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Background Limited data is available regarding the vascular response after fluoropolymer paclitaxel-eluting stent (FP-PES) implantation. We sought to assess the vascular response at 6- and 12-month after FP-PES implantation for femoropopliteal artery lesions using serial optical coherence tomography (OCT) examination. Methods and results From the IMPERIAL trial, this study evaluated 10 de novo femoropopliteal lesions treated with FP-PES. The primary study endpoint was neointimal tissue coverage at 6- and 12-month follow-up as assessed by serial OCT examination. The incidence of peri-strut low-intensity area (PLIA) and extra-stent lumen (ESL) was also assessed. A total of 203 matched cross-sectional images were evaluated at 6- and 12-month (5,615 and 5,763 struts, respectively). From 6 to 12 months, the mean neointimal thickness tended to increase from 198 μm to 233 μm with a significant reduction in the incidence of malapposed struts (0.59% vs. 0.28%, p=0.039). Conversely, uncovered struts and PLIA were more frequently observed at 12 months (4.4% vs. 7.8%, p=0.01; 12.7% vs. 21.0%, p Conclusions Neointimal proliferation was markedly inhibited from 6 to 12 months after FP-PES implantation, whereas the incidence of uncovered struts and PLIA significantly increased over time with the enlargement of ESL. Funding Acknowledgement Type of funding sources: None.

Background Impact of SYNTAX score II (SSII) on long-term clinical outcomes after cobalt-chromium everolimus-eluting stent (CoCr-EES) implantation remains unclear. Methods Between February 2010 and May 2011, 1064 consecutive patients with 1440 lesions were treated only with CoCr-EES implantation. Of these, the SSII was calculated in 1013 patients with 1345 lesions. Patients were divided into the tertile group: Tertiles for SSII (low SSII [12–28.9], n=334; intermediate SSII [29–39.1], n=339; and high SSII [39.2–80.8], n=340). We assessed the cumulative 7-year incidences of major adverse cardiac events (MACE), defined as a composite of cardiac death, myocardial infarction, definite stent thrombosis, and clinically driven target lesion revascularization (CDTLR) based on SSII groupings. Results Cumulative 7-year incidence of MACE was significantly higher in the high SSII group than in the other groups (34.1% vs. 18.6% vs. 17.2%, p Conclusions SSII has significantly impact on 7 years clinical outcomes after CoCr-EES implantation. Cumulative incidence of MACE Funding Acknowledgement Type of funding source: None

We experimentally demonstrate Cooper pairs' drastic enhancement of the band-to-band radiative recombination rate in a semiconductor. Electron Cooper pairs injected from a superconducting electrode into an active layer by the proximity effect recombine with holes injected from a $p$-type electrode. The recombination of a Cooper pair with $p$-type carriers dramatically increases the photon generation probability of a light-emitting diode in the optical-fiber communication band. The measured radiative decay time rapidly decreases with decreasing temperature below the superconducting transition temperature of the niobium electrodes. Our results indicate the possibility to open up new interdisciplinary fields between superconductivity and optoelectronics.

We experimentally demonstrate Cooper pairs' drastic enhancement of the band-to-band radiative recombination rate in a semiconductor. Electron Cooper pairs injected from a superconducting electrode into an active layer by the proximity effect recombine with holes injected from a p-type electrode. The recombination of a Cooper pair with p-type carriers dramatically increases the photon generation probability of a light-emitting diode in the optical-fiber communication band. The measured radiative decay time rapidly decreases with decreasing temperature below the superconducting transition temperature of the niobium electrodes. Our results indicate the possibility to open up new interdisciplinary fields between superconductivity and optoelectronics.

Application fields of light emitting diodes (LEDs) are expanding in various fields. Development of LED-based single photon sources is expected to open a new possibility to expand the applications to quantum information communication and processing. The authors have proposed a photon-emitting LED combined with superconducting electrodes, which is expected to be an on-demand entangled photon pair source. The main mechanism is based on the coherent spatial extension of the Cooper-pair states to the photon emitting layer, which is expected to enhance the oscillator strength of the radiative recombination processes by the Cooper-pair superradiance effect. The preliminary operation was demonstrated with InGaAs quantum well (QW) LEDs and about 20-times enhancement of the electroluminescence (EL) was observed under the low-injection current regime. This is the demonstration of the improved internal quantum efficiency (QE) under the low internal QE operation of the LED. In this paper, the enhancement of the radiative recombination processes with the superconducting (SC) effect is demonstrated by the lifetime measurements under the operation with high (~100%) internal quantum efficiency (QE).

UvrA and UvrB proteins play key roles in the damage recognition step in the nucleotide excision repair. However, the molecular mechanism of damage recognition by these proteins is still not well understood. In this work we analyzed the interaction between single-stranded DNA (ssDNA) labeled with a fluorophore tetramethylrhodamine (TMR) and Thermus thermophilus HB8 UvrA (ttUvrA) and UvrB (ttUvrB) proteins. TMR-labeled ssDNA (TMR-ssDNA) as well as UV-irradiated ssDNA stimulated ATPase activity of ttUvrB more strongly than did normal ssDNA, indicating that this fluorescent ssDNA was recognized as damaged ssDNA. The addition of ttUvrA or ttUvrB enhanced the fluorescence intensity of TMR-ssDNA, and the intensity was much greater in the presence of ATP. Fluorescence titration indicated that ttUvrA has higher specificity for TMR-ssDNA than for normal ssDNA in the absence of ATP. The ttUvrB showed no specificity for TMR-ssDNA, but it took over 200 min for the fluorescence intensity of the ttUvrB-TMR-ssDNA complex to reach saturation in the presence of ATP. This time-dependent change could be separated into two phases. The first phase was rapid, whereas the second phase was slow and dependent on ATP hydrolysis. Time dependence of ATPase activity and fluorescence polarization suggested that changes other than the binding reaction occurred during the second phase. These results strongly suggest that ttUvrB binds ssDNA quickly and that a conformational change in ttUrvB-ssDNA complex occurs slowly. We also found that DNA containing a fluorophore as a lesion is useful for directly investigating the damage recognition by UvrA and UvrB.

Pyridoxal reductase (PL reductase), which catalyzes reduction of PL by NADPH to form pyridoxine and NADP(+), was purified from Schizosaccharomyces pombe. The purified enzyme was very unstable but was stabilized by low concentrations of various detergents such as Tween 40. The enzyme was a monomeric protein with the native molecular weight of 41,000 +/- 1,600. The enzyme showed a single absorption peak at 280 nm (E(1%) = 10.0). PL and 2-nitrobenzaldehyde were excellent substrates, and no measurable activity was observed with short chain aliphatic aldehydes; substrate specificity of PL reductase was obviously different from those of yeast aldo-keto reductases (AKRs) so far purified. The peptide sequences of PL reductase were identical with those in a hypothetical 333-amino acid protein from S. pombe (the DDBJ/EMBL/GenBank(TM) accession number D89205). The gene corresponding to this protein was expressed in Escherichia coli, and the purified protein was found to have PL reductase activity. The recombinant PL reductase showed the same properties as those of native PL reductase. PL reductase showed only low sequence identities with members of AKR superfamily established to date; it shows the highest identity (18.5%) with human Shaker-related voltage-gated K(+) channel beta2 subunit. The elements of secondary structure of PL reductase, however, distributed similarly to those demonstrated in the three-dimensional structure of human aldose reductase except that loop A region is lost, and loop B region is extended. Amino acid residues involved in substrate binding or catalysis are also conserved. Conservation of these features, together with the major modifications, establish PL reductase as the first member of a new AKR family, AKR8.

Thermostable RecA protein (ttRecA) from Thermus thermophilus HB8 showed strand exchange activity at 65 degrees C but not at 37 degrees C, although nucleoprotein complex was observed at both temperatures. ttRecA showed single-stranded DNA (ssDNA)-dependent ATPase activity, and its activity was maximal at 65 degrees C. The kinetic parameters, K(m) and kcat, for adenosine triphosphate (ATP) hydrolysis with poly(dT) were 1.4 mM and 0.60 s-1 at 65 degrees C, and 0.34 mM and 0.28 s-1 at 37 degrees C, respectively. Substrate cooperativity was observed at both temperatures, and the Hill coefficient was about 2. At 65 degrees C, all tested ssDNAs were able to stimulate the ATPase activity. The order of ATPase stimulation was: poly(dC)poly(dT)M13 ssDNApoly(dA). Double-stranded DNAs (dsDNA), poly(dT).poly(dA) and M13 dsDNA, were unable to activate the enzyme at 65 degrees C. At 37 degrees C, however, not only dsDNAs but also poly(dA) and M13 ssDNA showed poor stimulating ability. At 25 degrees C, poly(dA) and M13 ssDNA gave circular dichroism (CD) peaks at around 192 nm, which reflect a particular structure of DNA. The conformation was changed by an upshift of temperature or binding to Escherichia coli RecA protein (ecRecA), but not to ttRecA. The dissociation constant between ecRecA and poly(dA) was estimated to be 44 microM at 25 degrees C by the change in the CD. These observations suggest that the capability to modify the conformation of ssDNA may be different between ttRecA and ecRecA. The specific structure of ssDNA was altered by heat or binding of ecRecA. After this alteration, ttRecA and ecRecA can express their activities at each physiological temperature.

MutS protein binds to DNA and specifically recognizes mismatched or small looped out heteroduplex DNA. In order to elucidate its structure-function relationships, the domain structure of Thermus thermophilus MutS protein was studied by performing denaturation experiments and limited proteolysis. The former suggested that T. thermophilus MutS consists of at least three domains with estimated stabilities of 12.3, 22.9 and 30.7 kcal/mol and the latter revealed that it consists of four domains: A1 (N-terminus to residue 130), A2 (131-274), B (275-570) and C (571 to C-terminus). A gel retardation assay indicated that T.thermophilus MutS interacts non-specifically with double-stranded (ds), but not single-stranded DNA. Among the proteolytic fragments, the B domain bound to dsDNA. On the basis of these results we have proposed the domain organization of T. thermophilus MutS and putative roles of these domains.

The mutM (fpg) gene, which encodes a DNA glycosylase that excises an oxidatively damaged form of guanine, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence encoded a 266 amino acid protein with a molecular mass of approximately 30 kDa. Its predicted amino acid sequence showed 42% identity with the Escherichia coli protein. The amino acid residues Cys, Asn, Gln and Met, known to be chemically unstable at high temperatures, were decreased in number in T.thermophilus MutM protein compared to those of the E.coli one, whereas the number of Pro residues, considered to increase protein stability, was increased. The T.thermophilus mutM gene complemented the mutability of the E.coli mutM mutY double mutant, suggesting that T. thermophilus MutM protein was active in E.coli. The T.thermophilus MutM protein was overproduced in E.coli and then purified to homogeneity. Size-exclusion chromatography indicated that T. thermophilus MutM protein exists as a more compact monomer than the E.coli MutM protein in solution. Circular dichroism measurements indicated that the alpha-helical content of the protein was approximately 30%. Thermus thermophilus MutM protein was stable up to 75 degrees C at neutral pH, and between pH 5 and 11 and in the presence of up to 4 M urea at 25 degrees C. Denaturation analysis of T.thermophilus MutM protein in the presence of urea suggested that the protein had at least two domains, with estimated stabilities of 8.6 and 16.2 kcal/mol-1, respectively. Thermus thermophilus MutM protein showed 8-oxoguanine DNA glycosylase activity in vitro at both low and high temperatures.

To investigate the role of alpha helices in protein thermostability, we compared energy characteristics of alpha helices from thermophilic and mesophilic proteins belonging to four protein families of known three-dimensional structure, for at least one member of each family. The changes in intrinsic free energy of alpha-helix formation were estimated using the statistical mechanical theory for describing helix/coil transitions in peptide helices [Munoz, V., Serrano, L. Nature Struc. Biol. 1:399-409, 1994; Munoz, V., Serrano, L. J. Mol. Biol. 245:275-296, 1995; Munoz, V., Serrano, L. J. Mol. Biol. 245:297-308, 1995]. Based on known sequences of mesophilic and thermophilic RecA proteins we found that (1) a high stability of alpha helices is necessary but is not a sufficient condition for thermostability of RecA proteins, (2) the total helix stability, rather than that of individual helices, is the factor determining protein thermostability, and (3) two facets of intrahelical interactions, the intrinsic helical propensities of amino acids and the side chain-side chain interactions, are the main contributors to protein thermostability. Similar analysis applied to families of L-lactate dehydrogenases, seryl-tRNA synthetases, and aspartate amino transferases led us to conclude that an enhanced total stability of alpha helices is a general feature of many thermophilic proteins. The magnitude of the observed decrease in intrinsic free energy on alpha-helix formation of several thermoresistant proteins was found to be sufficient to explain the experimentally determined increase of their thermostability. Free energies of intrahelical interactions of different RecA proteins calculated at three temperatures that are thought to be close to its normal environmental conditions were found to be approximately equal. This indicates that certain flexibility of RecA protein structure is an essential factor for protein function. All RecA proteins analyzed fell into three temperature-dependent classes of similar alpha-helix stability (delta G(int) = 45.0 +/- 2.0 kcal/mol). These classes were consistent with the natural origin of the proteins. Based on the sequences of protein alpha helices with optimized arrangement of stabilizing interactions, a natural reserve of RecA protein thermoresistance was estimated to be sufficient for conformational stability of the protein at nearly 200 degrees C.

We have recorded 500-MHz 1H NMR spectra in the 10-18-ppm range for aspartate aminotransferase from Escherichia coli and for three specific mutant forms. Histidine 143 has been replaced by either alanine or asparagine. In the third mutant, tryptophan 140 has been replaced by phenylalanine. The NMR spectrum of the native enzyme is very similar to that of porcine cytosolic aspartate aminotransferase in the most downfield region. However, the resonances of the proton on the ring nitrogen of the pyridoxal 5'-phosphate (peak A) and on the His-143 imidazole ring (peak B) of the E. coli enzyme are broader and more readily lost at low pH or higher temperatures than those of the porcine enzyme. The possible role of tautomerism in promoting such broadening is discussed. In the histidine mutant proteins, peak A of the pyridoxal 5'-phosphate form is too broad to see under most conditions but is clearly present in the pyridoxamine phosphate form. Peak B is missing in the 2 histidine mutants. Observation of nuclear Overhauser effects further confirms the identity of B as the resonance of HN epsilon 2 of His-143 and that of peak D at approximately 11.8 ppm as HN epsilon 2 of His-189. The mutant spectra also provide insight into electronic interactions between groups in and near the active site which confirm and supplement conclusions drawn from spectra of porcine cAspAT. While no clear loss of a peak was observed for the Trp-140 mutant in its free form, the spectrum of the succinate complex lacked a strong band at 11.26 ppm. This may represent the Trp-140 indole NH proton which has been shifted downfield by binding to a succinate carboxylate group. While our results confirm the basic similarity of cytosolic aspartate aminotransferase and E. coli aspartate aminotransferase 1H NMR spectra, they also point out differences that may be useful in identifying resonances. A large number of mutant proteins have been prepared for the E. coli enzyme. The present results provide essential information for future study of these mutants and for study of NMR spectra of isotopically labeled enzyme.

Interactions of Escherichia coli RecA protein with ATP and its analogues in the absence of DNA were studied by circular dichroic (CD) spectroscopy. The binding of RecA protein to ATP increased the CD band of ATP at around 260 nm. The positive CD band of the RecA protein-ATP complex suggested that the bound ATP was in the anti conformation, in accord with X-ray crystallographic data [Story, R.M. and Steitz, T.A. (1992) Nature 355, 374-376]. At pH 7.5 and at 25 degrees C the dissociation constant (Kd) and thermodynamic parameters for the binding of ATP to RecA protein were 18 microM (delta G = -6.5 kcal.mol-1), delta H = 0 kcal.mol-1, and delta S = 22 cal.mol-1.K-1. A non-hydrolyzable ATP analogue, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), gave a spectral change similar to that of ATP. The Kd for this analogue, 22 microM, was very close to the Km of ATP. These results in the absence of single-stranded DNA were different from those obtained by kinetic analysis [Weinstock, G.M. et al. (1981) J. Biol. Chem. 256, 8850-8855], which indicated that the inhibition constant of ATP gamma S was much smaller than the Km of ATP in the presence of DNA. For other ATP analogues (dATP, ADP, and dADP), similar spectral changes were observed, and their Kd values ranged from 19 to 54 microM. UTP, dUTP, and TTP also gave CD spectral changes, but not AMP, GTP, dGTP, CTP, and dCTP.(ABSTRACT TRUNCATED AT 250 WORDS)

We determined the three-dimensional structures of aspartate aminotransferase (AspAT) from Escherichia coli and its complex with inhibitor (2-methyl-L-aspartate) at 1.8A resolution. This enzyme reversibly catalyzes the transamination reaction and is a dimer of two identical subunits. Each subunit has 396 amino acid residues and one pyridoxal 5'-phosphate as a cofactor, and is divided into two domains, one large and the other small. Upon binding of the inhibitor, the small domain rotates by 5 degrees toward the large domain to close the active site. This domain movement is caused mainly by small but important main-chain conformational changes in the residues located over the domain interface of the small domain. In chicken mitochondrial AspAT, the domain movement was larger, with a rotational angle of 13 degrees. By comparison of these two structures, the difference in the rotational angles was found to be caused by the larger opening of the domain in the open form of chicken mitochondrial AspAT. Although the overall structures of these two enzymes were almost identical, the surface area of the domain interface in the E. coli enzyme was larger than that in mitochondrial AspAT, suggesting that the structure of the domain interface is responsible for the degree of movement of the small domain.

Escherichia coli aspartate aminotransferase (AspAT) and E. coli aromatic amino acid aminotransferase (AroAT) have almost identical and high activities toward acidic amino acid substrates. AroAT also has high activity toward aromatic amino acid substrates. The two proteins have 44% amino acid sequence homology. In order to study the mechanism responsible for the different substrate specificities of these aminotransferases, chimeric enzymes of AspAT and AroAT were constructed using homologous recombination in E. coli cells. Five chimeric enzymes were obtained, even though the nucleotide sequence homology between the two parent enzymes was as low as about 50%. The yields of the legitimate chimeric genes were related to the lengths of the homologous region between the two parent genes. Homologous recombination occurred in the region where more than eight nucleotides out of ten were identical. The substrate specificity of the chimeric enzymes suggest that not only the amino acid residues in the active site but also those distant from the active site contribute to the substrate specificity of the parental aminotransferases.

The active-site lysine residue of thermostable aspartate aminotransferase, Lys-239, to which the cofactor, pyridoxal 5'-phosphate (PLP), is bound, has been converted to Cys by site-directed mutagenesis. The thiol group of Cys-239 was chemically aminoethylated with ethylenimine. Amino acid analysis of the modified enzyme showed that it contained about 1 mol of S-(2-aminoethyl)cysteine (SAEC) per mol subunit. The activity of the mutant enzyme (K239SAEC) was about 14% of that of the wild-type enzyme. No significant difference in thermostability was found between the wild-type and K239SAEC enzymes. The UV-visible spectrum of K239SAEC showed a peak (lambda max 380 nm), due to absorption by the cofactor, at a 20 nm longer wavelength than that of the wild-type enzyme. The circular dichroism band due to the bound cofactor of K239SAEC also shifted toward a 20 nm longer wavelength. We determined kinetic parameters (rate constants, kmax, and dissociation constants, Kd, for the substrates) for each half transamination catalyzed by the wild-type and K239SAEC mutant enzymes by the stopped-flow method. The kmax values for the mutant enzyme reactions were 2.6-24 times lower than those for the wild-type enzyme ones. The two enzymes showed similar Kd values for the same substrates except glutamate; the mutant enzyme showed higher affinity for glutamate than the wild-type enzyme.

The recA gene of a thermophilic eubacterial strain, Thermus thermophilus (T.th.) HB8, was cloned from a genomic DNA library by Southern hybridization using a gene-internal fragment amplified by the polymerase chain reaction (PCR) method as the probe. The gene encoded a 36 kDa polypeptide whose amino acid sequence showed 61% identity with that of the Escherichia coli RecA protein. Characteristic amino acid changes between the two RecA proteins were found. In the amino acid composition of the T.th. RecA protein, the number of Pro residues was increased, the number of Cys residues was decreased, and Lys residues were replaced by Arg, Asp by Glu, Thr by Val, and Ile by Val or Leu. These changes are supposed to stabilize the native protein conformation against heat denaturation. The amino acid residues in the nucleotide binding site of the protein and in the protein-protein interaction site responsible for the oligomer formation were well conserved. The T.th. recA gene has the ability to complement the ultraviolet light (UV) sensitivity of a E. coli recA deletion mutant. Thus, the thermophilic bacterium has a RecA protein whose function will be common to the E. coli RecA protein.

A 61-year-old man suffering from compression fracture of the first lumbar vertebra was scheduled for anterior and posterior spinal fusion. Anesthesia was maintained with enflurane, nitrous oxide in oxygen and fentanyl. When his position was changed from right lateral to supine position, ventricular fibrillation (VF) occurred. The operation was discontinued and he was taken into ICU receiving cardiopulmonary resuscitation. Despite ordinary therapies including DC-shock and lidocaine infusion, Vf and defibrillation recurred more than 20 times for the first two hours. Electrocardiogram showed elevation of ST-T segments in II, III, aVF and V1-V3 leads. Because no effective treatment was found, we attempted to use glucose-insulin-potassium mixture, by which Vf stopped. Electrocardiogram taken on the following day showed abnormal Q wave in II, III and aVF leads, and the patient was diagnosed as having had acute myocardial infarction. On the fifth postoperative day, the patient was returned to the ward without neurological deficits. We conclude that glucose-insulin-potassium infusion is a beneficial therapy for Vf, which is resistant to ordinary treatments.

Three mutant Escherichia coli aspartate aminotransferases in which Val39 was changed to Ala, Leu, and Phe by site-directed mutagenesis were prepared and characterized. Among the three mutant and the wild-type enzymes, the Leu39 enzyme had the lowest Km values for dicarboxylic substrates. The Km values of the Ala39 enzyme for dicarboxylates were essentially the same as those of the wild-type (Val39) enzyme. These two mutant enzymes showed essentially the same kcat values for dicarboxylic substrates as did the wild-type enzyme. On the other hand, incorporation of a bulky side-chain at position 39 (Phe39 enzyme) decreased both the affinity (1/Km) and catalytic ability (kcat) toward dicarboxylic substrates. These results show that the position 39 residue is involved in the modulation of both the binding of dicarboxylic substrates to enzyme and the catalytic ability of the enzyme. Although the replacement of Val39 with other residues altered both the kcat and Km values toward various substrates including dicarboxylic and aromatic amino acids and the corresponding oxo acids, it did not alter the ratio of the kcat/Km value of the enzyme toward a dicarboxylic substrate to that for an aromatic substrate. The affinity for aromatic substrates was not affected by changing the residue at position 39. These data indicate that, although the side chain bulkiness of the residue at position 39 correlates well with the activity toward aromatic substrates in the sequence alignment of several aminotransferases [Seville, M., Vincent M.G.,Hahn, K. (1988) Biochemistry 27, 8344-8349], the residue does not seem to be involved in the recognition of aromatic substrates.

Tyr225 in the active site of Escherichia coli aspartate aminotransferase (AspAT) was replaced by phenylalanine or arginine by site-directed mutagenesis. X-ray crystallographic analysis of Y225F AspAT showed that the benzene ring of Phe225 was situated at the same position as the phenol ring of Tyr225 in wild-type AspAT. The mutations resulted in a great decrease in the rate of the transamination reaction, suggesting that Tyr225 is important for efficient catalysis. The kinetic analysis of half-transamination reactions of Y225F AspAT with four substrates (aspartate, glutamate, oxalacetate, and 2-oxoglutarate) and some analogues (2-methylaspartate, succinate, and glutarate) revealed a considerable increase in the affinities for all these compounds. In contrast, affinity for the amino acid substrates was decreased by mutation to arginine, but affinities for the keto acid substrates and the two dicarboxylates (succinate and glutarate) were increased. The electrostatic interaction between O(3') of the coenzyme [pyridoxal 5'-phosphate (PLP)] and the residue at position 225 affected the pKa value of the Schiff base, which is formed between the epsilon-amino group of Lys258 and the aldehyde group of PLP; based on the spectrophotometric titration the pKa values were determined to be 6.8 for wild-type AspAT, 8.5 for Y225F AspAT, and 6.1 for Y225R AspAT in the absence of substrate. The absorption spectra of the three AspATs were almost identical in the acidic pH region, but the spectrum of Y225F AspAT differed from that of wild-type or Y225R AspAT in the alkaline pH region.(ABSTRACT TRUNCATED AT 250 WORDS)

The gene encoding aspartate aminotransferase of a thermophilic Bacillus species, YM-2, has been cloned and expressed efficiently in Escherichia coli. The primary structure of the enzyme was deduced from nucleotide sequences of the gene and confirmed mostly by amino acid sequences of tryptic peptides. The gene consists of 1,176 base pairs encoding a protein of 392 amino acid residues; the molecular mass of the enzyme subunit is estimated to be 42,661 daltons. The active site lysyl residue that binds the coenzyme, pyridoxal phosphate, was identified as Lys-239. Comparison of the amino acid sequence with those of aspartate aminotransferases from other organisms revealed very low overall similarities (13-14%) except for the sequence of the extremely thermostable enzyme from Sulfolobus solfataricus (34%). Several amino acid residues conserved in all the compared sequences include those that have been reported to participate in binding of the coenzyme in three-dimensional structures of the vertebrate and E. coli enzymes. However, the strictly conserved arginyl residue that is essential for binding of the distal carboxyl group of substrates is not found in the corresponding region of the sequences of the thermostable enzymes from the Bacillus species and S. solfataricus. The Bacillus aspartate aminotransferase has been purified from the E. coli clone cell extracts on a large scale and crystallized in the buffered ammonium sulfate solution by the hanging drop method. The crystals are monoclinic with unit cell dimensions a = 121.2 A, b = 110.5 A, c = 81.8 A, and beta = 97.6 degrees, belonging to space group C2, and contain two molecules in the asymmetric unit. The crystals of the enzyme-alpha-methylaspartate complex are isomorphous with those without the substrate analog.

Trp140 of E. coli aspartate aminotransferase has been converted to Phe or Gly by site-directed mutagenesis. As compared to the wild-type enzyme, either of the mutant enzymes showed 10- to 100-fold increase in Km's for natural dicarboxylic substrates, but did not show appreciable changes in Km's for aromatic substrates. Teh kcat values for dicarboxylic and aromatic substrates were greatly decreased by [Trp140----Gly] mutation, but were decreased to lesser extents by [Trp140----Phe] mutation. These findings suggested that N(1) of Trp140 may not be essential for catalysis, but may be partly involved in the binding of the distal carboxylate group of the dicarboxylic substrates.

The catalytic site for C4 of C1s has been presumed to consist of a C4-binding domain and a proteolytic domain. A mAb to C1s, M81, blocked C4 activation and C4 binding to C1s. M81 recognized the H chain of C1s. Using M81 as a probe, we tried to define C4-binding site on C1s. Plasmin digestion of C1s generated four products of Mr 58,000 (P1), 48,000 (P2), 37,000 (P3), and 27,000 (P4). These products, except for P2, all possessed a 26,000-Da H chain fragment (26k-HF) connected to variable-sized L chain pieces. 26k-HF alone had an ability to interact with M81. Amino-terminal amino acid analysis of 26k-HF mapped the epitope for M81 to domain IV and/or V of gamma-domain of C1s. The gamma-domain therefore contains the C4-binding site. The confirm and further elucidate the role of the C4-binding site for C4, we used a substrate-blotting technique in which labeled C4 was incubated with nitrocellulose membrane-fixed C1s and its fragments. C4 was successfully blotted onto C1s and P1, but not P2-P4; i.e., further degradation of the L chain led to the loss of C4-binding. During the incubation, most of the added C4 was converted to C4b. The binding was augmented, if the proteolytic activity of C1s and P1 was blocked, so that the added C4 remained intact. Although C4b also bound to C1s and P1, its binding was less effective and abolished by the addition of cold C4. Based on these results, the gamma-domain and the L chain constitute the catalytic site of C1s to activate C4 to C4b. Moreover, the generated C4b, although it still has weak affinity for C1s, can be replaced by newly coming C4.

Cytosolic and mitochondrial isozymes of aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase [EC 2.6.1.1] ) were purified to homogeneity from rabbit liver. The rabbit liver isozymes were closely similar to the corresponding isozymes from other sources, including human heart, pig heart, chicken heart, and rat liver, in their molecular weights, absorption spectra, amino acid compositions, isoelectric points, and Michaelis constants for the substrates. The NH2-terminal amino acid sequences of rabbit liver isozymes were identified up to 30 residues, and showed some differences from those of the corresponding isozymes obtained from other animals so far studied.

The pH difference absorption spectra of human lysozyme [EC 3.2.1.17] were measured. The difference spectra in the acidic region had a peak at 300 nm, as observed for hen and turkey lysozymes. The pH dependence curve of the extinction difference at 300 nm was well interpreted in terms of the pK values of the catalytic groups (3.4 for Asp 52 and 6.8 for Glu 35 at 0.1 ionic strength and 25 degrees C) determined from the pH dependence of the circular dichroism at 303.5 nm (Kuramitsu et al. (1974) J. Biochem. 76, 671--683) and the fluorescence excited at 305 nm (Kuramitsu et al. (1978) J. Biochem. 83, 159--170). The difference spectra of human lysozyme in the alkaline pH region were characteristic of tyrosyl ionization. The perturbation of tryptophyl residues, which had been observed for hen and turkey lysozymes (KuramitsuHamaguchi (1979) J. Biochem. 85, 443--456), was not observed for human lysozyme. On the basis of the pH dependence curves of the extinction difference at 245 and and 295 nm, we roughly estimated the apparent pK values of the six tyrosyl residues as 9.2 9.2, 10.5, 10.9, 12.4, and 12.5. A time-dependent spectral change observed above pH 11 was not due to the exposure of buried tyrosyl residues on alkali denaturation but was due mainly to disulfide cleavage and exposure of buried tryptophyl residues.

ilvE gene of Escherichia coli was inserted into the region downstream of the tac promotor. As a result, the branched-chain amino acid aminotransferase was overproduced by about a hundred-fold in E. coli W3110. The overproduced aminotransferase was purified from cell extracts about 40-fold to homogeneity. Chemical and physicochemical analyses confirmed that it was a product of the ilvE gene. The enzyme existed in a hexamer with a subunit molecular weight of 34,000; the double trimer model of the enzyme presumed by the previous chemical cross-linking experiments (Lee-Peng, F.-C. et al. (1979) J. bacteriol. 139, 339-345) was supported by electron micrographs. The circular dichroic (CD) spectrum of branch-chain amino acid aminotransferase had double negative maxima at 210 and 220 nm. The alpha-helical content was estimated to be about 40% from the CD spectrum in the region of 200 to 250 nm. The absorption spectrum of the enzyme showed two peaks at 330 and 410 nm. There was no pH-dependent spectral shift. The CD spectrum of the coenzyme, pyridoxal 5'-phosphate, had negative peaks at 330 and 410 nm. These spectral properties of branched-chain amino acid aminotransferase were quite different from those of E. coli aspartate aminotransferase. Each subunit bound approximately 1 mol of pyridoxal 5'-phosphate. A lysyl residue, which forms a Schiff base with the aldehyde group of the pyridoxal 5'-phosphate, was identified in the primary structure of the enzyme.

The interactions of deoxy derivatives of GlcNAc, 6-deoxy-GlcNAc, and 3-deoxy-GlcNAc with hen egg-white lysozyme [EC 3.2.1.17] were studied at various pH's by measuring the changes in the circular dichroic (CD) band at 295 nm. It was shown that 6-deoxy-GlcNAc and 3-deoxy-GlcNAc bind at subsite C of lysozyme and compete with GlcNAc. The pH dependence of the binding constant of 6-deoxy-GlcNAc was the same as that of GlcNAc. On the other hand, the binding constants of 3-deoxy-GlcNAc were 3--10 times smaller than those of GlcNAc in the pH range from 3 to 9. X-ray crystallographic studies show that O(6) and O(3) of GlcNAc at subsite C are hydrogen-bonded to the indole NH's of Trp 62 and Trp 63, respectively, but the above results indicate that Trp 63, not Trp 62, is important for the interaction of GlcNAc with lysozyme.

The tyrB gene of E. coli K-12, which encodes aromatic amino acid aminotransferase (EC 2.6.1.57) was cloned. The nucleotide sequence of about 2 kilobase pairs containing the gene was determined. The coding region of the tyrB gene and the deduced amino acid sequence revealed that the aromatic amino acid aminotransferase of E. coli is homologous with the aspartate aminotransferase.

The pH dependence of the binding constant of (GlcNAc)3 to Asp 52-esterified lysozyme was determined by the fluorescence technique. The pK values of Asp 101 in the modified lysozyme and its complex with (GlcNAc)3 were determined to be 4.5 and 3.6, respectively, at 25 degrees C and 0.1 ionic strength. This result is different from that obtained by Parsons and Raftery ((1972) Biochemistry 11, 1633--1638), who observed no pK shift of Asp 101. The macroscopic pK value of Asp 52 in intact lysozyme determined by them using the pH difference titration data of Asp 52-esterified lysozyme relative to intact lysozyme ((1972) Biochemistry 11, 1623--1629) was 4.5, which is higher by about one pH unit than the pK value determined by our group (Kuramitsu et al. (1974) J. Biochem. 76, 671--683; (1977) ibid. 82, 585--597; (1978) ibid. 83, 159--170. We found that their pH difference titration data in the absence and presence of saccharides can be consistently interpreted in terms of our pK values of Asp 52, Glu 35, and Asp 101, if we assume that the pK value of another ionizable group (probably Asp 48) is perturbed on esterification of Asp 52.

The interactions of beta-methyl-GlcNAc, (GlcNAc)2, and (GlcNAc)3 with hen egg-white lysozyme [EC 3.2.1.17] in which Trp 62 is modified to kynurenine (Kyn 62-lysozyme) were studied by measuring the changes in the CD band and fluorescence due to the kynurenine at various pH values. The pH profiles of the binding constants of these saccharides to Kyn 62-lysozyme were very similar to those to intact lysozyme, although the binding constants were lower for the modified lysozyme than for intact lysozyme. The pK values of Asp 52, Glu 35, Asp 48, and Asp 101 in Kyn 62-lysozyme and in its complexes with beta-methyl-GlcNAc and with (GlcNAc)2 were in agreement with those of intact lysozyme and its complexes. The pK values of Asp 52 and Glu 35 in the modified lysozyme-(GlcNAc)3 complex were also in agreement with those of the complex with intact lysozyme, but the pK shift of Asp 101 was smaller for Kyn 62-lysozyme than for intact lysozyme. The significance of the decreased binding constants to Kyn 62-lysozyme is discussed. The pH dependence of the CD band due to the kynureinine in Kyn 62-lysozyme was interpreted in terms of the participation of the catalytic groups, Asp 52 (apparent pK 3.5) and Glu 35 (apparent pK 6.0), and the amino group of the kynurenine (apparent pK 0.75). This indicates that the ionization of the catalytic groups affects the state around Trp 62 and supports the previous proposal that there is a relation between the state around Trp 62 and the ionization of Glu 35 (Ikeda, K.Hamaguchi, K. (1973) J. Biochem. 74, 221--230; (1975) ibid. 77, 1--16; Nakae et al. (1975) J. Biochem. 77, 993--1006). The intrinsic pK value of the amino group of the kynurenine at position 62 shifted from 2.1 to 0.8 on complexing with (GlcNAc)3, indicating between the kynurenine and the sugar residue at subsite B.

The ilvE gene of the Escherichia coli K-12 ilvGEDA operon, which encodes branched-chain amino acid aminotransferase [EC 2.6.1.42], was cloned. The nucleotide sequence of 1.5 kilobase pairs containing the gene was determined. The coding region of the ilvE gene contained 927 nucleotide residues and could encode 309 amino acid residues. The predicted molecular weight, amino acid composition and the sequence of the N-terminal 15 residues agreed with the enzyme data reported previously (Lee-Peng, F.-C., et al. (1979) J. Bacteriol. 139, 339-345). From the deduced amino acid sequence, the secondary structure was predicted.

The nucleotide sequence of the aspartate aminotransferase [EC 2.6.1.1] structural gene, aspC, of Escherichia coli K-12 was determined. The coding region of the aspC gene contained 1,188 nucleotide residues and encoded 396 amino acid residues. The amino acid sequence deduced from the nucleotide sequence agreed perfectly with that of the protein recently determined for the aspartate aminotransferase of E. coli B (Kondo, K., Wakabayashi, S., Yagi, T.,Kagamiyama, H. (1984) Biochem. Biophys. Res. Commun. 122, 62-67).