Dubiumin, a chymotrypsin-like serine protease from the seeds of Solanum dubium Fresen.

A serine protease was purified 6.7-fold and with 35% recovery from the seeds Solanum dubium Fresen by a simple purification procedure that combined ammonium sulfate fractionation, cation exchange and gel filtration chromatographies. The enzyme, named dubiumin, has a molecular mass of 66kDa as estimated by gel filtration and SDS-PAGE.

Dehydration of main-chain amides in the final folding step of single-chain monellin revealed by time-resolved infrared spectroscopy.

Kinetic IR spectroscopy was used to reveal beta-sheet formation and water expulsion in the folding of single-chain monellin (SMN) composed of a five-stranded beta-sheet and an alpha-helix. The time-resolved IR spectra between 100 mus and 10 s were analyzed based on two consecutive intermediates, I(1) and I(2), appearing within 100 mus and with a time constant of approximately 100 ms, respectively. The initial unfolded state showed broad amide I' corresponded to a fluctuating conformation.

Expression of the Ha-ras suppressor family member 5 gene in the maturing rat testis.

We analyzed the gene expression of Ha-ras suppressor family member 5 (Hrasls5), which is considered to modulate the Ha-ras signaling cascade, from maturing rat testis. Expression was detected primarily in the spermatocytes in the maturing rat testis. The Hrasls5 gene product might function as a tumor suppressor as well as in spermatogenesis, as deduced from its amino acid sequence.

Cloning and expression of a gene encoding gallerimycin, a cysteine-rich antifungal peptide, from eri-silkworm, Samia cynthia ricini.

A cDNA clone encoding gallerimycin was isolated from larval fat body of immunized Samia cynthia ricini and named as Scr-gallerimycin. In naive larvae, no gene expression was detected, but strongly induced in fat body and hemocytes following immune challenge with bacteria or entomopathogenic fungus Beauveria bassiana. Strong expression of the gene was also induced by injection of peptidoglycan and zymosan, but very weakly by non-pathogenic fungus Aspergillus oryzae.

Induction of tyrosine hydroxylase gene expression by bacteria in the fat body of eri-silkworm, Samia cynthia ricini.

A cDNA clone encoding tyrosine hydroxylase (TH) was isolated from larval fat body of immunized Samia cynthia ricini. In naive larvae, the TH gene was expressed only in the brain, but strongly induced in fat body and hemocytes after injecting UV-killed bacteria. The induction of the gene was rather short-lived compared to that of antibacterial protein genes, reaching the maximum levels 6h after bacterial challenge, and then quickly diminished.

Three peptidoglycan recognition protein (PGRP) genes encoding potential amidase from eri-silkworm, Samia cynthia ricini.

Three cDNA clones encoding peptidoglycan recognition proteins (PGRP-B, -C and -D) were isolated from larval fat body of immunized Samia cynthia ricini. The deduced amino acid sequences show high homology to each other and also to Drosophila PGRP-LB, but rather lower homology to all of the known lepidopteran PGRPs including Samia PGRP-A, a receptor-type PGRP. The three PGRPs conserve the five amino acid residues which form the catalytic site of N-acetylmuramoyl L-alanine amidase as in Drosophila LB.

Peptidoglycan recognition protein (PGRP) from eri-silkworm, Samia cynthia ricini; protein purification and induction of the gene expression.

Peptidoglycan recognition protein (PGRP) was isolated from immunized hemolymph of the wild silkworm, Samia cynthia ricini, detecting the biding activity with (125)I-labeled peptidoglycan (PGN). The binding specificity of PGRP was tested by competitive inhibition of the binding to (125)I-labeled-PGN by a large excess amount of non-labeled-PGN or other glucans. The binding to labeled uncross-linked Lys-type PGN from Micrococcus luteus was strongly inhibited by non-labeled-PGN of the same structure and meso-diaminopimelic acid (DAP)-type cross-linked PGN from Bacillus cell wall, but only a little by cross-linked PGN from M.

Unique peroxidase reaction mechanism in prostaglandin endoperoxide H synthase-2: compound I in prostaglandin endoperoxide H synthase-2 can be formed without assistance by distal glutamine residue.

Prostaglandin-endoperoxide H synthase-2 (PGHS-2) shows peroxidase activity to promote the cyclooxygenase reaction for prostaglandin H2, but one of the highly conserved amino acid residues in peroxidases, distal Arg, stabilizing the developing negative charge on the peroxide through a hydrogen-bonding interaction, is replaced with a neutral amino acid residue, Gln. To characterize the peroxidase reaction in PGHS-2, we prepared three distal glutamine (Gln-189) mutants, Arg (Gln-->Arg), Asn (Gln-->Asn), and Val (Gln-->Val) mutants, and examined their peroxidase activity together with their structural characterization by absorption and resonance Raman spectra. Although a previous study (Landino, L.

Beta-1,3-glucan inducible expression of prophenoloxidase-activating proteinase from eri-silkworm, Samia cynthia ricini.

A cDNA clone encoding possible prophenoloxidase-activating serine protease (PAP) was isolated by screening the cDNA library from immunized larval fat body of the wild silkmoth, Samia cynthia ricini. The cDNA encodes a 438 amino acid open reading frame with a predicted 20 residue signal peptide. Samia PAP has high sequence similarity to Bombyx mori and Manduca sexta PAPs, which contain two amino terminal clip domains followed by a carboxyl-terminal catalytic domain.

Induction of hemolin gene expression by bacterial cell wall components in eri-silkworm, Samia cynthia ricini.

A cDNA clone encoding hemolin was isolated from fat body of immunized Samia cynthia ricini larvae based on subtractive suppression hybridization method. The cDNA encodes 413 amino acid residue open reading frame with an 18 residue predicted signal peptide. The expression of the gene was strongly induced in fat body and midgut by an injection of bacterial cells or peptidoglycans, but very weakly by lipopolysaccharide.

Time-resolved small-angle X-ray scattering investigation of the folding dynamics of heme oxygenase: implication of the scaling relationship for the submillisecond intermediates of protein folding.

Polypeptide collapse is generally observed as the initial folding dynamics of proteins with more than 100 residues, and is suggested to be caused by the coil-globule transition explained by Flory's theory of polymers. To support the suggestion by establishing a scaling behavior between radius of gyration (Rg) and chain length for the initial folding intermediates, the folding dynamics of heme oxygenase (HO) was characterized by time-resolved, small-angle X-ray scattering. HO is a highly helical protein without disulfide bridges, and is the largest protein (263 residues) characterized by the method.

Dehydration in the folding of reduced cytochrome c revealed by the electron-transfer-triggered folding under high pressure.

We determined the activation volume associated with protein folding of reduced cytochrome c from the collapsed intermediate to the native state. The folding rate was followed by a change in the absorption (420 nm) at various pressures between 0.1 and 200 MPa and at various concentrations of denaturant (guanidine hydrochloride) between 3.

Expression of small stress protein hsp20 gene in the maturing rat testis.

In this study, we cloned a cDNA that encodes a small heat shock protein, Hsp20 (alphaB crystallin-related protein), from a maturing rat testis by means of differential display. The full-length cDNA sequence was completely identical to that registered in the DNA databank. The expression of Hsp20 gene was detected strongly in the heart and slightly in the testis of a 9-week-old rat.

Promoting protein, a silkworm hemolymph protein promoting in vitro replication of nucleopolyhedrovirus, binds to beta-glucans.

A protein which bound to 125I-labeled peptidoglycan (PGN) was isolated from hemolymph of silkworm larvae. The N-terminal amino acid sequence and the molecular weight of the protein were in accord with those described for Promoting Protein (PP) from the silkworm. The binding of the protein to [125I]PGN was competitively inhibited by various beta-glucans.

Absence of a detectable intermediate in the compound I formation of horseradish peroxidase at ambient temperature.

A microsecond-resolved absorption spectrometer was developed to investigate the elementary steps in hydrogen peroxide (H(2)O(2)) activation reaction of horseradish peroxidase (HRP) at ambient temperature. The kinetic absorption spectra of HRP upon the mixing with various concentrations of H(2)O(2) (0.5-3 mm) were monitored in the time range from 50 to 300 mus.

Possible neuroprotective mechanism of human neuroglobin.

Neuroglobin (Ngb) is a newly discovered hexacoordinate globin that is expressed in vertebrate brain and can reversibly bind oxygen. Expression of Ngb increases in response to oxygen deprivation and protects neurons from hypoxia in vitro and in vivo. Recent work on human Ngb has shed light on the mechanism of this neuroprotection by human Ngb, as discussed in this review.

Specifically collapsed intermediate in the early stage of the folding of ribonuclease A.

Nature of the burst-phase signals of protein folding has been the subject of much debate as to whether the signals represent the formation of early intermediates or the non-specific collapse of unfolded polypeptides. To distinguish the two possibilities, the submillisecond folding dynamics of ribonuclease A (RNase A) was examined, and compared with those of the disulfide bond-ruptured analog of RNase A (r-RNase A). The circular dichroism measurements on RNase A showed the burst-phase signal within 320 micros after the initiation of the folding reaction, which was identical to that observed for r-RNase A.

Preparation and characterization of a chimeric zebrafish-human neuroglobin engineered by module substitution.

Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that can reversibly bind oxygen that is expressed in the brain. Zebrafish and human Ngb share about 50% amino acid sequence identity. These Ngb proteins consist of four compact protein structural unit "modules" referred to as M1-M4.

A novel spermatogenesis related factor-2 (SRF-2) gene expression affected by TCDD treatment.

We have cloned a gene which is specifically expressed at the stage of sexual maturation in the rat testis by means of differential display, and have named it spermatogenesis-related factor-2 (SRF-2). Testicular expression was first detected at 5 weeks of age, and its level of the expression increased up to 7 weeks, and was maintained even at 63 weeks. Its cDNA was 2,789 bp in length and encoded an open reading frame of 718 amino acids.

Identification of residues in human neuroglobin crucial for Guanine nucleotide dissociation inhibitor activity.

Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. We previously demonstrated that ferric human Ngb binds to the alpha-subunits of heterotrimeric G proteins (Galpha) and acts as a guanine nucleotide dissociation inhibitor (GDI) for Galpha. Here we have investigated the interaction between Ngb and Galpha in more detail.

Electron transfer reaction in a single protein molecule observed by total internal reflection fluorescence microscopy.

To observe an electron transfer (ET) process in a single protein molecule, we constructed a model system, Alexa-HCytb5, in which cytochrome b5 (Cytb5) is modified with a fluorescent probe, Alexa Fluor 647 dye. In this model system, intramolecular transfer of an electron from the Alexa dye to heme in Cytb5 is supposed to oxidize the probe and quench its fluorescence, and the ET reaction at the single-molecule level can be monitored as the intermittent change in the fluorescence intensity. Alexa-HCytb5 was fixed on the glass surface, and illumination of laser light by the total internal reflection resulted in blinking of the fluorescence from the single Alexa-HCytb5 molecule in the time scale of several hundred milliseconds.

Specific collapse followed by slow hydrogen-bond formation of beta-sheet in the folding of single-chain monellin.

Characterization of the conformational landscapes for proteins with different secondary structures is important in elucidating the mechanism of protein folding. The folding trajectory of single-chain monellin composed of a five-stranded beta-sheet and a helix was investigated by using a pH-jump from the alkaline unfolded to native state. The kinetic changes in the secondary structures and in the overall size and shape were measured by circular dichroism spectroscopy and small-angle x-ray scattering, respectively.

Oxidative stress-responsive intracellular regulation specific for the angiostatic form of human tryptophanyl-tRNA synthetase.

Tryptophanyl-tRNA synthetase (TrpRS) exists in two forms in human cells, i.e., a major form which represents the full-length protein and a truncated form (mini TrpRS) in which an NH(2)-terminal extension is deleted because of alternative splicing of its pre-mRNA.

A novel lebocin-like gene from eri-silkworm, Samia cynthia ricini, that does not encode the antibacterial peptide lebocin.

A cDNA clone with homology to lebocin gene was isolated from fat body of immunized Samia cynthia ricini larvae. The cDNA has an open reading frame encoding 162 amino acid residues. The deduced amino acid sequence shows significant homology to lebocin precursor proteins from Bombyx mori and Trichoplusia ni only in the "prosegment" region, but no homology to mature lebocin, a proline-rich antibacterial peptide, indicating the protein is not a precursor for lebocin antibacterial peptide.

Structural diversities of active site in clinical azole-bound forms between sterol 14alpha-demethylases (CYP51s) from human and Mycobacterium tuberculosis.

To gain insights into the molecular basis of the design for the selective azole anti-fungals, we compared the binding properties of azole-based inhibitors for cytochrome P450 sterol 14alpha-demethylase (CYP51) from human (HuCYP51) and Mycobacterium tuberculosis (MtCYP51). Spectroscopic titration of azoles to the CYP51s revealed that HuCYP51 has higher affinity for ketoconazole (KET), an azole derivative that has long lipophilic groups, than MtCYP51, but the affinity for fluconazole (FLU), which is a member of the anti-fungal armamentarium, was lower in HuCYP51. The affinity for 4-phenylimidazole (4-PhIm) to MtCYP51 was quite low compared with that to HuCYP51.