The vast amount of sequence information becoming available on genes from man and from other species calls for corresponding increases in the rate of collection for data of a more functional nature. Expression measurements often constitute a first step in this direction, and can be performed on a reasonably large scale using highly parallel hybridization methods. Large sets of targets (clones, inserts, oligonucleotides) are hybridized with labeled complex probes prepared from total cell or organ mRNA; under the proper conditions, signals measure the relative abundance of each sequence species, and can be acquired quantitatively. These techniques are presently available in three formats: high-density membranes to be hybridized with radioactive complex probes, microarrays of DNA spots (a miniaturized version of the former technique) using fluorescent complex probes, and oligonucleotide chips that, although developed originally for mutation detection, can be adapted to perform expression measurements. The miniaturized formats clearly represent the future, since they allow higher sensitivity, assay of large numbers of entities and hopefully provide the opportunity to use small amounts of starting material.

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Eukaryotic cells have developed an array of endomembrane systems that have differentiated to carry out various functions. They are involved in the pathways of endocytosis and exocytosis, and have an acidic lumenal pH ranging from 4.5 to 6.5. This review describes recent studies on the animal cell organelles and how they relate to the well studied systems of yeast. We focus mainly on (i) the primary proton pump (vacuolar type H⁺-ATPase) and other factors that establish acidic pH, and (ii) functions of the organelles as related to lumenal acidity.

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The rice blast fungus Pot 2 is an inverted repeat transposon which shares structural features with the Tc1-mariner superfamily. In a search for the transposition regulatory mechanism of presently quiescent Pot 2, we found truncated transcripts for the sense and antisense strands of the transposase (TPase) gene. Reverse transcription-PCR analysis identified the convergent overlapping transcription units that contain the entire coding region of the Tease gene. DNA fragments carrying presumed TATA box motifs at both ends of Pot 2 proved to be functional as promoters in transient expression assays. Using these promoters on transformation vectors, stable transformants of the fungus were obtained at a frequency comparable to that with the established system. Furthermore, the antisense promoter transcribed a marker gene from one of the original sites on the chromosome. The above results indicate that the antisense RNA is transcribed by the antisense promoter at the 3' end of the element, which has implications for a possible regulatory mechanism. This is the first example of the identification of an antisense promoter within a class II transposon from a eukaryote.

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Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein strongly related to α₂-macroglobulin (α₂-M). Interactions of tissue plasminogen activator (t-PA) with PZP and α₂-M were both investigated in vitro and the complexes were analyzed by polyacrylamide gel electrophoresis (PAGE). The results demonstrated that PZP-t-PA complex formation was evident within 1h of incubation, whereas α₂-M-t-PA complexes were formed after 18 h. Conclusions were supported by the following evidence: (i) PZP and α₂-M complexes revealed changes of the mobility rate in non-denaturing PAGE, similar to those observed with α-Ms-chymotrypsin; (ii) both PZP and α₂-M formed complexes of molecular size >360 kDa by SDS-PAGE, in accordance with the covalent binding of t-PA, which was previously reported for other proteinases; and (iii) PZP underwent a specific cleavage of the bait region with appearence of fragments of 85-90 kDa as judged by reducing SDS-PAGE. In contrast, the proteolytic attack on α₂-M was found to occur more slowly, requiring several hours of incubation with t-PA for generation of an appreciable amount of fragments of 85-90 kDa. The appearance of free SH-groups of α-Ms was further investigated by titration with 5, 5'-dithiobis (2-nitrobenzoic acid). The maximal level of SH-groups raised was 3.9 mol/mol of PZP and 3.5 mol/mol of α₂-M, indicating approximately one SH-group for each 180-kDa subunit. Finally, t-PA activity in PZP-t-PA complex was evaluated by measuring the hydrolysis of the chromogenic substrate Flavigen t-PA. Our results revealed that prolongation of the incubation period of this complex increased t-PAmediated hydrolysis of Flavigen t-PA until a plateau was reached, approximately between 60 and 120min. The present study suggests that PZP, by binding to t-PA, may contribute to the control of the activity of proteinases derived from fibrinolytic systems.

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Tetrameric, NADP-containing glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis catalyzes the oxidation of glucose into glucono-δ-lactone coupled to the reduction of fructose to sorbitol. GFOR is inactivated during substrate turnover in vitro, the long-term stability of the enzyme during conversions in biochemical reactors thereby being drastically reduced. The process of inactivation is triggered by structural transitions that are induced by the lactone product and involves aggregation as the ultimate cause of irreversible inactivation. Guanidinium hydrochloride-induced changes in the conformation of GFOR seem to be similar to those observed in the presence of lactone, and are manifested by incubation time-dependent increases in protein fluorescence and the solventexposed hydrophobic surface. The formation of high-order protein associates in solution in the presence of this denaturant proceeds from the native tetramer to a reversibly inactivated octamer and then to a dodecameric form that cannot be reactivated through spontaneous or assisted refolding. Therefore, stabilization of GFOR during turnover requires that the marked tendency of the enzyme to form aggregates is prevented efficiently. This goal has been accomplished in the presence of low urea concentrations (1.0M), which led to a 10-fold increase in the half-life of GFOR under operational conditions.

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The main pepsinogen from the mucosa of the shark, Centroscymnus coelolepis, has been purified and characterized. This zymogen, the most abundant protein in terms of quantity and activity (yield 72%), is a homogeneous monomer of molecular weight 42±0.7 kDa, as determined by electrophoresis. The aspartyl proteinase nature of this enzyme was confirmed by the considerable inhibition by pepstatin. Its specificity as to the oxidized B-chain of bovine insulin was determined using electrospray ionization mass spectrometry (ESI-MS) coupled with reversed phase high pressure liquid chromatography (RP-HPLC). The 15-16 Leu-Tyr bond was rapidly cleaved in this substrate, followed by the 24-25 Phe-Phe, 25-26 Phe-Tyr, and 11-12 Leu-Val bonds.

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The linoleic acids embedded in the SUVs of soy-PC, DMPC, and DPPC served as substrate for soybean lipoxygenase-1 (L-1). The initial velocity of the catalytic reaction and the concentration of the substrate showed a hyperbolic relation. The K_m values of L-1 for the linoleic acids in soy-PC, DMPC, and DPPC vesicles were 0.07, 0.09, and 0.11mM, respectively, being comparable with that for Tween-20 micellar linoleic acid. Soy-PC and DMPC competitively inhibited the enzyme with K₁ values of 0.20 and 0.13mM, respectively, whereas DPPC had no effect. DSC analysis revealed the phase separation of linoleic acid and DPPC in vesicles in the temperature range in which the enzyme reaction was carried out. This may account for the lack of inhibitory effect of DPPC on the enzyme. From the temperature dependence of the specific activity of the enzyme, the E_a values of the catalytic reaction were estimated to be 26.7 and 35.3 kJ•mol^-1 for soy-PC and DPPC vesicles, respectively. For linoleic acid-DMPC vesicles, a two-phase temperature dependence of the activity across the transition temperature of the mixed vesicles was suggested.

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Geranylgeraniol (GGO) at 50 μM induces apoptosis in HL-60 cells. We examined the effects of Zn²⁺ ions on this process. Treatment of HL-60 cells with Zn²⁺ ions inhibited subsequent GGO-induced fragmentation of DNA. In a cell-free system that consisted of a specific substrate for caspase-3 and a lysate of HL-60 cells that had been treated with 50 μM GGO, Zn²⁺ ions at concentrations above 0.1mM inhibited the activity of caspase-3. The effect of Zn²⁺ ions on the processing of caspase-3 during GGO-induced apoptosis was investigated by Western blotting, which revealed that an inactive 32-kDa precursor of caspase-3 was cleaved, in response to GGO, to yield an activated 17-kDa enzyme. Treatment of HL-60 cells with Zn²⁺ ions inhibited the cleavage of the precursor by a protease that was induced by treatment with GGO, and inhibition of this processing was well correlated with the inhibition by Zn²⁺ ions of caspase-3 activity in the cell-free system. In cell-extracted cytosols, Zn²⁺ ions inhibited the cleavage of the 32-kDa precursor by caspase-9 (Aapf-3) that was activated by addition of cytochrome c and dATP. These results indicate that inhibition of GGO-induced apoptosis in HL-60 cells by Zn²⁺ ions might be due to inhibition by Zn²⁺ ions of the processing of a precursor to caspase-3.

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The complete amino acid sequence of Akazara scallop, Chlamys nipponensis akazara, troponin-I was determined by automated Edman degradation. It is composed of 292 amino acid residues with a blocked N-terminus. The M_r is calculated to be 34, 678, about 14, 000 larger than that of vertebrate skeletal troponin-I but significantly smaller than the 52, 000 that had been estimated by SDS-polyacrylamide gel electrophoresis. The homologous sequence to vertebrate and arthropoda troponin-Is is found in the C-terminal region. In particular, the sequence of the regions essential for binding to actin and troponin-C is highly conserved. On the other hand, Akazara scallop troponin-I has 100-133 extra residues at the N-terminus compared with vertebrate troponin-I. This extra region is rich in Glu and Arg and has a unique sequence, that shows in part a high sequence homology with the tropomyosin-binding site of troponin-T and caldesmon.

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HPC-1/syntaxin 1A (HPC-1), which plays an important role in vesicular transport to the plasma membrane, possesses a hydrophobic sequence at its C terminus. When expressed from cDNA in COS cells, wild-type HPC-1 was localized in the Golgi complex and the plasma membrane. Truncation of the hydrophobic domain resulted in the cytoplasmic localization of the mutant, thus indicating that the domain indeed functions as a membrane anchor. A fusion protein with the C-terminal glycosylation sites was glycosylated in transfected cells, providing evidence that HPC-1 has a transmembrane structure, and that the protein is first inserted into the endoplasmic reticulum and then transported to the plasma membrane. A chimeric protein consisting of Escherichia coli maltose-binding protein with the last 24 amino acids of HPC-1 was inserted into the endoplasmic reticulum in a transmembrane topology and localized along the exocytic pathway of transfected cells similar to HPC-1. These results indicate that the portion is important for intracellular localization of HPC-1.

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The binding constants between disulfide-intact or various disulfide-reduced bovine α-lactalbumins and an Escherichia coli chaperonin, GroEL, were determined by using the equilibrium dialysis method. The disulfide-intact and one-disulfide (Cys6-Cys120)-reduced a-lactalbumins were shown not to bind with GroEL both in the presence and absence of Call. The two-disulfide (Cys6-Cys120 and Cys28-Cys111)-reduced α-lactalbumin, which has the native-like tertiary structure in its β-domain region and an unfolded α-domain in the presence of Call, showed considerable binding with GroEL. The binding free energy of the two-disulfide-reduced a-lactalbumin in the presence of Call is close to that of the molten globule state of disulfide-intact α-lactalbumin. This result suggests that GroEL binds to the unfolded α-domain of α-lactalbumin regardless of the conformation of the β-domain. The fully disulfide-reduced and two-disulfide-reduced α-lactalbumins were found to bind more strongly with GroEL in the absence of Ca²⁺ than the two-disulfidereduced α-lactalbumin in the presence of Ca²⁺, thus indicating that the unfolding of the β-domain of a-lactalbumin leads to stronger interaction with GroEL.

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When exposed to various stresses including heat shock, myxoamoebae, growing haploid cells of Physarum polycephalum, show marked morphological changes and consequently become disk-shaped microcysts. We have found that p 66 is induced exclusively in the course of microcyst formation and has an actin-binding activity. In this study, we purified p 66 to homogeneity and isolated a p 66 cDNA. The deduced protein sequence contained 601 amino acids and showed 31% identity to a yeast actin-interacting protein, AIP1. Northern blot analysis revealed that the amount of p 66 mRNA was significantly increased by heat shock in myxoamoebae but not in plasmodia. Thus, p 66 seems to be a developmentally-expressed stress protein which regulates the rearrangement of actin organization during microcyst formation P. polycephalum.

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Achromobacter β-lytic protease (blp), one of the bacteriolytic proteases secreted by Achromobacter lyticus, exhibited both peptidase and bacteriolytic activities at alkaline pH. The protease was strongly inhibited by 1, 10-phenanthroline, and one zinc atom was detected in the molecule by ion-spray mass spectrometry. The zinc-protease specifically cleaved Gly-X bonds in peptides and possibly possessed subsites S 2, S 1, S 1', and S 2' for binding substrate [Schecter, I. and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162]. Blp lysed Staphylococcus aureus and Micrococcus luteus cells more efficiently than Achromobacter α-lytic protease (alp) and lysozyme, thus being responsible for the high bacteriolytic activity of A. lyticus. In the lysis of bacterial cell walls, blp hydrolyzed both the D-Ala-Gly/Ala bond at the linkage between the peptide subunit and the interpeptide and the Gly-Gly bond in the interpeptide bridge. These results indicate that blp is a highly active bacteriolytic enzyme with a broad bacteriolytic spectrum, which acts primarily by splitting the linkage between the peptide subunit and the interpeptide in the peptidoglycan.

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Wild-type and several mutant human manganese superoxide dismutases (Mn-SODs) were produced in a baculovirus/insect cell system and characterized. The enzymatic activity of a homogenate of Sf 21 cells, infected with baculovirus carrying wild-type Mn-SOD and grown in the conventional medium, was indistinguishable from that of control cells, but was augmented by supplementation with Mn²⁺. The protein produced was largely imported into the mitochondria, as judged from the enrichment in the mitochondrial fraction, the mobility of the protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the results of N-terminal processing, which was confirmed by sequencing of the purified enzyme. However, a significant amount of precursor was also detected by an antibody raised against the human Mn-SOD signal peptide. While both Mn²⁺ and Fe³⁺ stimulated Mn-SOD accumulation within mitochondria, the active form was produced in the presence of submillimolar Mn²⁺ only. Amino acid substitutions at a signal peptide-cleavage site, His-Ser-Leu⁴ to Pro-Met-Val⁴, in the mature Mn-SOD prevented the processing of the precursor protein, and thus resulted in the accumulation of the precursor protein within mitochondria, as judged on immunostaining with an anti-Mn-SOD antibody. Mutant Mn-SODs with a truncated signal peptide or carboxyl region (8, 13, and 42 amino acid residues in the mature form) were barely solubilized, even with a nonionic detergent, and exhibited no activity, suggesting inappropriate folding of these mutant SODs. They were also susceptible to proteolytic degradation, while the wild-type and precursor forms were resistant. Thus, the baculovirus/insect cell expression system appears to be adequate for the analysis of mitochondrial import using intact cells as well as for the large scale production of active Mn-SOD.

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The Hsc70t gene is a HSP70 homolog gene expressed constitutively in spermatids in mice. This gene is linked to two heat-inducible HSP70 genes, HSP70.1 and HSP70.3, located in the MHC class III region. The syntenic region of human chromosome 6 contains the HSPA1B, HSPA1A, and HSPA1L genes. Here, we have isolated a HSPA1L cDNA clone from human testicular cells. The HSPA1L gene contained an intron 13 by upstream of the initiating ATG. A similar genomic structure was found in the Hsc70t gene. The transcription initiation site of the Hsc70t gene was located at ca. 600 by upstream of the heat-inducible Hsp70.3 gene, linked head-to-head. Sequence alignment of the mouse and human genes revealed that the human HSPA1L and HSPA1A genes were orthologous to the mouse Hsc70t and Hsp70.3 genes, respectively. Conserved sequence stretches observed in the 5' flanking region and the first exon of the spermatid-specific Hsp70 gene may be involved in regulation of the specific gene expression.

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Phenylalanine at position 2 of thrombin receptor-tethered ligand peptide (SFLLRNP) is crucially important for the activation of thrombin receptor. Its substitution by parafluorophenylalanine [(p-F) Phe] enhanced several times the activity in human epitheliallike SH-EP cells [Nose et al. (1993) Biochem. Biophys. Res. Commun. 193, 694-699]. To clarify the interaction mode of Phe-2-phenyl in receptor activation, a series of analogs having chemical modifications on the benzene ring of Phe-2 were synthesized and examined for their ability to induce the aggregation of human platelets. When the fluorine atom was placed at the meta or ortho position, the resulting analogs exhibited considerably diminished activity (about 10-20% of para-derivative), indicating that the substitution is allowed only at the para position. The derivative with pentafluorophenylalanine was totally devoid of activity. These results suggested that Phe-2 requires hydrogen atom (s) on the benzene ring presumably for interaction with the receptor. No activity enhancement was observed for analogs with para-chloro-, bromo-, or iodophenylalanine, indicating the importance of the high electronegativity of fluorine to intensify the dipole of CH (s) remaining in the Phe-2-benzene ring. Inactivity of analogs havingpara-iodophenylalanine and homophenylalanine indicated the importance of the size of para substituents, and the placement of hydroxyl, nitro, and trifluoromethyl groups at the para position led to no activity. The interaction of Phe-2 of SFLLRNP appeared to be structurally restricted to a limited space in the receptor. The results suggested the presence of face-to-edge π-π interaction based upon the CH/π interaction between the ligand Phe-2-phenyl group and the receptor aromatic group.

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Two types of active chimeric enzymes have been constructed by genetic engineering of chicken cytosolic adenylate kinase (AK) and porcine brain UMP/CMP kinase (UCK): one, designated as UAU, carries an AMP-binding domain of AK in the remaining body of UCK; and the other, designated as AUA, carries a UMP/CMP-binding domain of UCK in the remaining body of AK. Steady-state kinetic analysis of these chimeric enzymes revealed that UAU is 4-fold more active for AMP, 40-fold less active for UMP, and 4-fold less active for CMP than the parental UCK, although AUA has considerably lowered reactivity for both AMP and UMP. Circular dichroism spectra of the two chimeric enzymes suggest that UAU and AUA have similar folding structures to UCK and AK, respectively. Furthermore, proton NMR measurements of the UCK and UAU proteins indicate that significant differences in proton signals are limited to the aromatic region, where an imidazole C2H signal assigned to His 31 shows a downfield shift upon conversion of UCK to UAU, and the signals assigned to Tyr 49 and Tyr 56 in the UMP/CMP-binding domain disappear in UAU. In contrast, AUA has a T_m value about 11°C lower than AK, whereas UAU and UCK have similar T_m values. These results together show that the substrate specificity of nucleoside monophosphate (NMP) kinases can be engineered by the domain exchange, even though the base moiety of NMP appears to be recognized cooperatively by both the NMP-binding domain and the MgATP-binding core domain.

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A protein product of the tissue kallikrein gene family was isolated from the submandibular gland of DBA/2N mice. Amino acid sequencing showed this protein to be highly homologous to two tissue kallikreins, mK13 and mK26, also known as prorenin-converting enzymes PRECE and PRECE-2, respectively. The cDNA corresponding to the present enzyme was cloned, and its complete nucleotide sequence was determined. The cloned cDNA was different in 6 and 12 bases out of 783 nucleotides from those of mKlk-13 and mKlk-26 cDNAs, respectively, the homologies being 99.2 and 98.5% (nucleotide), or 98.3 and 96.2% (amino acid). Upon incubation with either bovine kininogens or mouse Ren 2 prorenin, this tissue kallikrein generated bradykinin and renin, respectively, as judged by Western blotting and protein sequence analysis. Isoelectric focusing analysis of the submandibular gland tissue kallikreins suggested that the present enzyme was not xpressed in CD-1 or ICR mice and that no mK13 protein was present in DBA/2N mice. These data suggest that the enzyme is an allozyme of mK13, a prorenin-converting enzyme highly expressed in the submandibular gland of DBA/2N mice. The mKlk-13 gene in mice is therefore suggested to be polymorphic, having at least two allelic forms with a high sequence homology. The designation mK13^b and mKlk-13^b for the protein and gene of this tissue kallikrein is proposed.

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We previously prepared a monoclonal antibody, S4D5, specific to vanadocytes, vanadiumcontaining blood cells, in the vanadium-rich ascidian Ascidia sydneiensis samea. Here, we demonstrate that a 45-kDa antigen recognized by S4D5 is 6-phosphogluconate dehydrogenase (6-PGDH), an enzyme of the pentose phosphate pathway, based on cDNA isolation of RNA samples from blood cells of the ascidian. Western blot analysis confirmed an abundance of 6-PGDH protein in the vanadocytes and localization of 6-PGDH in the soluble extract of the blood cells. Soluble protein exhibited a correspondingly high level of 6-PGDH enzymatic activity. Ascidians are known to selectively accumulate high levels of vanadium in vanadocytes, and the highest recorded concentration of accumulated vanadium is 350 mM, which is 10⁷ times the concentration in sea water. Almost all vanadium ions are reduced to the +3 oxidation state via the +4 oxidation state in vanadocytes, indicating that reducing agents must participate in the accumulation. On the other hand, vanadium ions in the +5 oxidation state are reduced to the +4 oxidation state by the presence of NADPH in vitro. Together, these observations suggest that NADPH produced in the pentose phosphate pathway may conjugate the reduction of vanadium from the +5 oxidation state through the +4 oxidation state in vanadocytes of ascidians.

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Studies on human cell hybrids of a cervical carcinoma cell line, HeLa, and normal fibroblasts have indicated that the tumorigenicity of these cells is under the control of a putative tumor suppressor on chromosome 11, although the nature of this suppressor remains unknown. We examined the expression of caveolin-1, a protein component of caveolae of the plasma membrane in these cell hybrids. The non-tumorigenic cell hybrid, CGL1, and normal fibroblast W138 cells expressed 21-24 kDa caveolin-1, whereas in tumorigenic hybrid CGL4 as well as in the parental HeLa cells, the level of caveolin-1 was markedly reduced. Caveolin-1 expression was also reduced in γ-ray-induced tumorigenic clones (GIMs) isolated from CGL1 cells, whereas non-tumorigenic irradiated cells expressed the same level of caveolin-1 as CGL1 cells. In accordance with these changes, the cellular level of caveolin-1 mRNA was reduced in the tumorigenic CGL4 cells and GIMs without any detectable changes in the caveolin-1 gene. However, the in vivo tumor growth of CGL4 cells was not altered when caveolin-1 was stably overexpressed through the transfection of a human caveolin-1 cDNA. These results suggest that reduction of caveolin-1 expression is necessary but not sufficient for emergence of the tumorigenic phenotypes of HeLa cell hybrids. Possible roles of the putative tumor suppressor in the control of gene expression are also discussed.

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Human immunodeficiency virus (HIV) and its clinical syndrome, acquired immune deficiency syndrome (AIDS), are one of the world's most prominent health problems. To understand the mechanisms underlying HIV transcription and thereby its propagation, we have focused on the molecular interactions at the GC-rich binding sites of the HIV-1 core promoter, a region important for HIV-1 transcription. Previous biochemical studies have shown that Spl, a zinc finger transcription factor initially isolated as a cellular factor binding that binds to the SV40 early promoter GC-rich sequence, binds to the HIV-1 GC-rich binding sites due to sequence similarities. However, the HIV-1 GC-rich binding sites are considerably different from the Spl consensus binding sequence, and recent genetic studies have shown the lack of regulation by Spl in numerous genes thought to be regulated by that factor in the past. We reasoned that other factors may bind to the HIV-1 GC-rich binding sites. Using the native HIV-1 GC-rich binding sequence as the bait, genetic screening for interacting factors was performed by the yeast one-hybrid method. A cDNA encoding a novel zinc finger protein named GBF, G_-C-rich sites b_-inding f_-actor, was isolated from a human peripheral blood leukocyte library. Primary structure analysis of GBF revealed a C₂H₂ Krüppel-type zinc finger at its C-terminus, and putative acidic and proline-rich domains at its N-terminus. We also show that GBF belongs to a subgroup of Krüppel-type zinc fingers distinct from Spl. By directly addressing interactions at the HIV-1 GC-rich binding sites, our present study sheds new light on molecular interactions at the HIV-1 promoter.

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The role of the NH₂-terminal hydrophobic patch of cytochrome P4502B4 (CYP2B4) in interactions with NADPH-cytochrome P450 reductase (P450R) and cytochrome b₅ (b₅) was assessed using a variant lacking the signal anchor sequence (Δ2-27). CD, second-derivative, and fluorescence emission spectra indicated that the structure of the deletion mutant slightly differed from that of the native CYP2B4. Fitting of the initial-velocity patterns for P450R- and b₅-directed electron transfer to the ferric CYP2B4 forms to Michaelis-Menten kinetics revealed an approximately 2.3-fold decrease in the affinity of the two electron donors for the engineered enzyme, while the reductive efficiency remained unaffected. Circumstantial analysis suggested that impaired association of the redox proteins with P4502B4(Δ2-27) accounted for this phenomenon. Interestingly, spectral docking of P450R to the truncated pigment was not hampered, while the binding of b₅ was blocked. The rates of substrate-triggered aerobic NADPH consumption in systems containing CYP2B4(Δ2-27) and P450R were 16 to 56% those obtained with the unchanged hemoprotein. Decelerated cofactor oxidation did not arise on defective substrate binding or perturbed utilization of the substrate-bound oxy complex. Experiments with b₅ as the ultimate electron donor hinted at some damage to second-electron transfer to the truncated enzyme. The results are consistent with the proposal that the NH₂-terminal hydrophobic region of CYP2B4 might be of importance in preservation of the catalytic competence of the enzyme.

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Crystallographic data reveal that Met-118 in bacteriorhodopsin (bR) contacts directly with the C₉ methyl group of retinal, and Khorana et al. [J. Biol. Chem. 268, 20305-20311 (1993)] suggest that this contact may regulate the absorption maximum (λ_max). We have replaced the amino acid (Val-108) corresponding to Met-118 of bR by methionine in pharaonis phoborhodopsin (ppR), whose λ_max is ca. 500 nm, while those of other bacterial rhodopsins such as bR, halorhodopsin, and sensory rhodopsin are red-shifted by 60-90 nm. By flashphotolysis measurement, we could not recognize a large spectral red-shift of the V108M mutant. On the other hand, the decay of ppR_M (M-intermediate) of the mutant was approximately three times as fast as that of wild-type, and an M-like intermediate (M') whose λ_max is blue-shifted by 60 nm from that of M became appreciable. The replacement abolished the shoulder of the ppR_M spectrum. From these findings, we infer that the distance between the retinal and the 108-position in ppR is relatively long, and that in the M-state this distance is shortened.

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Phenylalanine 120 is a candidate residue juxtaposing catalytic His 12 and His 119 in ribonuclease A (RNase A). To clarify its role in construction of the catalytic center, Phe 120 was replaced by alanine, tryptophan, leucine, or glutamic acid by site-directed mutagenesis. The transphosphorylation and hydrolysis activities of the mutant RNase As, respectively, toward cytidinyl 3', 5' adenosine (CpA) and cytidine 2', 3' cyclic monophosphate (C>p) were compared with those of the wild type enzyme. The K_m values of the two reactions increased markedly with slight changes in the k_cat values. The pK_e values of His12 and His119 in the wild type and mutant enzymes, estimated from the pH dependence of the k_cat/K_m values, showed little change. The rate of carboxymethylation was reduced markedly by the mutations. The K_i values of the phosphate anion as to hydrolysis activity increased only slightly when Phe 120 was replaced by leucine, tryptophan, or alanine. These findings suggest that Phe 120 participates in the binding of the substrate, juxtaposing His 12 and His 119, and in stabilizing the transition state intermediate in the hydrolysis reaction. Furthermore, the decreases in the thermal denaturation temperatures of all the mutants, particularly F120E, indicate that Phe 120 also helps maintain the conformational stability of RNase A.

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We have demonstrated that N-terminal sequencing can be performed successfully despite boiling protein samples in the presence of urea under precise conditions, before loading them onto SDS-PAGE and transfer to polyvinylidene difluoride membrane. Using myoglobin as a test protein, we found that its ability to undergo N-terminal sequencing was not affected by the presence of urea provided “ultra-pure” urea was used. Consistent with this result, we verified that urea did not carbamylate myoglobin since its molecular mass was measured by mass spectrometry after electroelution of the protein band from the gel. These observations are useful for the study of integral membrane proteins, in particular to study their topology from proteolysis experiments, since heating in the presence of urea before SDS-PAGE reduces membrane protein aggregation [Soulié, S., Møller, J. V., Falson, P., and le Maire, M. (1996) Anal. Biochem. 236, 363-364]. We show that the sequencing yield of a hydrophobic peptide from reticulum Ca²⁺-ATPase was more than doubled in the presence of urea in accord with the quantification of the Coomassie Blue staining of the gel and of the amount present on the polyvinylidene difluoride membrane. For three peptides of the gastric H⁺K⁺-ATPase, the sequencing yield after urea treatment increased almost threefold.

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We isolated and identified a 110-kDa myosin I from porcine aorta media smooth muscle [Y. Hasegawa et al. (1996) J. Biochem. 120, 971-976]. Partial peptide sequences of the 110-kDa myosin I fragments were homologous to amino acid sequences deduced from myosin Iβ of bovine brain and adrenal gland. We investigated biochemically the distribution of the 110-kDa myosin I by cell fractionation methods. About 10% of myosin I present in whole cells could be extracted by treatment with 0.02% saponin, which does not liberate organelles, indicating that at least 10% of myosin I present in A10 cells is associated with neither organelles nor cytoskeleton. After treatment of A10 cells with 0.5% Triton X-100, the insoluble cytoskeleton contained 45% of myosin I present in whole cells. Treatment with MgATP extracted most of myosin I from the cytoskeleton, indicating that the distribution of myosin I is maintained by binding of the myosin I head to an actin filament. On the other hand, when the cell homogenate was fractionated on sucrose density step gradients, about 80% of myosin I was associated with membranes of various densities. An attempt to dissociate the myosin I from the membranes in the presence of MgATP was not successful. These results show that about 80% of total myosin I is associated directly with membranes, not through F-actin. The amounts of myosin I associated with membranes or cytoskeleton provide evidence that myosin I in A10 cells is associated in part with only membrane and in part with both cytoskeleton and membranes. Our results lead to conclusion that myosin I exist in several states: membrane-and-cytoskeleton-associated, membrane-associated, and membrane-and-cytoskeleton-free. These states may be in dynamic equilibrium, allowing myosin I to respond to the cellular requirements.

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Dipeptidyl peptidase IV (DPP IV) in normal human serum was purified 14, 400-fold with a 25% yield to homogeneity. The molecular weight of the purified enzyme was approximately 110, 000 on SDS-PAGE, almost the same as that of human kidney membrane-bound DPP IV. No difference was found between the two enzymes enzymologically and immunologically, either in substrate specificity, susceptibility to inhibitors, or cross-reactivity with an antirat kidney DPP IV antibody, or in their ability to bind adenosine deaminase. However, the N-terminal amino acid sequence of serum DPP IV lacked the transmembrane domain of the membrane-bound enzyme and started at the 39 th position, serine, from the N-terminus predicted from the cDNA nucleotide sequence. These results suggest that membrane-bound DPP IV loses its transmembrane domain upon release into the serum, and that its structure on the plasma membrane is not required for its binding to adenosine deaminase.

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A fundamental dogma has developed over the past 20 years that non-enzymatic glycation involving saccharide chains of greater than 3 to 4 residues is an extremely unlikely reaction. Our investigations using glycosaminoglycans have shown that, given sufficient time, polypeptide-polysaccharide conjugates form via the Schiff base-Amadori rearrangement mechanism. Further, even though these straight chain polysaccharides are relatively charged and sterically hindered, spontaneous glycation can also occur in vivo. A complete reinvestigation of all aldose terminating polysaccharides is required to elucidate this new class of macromolecules, which is likely to contain unusual polypeptide-polysaccharide combinations and functions.

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The Escherichia coli ArcB sensor is involved in anaerobic signal transduction. ArcB is an unorthodox His-kinase, in that it contains three types of phosphotransfer signaling domains in its primary amino acid sequence, namely, transmitter (or His-kinase), receiver, and histidine-containing phosphotransfer (HPt) domains. In this study, we first conducted an in vivo experiment to determine whether or not the phosphorylation of the HPt domain is crucial for ArcB/ArcA-mediated anaerobic signal transduction. The results are best interpreted as meaning that the HPt domain of AreB is important for the anaerobic signal transduction, as far as the expression of the succinate dehydrogenase (sdh) operon is concerned. We then isolated a set of ArcB mutant each with a single amino acid substitution in the HPt domain, which has lost the ability to function as a phospho-transmitter. The results of such mutational analyses, together with the three-dimensional crystal structure of the HPt domain, provided an insight into the structure and function of the HPt domain of ArcB.

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His397 was replaced with alanine by site-directed mutagenesis of the cloned PRC1 gene in order to confirm the role of this residue in the proton-relay system of carboxypeptidase Y (CPY). The expressed and purified H397A showed a CD spectrum almost identical to that of the wild type enzyme, but its heat stability and conformation on heating differed somewhat. Kinetic analysis showed that the k_cat values of the purified H397A toward the peptide substrates, Z-Phe-Leu and Z-Gly-Phe, were reduced to approximately 4×10^-5-fold, whereas the K_m values remained almost unchanged. The activity of the H397A preparation with the ester substrate, Ac-Phe-OEt, was negligible. The low activity of our H397A was lost on treatment with DFP and Z-Phe-CH₂Cl, site-specific inhibitors, respectively, for Ser146 and His397, and with the HgCl₂ and PCMB, SH-reagents for Cys341. After treatment with these inhibitors, the k_cat value for the H397A preparation toward Z-Phe-Leu decreased 1×10³-fold or more. The value was approximately 10^-8 for the wild type enzyme. This level of activity is 10³-fold lower than the reported value for the same mutant of CPY [Carlsberg Res. Commun. 54, 165-171 (1989)], and more than 10-fold lower than the values for the corresponding His-to-Ala mutants of trypsin [J. Am. Chem. Soc. 114, 1784-1790 (1992)] and subtilisin [Nature 332, 564-568 (1988)]. These findings, together with the pH profiles and chromatographic behavior, are evidence that the low activity of the H397A preparation is due to contamination by wild type CPY. The decreased k_cat value of our H397A mutant is the lowest reported among the corresponding histidine mutants of serine proteases. We conclude that the proton-relay system composed of Ser146 and His397 is the sole catalytic center of CPY, and that its destruction leads to complete inactivation.

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The apolipoprotein E receptor 2 (apoER2) gene consists of a mosaic of exons, which may have been assembled by “exon shuffling.” Analysis of apoER2 transcripts in several species reveals a lost repeat in the ligand-binding domain of primate apoER2. A pseudo-exon found in the primate apoER2 genes corresponds to the lost repeat but contains a crucial deletion that leads to a translational frameshift. The pseudo-exon sequence in primary transcripts of the human apoER2 gene is shown to be abolished by exon skipping due to two nucleotide substitutions at the 5'-splice donor adjacent to the pseudo-exon. These data suggest the occurrence of exon loss in the evolution of the primate apoER2 gene.

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Eight bromelain inhibitor (BI) isoform fractions were isolated from pineapple stem, and then submitted to conventional amino acid sequencing after performic acid oxidation and subsequent separation of the resulting light and heavy chains. The results revealed that all fractions exhibited microheterogeneity, containing at least two major components, but that all the isoinhibitors have a common double-chain structure (M_r=ca. 5, 700-5, 900) with five disulfide bonds and similar amino acid sequences. Notably, Fraction BI-VIII exhibited less than 40% of the specific inhibitory activity toward stem bromelain as compared with the other inhibitor fractions. This fraction was a mixture of two isoforms, BI-VIII (1) and BI-VIII (2), the latter lacking the arginine or glutamine residue at the COOH-terminus of the light chain. Furthermore, the oxidized light chain of BI-III, used as a representative normal isoinhibitor, was found to exhibit significant inhibitory activity, whereas the oxidized light chain of BI-VIII (2) lacking the COOH-terminal Arg or Gln showed only very low inhibitory activity. Therefore, the major bromelain-inhibitory site was indicated to be the COOH-terminal residue, Arg or Gln, of the light chain. This is consistent with the three-dimensional structure model constructed by computer modeling for the hypothetical complex between BI-VI and papain, a close homolog of bromelain.

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To clarify the regulatory mechanism of pro-gelatinase A (proGelA) activation at a cellular level, expression of gelatinase A (GelA), three MT-MMPs, and TIMP-2 was examined with 11 human cancer cell lines cultured in the presence and absence of stimulants. MT1-MMP mRNA was expressed in 8 cell lines, while MT2-MMP and MT3-MMP mRNAs were expressed in fewer cell lines. The cells with high proGelA activation strongly expressed MT1-MMP mRNA but not MT2-MMP and MT3-MMP mRNAs, suggesting that MT1-MMP was responsible for the proGelA activation in the cancer cells. Treatments with concanavalin A (Con A) and a phorbor ester (TPA) enhanced the MT1-MMP expression, but only Con A stimulated the proGelA activation in many cell lines. In HT1080 fibrosarcoma cells, however, TPA also stimulated the activation. The level of TIMP-2 secreted into culture medium inversely correlated with proGelA activation. For example, 2 squamous cell carcinoma lines (HSC-3 and HSC-4) and 3 HT1080 clones, which efficiently activated proGelA, secreted little TIMP-2 into medium, whereas other cell lines and other HT1080 clones, which hardly activated proGelA, secreted TIMP-2 at high levels. When HSC-3 cells were incubated with TIMP-2 protein or transfected with TIMP-2 cDNA, the proGelA activation was strongly inhibited. These results indicated that extracellular TIMP-2 was an important negative regulator of proGelA activation. However, the level of extracellular TIMP-2 was not consistent with that of TIMP-2 mRNA in some cell lines. Other experimental results suggested that TIMP-2 might be rapidly metabolized after binding to MT1-MMP, and Con A treatment might stabilize the complex of TIMP-2 and MT1-MMP on cell membranes.

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