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Proteinase inhibitors are important for the regulation of the activity of enzymes essential for the survival and maintenance of all organisms, and they may hold medicinal and agricultural value. Hyacinthus orientalis L. serine protease inhibitors (HOSPIs), belonging to the Bowman-Birk type inhibitor (BBI) family, have strong inhibitory activities against mammalian serine proteinases. This study explored the relationship between gene structure and multiple isoinhibitor production of these diversified BBIs by analyzing sequences of HOSPI precursor genes.Genomic DNA of H. orientalis roots was obtained and fragmented using 13 specific restriction enzymes, which were amplified by inverse and nested polymerase chain reactions, cloned into the pBluescript II SK (+) vector, and directly sequenced using specific primers. HOSPI gene and protein expression were assessed by quantitative real-time PCR and western blot, respectively. Proteinase inhibitory activity of hyacinth bulb extracts was evaluated by fluorescein isothiocyanate-labeled casein.Four distinct HOSPI precursor genes were identified, encoding 2-4 different HOSPI domains that were surrounded by additional sequences (named head, linker, and tail sequences) and some introns. Moreover, 3' splicing of the linker sequence may occur through introns inserted between linker sequences. HOSPI gene and protein expression was higher during the stem elongation and the flowering periods.These results indicate that gene duplication of the HOSPI precursor as a single set, including tandem repeated HOSPI domains, leads to diversity and effective production of mature HOSPIs by posttranslational processing.These findings shed light on the diversity of proteinase inhibitors.

Bowman–Birk inhibitors (BBIs) are plant-derived serine proteinase inhibitors. Endogenously, they function as defense molecules against pathogens and insects, but they also have been explored for applications in cancer treatment and inflammatory disorders. Here, we isolated 15 novel BBIs from the bulb of Hyacinthus orientalis (termed HOSPIs). These isoinhibitors consisted of two or three chains, respectively, that are linked by disulfides bonds based on proposed cleavage sites in the canonical BBI reactive site loop. They strongly inhibited trypsin (Ki = 0.22–167 nM) and α-chymotrypsin (Ki = 19–1200 nM). Notably, HOSPI-B4 contains a six-residue reactive loop, which appears to be the smallest such motif discovered in BBIs to date. HOSPI-A6 and -A7 contain an unusual reactive site, i.e. Leu–Met at the P1–P1′ position and have strong inhibitory activity against trypsin, α-chymotrypsin, and elastase. Analysis of the cDNA encoding HOSPIs revealed that the precursors have HOSPI-like domains repeated at least twice with a defined linker sequence connecting individual domains. Lastly, mutational analysis of HOSPIs suggested that the linker sequence does not affect the inhibitory activity, and a Thr residue at the P2 site and a Pro at the P3′ site are crucial for elastase inhibition. Using mammalian proteases as representative model system, we gain novel insight into the sequence diversity and proteolytic activity of plant BBI. These results may aid the rational design of BBI peptides with potent and distinct inhibitory activity against human, pathogen, or insect serine proteinases.

Habu serum factor (HSF) and HSF-like protein (HLP) are fetuin family proteins isolated from Protobothrops flavoviridis (habu snake) serum with different physiological activities. A comparison of their cDNAs and intronic sequences revealed that nucleotide substitutions were primarily in protein-coding regions, and the substitution patterns indicated accelerated evolution of these proteins. Genomic DNA fragment analysis, including intron 1, revealed a 6.6-kb insertion homologous to the full-length mammalian LINE1 (L1) retrotransposable element (PfL1) only in the HLP gene. This segment retains an open reading frame (ORF) that encodes a reverse transcriptase (RT)-like protein (PfRT). We further found that a large number of homologous segments have dispersed in the habu snake genome, although we could not determine the enzymatic activities of their products. Moreover, an analysis of habu snake liver RNA indicated active transcription of the PfRT genes, suggesting that high levels of RT activity in this snake have driven the evolution of unique phenotypes of venom enzymes and serum inhibitors of them.

Viperidae snakes containing various venomous proteins also have several anti-toxic proteins in their sera. However, the physiological function of serum protein has been elucidated incompletely. Small serum protein (SSP)-1 is a major component of the SSPs isolated from the serum of a Japanese viper, the habu snake (Trimeresurus flavoviridis). It exists in the blood as a binary complex with habu serum factor (HSF), a snake venom metalloproteinase inhibitor. Affinity chromatography of the venom on an SSP-1-immobilized column identified HV1, an apoptosis-inducing metalloproteinase, as the target protein of SSP-1. Biacore measurements revealed that SSP-1 was bound to HV1 with a dissociation constant of 8.2 × 10−8 M. However, SSP-1 did not inhibit the peptidase activity of HV1. Although HSF alone showed no inhibitory activity or binding affinity to HV1, the SSP-1–HSF binary complex bound to HV1 formed a ternary complex that non-competitively inhibited the peptidase activity of HV1 with a inhibition constant of 5.1 ± 1.3 × 10−9 M. The SSP-1–HSF complex also effectively suppressed the apoptosis of vascular endothelial cells and caspase 3 activation induced by HV1. Thus, SSP-1 is a unique protein that non-covalently attaches to HV1 and changes its susceptibility to HSF.

Novel proteins were isolated from the sera of Chinese Mamushi (Gloydius blomhoffi brevicaudus) and Habu (Trimeresurus flavoviridis). The primary structures of these proteins were determined by protein sequencing, and the nucleotide sequences were established by cDNA cloning from the liver mRNAs. They belonged to the fetuin family having a double-headed cystatin-like domain and a His-rich domain, akin to HSF, an antihemorrhagic factor isolated from Habu serum. They showed no antihemorrhagic activity and were designated HSF-like proteins (HLPs). Mamushi serum contained two different HLPs termed HLP-A and HLP-B. Both HLP-A and Habu HLP had a unique 17-residue deletion in their His-rich domains. HLP-B comprised two glycosylated polypeptide chains and inhibited the precipitation of calcium phosphate as potently as does bovine fetuin. HLP-B was hence identified as a snake fetuin. The phylogenetic analysis of the fetuin family of proteins showed that antihemorrhagins and HLPs have evolved from this snake fetuin.

A series of analogs of serum thymic factor (Facteur Thymique Sérique; FTS) devoid of C- and N-terminal few residues were synthesized and their ability to induce Thy-l (theta) antigen in vitro on mouse T lymphocyte precursors was examined. The pentapeptide moiety (Lys-Ser-Gln-Gly-Gly) seems to be important for the expression of agonistic activity. Some of the short chain peptides showed antagonistic properties. Synergistic activation by two inactive analogs was observed.

Five small serum proteins (SSPs) with molecular masses of 6.5-10 kDa were detected in Habu (Trimeresurus flavoviridis) serum; this included two novel proteins SSP-4 and SSP-5. The amino acid sequences of these proteins and of SSP-1, SSP-2, and SSP-3, which were reported previously, were determined on the basis of the nucleotide sequences of their cDNAs. Although these proteins exhibited only limited sequence identity to mammalian prostatic secretory protein of 94 amino acids (PSP94), the topological pattern of disulfide bonds in SSPs was identical to that of the mammalian proteins. SSP-3 and SSP-4 lacked approximately 30 residues at the C-terminal. Each of the full-length cDNAs encoded a mature protein of 62-90 residues and a highly conserved signal peptide. The evolutionary distances between SSPs estimated on the basis of the amino acid changes were significantly greater than those of the synonymous nucleotide substitutions; these finding, together with results from analyses of nonsynonymous to synonymous rates of change (dN/dS) suggest that snake SSPs have endured substantial accelerated adaptive protein evolution. Such accelerated positive selection in SSPs parallels other findings of similar molecular evolution in snake venom proteins and suggests that diversifying selection on both systems may be linked, and that snake SSP genes may have evolved by gene duplication and rapid diversification to facilitate the acquisition of various functions to block venom activity within venomous snakes.

An antihemorrhagic protein has been isolated from the serum of Chinese mamushi (Gloydius blomhoffi brevicaudus) by using a combination of ethanol precipitation and a reverse-phase high-performance liquid chromatography (HPLC) on a C8 column. This protein-designated Chinese mamushi serum factor (cMSF)-suppressed mamushi venom-induced hemorrhage in a dose-dependent manner. It had no effect on trypsin, chymotrypsin, thermolysin, and papain but inhibited the proteinase activities of several snake venom metalloproteinases (SVMPs) including hemorrhagic enzymes isolated from the venoms of mamushi and habu (Trimeresurus flavoviridis). A similar protein (Japanese MSF, jMSF) with antihemorrhagic activity has also been purified from the sera of Japanese mamushi (G. blomhoffi). The N-terminal 70 and 51 residues of the intact cMSF and jMSF were directly analyzed; a similarity between the sequences of two MSFs to that of antihemorrhagic protein (HSF) from habu serum was noticed. To obtain the complete amino acid sequences of MSFs, cDNAs encoding these proteins were cloned from the liver mRNA of Chinese and Japanese vipers based on their N-terminal amino acid sequences. The mature forms of both MSFs consisted of 305 amino acids with a 19-residue signal sequence, and a unique 17-residue deletion was detected in their His-rich domains.

Certain snakes have antihemorrhagic proteins in their sera. Habu serum factor (HSF), an antihemorrhagic protein isolated from the serum of the Japanese habu snake (Trimeresurus flavoviridis) is composed of two cystatin-like domains (D1 and D2) and a His-rich domain, and it inhibits several snake venom metalloproteinases (SVMPs). The activity of HSF can be abolished by trinitrophenylation of Lys residues with 2,4,6-trinitrobenzene sulphonic acid. Upon complex formation of HSF with SVMP, however, the loss of its inhibitory activity by the chemical modification was suppressed, and Lys(15), Lys(41), and Lys(103) residues in HSF were not trinitrophenylated. In order to identify the domain that is critical to the inhibitory activity on SVMPs, native HSF was digested with papain followed by cleavage with cyanogen bromide, yielding a low-molecular mass fragment that was composed of two peptide chains (residues 5-89 and 312-317) linked by a disulfide bond. This fragment inhibited several SVMPs and showed significant antihemorrhagic activity. This indicates that the N-terminal half of D1 is indispensable for the antihemorrhagic activity of HSF. Furthermore, a three-dimensional model of two cystatin-like domains constructed by the homology modeling has indicated that three Lys residues (15, 41, and 103) are exposed to the same surface of HSF molecule.

The cytoskeleton plays an important role in the stability and function of the membrane. Spectrin release from erythrocyte ghosts makes the membrane more fragile. However, the detail of membrane fragility has remained unclear. In the present study, the effects of incubation temperatures and polyamines on the membrane structure of ghosts under hypotonic conditions have been examined. Upon exposure of ghosts to a hypotonic buffer at 0-37 degrees C, reduction of ghost volume, spectrin release and decrease of band 3-cytoskeleton interactions were clearly observed above 30 degrees C. However, such changes were completely inhibited by spermine and spermidine. Interestingly, conformational changes of spectrin induced at 37 degrees C or 49 degrees C were not suppressed by both polyamines. Flow cytometry of fluorescein isothiocyanate-labelled ghosts exposed to 37 degrees C demonstrated the two peaks corresponding to ghosts with normal spectrin content and decreased one. Taken together, these results indicate that the degree of spectrin release from the membrane under hypotonic conditions is not same in all ghosts, and that polyamines inhibit the spectrin release followed by changes in the membrane structure, but not conformational changes of spectrin.

Upon exposure of human erythrocytes to a high pressure of 200 MPa, both hemolysis and vesiculation occur. The hemolysis of erythrocytes at 200 MPa was enhanced by removal of sialic acids from the membrane surface with papain. However, such enhancement was suppressed by cross-linking of band 3 via an anti-band 3 antibody (AB3A), which recognizes the exofacial domain of band 3, or by clustering of band 3 via Zn 2 + . On the other hand, the size of high-pressure-induced vesicles increased from 423 to 525 nm in diameter upon exposure to papain of erythrocytes, but decreased to 444 nm with following treatment with AB3A. In these vesicles, the content of spectrin relative to band 3 was almost the same. Furthermore, the band 3-cytoskeleton interactions in erythrocyte membranes remained unaltered upon treatment with papain and AB3A. Flow cytometric analysis demonstrated that papain-pretreated erythrocytes mainly produce open ghosts at 200 MPa and that the production of such open ghosts is suppressed by AB3A. Thus, upon removal of negative charges from the membrane surface, open ghosts are readily produced due to the release of larger vesicles under pressure. Upon cross-linking of band 3 via AB3A, however, the release of smaller vesicles at 200 MPa is facilitated so that high-pressure-induced hemolysis is suppressed.

Brevilysin L4 (L4) is a non-hemorrhagic P-I class metalloprotease (MP) isolated from Gloydius halys brevicaudus venom. Its complete amino acid sequence has been determined. L4 is a single-chain polypeptide and highly homologous to those of other snake venom MPs. A zinc-binding motif, HExxHxxGxxH, is located at residues 142-152. A characteristic feature of L4 is the presence of a spacer sequence (LRTDTVS) at the C-terminal that links metalloprotease and disintegrin domains and is usually removed by post-translational proteolysis, suggesting that L4 is expressed together with a spacer region and a disintegrin domain at the C-terminal. The nucleotide sequence of a cDNA clone encoding L4 has revealed that L4 is a disintegrin precursor and produced as a P-II class MP. The disintegrin coded after L4 sequence was brevicaudin 1, a disintegrin previously isolated from the same venom. P-II class MPs have been suspected to undergo autoproteolysis to release disintegrins. Although being P-I class MP, L4 itself autocatalytically degrades with a half-life of 30min at pH 8.5 and 37 degrees C in the absence of Ca(2+). Sequence analysis of several fragment peptides produced during the autolysis of L4 indicated that more than 40 peptide bonds were split, and the cleavages of Ser(60)-Asn(61), Thr(99)-Ala(100), and Phe(103)-Asp(104) bonds may trigger the autoproteolysis. Addition of Ca(2+) completely suppressed the cleavage of these particular bonds, resulting in a marked prevention of autoproteolysis. Thus, L4 provides a good model for the investigation of autolysis of some MPs.

Some venomous snakes possess anti-toxic proteins in their sera that may play a role in neutralizing the haemorrhagic factors or toxins in their own venom. Five small serum proteins (SSP-1-SSP-5) were isolated from the serum of Japanese viper (Protobothrops flavoviridis), and were found to act as self-defence proteins against the viper's own toxic components. However, the physiological function of SSP-3 has not been completely elucidated. Affinity chromatography of the venom on an SSP-3-immobilized column identified a novel 55-kDa protein as the target molecule of SSP-3. Sequences of internal fragments of this SSP-3-binding protein showed high homology to those of metalloproteinases from the P. flavoviridis venom. The cDNA sequence revealed that this protein, termed flavorase, is a P-III class metalloproteinase consisting of 423 amino acid residues. The purified protein did not show haemorrhagic and cytotoxic activity. Biacore measurements revealed that SSP-3 was bound to flavorase with a dissociation constant of 6.4 × 10(-9) M. SSP-3 non-competitively inhibited the peptidase activity of flavorase with an inhibition constant of 6.6 × 10(-9) M.

Some snakes have several anti-toxic proteins in their sera that neutralize their own venom. Five new small serum proteins (SSPs) were isolated from Japanese mamushi (Gloydius blomhoffii) serum by gel-filtration and RP-HPLC, and their N-Terminal sequences were determined. The amino acid sequences of the precursor proteins were deduced from the nucleotide sequences of cDNAs encoding them. Due to the sequence similarity to those of SSPs in habu snake (Protobothrops flavoviridis) serum (>75% identity), these proteins were designated mSSP-1 to mSSP-5 as the homologs of habu proteins. mSSP-1 was stable at 100 °C and in the pH range of 1–10, and inhibited the proteolytic activity of a certain snake venom metalloproteinase. The inhibitory activity was extinguished by modifying the amino groups of mSSP-1. mSSP-1 is the first prostate secretory protein of the 94 amino acid-family protein with a carbohydrate chain in the Asn37 residue.

When human erythrocytes were preincubated at 37-52 degrees C under atmospheric pressure before exposure to a pressure of 200 MPa at 37 degrees C, the value of hemolysis was constant (about 43%) up to 45 degrees C but became minimal at 49 degrees C. The results from anti-spectrin antibody-entrapped red ghosts, spectrin-free vesicles, and N-(1-pyrenyl)iodoacetamide-labeled ghosts suggest that the denaturation of spectrin is associated with such behavior of hemolysis at 49 degrees C. The vesicles released at 200 MPa by 49 degrees C-preincubated erythrocytes were smaller than those released by the treatment at 49 degrees C or 200 MPa alone. The size of vesicles released at 200 MPa was independent of preincubation temperature up to 45 degrees C, and the vesicles released from 49 degrees C-preincubated erythrocytes became smaller with increasing pressure up to 200 MPa. Thus, hemolysis and vesiculation under high pressure are greatly affected by the conformation of spectrin before compression. Since spectrin remains intact up to 45 degrees C, the compression of erythrocytes at 200 MPa induces structural changes of spectrin followed by the release of large vesicles and hemolysis. On the other hand, in erythrocytes that are undergoing vesiculation due to spectrin denaturation at 49 degrees C, compression produces smaller vesicles, so that the hemolysis is suppressed.

Murine erythroleukemia cells were subjected to physicochemical stresses such as high pressure (60--110 MPa), heating (42--45 degrees C), and ultraviolet (UV) irradiation (5--25 kJ/cm(2)). After exposure to these stresses, the cells were cultured at 37 degrees C and atmospheric pressure. The number of the cells treated at 100 MPa, 45 degrees C, or 20 kJ/cm(2) remained constant or decreased at an early stage of culture. The nuclear morphology, agarose gel electrophoresis, and flow cytometry of these cells showed that they undergo apoptosis. The activity of caspase-3 was observed in cells subjected to each stress. In particular, caspase-3 was most readily activated by high pressure under our conditions. The caspase-3 activity and apoptosis exhibited a similar pressure dependence. It is important that both caspase-3 activation and apoptosis induced by high pressure were significantly suppressed by a caspase-3 inhibitor. These results suggest that high-pressure-induced apoptosis is also characterized by the activation of caspase-3, as seen with heat- and UV-induced apoptosis.

The complete amino acid sequence of brevilysin H6 (H6), a zinc-protease isolated from Gloydius halys brevicaudus venom, was determined by a manual Edman degradation method. H6 has an amino-terminal pyroglutamic acid and consists of a total of 419 residues. An N-linked sugar chain is attached at Asn-181. The molecule is composed of three domains (metalloprotease, disintegrin-like and cysteine-rich domains), as commonly found in other high molecular mass metalloproteases from snake venoms. In the absence of calcium ions, H6 is autocatalytically degraded with a half-life of 47 min to give 29 and 45 kDa fragments, which correspond to residues 208-419 and 99-419 of H6, respectively. Thus, the autoproteolysis seemed to start from the cleavage of either the Leu(98)-Leu(99) or Asp(207)-Ile(208) bond. Calcium ions suppressed both the formation of the 45 kDa fragment and the rate of autoproteolysis. Calcium ions also contributed to the stability of H6 against pH, heating, urea and cysteine. More than twenty-five peptide bonds adjacent to hydrophobic residues in the metalloprotease domain were progressively cleaved during the autoproteolysis.

A peptidase was purified from seeds of Canavalia ensiformis by extraction with water, ammonium sulfate precipitation, and successive chromatographies on DEAE-Toyopearl 650M, butyl-Toyopearl 650M, and G-3000 SW columns. The enzyme has an apparent molecular weight of 41,000. Activity is maximal at pH 9 and 60 degrees C. The enzyme hydrolyzed synthetic substrates at Arg-X and Lys-X bonds more rapidly than bovine trypsin did, and did not cleave protein or ester substrates. The enzyme was inhibited by alkylamines and several serine protease inhibitors such as diisopropylfluorophosphate, chymostatin, leupeptin, and benzamidine. Cysteine protease-, metalloprotease-, and proteinous trypsin inhibitors were ineffective. Inhibition by alkylamines was dependent on length of the alkyl chains. From the substrate specificity and susceptibility to chemicals, the enzyme is a unique peptidase with trypsin-like specificity.

The structure of spectrin in aqueous solutions or erythrocyte ghosts was characterized by using a fluorophore, N-(1-pyrenyl)iodoacetamide (NPIA). The excimer fluorescence of NPIA was clearly observed in isolated spectrin, but not in other proteins such as actin, bovine serum albumin, and lysozyme. The excimer disappeared upon addition of sodium dodecyl sulfate, but reappeared by dialysis. Further, the excimer appeared in spectrin extracts from ghosts labeled at 37 °C with NPIA. Interestingly, no such excimer in spectrin extracts appeared upon pretreatment of ghosts with diamide, and then N-ethylmaleimide. However, when such double-treated ghosts were exposed to dithiothreitol before NPIA labeling, the excimer fluorescence reappeared. Further, excimer fluorescence was observed in ghosts which were pretreated above 30 °C, especially at 50 °C, and then labeled at 0 °C with NPIA. The data of nondenaturing polyacrylamide gel electrophoresis and CD spectra showed marked changes in the structure of spectrin at 5...

A non-hemorrhagic proteinase, brevilysin L6 (L6), has been purified to homogeneity from Agkistrodon halys brevicaudus venom by gel filtration and DEAE-Toyopearl 650M chromatography. It is an acidic protein with an isoelectric point of 4.8, and its molecular mass was estimated to be 21.5 kDa by SDS-PAGE. The optimum pH of L6 was about 9. EDTA and o-phenanthroline inhibited the proteolytic activity, suggesting that L6 is a metalloprotease. Cysteine also inhibited the activity of L6, but glutathione did not. The protein was stable in the pH range of 5-8.5 and below 40 degreesC. Calcium ions had no effect on the proteolytic activity of L6 or on its thermal stability. The enzyme preferentially cleaved X-Leu, X-Phe, X-Val, and X-His bonds. L6 showed weak alpha-fibrinogenase activity. The complete amino acid sequence of L6 was also determined by manual Edman degradation. L6 is a non-glycosylated single-chain polypeptide consisting of 203 residues with an N-terminal pyroglutamic acid and a calculated molecular weight of 22,713 Da. Its entire sequence is highly homologous to those of other metalloproteases from various snake venoms. A zinc-binding motif, HEXXHXXGXXH, is located at residues 143-153 in the sequence of L6.

The effect of high pressure on proliferation and cell cycle progression was examined using murine erythroleukemia (MEL) cells. The MEL cells were exposed to high pressures (0.1-130 MPa) and then cultured for 5 days at atmospheric pressure. The proliferation of MEL cells was unaffected up to 60 MPa, but was suppressed at 80-110 MPa. Above 120 MPa, the cells were fragmented. The cell cycle analysis of 80 MPa-treated MEL cells showed that the cells in S phase are most sensitive to high pressure and they arrest in G2 phase. Interestingly, G2-arrested cells reinitiated DNA synthesis, resulting in giant cells with high DNA contents. Furthermore, when such G2-arrested cells were exposed to caffeine, premature mitosis, characterized by chromosome pulverization, was observed. These results suggest that the suppression of proliferation in high-pressure-treated MEL cells is associated with G2 arrest following S phase delay. Thus, it seems valuable to apply high pressure to the investigation of the cell cycle.

In the membrane stability of human erythrocytes, the role of two cysteine residues (Cys-201 and Cys-317) in the cytoplasmic domain of band 3 is not clear. So we tried to resolve this problem by examining hemolytic properties under high pressure. From SH contents and spin labeling, it was found that Cys-201 and Cys-317 of band 3 were modified with N-ethylmaleimide (NEM). The hemolysis of intact erythrocytes at 200 MPa was suppressed by the binding of 4, 4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), anion transport inhibitor, to band 3. Similarly, the suppressive effect of DIDS was observed in the erythrocyte that Cys-201 and Cys-317 were modified with NEM. These results suggest that the cysteine residues in the cytoplasmic domain of band 3 are not essential for the DIDS-induced membrane stabilization.

Hemolytic properties of human erythrocytes in hereditary spherocytosis (HS) were examined under hydrostatic pressure or hypotonic conditions. In the hypotonic buffer, HS erythrocytes were more fragile than normal erythrocytes, and the osmotic fragility was similarly enhanced if both erythrocytes were treated with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), an anion transport inhibitor. On the other hand, the hemolysis of HS erythrocytes at 200 MPa was almost the same degree as that of normal cells. Upon DIDS treatment, the hemolysis of normal erythrocytes at 200 MPa was suppressed by about 35%, whereas that of HS erythrocytes was not affected. The absence of a suppressive effect of DIDS on high-pressure-induced hemolysis is likely to be HS-specific and may be a reflection of the underlying defects of band 3-cytoskeleton interactions in HS erythrocytes.

The effects of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) on the hemolysis of human erythrocytes were examined. To hemolyze, the erythrocytes were exposed to high pressure (200 MPa), heating (52 °C), or a hypotonic medium. Upon EDC treatment, the hemolysis induced by high pressure or heating was completely suppressed, whereas osmotic hemolysis was enhanced. In EDC-treated erythrocytes, high-molecular-weight aggregates of membrane proteins were formed by cross-linking of the membrane with the cytoskeleton, in addition to that of membrane proteins themselves. In such a cross-linking reaction, however, iron(II) in hemoglobin and thiol groups in membrane proteins remained stable. Although phosphate transport via band 3 was considerably suppressed by EDC, the intracellular K+ concentration was almost constant. These results suggest that high-pressure- or heat-induced hemolysis, compared with osmotic hemolysis, is suppressed by a stabilization of the cytoskeletal network.

Effects of anion transport inhibitors such as 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS) and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonate on hemolysis of human erythrocytes at 200 MPa were examined by changing intracellular pH (7.2–7.9). These inhibitors suppressed the hemolysis at neutral pH but enhanced it at alkaline pH. However, such an enhancement was suppressed by cross-linking of membrane proteins using diamide. From the near-UV CD spectra of band 3 and the relation between hemolysis and anion transport in intact or trypsin-treated erythrocytes, it was found that such hemolytic properties were characterized by the binding of inhibitors to band 3. In addition, spectrin detachment from the erythrocyte membrane by high pressure was considerably suppressed by DIDS treatment at neutral pH, but not by DIDS labeling at alkaline pH. These results suggest that the interaction of the cytoplasmic domain of band 3 with the cytoskeleton, which is induced by the binding of ligands to the exofacial domain of band 3, is dependent on the intracellular pH, i.e., the linking is tightened at neutral pH but relaxed at alkaline pH.

The amino acids of two Bowman-Birk type proteinase inhibitors (CLTI-I and -II) from the seeds of Canavalia lineata were sequenced by a manual Edman degradation using the DABITC/PITC double coupling method after enzymatic digestions with Achromobacter lyticus lysyl endopeptidase, Staphylococcus aureus V8 protease, and chymotrypsin. CLTI-I contains 75 amino acid residues. CLTI-II has an identical sequence to CLTI-I except an extra Asp residue attached at the C-terminus. The inhibitors showed a homology (40-70%) to other Bowman-Birk inhibitors. The reactive-site peptide bonds were estimated to be Lys21-Ser22 and Leu48-Ser49 against trypsin and chymotrypsin, respectively. An inhibitory active fragment containing only the chymotrypsin-reactive site was also described.

A subtilisin inhibitor was purified from the seeds of Canavalia lineata by ammonium sulfate precipitation, ultrafiltration on a YM-30 membrane, column chromatography on DEAE-Toyopearl and SP-Toyopearl, followed by reverse-phase HPLC. The inhibitor (CLSI-I) is a low molecular weight protein (M(r) about 6500) containing no half-cystine residue, and quite stable as to extreme heat and pH treatment. CLSI-I inhibited subtilisin-type serine proteases including S. griseus alkaline protease. The amino acids of CLSI-I were sequenced by manual Edman degradation after enzymatic digestion with Achromobacter lyticus lysyl endopeptidase and Staphylococcus aureus V8 protease. CLSI-I contains 65 amino acid residues and showed a high homology to potato inhibitor I family proteins.

We previously demonstrated that caspase-3, an executioner of apoptosis, is activated in the pressure-induced apoptosis of murine erythroleukemia (MEL) cells (at 100 MPa). Here, we examined the pathway of caspase-3 activation using peptide substrates and caspase inhibitors. Using the substrates of caspases-8 and -9, it was found that both are activated in cells under high pressure. The production of nuclei with sub-G1 DNA content in 100 MPa-treated MEL cells was suppressed by inhibitors of caspases-8 and -9, and pan-caspase. In 100 MPa-treated cells, pan-caspase inhibitor partially prevented the cytochrome c release from the mitochondria and the breakdown of mitochondrial membrane potential. These results suggest that the intrinsic and extrinsic pathways are activated in apoptotic signaling during the high pressure-induced death of MEL cells.

Habu serum factor (HSF) is a metalloproteinase inhibitor that is isolated from the serum of habu snake (Trimeresurus flavoviridis), and it can suppress snake venom-induced hemorrhage. In the present study, the inhibitory property and fundamental structure of HSF were analyzed in detail. HSF inhibited all the hemorrhagic and most of the non-hemorrhagic metalloproteinases tested from the venoms of T. flavoviridis and Gloydius halys brevicaudus. HSF was extremely stable in a broad range of temperature and pH, and the treatments with a temperature of 100 degrees C or pH ranging from 1 to 13 barely affects its reactivity against G. halys brevicaudus H6 protease. Gel filtration chromatography revealed that HSF binds to the H6 protease with a 1:1 molar ratio. A secondary structure profile of HSF that was monitored by circular dichroism spectrum remained unvaried up to 2 M urea. The activity of HSF was stoichiometrically abolished by chemical modification with 2,4,6-trinitrobenzene sulfonic acid and N-bromosuccinimide; this indicates that Lys and Trp residues in its sequence play a role in the inhibitory mechanism. In this study, the amino acid sequence of HSF that was obtained by cDNA cloning was identical to that reported previously, except for five substitutions. We concluded that these discrepancies reflect a difference in the places of capture of the snake specimens.

Previously, we demonstrated that when mouse erythroleukemia cells are exposed to a pressure of 80 MPa, the cell-cycle progression of S-phase cells is retarded. To examine the effects of high pressure on DNA replication, we used a Xenopus cell-free system. From cell-cycle progression of sperm nuclei, it was found that sperm nuclei are stable to a pressure of 80 MPa, whereas egg extracts are susceptible to high pressure. Similarly, biotin-16-dUTP was incorporated into 80 MPa-treated sperm nuclei in pressure-untreated extracts, but not into naive sperm nuclei in 80 MPa-treated extracts. These results indicate that DNA replication in Xenopus cell-free system is suppressed by the susceptibility of the extracts to a pressure of 80 MPa.

Genes for Bowman–Birk type protease inhibitors (BBIs) of wild soja (Glycine soja) and soybean (Glycine max) comprise a multigene family. The organization of the genes for wild soja BBIs (wBBIs) was elucidated by an analysis of their cDNAs and the corresponding genomic sequences, and compared with the counterparts in the soybean. The cDNAs encoding three types of wild soja BBIs (wBBI-A, -C, and -D) were cloned. Two subtypes of cDNAs for wBBI-A, designated wBBI-A1 and -A2, were further identified. Similar subtypes (sBBI-A1 and -A2) were also found in the soybean genome. cDNA sequences for wBBIs were highly homologous to those for the respective soybean homologs. Phylogenetic analysis of these cDNAs demonstrated the evolutional proximity between these two leguminae strains.

Publisher Summary This chapter describes the activity, specificity and structural chemistry of Brevilysin H6. Snake venom metalloproteases belong to the reprolysin subfamily, which includes many zinc-containing metalloproteases. Brevilysin H6 is a main component in Gloydius halys brevicaudus venom. Brevilysin H6 has an optimum pH of 8.5. The protein is stable in the pH range of 6–8 and below 40°C. Calcium ion enhances the proteolytic activity up to about 250%, and increasing the thermal and pH stability. The protease activity is inhibited by several metal chelating agents, 1M urea and some thiol compounds, but not by phosphoramidon, an inhibitor of bacterial metalloproteases. Strong α-fibrinogenase activity is notable. H6 is an endoprotease and cleaves preferentially at the amino side of hydrophobic residues such as -Leu, -Val, -Met and -Phe. H6 is a metalloprotease with molecular weight of about 60,000 and 46,439 from the primary structure, and its isoelectric point is 4.8. It contains one zinc and three calcium ions and is composed of 419 amino acid residues. The N-terminus is a pyroglutamyl residue and an N-linked sugar chain is attached at Asn 181.

High-pressure-induced hemolysis is suppressed by pretreating human erythrocytes at 49 degrees C, or enhanced by pretreatment with trypsin. So, the response of these pretreated cells to a pressure of 200 MPa was examined using flow cytometry. In the case of intact erythrocytes, a major product was fragmented particles. From 49 degrees C-pretreated cells, vesicles were mainly released. Trypsin-pretreated cells mainly produced open ghosts. Additionally, intact erythrocytes, 49 degrees C-pretreated ones, and trypsin-pretreated ones also released at 200 MPa vesicles of diameter 464 +/- 9, 259 +/- 18, and 574 +/- 16 nm, respectively. These results suggest that mother cells, fragmented particles, vesicles, and open ghosts from 200 MPa-treated erythrocytes are easily monitored by flow cytometry and that the size of released vesicles may also be an important factor in high-pressure-induced hemolysis.

Three protease inhibitors (OTI-1-3) have been purified from onion (Allium cepa L.) bulbs. Molecular masses of these inhibitors were found to be 7,370.2, 7,472.2, and 7,642.6 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. Based on amino acid composition and N-terminal sequence, OTI-1 and -2 are the N-terminal truncated proteins of OTI-3. All the inhibitors are stable to heat and extreme pH. OTI-3 inhibited trypsin, chymotrypsin, and plasmin with dissociation constants of 1.3 x 10(-9) M, 2.3 x 10(-7) M, and 3.1 x 10(-7) M, respectively. The complete amino acid sequence of OTI-3 showed a significant homology to Bowman-Birk family inhibitors, and the first reactive site (P1) was found to be Arg17 by limited proteolysis by trypsin. The second reactive site (P1) was estimated to be Leu46, that may inhibit chymotrypsin. OTI-3 lacks an S-S bond near the second reactive site, resulting in a low affinity for the enzyme. The sequence of OTI-3 was also ascertained by the nucleotide sequence of a cDNA clone encoding a 101-residue precursor of the onion inhibitor.

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.

It is reported that when mouse cells are exposed to a pressure of 80 MPa, the cells in S-phase are sensitive to the pressure. To analyze the sensitivity of S-phase cells to pressure, we used a Xenopus egg cell-free system. The DNA replication was monitored by the incorporation of biotin-16-dUTP into sperm nuclei. The progress of DNA replication was completely inhibited by exposure to a pressure (80MPa) of a mixture of sperm nuclei and egg extracts or egg extracts only. On the other hand, 80 MPa-treated sperm nuclei incorporated biotin-16-dUTP, and entered M-phase. These results suggest that sperm nuclei are insensitive to a pressure of 80 MPa and some components in egg extracts are sensitive to the pressure.

Upon exposure of human erythrocytes to high pressures (0.1-200 MPa), hemolysis and vesiculation start to occur at about 140 MPa. At 200 MPa, the value of hemolysis is about 45%, and the diameter of released vesicles is about 460 nm. Furthermore, when transmembrane proteins are cross-linked with cytoskeletal ones using diamide, high-pressureinduced hemolysis is greatly suppressed. In this case, the diameter of vesicles is about 230 nm. These results suggest that high-pressure-induced hemolysis may be associated with the size of released vesicles.

Murine erythroleukemia (MEL) cells were exposed to a high pressure of 80 MPa or aphidicolin (APH), DNA polymerase inhibitor. The effects of caffeine on cell cycle were examined using these cells. During the culture of 80 MPa-treated MEL cells at atmospheric pressure, the cells arrested in the G2 phase, and cyclin B and hyperphosphorylated p34(cdc2) were accumulated. Namely, maturation promoting factor (MPF) composed of p34(cdc2) and cyclin B was inactive. However, upon exposure to caffeine, these cells entered prematurely into mitosis by activating MPF. Caffeine-induced premature mitosis was suppressed by butyrolactone I and orthovanadate. On the other hand, APH-treated MEL cells, which were not exposed to 80 MPa, were not so sensitive to caffeine-induced premature mitosis despite cyclin B accumulation. In this case, dephosphorylation of p34(cdc2) was not induced by caffeine. Interestingly, caffeine-induced premature mitosis in the 80 MPa-treated cells was also suppressed by APH. These results suggest that the premature mitosis of 80 MPa-treated MEL cells by caffeine is induced by active MPF, and that APH-sensitive molecules such as DNA polymerase may also play an important role in the checkpoint that controls the transition from G2 to M phase.

The effects of high pressure (0.1-80 MPa) on cell adhesion were examined using Chinese hamster ovary cells. The cells detached by EDTA or trypsin were cultured for 4 h at atmospheric pressure. In each case, the rate of the adhesive cells was about 85%. Upon the addition of cycloheximide, such cell adhesion was almost completely inhibited in trypsin-treated cells but less affected in EDTA-treated cells. When the EDTA-detached cells were subjected to high pressures and cultured at atmospheric pressure, the cell adhesion was unaffected up to 40 MPa, but was suppressed significantly at higher pressures although the integrins were expressed on the plasma membranes. The suppressive effects induced by high pressure (80 MPa) were marked in trypsin-treated cells, in which the integrins were digested. Furthermore, actin stress fibers in spread cells disappeared due to cell rounding upon exposure to 80 MPa, and such rounded cells were partially detached from the substratum. These results suggest that the retardation of adhesion by high pressure in EDTA-detached cells may be due to the perturbed interactions between integrin and its associated proteins, whereas that of trypsin-detached cells is due to the delayed expression of integrins toward the membrane surface, in addition to the cause seen in the case of EDTA.

Two platelet aggregation inhibitors, ussuristatin 1 (US-1) and 2 (US-2), were newly isolated from the venom of Chinese viper (Agkistrodon ussuriensis) by SP-Toyopearl 650M column chromatography and reverse-phase HPLC. The Mrs of these polypeptides were estimated to be about 8,000 by SDS-PAGE. Analytical gel filtration revealed that US-2 exists as a dimer. Both polypeptides comprised 71 amino acids, whose sequences showed high similarities to those of other disintegrins. US-1 had a typical Arg-Gly-Asp (RGD) sequence, which is responsible for blocking the binding of fibrinogen to the receptor. In US-2, the corresponding sequence was Lys-Gly-Asp (KGD). US-1 strongly suppressed platelet aggregation induced by ADP, collagen, thrombin, and epinephrine with IC50 = 17-33 nM. US-2 also inhibited the platelet aggregation, but the IC50s were about ten times higher. US-1 also dose-dependently inhibited the adhesion of human melanoma cells to fibrinogen and fibronectin, while US-2 did not inhibit the cell adhesion to fibronectin. This indicates that the KGD-bearing disintegrin is a specific inhibitor for the fibrinogen receptor.

Publisher Summary This chapter focuses on the analysis of ascorbic acid, dehydroascorbic acid, and transformation products using ion-pairing high-performance liquid chromatography with multiwavelength ultraviolet and electrochemical detection. Preparative separation of transformation products is carried out by DEAE-Sepharose column chromatography. Elution is carried out with 10 m M sodium phosphate buffer, pH 6.0. The eluates are examined by absorption spectrum and electrochemical detection–high-performance liquid chromatography (ECD–HPLC) analysis. L -ascorbic acid (AsA), erythro- L -ascorbic acid (EAsA), and the reductant R-345, possess an absorption maximum at 345 nm. Analysis by ECD–HPLC reveals three main ECD-active spots—AsA, EAsA, and a reductant possessing an absorption maximum at 290 nm. The latter reductant has a retention time a little less than that of AsA, and it is eluted in the void volume on a DEAE-Sepharose column, showing a less negative charge. Its absorption maximum at 290 nm in neutral aqueous solution shifts to 335 nm above pH 9.0.

A non-hemorrhagic metalloprotease (protease L4) was purified from the venom of Chinese Mamushi ( Agkistrodon halys brevicaudus ) by gel filtration and anion-exchange chromatography. Protease L4 has the molecular weight of 22 000 and its optimum pH was 8.5. The protein was stable in the pH range of 5–9 and below 40°C. The proteolytic activity was inhibited by metal-chelating agents and some metal ions. Calcium ion activated the activity dose-dependently, but had only a minor effect on the thermal and pH stability. L4 showed fibrinogenase activity, hydrolyzing only the A a chain of fibrinogen. The protease cleaved preferentially at the N-terminal of Leu and His residues of some peptides.

Two subtilisin inhibitors (CLSI-II and -III) were purified from seeds of Canavalia lineata by extraction with water, ammonium sulfate precipitation, and chromatographies on DEAE-Toyopearl and hydroxyapatite. The two inhibitors have the same molecular weight of about 22,000, and quite similar amino acid compositions. They contain five half-cystine residues and tend to dimerize through an intermolecular disulfide bridge due to the presence of a single cysteine residue. CLSI-III only inhibited subtilisin-type serine proteases, while CLSI-II showed a wider inhibitory specificity. Though the two inhibitors have almost identical thermal labilities, CLSI-II is more stable as to extreme pH than CLSI-III. They are considered to be Kunitz type inhibitors on the basis of several properties.

The amino acid sequences of two subtilisin inhibitors (CLSI-II and -III) from Canavalia lineata seeds were determined by manual Edman degradation using the DABITC/PITC double coupling method after enzymatic digestions and CNBr degradation. CLSI-II and -III consist of 190 and 183 amino acids, respectively, and have identical amino acid sequences except for in the C-terminal regions: an elongated sequence, Gly-Thr-Ile-Arg- Ser-Asp-Gly, was found at the C-terminus of CLSI-II. A short-chain analog protein without an N-terminal Asn residue was also detected in each inhibitor preparation. The inhibitors showed significant homology to Kunitz type inhibitors, but differed from them with respect to the half-cystine content. Among five half-cystine residues present in CLSI-III, two disulfide bonds link Cys44 to Cys88 and Cys142 to Cys149, Cys106 being present as a free cysteine residue. Phenylglyoxal treatment abolished the inhibitory activity of CLSI-III, indicating the participation of an Arg residue in the interaction with the enzyme. The reactive-site peptide bond was deduced to be Arg68-Gly69.

The amino acid sequences of two lectins from the seeds of Canavalia lineata and C. virosa have been determined by the manual Edman degradation method. Both proteins were found to be highly homologous to concanavalin A, a lectin from C. ensiformis. All the residues suggested to participate in binding to carbohydrates and metal ions are completely conserved in the proteins.