 7, 8, and 10. In the present study, we hypothesized that specific cysteine peptidase genes could be associated with digestive functions for food processing based on comparison of gene expression profiles in different developmental stages, feeding and non-feeding. RNA-Seq was used to determine the relative expression of cysteine peptidase genes among four major developmental stages (egg, larvae, pupae, and adult) of T. castaneum. We also compared cysteine peptidase genes in T. castaneum to those in other model insects and coleopteran pests. By combining transcriptome expression, phylogenetic comparisons, response to dietary inhibitors, and other existing data, we identified key cysteine peptidases that T. castaneum larvae and adults use for food digestion, and thus new potential targets for biologically-based control products.

Background: Larvae of the tenebrionids Tenebrio molitor and Tribolium castaneum have highly compartmentalized guts, with primarily cysteine peptidases in the acidic anterior midgut that contribute to the early stages of protein digestion., Results: High throughput sequencing was used to quantify and characterize transcripts encoding cysteine peptidases from the C1 papain family in the gut of tenebrionid larvae. For T. castaneum, 25 genes and one questionable pseudogene encoding cysteine peptidases were identified, including 11 cathepsin L or L-like, 11 cathepsin B or B-like, and one each F, K, and O. The majority of transcript expression was from two cathepsin L genes on chromosome 10 (LOC659441 and LOC659502). For cathepsin B, the major expression was from genes on chromosome 3 (LOC663145 and LOC663117). Some transcripts were expressed at lower levels or not at all in the larval gut, including cathepsins F, K, and O. For T. molitor, there were 29 predicted cysteine peptidase genes, including 14 cathepsin L or L-like, 13 cathepsin B or B-like, and one each cathepsin O and F. One cathepsin L and one cathepsin B were also highly expressed, orthologous to those in T. castaneum. Peptidases lacking conservation in active site residues were identified in both insects, and sequence analysis of orthologs indicated that changes in these residues occurred prior to evolutionary divergence. Sequences from both insects have a high degree of variability in the substrate binding regions, consistent with the ability of these enzymes to degrade a variety of cereal seed storage proteins and inhibitors. Predicted cathepsin B peptidases from both insects included some with a shortened occluding loop without active site residues in the middle, apparently lacking exopeptidase activity and unique to tenebrionid insects. Docking of specific substrates with models of T. molitor cysteine peptidases indicated that some insect cathepsins B and L bind substrates with affinities similar to human cathepsin L, while others do not and have presumably different substrate specificity., Conclusions: These studies have refined our model of protein digestion in the larval gut of tenebrionid insects, and suggest genes that may be targeted by inhibitors or RNA interference for the control of cereal pests in storage areas.

This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases of the C1 papain family, important in many biological processes. The structure of the newly synthesized substrates is Glp-Xaa-Ala-Y (where Glp=pyroglutamyl; Xaa=Phe or Val; and Y=pNA [p-nitroanilide], AMC [4-amino-7-methylcoumaride], or AFC [4-amino-7-trifluoromethyl-coumaride]). Substrates were synthesized enzymatically to guarantee selectivity of the reaction and optical purity of the target compounds, simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized substrates was evaluated by C1 cysteine peptidases from different organisms and with different functions, including plant enzymes papain, bromelain, ficin, and mammalian lysosomal cathepsins B and L. The new substrates were selective for C1 cysteine peptidases and were not hydrolyzed by serine, aspartic, or metallo peptidases. We demonstrated an application of the selectivity of the synthesized substrates during the chromatographic separation of a multicomponent set of digestive peptidases from a beetle, Tenebrio molitor. Used in combination with the cysteine peptidase inhibitor E-64, these substrates were able to differentiate cysteine peptidases from peptidases of other classes in midgut extracts from T. molitor larvae and larvae of the genus Tribolium; thus, they are useful in the analysis of complex mixtures containing peptidases from different classes., (Published by Elsevier Inc.)

Prolyl carboxypeptidase (PRCP) is a lysosomal proline specific serine peptidase that also plays a vital role in the regulation of physiological processes in mammals. In this report, we isolate and characterize the first PRCP in an insect. PRCP was purified from the anterior midgut of larvae of a stored product pest, Tenebrio molitor, using a three-step chromatography strategy, and it was determined that the purified enzyme was a dimer. The cDNA of PRCP was cloned and sequenced, and the predicted protein was identical to the proteomic sequences of the purified enzyme. The substrate specificity and kinetic parameters of the enzyme were determined. The T. molitor PRCP participates in the hydrolysis of the insect's major dietary proteins, gliadins, and is the first PRCP to be ascribed a digestive function. Our collective data suggest that the evolutionary enrichment of the digestive peptidase complex in insects with an area of acidic to neutral pH in the midgut is a result of the incorporation of lysosomal peptidases, including PRCP., (Published by Elsevier Ltd.)

The yellow mealworm, Tenebrio molitor, is a pest of stored grain products and is sensitive to the Bacillus thuringiensis (Bt) Cry3Aa toxin. As digestive peptidases are a determining factor in Cry toxicity and resistance, we evaluated the expression of peptidase transcripts in the midgut of T. molitor larvae fed either a control or Cry3Aa protoxin diet for 24 h (RNA-Seq), or in larvae exposed to the protoxin for 6, 12, or 24 h (microarrays). Cysteine peptidase transcripts (9) were similar to cathepsins B, L, and K, and their expression did not vary more than 2.5-fold in control and Cry3Aa-treated larvae. Serine peptidase transcripts (48) included trypsin, chymotrypsin and chymotrypsin-like, elastase 1-like, and unclassified serine peptidases, as well as homologs lacking functional amino acids. Highly expressed trypsin and chymotrypsin transcripts were severely repressed, and most serine peptidase transcripts were expressed 2- to 15-fold lower in Cry3Aa-treated larvae. Many serine peptidase and homolog transcripts were found only in control larvae. However, expression of a few serine peptidase transcripts was increased or found only in Cry3Aa-treated larvae. Therefore, Bt intoxication significantly impacted the expression of serine peptidases, potentially important in protoxin processing, while the insect maintained the production of critical digestive cysteine peptidases., (Published by Elsevier Inc.)

The major storage proteins in cereals, prolamins, have an abundance of the amino acids glutamine and proline. Storage pests need specific digestive enzymes to efficiently hydrolyze these storage proteins. Therefore, post-glutamine cleaving peptidases (PGP) were isolated from the midgut of the stored-product pest, Tenebrio molitor (yellow mealworm). Three distinct PGP activities were found in the anterior and posterior midgut using the highly-specific chromogenic peptide substrate N-benzyloxycarbonyl-L-Ala-L-Ala-L-Gln p-nitroanilide. PGP peptidases were characterized according to gel elution times, activity profiles in buffers of different pH, electrophoretic mobility under native conditions, and inhibitor sensitivity. The results indicate that PGP activity is due to cysteine and not serine chymotrypsin-like peptidases from the T. molitor larvae midgut. We propose that the evolutionary conservation of cysteine peptidases in the complement of digestive peptidases of tenebrionid stored-product beetles is due not only to the adaptation of insects to plants rich in serine peptidase inhibitors, but also to accommodate the need to efficiently cleave major dietary proteins rich in glutamine., (Published by Elsevier Inc.)

The complex of digestive proteinases in caterpillars of the greater wax moth Galleria mellonella was studied. Using chromogenic substrates and inhibitor analysis, it was found that serine proteinases play a key role in this complex. Three anionic and two cationic forms of trypsin and one anionic and one cationic form of chymotrypsin were identified by zymography in the midgut extract of G. mellonella. The most active trypsin was purified to electrophoretic homogeneity, and its N-terminal amino acid sequence was shown to be identical to that of mature trypsin from Plodia interpunctella. Midgut extract from G. mellonella was capable of processing Cry-proteins from Bacillus thuringiensis ssp. galleriae. Enzymes with tryptic and chymotryptic activities participate in this process, and activation of protoxin Cry9A is not the rate-limiting stage in the toxic action of this protein on the greater wax moth.

The transcriptome response of Tribolium castaneum larvae to dietary protease inhibitors was evaluated by whole-genome microarray analysis. RNA was isolated from guts of larvae fed control diet (no inhibitor), or diets containing 0.1% E-64 (cysteine protease inhibitor), 5.0% soybean trypsin inhibitor (STI, serine protease inhibitor), or a combination of 0.1% E-64 and 5.0% STI. Data were analyzed by pairwise analysis, in which each inhibitor treatment group was compared to control, or ANOVA of all treatment groups. In pairwise analysis, the expression of only 253 genes was significantly altered (p<0.05) in response to STI treatment, whereas E-64 and combination treatments resulted in 1574 and 1584 differentially regulated genes. The data indicate that treatments containing E-64, whether alone or in combination, significantly impacts gene expression in T. castaneum larvae. ANOVA analysis revealed 2175 genes differentially expressed in inhibitor-treated larvae compared to control (p<0.05), including genes related to proteases that were mostly up-regulated, namely cathepsins B and L, chymotrypsins, and nonproteolytic cysteine cathepsin or serine protease homologs. Inhibitor treatments induced the differential expression of other gut-related genes, as well as genes encoding proteins of unknown function. These data suggest that T. castaneum larvae compensate for dietary cysteine protease inhibitors by altering large-scale gene expression patterns., (Published by Elsevier Inc.)

Sequences of peptidases with conserved motifs around the active site residues that are characteristic of trypsins (similar to trypsin peptidases, STP) were obtained from publicly-available fungal genomes and related databases. Among the 75 fungal genomes, 29 species of parasitic Ascomycota contained genes encoding STP and their homologs. Searches of non-redundant protein sequences, patented protein sequences, and expressed sequence tags resulted in another 18 STP sequences in 10 fungal species from Ascomycota, Basidiomycota, and Zygomycota. A comparison of fungi species containing STP sequences revealed that almost all are pathogens of plants, animals or fungi. A comparison of the primary structure of homologous proteins, including the residues responsible for substrate binding and specificity of the enzyme, revealed three groups of homologous sequences, all presumably from S1 family: trypsin-like peptidases, chymotrypsin-like peptidases and serine peptidases with unknown substrate specificity. Homologs that are presumably functionally inactive were predicted in all groups. The results in general support the hypothesis that the expression of trypsin-like peptidases in fungi represents a marker of fungal phytopathogenicity. A phylogenetic tree was constructed using peptidase and homolog amino acid sequences, demonstrating that all have noticeable differences and almost immediately deviate from the common root. Therefore, we conclude that the changes that occurred in STP of pathogenic fungi in the course of evolution represent specific adaptations to proteins of their respective hosts, and mutations in peptidase genes are important components of life-style changes and taxonomic divergence.

Tribolium castaneum is an important agricultural pest and an advanced genetic model for coleopteran insects. We have taken advantage of the recently acquired T. castaneum genome to identify T. castaneum genes and proteins in one of the more critical environmental interfaces of the insect, the larval alimentary tract. Genetic transcripts isolated from the T. castaneum larval gut were labeled and hybridized to a custom array containing oligonucleotides from predicted genes in the T. castaneum genome. Through a ranking procedure based on relative labeling intensity, we found that approximately 17.6% of the genes represented in the array were predicted to be highly expressed in gut tissue. Several genes were selected to compare relative expression levels in larval gut, head, or carcass tissues using quantitative real-time PCR, and expression levels were, with few exceptions, consistent with the gut rankings. In parallel with the microarrays, proteins extracted from the T. castaneum larval gut were subjected to proteomic analysis. Two-dimensional electrophoretic analysis combined with MALDI-TOF resulted in the identification of 37 of 88 selected protein samples. As an alternative strategy, one-dimensional electrophoretic separation of T. castaneum larval gut proteins followed by two-dimensional nano-HPLC and ESI-MS/MS resulted in the identification of 98 proteins. A comparison of the proteomic studies indicated that 16 proteins were commonly identified in both, whereas 80 proteins from the proteomic analyses corresponded to genes with gut rankings indicative of high expression in the microarray analysis. These data serve as a resource of T. castaneum transcripts and proteins in the larval gut and provide the basis for comparative transcriptomic and proteomic studies related to the gut of coleopteran insects.

The spectra of Tribolium castaneum and T. confusum larval digestive peptidases were characterized with respect to the spatial organization of protein digestion in the midgut. The pH of midgut contents in both species increased from 5.6-6.0 in the anterior to 7.0-7.5 in the posterior midgut. However, the pH optimum of the total proteolytic activity of the gut extract from either insect was pH 4.1. Approximately 80% of the total proteolytic activity was in the anterior and 20% in the posterior midgut of either insect when evaluated in buffers simulating the pH and reducing conditions characteristic for each midgut section. The general peptidase activity of gut extracts from either insect in pH 5.6 buffer was mostly due to cysteine peptidases. In the weakly alkaline conditions of the posterior midgut, the serine peptidase contribution was 31 and 41% in T. castaneum and T. confusum, respectively. A postelectrophoretic peptidase activity assay with gelatin also revealed the important contribution of cysteine peptidases in protein digestion in both Tribolium species. The use of a postelectrophoretic activity assay with p-nitroanilide substrates and specific inhibitors revealed a set of cysteine and serine endopeptidases, 8 and 10 for T. castaneum, and 7 and 9 for T. confusum, respectively. Serine peptidases included trypsin-, chymotrypsin-, and elastase-like enzymes, the latter being for the first time reported in Tenebrionid insects. These data support a complex system of protein digestion in the Tribolium midgut with the fundamental role of cysteine peptidases.

Two post-proline cleaving enzymes PRE1 and PRE2 with molecular masses of 101 and 62 kDa, respectively, capable of hydrolyzing Z-AlaAlaPro-pNA were isolated for the first time from the midgut of the flour beetle Tenebrio molitor and characterized. PRE1 is active only in acidic media, with a maximum at pH 5.6, whereas PRE2, both in acidic and alkaline media with a maximum at pH 7.9. Using inhibitory analysis, both PRE1 and PRE2 were shown to belong to serine peptidases. Some data indicate that a Cys residue is located close to the PRE2 active site. Z-Pro-prolinal, a specific inhibitor of prolyl oligopeptidases, inhibits completely PRE2 and partially PRE1. The substrate specificities of the isolated enzymes were studied. It was shown that Z-AlaAla-Pro-pNA was the best substrate for PRE1, and Z-AlaPro-pNA, for PRE2. The combination of the studied properties allowed characterization of PRE2 as a prolyl oligopeptidase.

Two soluble post-proline cleaving peptidase activities, PPCP1 and PPCP2, were demonstrated in Tenebrio molitor larval midgut with the substrate benzyloxycarbonyl-L-alanyl-L-proline p-nitroanilide. Both activities were serine peptidases. PPCP1 was active in acidic buffers, with maximum activity at pH 5.3, and was located mainly in the more acidic anterior midgut lumen. The dynamics of PPCP1 activity and the total activity of soluble digestive peptidases in the course of food digestion were similar, suggesting that the enzyme participates in protein digestion. PPCP2 is a nondigestive soluble tissue enzyme evenly distributed along the midgut. An increase in the activity of PPCP2 was observed in buffers of pH 5.6-8.6 and was maximal at pH 7.4. The sensitivity of PPCP2 to inhibitors and the effect of pH are similar to prolyl oligopeptidases with a cysteine residue near the substrate binding site.

Peptidase sequences were analysed in randomly picked clones from cDNA libraries of the anterior or posterior midgut or whole larvae of the yellow mealworm, Tenebrio molitor Linnaeus. Of a total of 1528 sequences, 92 encoded potential peptidases, from which 50 full-length cDNA sequences were obtained, including serine and cysteine proteinases and metallopeptidases. Serine proteinase transcripts were predominant in the posterior midgut, whereas transcripts encoding cysteine and metallopeptidases were mainly found in the anterior midgut. Alignments with other proteinases indicated that 40% of the serine proteinase sequences were serine proteinase homologues, and the remaining ones were identified as either trypsin, chymotrypsin or other serine proteinases. Cysteine proteinase sequences included cathepsin B- and L-like proteinases, and metallopeptidase transcripts were similar to carboxypeptidase A. Northern blot analysis of representative sequences demonstrated the differential expression profile of selected transcripts across five developmental stages of Te. molitor. These sequences provide insights into peptidases in coleopteran insects as a basis to study the response of coleopteran larvae to external stimuli and to evaluate regulatory features of the response.

The spectrum of Tenebrio molitor larval digestive proteinases was studied in the context of the spatial organization of protein digestion in the midgut. The pH of midgut contents increased from 5.2-5.6 to 7.8-8.2 from the anterior to the posterior. This pH gradient was reflected in the pH optima of the total proteolytic activity, 5.2 in the anterior and 9.0 in the posterior midgut. When measured at the pH and reducing conditions characteristic of each midgut section, 64% of the total proteolytic activity was in the anterior and 36% in the posterior midgut. In the anterior midgut, two-thirds of the total activity was due to cysteine proteinases, whereas the rest was from serine proteinases. In contrast, most (76%) of the proteolytic activity in the posterior midgut was from serine proteinases. Cysteine proteinases from the anterior were represented by a group of anionic fractions with similar electrophoretic mobility. Trypsin-like activity was predominant in the posterior midgut and was due to one cationic and three anionic proteinases. Chymotrypsin-like proteinases also were prominent in the posterior midgut and consisted of one cationic and four anionic proteinases, four with an extended binding site. Latent proteinase activity was detected in each midgut section. These data support a complex system of protein digestion, and the correlation of proteinase activity and pH indicates a physiological mechanism of enzyme regulation in the gut.

Tenebrio molitor larval digestive proteinases were purified and characterized by gel filtration chromatography combined with activity electrophoresis. Cysteine proteinases, consisting of at least six distinct activities, were found in three chromatographic peaks in anterior and posterior midgut chromatographies. The major activity in the anterior midgut, peak cys II, consisted of cysteine proteinases with Mm of 23 kDa. The predominant peak in the posterior, cys I, was represented by 38 kDa proteinases. The activities of all cysteine proteinases were maximal in buffers from pH 5.0 to 7.0, with 80% stability at pH values from 4.0 to 7.0. In the conditions of the last third of the midgut, the activity and stability of cysteine proteinases was sharply decreased. Trypsin-like activity included a minor peak of "heavy" trypsins with Mm 59 kDa, located mainly in the anterior midgut. An in vitro study of the initial stages of digestion of the main dietary protein, oat 12S globulin, by anterior midgut proteinases revealed that hydrolysis occurred through the formation of intermediate high-Mm products, similar to those formed during oat seed germination. Cysteine proteinases from the cys III peak and heavy trypsins were capable of only limited proteolysis of the protein, whereas incubation with cys II proteinases resulted in substantial hydrolysis of the globulin.

A chymotrypsin-like proteinase was isolated from the posterior midgut of larvae of the yellow mealworm, Tenebrio molitor, by ion-exchange and gel filtration chromatography. The enzyme, TmC1, was purified to homogeneity as determined by SDS-PAGE and postelectrophoretic activity detection. TmC1 had a molecular mass of 23.0 kDa, pI of 8.4, a pH optimum of 9.5, and the optimal temperature for activity was 51 degrees C. The proteinase displayed high stability at temperatures below 43 degrees C and in the pH range 6.5-11.2, which is inclusive of the pH of the posterior and middle midgut. The enzyme hydrolyzed long chymotrypsin peptide substrates SucAAPFpNA, SucAAPLpNA and GlpAALpNA and did not hydrolyze short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SucAAPFpNA, with k(cat app) 36.5 s(-1) and K(m) 1.59 mM. However, the enzyme had a lower K(m) for SucAAPLpNA, 0.5 mM. Phenylmethylsulfonyl fluoride (PMSF) was an effective inhibitor of TmC1, and the proteinase was not inhibited by either tosyl-l-phenylalanine chloromethyl ketone (TPCK) or N(alpha)-tosyl-l-lysine chloromethyl ketone (TLCK). However, the activity of TmC1 was reduced with sulfhydryl reagents. Several plant and insect proteinaceous proteinase inhibitors were active against the purified enzyme, the most effective being Kunitz soybean trypsin inhibitor (STI). The N-terminal sequence of the enzyme was IISGSAASKGQFPWQ, which was up to 67% similar to other insect chymotrypsin-like proteinases and 47% similar to mammalian chymotrypsin A. The amino acid composition of TmC1 differed significantly from previously isolated T. molitor enzymes.

The review deals with analysis of the possibility of the use of genes of inhibitors of proteolytic enzymes of plants to increase plant tolerance to insect pests and phytopathogens. The idea of using protease inhibitors for plant defense is strongly supported, first, by their wide distribution in plant tissues and high activity towards various proteolytic enzymes of insects, bacteria and fungi. The results obtained for the last years indicate that the genetic engineering approach is perspective for solving of this kind of problems. The main losses and advantages of the discussed approach are also considered. The described approach for increase of plant tolerance to insects and pathogens has few advantages as compared to traditional ones and belongs to ecologically pure technologies.

A new trypsin-like proteinase was purified to homogeneity from the posterior midgut of Tenebrio molitor larvae by ion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Superdex-75. The isolated enzyme had molecular mass of 25.5 kD and pI 7.4. The enzyme was also characterized by temperature optimum at 55 degrees C, pH optimum at 8.5, and K(m) value of 0.04 mM (for hydrolysis of Bz-Arg-pNA). According to inhibitor analysis the enzyme is a trypsin-like serine proteinase stable within the pH range of 5.0-9.5. The enzyme hydrolyzes peptide bonds formed by Arg or Lys residues in the P1 position with a preference for relatively long peptide substrates. The N-terminal amino acid sequence, IVGGSSISISSVPXQIXLQY, shares 50-72% identity with other insect trypsin-like proteinases, and 44-50% identity to mammalian trypsins. The isolated enzyme is sensitive to inhibition by plant proteinase inhibitors and it can serve as a suitable target for control of digestion in this stored product pest.

Proteinase inhibitors were studied in the midgut of Nauphoeta cinerea Oliv. (Blattoptera: Blaberidae) in experimental conditions, excluding their nutritional origin. One trypsin inhibitor (TI) with M(r) 8,000 and two subtilisin inhibitors (SI1 and SI2) with M(r) 13,000 and 8,000 were detected after fractionation of total protein preparation on Sephadex G-50. Ninety-four percent of both types of inhibitors was located in anterior midgut (AM). TI was 120-fold purified by FPLC-chromatography on Mono Q. Its isoelectric point was 4.3. TI lost a large part of activity in acidic and especially in alkaline medium. TI, SI1, and SI2 effectively inhibited activities of endogenous proteinases from posterior midgut (PM) of the cockroach. A search for inhibitor of endogenous unusual SH-dependent proteinase from AM revealed in AM a new inhibitor with M(r) 18,000. It was also inactivated in alkaline medium and was effective against proteinases from PM along with unusual SH-dependent proteinase from AM. A mechanism of regulation of activity of midgut proteinases is proposed based on pH-stability of inhibitors., (Copyright 2001 Wiley-Liss, Inc.)

Compartmentalization of proteinases, amylases, and pH in the midgut of Nauphoeta cinerea Oliv. (Blattoptera:Blaberidae) was studied in order to understand the organization of protein and starch digestion. Total proteolytic activity measured with azocasein was maximal at pH 11.5 both in anterior (AM) and posterior (PM) halves of the midgut, but the bulk of activity (67%) was found in PM. Total AM and PM preparations were fractionated on a Sephadex G-50 column and further analysed by means of activity electrophoresis and specific inhibitors and activators. The major activity in PM was classified as an unusual SH-dependent proteinase with M(r) 24,000 and pH optimum with synthetic substrate BApNA at 10.0. The enzyme was 43-fold activated in the presence of 1 mM DTT, insensitive to synthetic inhibitors of serine (PMSF, TLCK, TPCK) and cysteine (IAA, E-64) proteinases, strongly inhibited by STI, and displayed four active bands on zymograms. In PM, activities of trypsin-like, chymotrypsin-like, subtilisin-like, and cysteine proteinases were observed. Aspartic and metalloproteinases were not detected. In AM, activity of unusual SH-dependent proteinase also dominated and activity of chymotrypsin-like proteinase was observed, but their levels were much lower than in PM. Distribution of amylase activity, exhibiting an optimum at pH 6.0, was quite the opposite. The major part of it (67%) was located in AM. Treatment of amylase preparation with proteinases from AM and PM reduced amylase activity twofold. pH of the midgut contents was 6.0-7.2 in AM, 6.4-7.6 in the first and 8.8-9.3 in the second halves of PM. Thus, pH in AM is in good agreement with the optimal pH of amylase, located in this compartment, but the activity of proteinases, including the ability to degrade amylase, in such an environment is low. Active proteolysis takes place in the second half of PM, where pH of the gut is close to the optimal pH of proteinases., (Copyright 2001 Wiley-Liss, Inc.)

Cotyledons of dry buckwheat (Fagopyrum esculentum Moench) seeds were used to study the cellular localization of a metalloproteinase which performs in vitro the initial limited proteolysis of the main storage protein of the seed, and of its proteinaceous inhibitor. Fractions of complex protein bodies (PB 1) and of the cytoplasm and membrane material (CMM) were obtained by fractionating cotyledons in a mixture of acetone and CCl4. The greater part of the metalloproteinase activity was found to be localized in the PB 1 fraction, with a lesser amount in the CMM fraction, whereas the metalloproteinase inhibitor was localized almost entirely in the PB 1 fraction. The data obtained indicate that the complex protein bodies of dry buckwheat seeds contain the components of the proteolytic system responsible for the initial degradation of the main storage protein - the 13S globulin - of buckwheat seeds, i.e. 13S globulin, the metalloproteinase, and its inhibitor. This confirms that it is possibile for the metalloproteinase to perform a controlled proteolysis of the 13S globulin in vivo. The effect of divalent cations on the degradation of the 13S globulin was also studied. A mechanism is discussed whereby the proteolysis of 13S globulin is initiated by divalent cations released as a result of phytin decationization during seedling growth.

A new procedure for isolation of purified nuclear fraction from the cells of the phytoflagellatum Astasia longa has been developed. The resulting preparations were characterized in terms of their chemical composition. A method of isolation to total histone preparations from the nuclear fractions of A. longa based on the metabolic peculiarities of the phytoflagellatum has been developed. Pure histone fractions were obtained by intensive disruption and thorough washing of the nuclear fraction from the non-histone proteins with weak solutions of salts. The intensive proteolysis of the histones in the course of washing was stopped by an addition of the specific inhibitors of proteinases--phenylmethylsulfanylfluoride and pCMB (pH 7,8--8,0). The use of NaHSO3 or boiling of the nuclear fraction to suppress the histone proteolysis were found to be ineffective. Extraction of the histones by 1 M CaCl2 appeared more productive than the use of mineral acids solutions. The total histone preparation isolated under optimal conditions was found in the chromatin in amounts equivalent to those of DNA; its amino acid composition was typical for histones. During polyacrylamide gel electrophoresis in an acidic system in the presence of urea the histones produced four main bands, whereas in the presence of DS-Na--five bands. In terms of the amino acid composition and electrophoretic properties in different systems the histones of A. longa are similar but not identical to calf thymus histones.

A preparation of total histones has been isolated for the first time from the purified fractions of T. lewisi cell nuclei and characterized in terms of its chemical composition and RNA-polymerase activity. A special attention during the isolation procedure was given to the repression of proteolytic degradation of the histones. The amount of protein in the chromatin is equivalent to that of DNA. The amino acid composition and heterogeneity of the protein during polyacrylamide gel electrophoresis in an acid system and in the presence of sodium dodecyl sulfate are typical for histones. Using two-dimensional electrophoresis, differential staining of electrophoregrams and ion-exchange chromatography on CM-cellulose the total preparation has been found to be made up of five fractions: two -- arginine-rich (one of them identical to histone H4, the other being similar to histone H3 from calf thymus); two -- moderately lysine-rich fractions, slightly differing in their properties from histones H2A and H2B from calf thymus, and one specific fraction with mol. weight of 16 000 and an extremely high positive charge. The above methods in combination with specific extraction have been used to demonstrate the absence of a typical lysine histone in the preparation, which is correlated with the absence of typical methaphase chromosomes during mitosis in T. lewisi.